Lactobacillus

Appl Microbiol Biotechnol . 2004 Jul 23; {Epub ahead of print}
Quantification by real-time PCR of Lactococcus lactis subsp . cremoris in milk fermented by a mixed culture; Grattepanche F et al.; During cheese making, interactions between different strains of lactic acid bacteria play an important role . However, few methods are available to specifically determine each bacterial population in mixed cultures, in particular for strains of the same species . The aim of this study was to develop a real-time PCR quantification method to monitor the population of Lactococcus cremoris ATCC 19257 in mixed culture with Lactobacillus rhamnosus RW-9595M and the bacteriocin-producing microorganism Lc . diacetylactis UL719 . The specificity of the two primers 68FCa33 and 16SR308 used to amplify a 240-bp fragment of DNA from Lc . cremoris was demonstrated by conventional PCR . Using these primers for real-time PCR, the detection limit was 2 cfu/reaction or 200 cfu of Lc . cremoris ATCC 19257 per millilitre of mixed culture in milk . In pure culture batch fermentation, good correlation was obtained between real-time PCR and the conventional plating method for monitoring Lc . cremoris growth . In mixed culture batch fermentation, Lb . rhamnosus and Lc . cremoris decreased due to nisin Z production by Lc . diacetylactis . The decrease of the Lc . cremoris cell population detected by real-time PCR was not possible to observe by the plate count method in the presence of a Lc . diacetylactis population that was 1 log higher.

Am J Clin Nutr, 2004 Aug, 80(2), 245 - 56
Yogurt and gut function; Adolfsson O et al.; In recent years, numerous studies have been published on the health effects of yogurt and the bacterial cultures used in the production of yogurt . In the United States, these lactic acid-producing bacteria (LAB) include Lactobacillus and Streptococcus species . The benefits of yogurt and LAB on gastrointestinal health have been investigated in animal models and, occasionally, in human subjects . Some studies using yogurt, individual LAB species, or both showed promising health benefits for certain gastrointestinal conditions, including lactose intolerance, constipation, diarrheal diseases, colon cancer, inflammatory bowel disease, Helicobacter pylori infection, and allergies . Patients with any of these conditions could possibly benefit from the consumption of yogurt . The benefits of yogurt consumption to gastrointestinal function are most likely due to effects mediated through the gut microflora, bowel transit, and enhancement of gastrointestinal innate and adaptive immune responses . Although substantial evidence currently exists to support a beneficial effect of yogurt consumption on gastrointestinal health, there is inconsistency in reported results, which may be due to differences in the strains of LAB used, in routes of administration, or in investigational procedures or to the lack of objective definition of "gut health." Further well-designed, controlled human studies of adequate duration are needed to confirm or extend these findings.

Arch Oral Biol, 2004 Sep, 49(9), 719 - 25
A cluster analysis model for caries risk assessment; Sanchez-Perez L et al.; Cluster analysis was applied to determine, the natural grouping of individuals, among sixty 8-10-year-old children, and to identify the most significant set of markers for risk assessment . The risk clusters were obtained with initial clinical and bacteriological measurements including dmf + DMFS, active caries, mutans streptococci and lactobacilli counts in plaque or saliva on two media, and Snyder's test results . The morbidity clusters were constructed with the final clinical indexes and incidence after 18 months (dependent variables) . A risk cluster was identified that included the following significant initial variables; dmf + DMFS, active caries, counts of mutans streptococci from plaque on TSY20B and lactobacilli in saliva, and Snyder's test results . This set of markers identified 86% of the children at high risk who developed high morbidity, as well as 94% of children in the low-risk cluster who developed low or no caries . The results of this investigation provide the basis to develop a system for caries risk assessment.

Am J Reprod Immunol, 2004 Aug, 52(2), 106 - 12
Relationship between cervical mucus interleukin-8 concentrations and vaginal bacteria in pregnancy; Sakai M et al.; PROBLEM: High interleukin (IL)-8 concentration in cervical mucus in the second trimester is a risk factor for premature birth . We investigated the relationship between vaginal pathogens and IL-8 in cervical mucus . METHOD OF STUDY: In 501 women with single pregnancy, vaginal secretions were cultured for bacteria and cervical mucus IL-8 concentrations were measured between 20 and 24 gestational weeks . RESULTS: Lactobacillus species were detected in 56.0% of 84 subjects with high IL-8 (> or =377 ng/mL), significantly less often than in 417 subjects with IL-8 below 377 ng/mL (84.7%; P < 0.0001) . Anaerobic pathogens were detected in 83.3% of high IL-8 subjects, significantly more often than in normal IL-8 subjects (43.9%; P < 0.0001) . By multivariate analysis, cervical IL-8 was significantly high only in subjects without Lactobacillus species; they showed a significantly higher prematurity rate than Lactobacillus-positive subjects . CONCLUSIONS: Absence of vaginal Lactobacilli was associated with increased cervical IL-8 and increased risk of premature delivery.

Biochemistry, 2004 Aug 3, 43(30), 9685 - 94
Multiple substrate binding states and chiral recognition in cofactor-independent glutamate racemase: a molecular dynamics study; Mobitz H et al.; Glutamate racemase (MurI) catalyzes the racemization of glutamate; two cysteine residues serve as catalytic acid and base . On the basis of the crystal structure of MurI from the hyperthermophilic bacterium Aquifex pyrophilus, we performed molecular dynamics (MD) simulations of six different systems to investigate stereochemistry, substrate ligation, and active site protonation state . The catalytic competence of individual systems was assessed by the abundance of reactive conformers . Only systems in which Cys70 is poised to deprotonate d-Glu were found to be catalytically competent (idem Cys178/l-Glu), in agreement with the experimentally observed stereochemistry of Lactobacillus fermentii MurI {Tanner, M . E . et al . (1993) Biochemistry 32, 3998-4006} . Only systems in which the alpha-amino group of l/d-Glu and the imidazole moiety of His are deprotonated are catalytically competent . The active site of MurI displays an unusual flexibility in substrate ligation, and several transitions between stable binding patterns were observed . In catalytically competent binding states, the conserved threonine residues 72, 114, and 117 ligate the alpha-carboxylate of Glu and the Asn71 amides ligate the alpha-amino group of Glu, whereas the delta-carboxylate of Glu is steered by electrostatic repulsion from the Asp7 and Glu147 side chain carboxylates . A network of hydrogen bonds controls the positioning of each thiol/thiolate . In what we term substrate flipping, Glu suddenly rotates into a binding pattern that resembles the post-racemization state of the other enantiomer, i.e., each enantiomer can be bound in two distinct states . Substrate flipping and unfavorable substrate binding successively trigger dissociation of the substrate, accompanied by an opening of the active site channel . We explain how the weak binding of Glu contributes to catalysis and suggest a mechanism by which binding mismatches are propagated into an opening of the active site.

Sex Transm Dis, 2004 Aug, 31(8), 465 - 8
Assay for establishing whether microbicide applicators have been exposed to the vagina; Wallace A et al.; OBJECTIVES: To develop an accurate, rapid, and inexpensive method for verifying vaginal applicator use . GOAL: To develop a method for assessing compliance in microbicide clinical trials . STUDY DESIGN: Single use Microlax applicators containing a placebo formulation either were or were not exposed to the vagina . Three assays were developed to determine whether the applicators had been used vaginally . RESULTS: Blinded examiners were able to discern 63% of the time whether or not applicator tips had been exposed to the vagina . Optical density (to measure lactobacilli), increased in media exposed to used applicators but not in media exposed to unused applicators . When tips of applicators were stained with trypan blue, used applicators could be distinguished easily from unused applicators . CONCLUSION: Staining of applicator is accurate, simple, rapid, and inexpensive . This method could be be used in clinical settings in the developing world . Dying applicator tips could prove useful in excluding non-compliant subjects, analyzing data, or developing social intervention strategies to improve compliance.

Clin Exp Immunol, 2004 Aug, 137(2), 393 - 401
Role of peptide antigen for induction of inhibitory antibodies to Streptococcus mutans in human oral cavity; Tsuha Y et al.; The alanine-rich repeating region (A-region) in the surface protein antigen (PAc) of Streptococcus mutans has received much attention as an antigenic component for vaccines against dental caries . The PAc (residue 361-386) peptide in the A-region possesses a multiple binding motif (L- -V-K- -A) to various HLA-DR molecules and a B-cell core epitope (- Y- - -L- -Y- - - -) that recognizes the inhibiting antibody to S . mutans . In the present study, we investigated the immunogenicity of the PAc (361-386) peptide in humans and regulators of induction of the anti-PAc (361-386) peptide IgA antibody (aPPA) in saliva . The PAc (361-386) peptide was confirmed as an ideal peptide antigen for induction of the inhibiting antibody to S . mutans in 151 healthy human subjects (36.6 +/- 12.6 years old) by quantitative analyses of oral bacteria and ELISA, as the aPPA titre in human saliva decreased significantly in an age-dependent manner . Homozygous DRB1*0405 and 1502, and heterozygous DRB1*0405/1502 showed a negative association with production of aPPA and tended to reduce the number of total streptococci in saliva . In contrast, the DRB1*1501 allele was significantly correlated with a high level of induction of the antibodies, and also tended to reduce lactobacilli and mutans streptococci . Further, peptide immunogenicity was confirmed in NOD-SCID mice grafted with human peripheral blood mononuclear cells . Our results indicate that the interplay between regulators such as age, DRB1 genotype, cytokines, and peptide immunogenicity may provide a potential means for developing a vaccine useful for the prevention of dental caries as well as their diagnosis.

J Food Prot, 2004 Jul, 67(7), 1429 - 32
Influence of calcium lactate on the fate of spoilage and pathogenic microorganisms in orange juice; Yeh JY et al.; Calcium lactate is used by the beverage industry as a source of calcium to fortify fruit juice . The objective of this study was to evaluate the influence of various concentrations of calcium lactate on the fate of pathogenic and spoilage microorganisms in orange juice . Commercial nonfortified orange juice was supplemented with calcium lactate at a concentration equivalent to 0, 5, 10, 15, 20, 25, or 30% dietary reference intake . The pH of each fortified juice was adjusted to 3.6 or 4.1 . The prepared juice samples were inoculated separately with a three-strain mixture of salmonellae, a three-strain mixture of spoilage yeasts, and three single strains of spoilage bacteria including Alicyclobacillus acidoterrestris, Lactobacillus plantarum, and Lactobacillus sake . The contaminated juice was stored at 4 and 10 degrees C, respectively, for 6 to 7 weeks and assayed once a week for populations of salmonellae, spoilage yeasts, or spoilage bacteria . The results indicated that A . acidoterrestris was inhibited in all juice stored at 4 degrees C and low-pH juice stored at 10 degrees C . The bacterium, however, was able to grow at 10 degrees C in the high-pH juice with calcium lactate concentrations equivalent to 0 and 5% dietary reference intake . The cells of L . sake declined and eventually died off in low-pH juice stored at 4 and 10 degrees C and in high pH stored at 4 degrees C . But the organism flourished at 10 degrees C in the high-pH juice containing 0, 10, and 20% dietary reference intake of calcium lactate . The populations of L . plantarum remained approximately stable in low- as well as in high-pH juice stored at both 4 and 10 degrees C . While inhibited at 4 degrees C, the spoilage yeasts grew at 10 degrees C . Salmonellae died off in all juice stored at 4 degrees C and in low-pH juice stored at 10 degrees C . However, they persisted in the high-pH juice stored at 10 degrees C except in the samples that contained 20 to 30% dietary reference intake of calcium lactate.

Life Sci, 2004 Aug 20, 75(14), 1727 - 34
Effects of Lactobacillus helveticus fermented milk on bone cells in vitro; Narva M et al.; Milk fermented with Lactobacillus helveticus (L . helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE) . We investigated the effects of L . helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro . An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells . An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells . The Lh-whey increased bone formation 1.3-1.4 times with the 1 x 10(-5), 1 x 10(-4) and 1 x 10(-3) solutions . The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect . No significant effects were observed on osteoclasts in vitro with any of the study products . L . helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro . The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L . helveticus fermented milk whey.

Bioconjug Chem, 2004 Jul-Aug, 15(4), 685 - 93
Synthesis and characterization of bioconjugates of S-layer proteins; Sampathkumar P et al.; The self-assembling proteins that form crystalline surface layers (S-layers) on many microbial species have found numerous applications due to their nanostructured nature . To devise a new method to construct surface displays that exploit S-layer self-assembly activity and nanostructural properties, we have constructed polymer bioconjugates of S-layer proteins . The conjugates formed are similar in function to the monomer alkanethiols that form self-assembled monolayers (SAMs) on gold surfaces . However, the self-assembly is driven by the protein "headgroup" that positions polymer-tethered endgroups on a surface . This paper examines the integration of protein purification, conjugation, and surface assembly that has led to the development of this new method for the formation of nanostructured surfaces . Purified S-layer proteins from Lactobacillus brevis were conjugated with small molecule probes and polymers using amine-based reactions . To keep multiple labeling of protein amine groups to acceptable levels, the conjugations were performed at pH 6.5, allowing for limited yields (24-39%) as determined by mass spectrometry and SDS-polyacrylamide gel electrophoresis . As the presence of high levels of unlabeled S-layer proteins is undesired, we have developed a protocol for further purification that employs monomeric avidin affinity chromatography . The surface self-assembly of the polymer bioconjugates onto amine-terminated microspheres was studied using epi-fluorescence, confocal, and scanning electron microscopy . The surfaces obtained exhibited homogeneous distributions of tethered molecules . Also, in cases where the modular assembly of two distinct types of tethered endgroups was accomplished, there was no evidence for phase separation in the surfaces . The modular assembly method will provide a potential route to controlling surface display density as the starting assembly conditions guide displayed endgroup concentrations in mixed molecular monolayers.

Arch Anim Nutr, 2004 Jun, 58(3), 245 - 54
The effects of lactic acid bacteria inoculants and formic acid on the formation of biogenic amines in grass silages; Steidlova S et al.; Silages were prepared in six laboratory experiments from four direct-cut grassland swards and pure swards of perennial ryegrass and false oat with dry matter contents ranging between 180 and 325 g/kg . Grass was fermented at 22 degrees C and silages were stored at the same temperature for 4 months . Untreated silages (negative control) and silages preserved with 3 g/kg of formic acid (positive control) were compared with silages inoculated with commercial strains of Lactobacillus plantarum, Lactobacillus buchneri and a mixed preparation Microsil . The inoculants were applied at a dose of 5.10(6) CFU/g of grass . Seven biogenic amines were extracted from silages with perchloric acid and determined as N-benzamides by micellar electrokinetic capillary chromatography . Common chemical quality parameters of silages were also determined . Tyramine, cadaverine and putrescine were the amines occurring at the highest concentration . As compared to untreated silages, formic acid was most effective to suppress formation of the main amines . Also the inoculants often decreased amine contents significantly (P < 0.05) . The inoculants decreased levels of polyamine spermidine more efficiently than formic acid . Contents of histamine, tryptamine and polyamine spermine were very low, commonly below the detection limits.

Eur J Obstet Gynecol Reprod Biol, 2004 Aug 10, 115(2), 148 - 53
Efficient prematurity prevention is possible by pH-self measurement and immediate therapy of threatening ascending infection; Hoyme UB et al.; In two prospective investigations, the effectiveness of the self care programme for prematurity prevention, developed by Saling, was investigated . Pregnant women in Erfurt have been offered to perform self measurements of their vaginal pH by means of test gloves twice a week in order to screen for any disturbances in the vaginal milieu . The women were instructed to see their physician immediately, if abnormal pH > or = 4.7 or other risk factors were present, in order to get them confirmed and to start lactobacillus acidophilus therapy or in case of bacterial vaginosis to treat with clindamycin cream i . vag . Patients who were not interested in the programme served as a control group . Seventy-three out of 381 women in the intervention group have been identified as risk cases . Fifty-eight of them were treated with a lactobacillus preparation, and 24 with clindamycin cream, three patients refused to have any therapy . In this study, the prematurity rate was 8.1% in the self measurement/intervention group versus 12.3% in the control group (P < 0.05, n = 2341) . 0.3% versus 3.3% of the neonates belonged to the group of very early prematures with a gestational age of <32 + 0 (P < 0.01) . PROM was registered in 22.8% versus 30.8% (P < 0.001), respectively . Starting 1 March 2000, a similar statewide pH screening programme was initiated in order to reduce prematurity in the State of Thuringia . According to the study design, a significant decrease of prematurity was hypothetically expected for the second half of 2000 . In Erfurt, an overall decrease of prematurity from 7.68 to 6.81% and a reduction of cases < or = 32 weeks from 3.22 to 2.39% was observed . Premature rupture of membranes was seen in 19 versus 0 pregnancies with early prematurity (n = 1600) . Data from 16,276 women are available for the state of Thuringia . On this basis, a significant reduction of early prematurity from 1.58 to 0.99% was seen, respectively (P < 0.001) . Comparing low birthweights a significant reduction of cases was achieved as well in all groups . On the basis of the data obtained we recommend the extension of the campaign in whole of Germany.

J Ind Microbiol Biotechnol, 2004 Sep, 31(8), 362 - 8 Epub 2004 Jul 15.
Inhibition of yeast by lactic acid bacteria in continuous culture: nutrient depletion and/or acid toxicity?
Bayrock DP, Ingledew WM.
Lactic acid was added to batch very high gravity (VHG) fermentations and to continuous VHG fermentations equilibrated to steady state with Saccharomyces cerevisiae . A 53% reduction in colony-forming units (CFU) ml(-1) of S . cerevisiae was observed in continuous fermentation at an undissociated lactic acid concentration of 3.44% w/v; and greater than 99.9% reduction was evident at 5.35% w/v lactic acid . The differences in yeast cell number in these fermentations were not due to pH, since batch fermentations over a pH range of 2.5-5.0 did not lead to changes in growth rate . Similar fermentations performed in batch showed that growth inhibition with added lactic acid was nearly identical . This indicates that the apparent high resistance of S . cerevisiae to lactic acid in continuous VHG fermentations is not a function of culture mode . Although the total amount of ethanol decreased from 48.7 g l(-1) to 14.5 g l(-1) when 4.74% w/v undissociated lactic acid was added, the specific ethanol productivity increased ca . 3.2-fold (from 7.42 x 10(-7) g to 24.0 x 10(-7) g ethanol CFU(-1) h(-1)), which indicated that lactic acid stress improved the ethanol production of each surviving cell . In multistage continuous fermentations, lactic acid was not responsible for the 83% (CFU ml(-1)) reduction in viable S . cerevisiae yeasts when Lactobacillus paracasei was introduced to the system at a controlled pH of 6.0 . The competition for trace nutrients in those fermentations and not lactic acid produced by L . paracasei likely caused the yeast inhibition.

Microbiology, 2004 Jul, 150(Pt 7), 2099 - 112
Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase; Kralj S et al.; Lactobacillus reuteri strain 121 uses sucrose for synthesis of a unique, soluble glucan ('reuteran') with mainly alpha-(1-->4) glucosidic linkages . The gene (gtfA) encoding this glucansucrase enzyme had previously been characterized . Here, a detailed biochemical and molecular analysis of the GTFA enzyme is presented . This is believed to be the first report describing reuteransucrase enzyme kinetics and the oligosaccharides synthesized with various acceptors . Alignments of the GTFA sequence with glucansucrases from Streptococcus and Leuconostoc identified conserved amino-acid residues in the catalytic core critical for enzyme activity . Mutants Asp1024Asn, Glu1061Gln and Asp1133Asn displayed 300- to 1000-fold-reduced specific activities . To investigate the role of the relatively large N-terminal variable domain (702 amino acids) and the relatively short C-terminal putative glucan-binding domain (267 amino acids, with 11 YG repeats), various truncated derivatives of GTFA (1781 amino acids) were constructed and characterized . Deletion of the complete N-terminal variable domain of GTFA (GTFA-Delta N) had little effect on reuteran characteristics (size, distribution of glycosidic linkages), but the initial transferase activity of the mutant enzyme increased drastically . Sequential C-terminal deletions (up to six YG repeats) in GTFA-Delta N also had little effect on reuteran characteristics . However, enzyme kinetics drastically changed . Deletion of 7, 8 or 11 YG repeats resulted in dramatic loss of total enzyme activity (43-, 63- and 1000-fold-reduced specific activities, respectively) . Characterization of sequential C-terminal deletion mutants of GTFA-Delta N revealed that the C-terminal domain of reuteransucrase has an important role in glucan binding.

J Gen Appl Microbiol, 2004 Apr, 50(2), 79 - 90
Isolation and characterization of lactobacilli from some traditional fermented foods and evaluation of the bacteriocins; Jamuna M et al.; Lactic acid bacteria (LAB) commonly used in food as starter cultures are known to produce antimicrobial substances such as bacteriocins and have great potential as food biopreservatives . LAB isolated from traditional fermented foods (appam batter and pickles) were screened for bacteriocin production . Two lactobacilli, LABB and LABP (one from each source) producing bacteriocins were characterized . Both the bacilli were homo-fermentative, catalase negative and micro-aerophilic in nature . LABB was found to be a thermobacterium growing at 45 degrees C while LABP was a streptobacterium growing at 15 degrees C . Both were able to grow at pH 4.5-8.6 but were intolerant to high salt concentration . They failed to produce gas from glucose as well as ammonia from arginine . Among the sugars examined they could not ferment arabinose, raffinose, rhamnose or xylose . Additionally, LABB could not ferment esculin, gluconate or mannose . LABB is identified as Lactobacillus acidophilus while LABP as Lb . casei . Their bacteriocins showed a broad inhibitory spectrum against the indicator organisms tested . They were active below pH 8.0 and after autoclaving as well . There was a complete loss of activity when treated with proteolytic enzymes such as trypsin indicating the proteinaceous nature of the active molecules . SDS-PAGE of partially purified bacteriocins indicated the molecular mass of the bacteriocin as 3.8 and 4.5 kDa for LABB and LABP respectively.

Int J Food Microbiol, 2004 Aug 1, 94(3), 287 - 99
Phenotypic and PCR-based characterization of the microflora in Norvegia cheese during ripening; Ostlie HM et al.; Microbiological sampling of Norvegia cheese from three cheese factories was done during ripening . The evolution of aerobic mesophilic bacteria, lactococci, lactobacilli, enterococci, presumptive leuconostoc and pediococci was investigated after 30, 90, 180 and 270 days of ripening . Isolates (135) of non-starter lactic acid bacteria (NSLAB) from nine Norvegia cheeses after 90, 180 and 270 days of ripening were examined . The isolates were tested by physiological and biochemical assays, species-specific PCR and 16S rDNA sequencing . After 90 days of ripening Leuconostoc spp., most probably from the starter, and the NSLAB specie Lactobacillus paracasei dominated among the isolates, however, after longer ripening Lb . paracasei dominated . The development and evolution of the microflora in Norvegia varied according to dairy and ripening time.

J Appl Microbiol, 1998 Jan, 84(1), 125 - 32
Selection of lactobacilli for chicken probiotic adjuncts; Garriga M et al.; During inhibitory activity screening of 296 strains of lactic acid bacteria from the gastro-intestinal tract of chicks, 77 strains showed inhibition against enteric indicator strains (Salmonella enteritidis and Escherichia coli) . Eight different strains identified as Lactobacillus salivarius were selected for the following attributes: their ability to inhibit all the indicator strains; a high adhesion efficiency to the epithelial cells of chickens and also their resistance to a number of antibiotics, monensin, bile salts and pH 3.0 . The inhibitory action was not affected by the addition of catalase and no inhibition was detected after neutralizing the supernatant culture fluid . The competitiveness of the most promising strains, Lact . salivarius CTC2183 and CTC2197, was assessed in chicken feed mixture and in vivo . It was concluded that both strains were capable of becoming predominant over the indigenous flora in the incubated chicken feed mixture . In vivo tests showed that Lact . salivarius CTC2197 was able to colonize and overcome Lact . salivarius CTC2183 and the indigenous flora in the crop and caecum of the inoculated chicks.

J Appl Microbiol, 1998 Jan, 84(1), 97 - 102
Cultural conditions for the production of bacteriocin by a native isolate of Lactobacillus delbruecki ssp . bulgaricus CFR 2028 in milk medium; Balasubramanyam BV et al.; The effect of growth parameters and the molecular basis for antibacterial activity by a natural isolate of Lactobacillus delbruecki ssp . bulgaricus CFR 2028 was studied . The inhibition was tested against a toxigenic strain of Bacillus cereus F 4810 . When grown in milk medium, the activity was highest at an incubation temperature of 37 degrees C in 48 h . The antibacterial activity appeared to be produced between late logarithmic and early stationary phases . The active principle was proteinaceous in nature (bacteriocin) and stable to low pH (3.8-5.0) and heat (75 degrees C for 30 min) . There was also the possible role of hydrogen peroxide in bringing about inhibition . The strain of Lact . delbruecki ssp . bulgaricus CFR 2028 revealed the presence of plasmid DNA bands of 9.4 and 6.5 kbp, respectively, in agarose gel electrophoresis . The above strain has the potential to be used as a biopreservative in popular Indian fermented foods.

J Appl Microbiol, 1998 Jan, 84(1), 72 - 80
Lactobacillus helveticus heterogeneity in natural cheese starters: the diversity in phenotypic characteristics; Fortina MG et al.; The study of wild strains from natural habitats is a useful means of understanding better the heterogeneity within a species of biotechnological importance, and of obtaining atypical isolates with unknown capabilities . In the present research carried out on different Lactobacillus helveticus strains isolated from natural cheese starters, it was observed that several biotechnologically important characteristics can differ greatly between strains . Biotypes were found which differ in terms of fructose, maltose and trehalose fermentation, acidifying activity, proteolytic and peptidase activity, and antibiotic and lysozyme resistance . The possibility of choosing Lact . heleveticus strains with specific biotechnological profiles will influence the quality and the variety of dairy products.

J Clin Microbiol, 2004 Jul, 42(7), 3128 - 36
Quantitative analysis of diverse Lactobacillus species present in advanced dental caries; Byun R et al.; Our previous analysis of 65 advanced dental caries lesions by traditional culture techniques indicated that lactobacilli were numerous in the advancing front of the progressive lesion . Production of organic acids by lactobacilli is considered to be important in causing decalcification of the dentinal matrix . The present study was undertaken to define more precisely the diversity of lactobacilli found in this environment and to quantify the major species and phylotypes relative to total load of lactobacilli by real-time PCR . Pooled DNA was amplified by PCR with Lactobacillus genus-specific primers for subsequent cloning, sequencing, and phylogenetic analysis . Based on 16S ribosomal DNA sequence comparisons, 18 different phylotypes of lactobacilli were detected, including strong representation of both novel and gastrointestinal phylotypes . Specific PCR primers were designed for nine prominent species, including Lactobacillus gasseri, L . ultunensis, L . salivarius, L . rhamnosus, L . casei, L . crispatus, L . delbrueckii, L . fermentum, and L . gallinarum . More than three different species were identified as being present in most of the dentine samples, confirming the widespread distribution and numerical importance of various Lactobacillus spp . in carious dentine . Quantification by real-time PCR revealed various proportions of the nine species colonizing carious dentine, with higher mean loads of L . gasseri and L . ultunensis than of the other prevalent species . The findings provide a basis for further characterization of the pathogenicity of Lactobacillus spp . in the context of extension of the carious lesion.

J Clin Microbiol, 2004 Jul, 42(7), 3023 - 9
Molecular analysis of the microflora associated with dental caries; Munson MA et al.; Molecular techniques have revealed many novel, presumed unculturable, taxa in oral infections . The aim of this study was to characterize the bacterial community of the middle and advancing front of carious dental lesions by cultural and molecular analyses . Samples were collected with a hand excavator from five teeth with carious lesions involving dentine . Samples were cultured on blood agar and Rogosa agar incubated in air plus 5% CO(2) and on fastidious anaerobe agar anaerobically . DNA was also extracted directly from the samples and 16S rRNA genes were amplified by PCR with universal primers . PCR products were singularized by cloning, and the cloned inserts and cultured isolates were identified by 16S rRNA gene sequence analysis . We identified 95 taxa among the 496 isolates and 1,577 clones sequenced; 44 taxa were detected by the molecular method alone; 31 taxa were previously undescribed . Only three taxa, Streptococcus mutans, Rothia dentocariosa, and an unnamed Propionibacterium sp., were found in all five samples . The predominant taxa by anaerobic cultivation were the novel Propionibacterium sp . (18%), Olsenella profusa (14%), and Lactobacillus rhamnosus (8%) . The predominant taxa in the molecular analysis were Streptococcus mutans (16%), Lactobacillus gasseri/johnsonii (13%), and Lactobacillus rhamnosus (8%) . There was no significant difference between the compositions of the microflora in the middle and advancing front samples (P < 0.05, Wilcoxon matched pairs, signed ranks test) . In conclusion, combined cultural and molecular analyses have shown that a diverse bacterial community is found in dentinal caries and that numerous novel taxa are present.

Clin Diagn Lab Immunol, 2004 Jul, 11(4), 675 - 9
Reduction of influenza virus titer and protection against influenza virus infection in infant mice fed Lactobacillus casei Shirota; Yasui H et al.; We investigated whether oral administration of Lactobacillus casei strain Shirota to neonatal and infant mice ameliorates influenza virus (IFV) infection in the upper respiratory tract and protects against influenza infection . In a model of upper respiratory IFV infection, the titer of virus in the nasal washings of infant mice administered L . casei Shirota (L . casei Shirota group) was significantly (P < 0.05) lower than that in infant mice administered saline (control group) (10(2.48) +/- 10(0.31) and 10(2.78) +/- 10(0.4), respectively) . Further, the survival rate of the L . casei Shirota group was significantly (P < 0.05) higher than that of the control group (14.3 versus 40.0%) . One day after infection, pulmonary NK cell activity and interleukin-12 production by mediastinal lymph node cells of mice in the L . casei Shirota group were significantly greater than those of mice in the control group . These findings suggest that oral administration of L . casei Shirota activates the immature immune system of neonatal and infant mice and protects against IFV infection . Therefore, oral administration of L . casei Shirota may accelerate the innate immune response of the respiratory tract and protect against various respiratory infections in neonates, infants, and children, a high risk group for viral and bacterial infections.

Lett Appl Microbiol, 2004, 39(2), 137 - 43
High-level gene expression in Lactobacillus plantarum using a pheromone-regulated bacteriocin promoter; Mathiesen G et al.; AIMS: To use promoters and regulatory genes involved in the production of the bacteriocin sakacin P to obtain high-level regulated gene expression in Lactobacillus plantarum . METHODS AND RESULTS: In a plasmid containing all three operons naturally involved in sakacin P production, the genes encoding sakacin P and its immunity protein were replaced by the aminopeptidase N gene from Lactococcus lactis (pepN) or the beta-glucuronidase gene from Escherichia coli (gusA) . The new genes were precisely fused to the start codon of the sakacin P gene and the stop codon of the immunity gene . This set-up permitted regulated (external pheromone controlled) overexpression of both reporter genes in L . plantarum NC8 . For PepN, production levels amounted to as much as 40% of total cellular protein . CONCLUSIONS: Promoters and regulatory genes involved in production of sakacin P are suitable for establishing inducible high-level gene expression in L . plantarum . SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a system for controllable gene expression in lactobacilli, giving some of the highest expression levels reported so far in this genus.

J Allergy Clin Immunol, 2004 Jul, 114(1), 131 - 6
Lactobacillus GG effect in increasing IFN-gamma production in infants with cow's milk allergy; Pohjavuori E et al.; BACKGROUND: Probiotic bacteria are potentially beneficial to maturation of the infant's immune system . OBJECTIVE: To examine the role of probiotic bacteria in treatment of cow's milk allergy (CMA) and IgE-associated dermatitis, we investigated the immunologic effects of Lactobacillus rhamnosus GG (LGG) and a mixture of 4 bacterial species (MIX) . METHODS: In a randomized, double-blind study design, concomitantly with elimination diet and skin treatment, LGG, MIX, or placebo was given for 4 weeks to infants with suspected CMA . After anti-CD3 (OKT3) and anti-CD28 stimulation of PBMCs, IFN-gamma, IL-4, IL-5, and IL-12 levels were measured in culture supernatants by ELISA . Intracellular IFN-gamma, IL-4, and IL-5 production on CD4 lymphocytes was analyzed with fluorescence-activated cell sorting . RESULTS: Secretion of IFN-gamma by PBMCs before the treatment was significantly lower in infants with CMA (P=.016) and in infants with IgE-associated CMA (P=.003) than in non-CMA infants . Among the infants who received LGG, the level of secreted IFN-gamma increased in those with CMA (P=.006) and in those with IgE-associated dermatitis (P=.017) when compared with the placebo group . Secretion of IL-4 increased significantly in infants with CMA in the MIX (P=.034) but not in the LGG group . CONCLUSION: Deficiency in IFN-gamma response appears to be related to CMA . LGG raises IFN-gamma production of PBMC in infants with CMA and in infants with IgE-associated dermatitis and may thus provide beneficial TH1 immunomodulatory signals . MIX, although containing LGG, appears to modulate the immune responses differently .

Appl Environ Microbiol, 2004 Jul, 70(7), 4286 - 92
Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds; Wisselink HW et al.; To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity . High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol . Moreover, the formed mannitol was not reutilized upon glucose depletion . Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L . lactis seemed to be the most promising strain for mannitol production.

Appl Environ Microbiol, 2004 Jul, 70(7), 3821 - 30
Specific response of a novel and abundant Lactobacillus amylovorus-like phylotype to dietary prebiotics in the guts of weaning piglets; Konstantinov SR et al.; Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp) . An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes . Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity . A higher number of DGGE bands in the colon (24.2 +/- 5.5) than in the ileum (9.7 +/- 4.2) was observed in all samples . In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control . Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene . This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates . Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH . The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.

Int J Food Microbiol, 2004 Aug 15, 95(1), 11 - 8
Spectrum of bacteriocin activity of Lactobacillus plantarum BS and fingerprinting by RAPD-PCR; Elegado FB et al.; The spectrum of antimicrobial activity of Lactobacillus plantarum BS against representative bacterial species was established through deferred assay and 'spot-on-lawn' assay using actively growing cells and partially purified bacteriocin extract, respectively . Only lactobacilli, pediococci, enterococci, bacilli and Listeria were inhibited from the test microorganisms . Slight bacteriocinogenic activity through 'spot-on-lawn' assay was detected against Staphylococcus aureus and Escherichia coli O157:H7 . Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis was used to compare the fingerprint of L . plantarum BS with other strains of L . plantarum . Using the 16S rRNA-based primer, P32, the bacteriocinogenic isolate exhibited identical RAPD-PCR fingerprints to L . plantarum ATCC 14917 . Dendrograms derived from the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) were constructed to show the similarity relationships among the investigated strains based on RAPD-PCR analysis . Bands differentiating L . plantarum BS from L . plantarum ATCC 14917 were also identified by varying the annealing temperature.

J Appl Microbiol, 2004, 97(2), 446 - 58
Polyphasic study of the genetic diversity of lactobacilli associated with 'Almagro' eggplants spontaneous fermentation, based on combined numerical analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoresis patterns; Sanchez I et al.; AIMS: The goal of this study was to assess the genetic diversity of lactic acid bacteria (LAB) from the complex natural ecosystem present in the spontaneous fermentation of 'Almagro' eggplants by a polyphasic approach based on molecular techniques . METHODS AND RESULTS: Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) were applied to 149 Lactobacillus isolates obtained from that fermentation process . Two random primers, OPL-05 and ArgDei-For, and two rare-cutting enzymes, SfiI and SmaI, chosen after preliminary testing on the basis of band intensity and distribution, were used . RAPD and PFGE generated electrophoretic patterns suitable for strain discrimination, but further discrimination was achieved when combined numerical analysis of the results from both methods and the results previously obtained by SDS-PAGE whole cell protein analysis, was carried out . The findings indicated a considerable degree of genomic diversity in the LAB microbiota studied and especially in the Lactobacillus plantarum isolates . In terms of species assignment, the polyphasic study allowed a definite and well-founded identification of 98.7% of the isolates . CONCLUSIONS: The combined numerical analysis of RAPD and PFGE patterns represented a useful tool to discriminate the diversity of the Lactobacillus strains responsible for the spontaneous fermentation of this pickle . The species identification and strain typing results from the polyphasic study were regarded as the most exact compromise yielding the fewest contradictions based on the available data . SIGNIFICANCE AND IMPACT OF THE STUDY: Combined numerical analysis of RAPD-PCR and PFGE patterns has not yet been employed to study the genetic diversity of LAB from an ecosystem like that found in fermenting vegetables.

J Appl Microbiol, 2004, 97(2), 314 - 22
Development of polythene films for food packaging activated with an antilisterial bacteriocin from Lactobacillus curvatus 32Y; Mauriello G et al.; AIMS: The aims of this work were to (i) use a bacteriocin produced by Lactobacillus curvatus 32Y active against Listeria monocytogenes to activate polythene films by different methods, (ii) implement a large-scale process for antilisterial polythene films production and (iii) verify the efficacy of the developed films in inhibiting the growth of L . monocytogenes during the storage of meat products . METHODS AND RESULTS: The film was made active by using the antilisterial bacteriocin 32Y by Lact . curvatus with three different procedures: soaking, spraying and coating . The antimicrobial activity of the activated films was tested in plate assays against the indicator strain L . monocytogenes V7 . All the used procedures yielded active polythene films although the quality of the inhibition was different . The coating was therefore employed to develop active polythene films in an industrial plant . The antimicrobial activity of the industrially produced films was tested in experiments of food packaging involving pork steak and ground beef contaminated by L . monocytogenes V7 at roughly 10(3) CFU cm(-2) and gram respectively . The results of the challenge tests showed the highest antimicrobial activity after 24 h at 4 degrees C, with a decrease of about 1 log of the L . monocytogenes population . CONCLUSIONS: Antimicrobial packaging can play an important role in reducing the risk of pathogen development, as well as extending the shelf life of foods . SIGNIFICANCE AND IMPACT OF THE STUDY: Studies of new food-grade bacteriocins as preservatives and development of suitable systems of bacteriocin treatment of plastic films for food packaging are important issues in applied microbiology and biotechnology, both for implementing and improving effective hurdle technologies for a better preservation of food products.

J Agric Food Chem, 2004 Jul 14, 52(14), 4577 - 81
Chemical moieties and interactions involved in the binding of zearalenone to the surface of Lactobacillus rhamnosus strains GG; El-Nezami H et al.; Viable, heat-and acid-killed Lactobacillus rhamnosus strain GG (LGG) has shown high binding properties with zearalenone (ZEN) . To identify the type of chemical moieties and interactions involved in binding with the ZEN, LGG was subjected to different chemical and enzymatical treatments, prior to the binding experiments . Pretreating the viable, heat- and acid-killed bacteria with m-periodate significantly decreased ZEN binding, suggesting that ZEN binds predominantly to carbohydrate components . Pretreatment with Pronase E had no effect on the ability of viable cells to bind ZEN, however, a reduction in the binding of ZEN by heat- and acid-killed cells, suggesting that the new binding sites exposed by heat or acid are proteins in nature . Pretreatment with urea also decreased binding, suggesting that hydrophobic interactions play a role in ZEN binding . The binding of ZEN in concentrations ranging from 0.79 to 62.82 microM and its subsequent dissociation by repetitive aqueous washes was also studied . The binding sites of the bacteria were not saturated by the maximum ZEN concentration studied.

J Bacteriol, 2004 Jul, 186(14), 4543 - 55
The Lactobacillus casei ptsHI47T mutation causes overexpression of a LevR-regulated but RpoN-independent operon encoding a mannose class phosphotransferase system; Maze A et al.; A proteome analysis of Lactobacillus casei mutants that are affected in carbon catabolite repression revealed that a 15-kDa protein was strongly overproduced in a ptsHI47T mutant . This protein was identified as EIIA of a mannose class phosphotransferase system (PTS) . A 7.1-kb DNA fragment containing the EIIA-encoding open reading frame and five other genes was sequenced . The first gene encodes a protein resembling the RpoN (sigma54)-dependent Bacillus subtilis transcription activator LevR . The following pentacistronic operon is oriented in the opposite direction and encodes four proteins with strong similarity to the proteins of the B . subtilis Lev-PTS and one protein of unknown function . The genes present on the 7.1-kb DNA fragment were therefore called levR and levABCDX . The levABCDX operon was induced by fructose and mannose . No "-12, -24" promoter typical of RpoN-dependent genes precedes the L . casei lev operon, and its expression was therefore RpoN independent but required LevR . Phosphorylation of LevR by P approximately His-HPr stimulates its activity, while phosphorylation by P approximately EIIBLev inhibits it . Disruption of the EIIBLev-encoding levB gene therefore led to strong constitutive expression of the lev operon, which was weaker in a strain carrying a ptsI mutation preventing phosphorylation by both P approximately EIIBLev and P approximately His-HPr . Expression of the L . casei lev operon is also subject to P-Ser-HPr-mediated catabolite repression . The observed slow phosphoenolpyruvate- and ATP-dependent phosphorylation of HPrI47T as well as the slow phosphoryl group transfer from the mutant P approximately His-HPr to EIIALev are assumed to be responsible for the elevated expression of the lev operon in the ptsHI47T mutant .

Folia Microbiol (Praha), 2004, 49(2), 169 - 71
Microflora of the honeybee gastrointestinal tract; Kacaniova M et al.; Microorganisms in the midgut and rectum of the honeybee were enumerated and characterized . Counts of aerobic microorganisms were distinctly lower than counts of anaerobes (10(5)-10(6) viable cells per g of intestinal content vs . 10(8)-10(9) per g) . Total numbers of anaerobic microorganisms were almost identical with the count of anaerobic Gram-positive acid resistant rods . A higher number of coliform bacteria and Bacillus spp . was detected in the rectum (10(5) per g) . Anaerobic and aerobic microorganisms, coliforms, enterococci, Bacillus spp., Pseudomonas spp . and yeasts were found in all bees; lactobacilli, staphylococci and moulds were not found.

Int J Antimicrob Agents, 2004 Jul, 24(1), 85 - 8
Microbiological/clinical characteristics and validation of topical therapy with kanamycin in aerobic vaginitis: a pilot study; Tempera G et al.; The term 'aerobic vaginitis' defines a 'new' vaginal pathology that is neither classifiable as specific vaginitis nor as bacterial vaginosis . We studied a sample of 30 women with a clinical and microbiological diagnosis of aerobic vaginitis and compared the efficacy and tolerability of kanamycin and meclocycline, two products commercially available in Italy in the form of vaginal pessaries . In chronological order of enrollment, the patients were alternately treated with kanamycin or meclocycline; the dose of administration in both groups was of one pessary per day for 6 days . The evaluation of the therapeutic efficacy was carried out both at the first check-up (7th-8th day) and at a second check-up (13th-16th day) . At the first follow-up carried out immediately at the end of therapy, the percentage of normalisation of clinical signs and symptoms was increased independently of the type of treatment in the case of moderate grade aerobic vaginitis, while kanamycin was produced a better effect in the group with severe aerobic vaginitis . Furthermore, at the second follow-up, a direct correlation with recovery of vaginal homeostasis was demonstrated by the normalisation of the vaginal pH and by the presence of lactobacilli, only in kanamycin treated group . In conclusion, our results showed the validity of the treatment with kanamycin intravaginally in this recently recognised disease.

Biometals, 2004 Jun, 17(3), 285 - 9
Neonatal small bowel epithelia: enhancing anti-bacterial defense with lactoferrin and Lactobacillus GG; Sherman MP et al.; BACKGROUND AND AIMS: Extremely preterm human infants have increased susceptibility to small bowel infection . We hypothesized that early colonization of the immature small intestine with Lactobacillus GG (LGG), and use of a recombinant lactoferrin (rhLF) to promote growth of LGG, would enhance gut defenses against enteroinvasive Escherichia coli . METHODS: Newborn rat pups were treated with nothing, intra-gastric LGG, or rhLF + LGG on days 3 and 4 of life . Gut colonization by LGG was quantified in lavaged jejunal and ileal fluid and gut wall homogenates on day 5 of life . Separate studies used similarly treated litters of newborn rats that were infected late on day 4 of life with E . coli {10(12) CFU/kg} . Sixteen hours later, the numbers of E . coli were measured in small bowel fluid and gut wall homogenates . RESULTS: Control pups initially had lactic acid bacteria colonize the bowel, but these bacteria were not LGG . Pups treated with LGG or rhLF + LGG had significantly higher numbers of LGG in the ileum versus jejunum . Contrary to our hypothesis, rhLF did not augment LGG colonization . After E . coli-related gut infection, planktonic {lavage fluid} and epithelia-adherent growth {gut wall homogenates} of E . coli in the small bowel were most effectively reduced by pre-treatment with rhLF and LGG (P < .05) . CONCLUSION: Prophylactic therapy with recombinant human lactoferrin and the probiotic, Lactobacillus GG, act to enhance defenses against invasive E . coli in the nascent small intestine . We suggest that rhLF and LGG are therapeutic agents that may reduce necrotizing enterocolitis and gut-related sepsis in preterm human infants.

J Clin Gastroenterol, 2004 Jul, 38(6 Suppl), S107 - 10
Utilization of the intestinal tract as a delivery system for urogenital probiotics; Morelli L et al.; Orally consumed viable bacteria with proposed beneficial health effects, the so-called probiotics, are increasingly used to treat disorders of the gastrointestinal tract . It has been recently suggested that specifically selected lactobacilli could have a therapeutic role in female urogenital tract infections . It has been also suggested that some of these bacteria could be not intravaginally instilled, but also orally delivered . The authors investigate the ability of lactobacilli isolated from the vagina to survive gastrointestinal tract transit, and establish a link between the rate of intestinal survival and vaginal colonization.

J Clin Gastroenterol, 2004 Jul, 38(6 Suppl), S104 - 6
Probiotics in the treatment of irritable bowel syndrome; Saggioro A; Irritable Bowel Syndrome (IBS) may be diagnosed on the presence of symptoms, according to Rome II criteria and some studies have shown that abnormal colonic fermentation may be an important factor in the development of symptoms in some patients with IBS . Since the fermentations of substrates by the intestinal flora may play a key role in the use of probiotics in the treatment of IBS, fifty patients (24 males, 26 females), mean age 40 years (range = 26-64 years) with IBS, according to Rome II criteria, were enrolled into the study after informed consensus . Patients were randomly assigned to receive either the active preparation containing Lactobacillus Plantarum LP0 1 and Bifidocterium Breve BR0 both at a concentration of 5 x 10 CFU/ml, or placebo powder containing starch identical to the study product, for 4 weeks . To evaluate treatment efficacy two different scores were considered: Pain score in different abdominal locations after treatment decreased in probiotics group of 38% versus 18% (P < 0.05) of placebo group after 14 days and of 52% versus 11% (P < 0.001) after 28 days . The severity score of characteristic IBD symptoms significantly decreased in probiotic group versus placebo group after 14 days 49.6% versus 9.9% (P < 0.001) and these data were confirmed after 28 days (44.4% versus 8.5%, P < 0.001) . In conclusion, short-term therapy with Lactobacillus PlantarumLP0 1 and Bifidocterium Breve BR0 may be considered a promising approach to the therapy for IBS.

Vet Parasitol, 2004 Jul 14, 122(3), 171 - 82
In vitro inhibition of Eimeria tenella invasion by indigenous chicken Lactobacillus species; Tierney J et al.; The aim of this study was to determine the effects of indigenous chicken Lactobacillus species isolates from different parts of the gastrointestinal tract on Eimeria tenella invasion in vitro and to characterise the nature of inhibition, if any . The effects of competitive exclusion, steric interference and bacterial extracellular factors on E . tenella invasion were examined in an MDBK cell model . Several Lactobacillus species were initially isolated from chickens and identified by biochemical characteristics and 16S-rRNA . All Lactobacillus species isolates tested, significantly inhibited E . tenella invasion . Steric interference did not affect parasite invasion . Extracellular metabolic factors secreted by Lactobacillus species isolates into the surrounding media were shown to inhibit parasite invasion and these factors appeared to be heat stable . These results show that the natural microflora of poultry can provide a source of E . tenella-inhibiting Lactobacillus species in vitro, and thus may contribute to the control of Eimeria infection.

Sex Transm Dis, 2004 Jul, 31(7), 393 - 400
Vaginal lactobacilli in adolescents: presence and relationship to local and systemic immunity, and to bacterial vaginosis; Alvarez-Olmos MI et al.; OBJECTIVE: The objective of this study was to survey middle adolescents for the presence of vaginal lactobacilli, lactobacilli-specific immune sensitization, and correlates of vaginal immunity with lactobacilli and bacterial vaginosis (BV) . METHODS: A cohort of 89 female adolescents were evaluated for the presence of vaginal lactobacilli species, H2O2-producing species, and the prevalence of BV . Cytokines and antibodies in cervicovaginal lavages were detected and peripheral blood lymphocyte (PBL) responses to Lactobacillus crispatus were evaluated . RESULTS: The majority of lactobacillus species were H2O2-producing and predominated by Lactobacillus acidophilus . PBL responses to lactobacilli were detectable in 50% of the cohort . BV was present in 36% of adolescents and negatively correlated with the presence of vaginal lactobacilli . The majority of locally associated cytokines and antibodies were similar in those with or without BV or lactobacilli . CONCLUSIONS: Adolescents harbor vaginal lactobacilli with relationships to BV along with lactobacilli-specific immune sensitization, but with few correlates of local immunity to lactobacilli or BV.

Lett Appl Microbiol, 2004, 38(4), 289 - 95
The effect of alpha-ketoglutaric acid on amino acid utilization by nonstarter Lactobacillus spp . isolated from Cheddar cheese; Williams AG et al.; AIMS: To examine the effect of alpha-ketoglutaric acid (alpha-KG) on the utilization and catabolism of amino acids by strains of nonstarter lactobacilli isolated from Cheddar cheese . METHODS AND RESULTS: The effect of alpha-KG in the growth medium of nonstarter lactobacilli on amino acid metabolism, catabolite levels, peptide hydrolase and aminotransferase activities was examined . The pattern of amino acid utilization, catabolite formation and aminotransferase activity was affected by keto acid . CONCLUSIONS: Amino acid conversion into cheese aroma and flavour compounds by nonstarter lactobacilli is enhanced in the presence of alpha-ketoglutarate . SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing the availability of alpha-ketoglutarate in cheese offers a possible method of reducing the maturation period by accelerating the rate of character compound formation from amino acids by the nonstarter lactobacilli.

Biotechnol Bioeng, 2004 Jul 5, 87(1), 34 - 42
A natural compound (reuterin) produced by Lactobacillus reuteri for hemoglobin polymerization as a blood substitute; Chen YC et al.; Stroma-free hemoglobin (Hb) has been modified by pyridoxylation and followed by polymerization with glutaraldehyde as a blood substitute . Nevertheless, the reaction rate of pyridoxylated Hb (PLP-Hb) with glutaraldehyde is too fast to control its molecular weight distribution . Additionally, it was reported that glutaraldehyde is cytotoxic even at low doses . To overcome these problems, another aldehyde, beta-hydroxypropionaldehyde (beta-HPA), was used in the study to polymerize hemoglobin (PLP-Hb) . beta-HPA is a natural compound (reuterin) produced by Lactobacillus reuteri . It was found that the maximum degree of PLP-Hb polymerization by reuterin (RR-PLP-Hb) was approximately 40% if the formation of high molecular (> 500 kDa) polymers should be prevented . In contrast, at the same reaction condition, the glutaraldehyde-polymerized PLP-Hb solution became gel-like, due to overpolymerization . This indicated that the rate of PLP-Hb polymerization by reuterin was significantly slower than that by glutaraldehyde . With increasing the reaction temperature, PLP-Hb concentration, or reuterin-to-PLP-Hb molar ratio, the time to reach the maximum degree of PLP-Hb polymerization by reuterin became significantly shorter . Removal of unpolymerized PLP-Hb from the RR-PLP-Hb solution can be effectively achieved by a gel-filtration column . The P(50) value of the unmodified Hb solution was 14 torr, while that of the RR-PLP-Hb solution was 20 torr, an indication of lower oxygen affinity . Additionally, the oxygen-Hb dissociation curves for both test solutions had a sigmodial shape and a nearly 100% saturation at 100 torr . In the in vivo study, it was found that the animals treated with the RR-PLP-Hb solution all survived and remained healthy more than 3 months . In contrast, only one out of six rats survived for the control group treated with the unmodified Hb solution . Furthermore, it was found that the RR-PLP-Hb solution resulted in a significantly longer circulation time ( approximately 12 h) than the unmodified Hb solution ( approximately 1.5 h) . These results suggest that the reuterin-polymerized PLP-Hb solution may be a new option in the development of blood substitutes .

Acta Odontol Latinoam, 2002, 15(1-2), 3 - 9
A comparative study in vivo of the therapeutic effect of triclosan, hexetidine and chitosan; Virga C et al.; The present clinical study was performed to comparatively assess the therapeutic effect of Low and High Molecular Weight Chitosan (LMWCh and HMWCh), hexetidine, triclosan . Plaque index, saliva buffering capacity and bacteriological controls for S . mutans and lactobacilli, were performed . The plaque and bacterial indices revealed statistically significant differences between groups . Buffering capacity was similar using, hexetidine, and triclosan, whereas it was maximum in 100% of the patients in the LMWCh and HMWCh groups . Only 0.5% HMWCh induced low activity of S . mutans in 100% of the patients and caused complete inhibition of lactobacilli growth . No changes were observed in the profile of salivary proteins . The present clinical study confirms the therapeutic efficacy of the chitosan as a bacteriostatic agent.

Mikrobiol Z, 2004 Mar-Apr, 66(2), 86 - 91
{Microflora of milk with prolonged storage term at different production process}; Chagarovskii VP et al.; New technologies have been studied for their effect on microbiologic indices of milk with prolonged storage term . It has been shown that the use of double pasterization decreases the number of Bacillus subtilis representatives to units of CFU/ml . The method of hot milk pouring into sealed package has 99.8% efficiency . Residual microflora was of dozens of CFU/ml and remained stable during 10 days . Lactobacillus acidophilus being introduced in pasterized milk, the amount of residual microflora decreased 2.3 times and storage term for milk increased to 14 days.

Mikrobiol Z, 2004 Mar-Apr, 66(2), 63 - 8
{Growth characteristics of Lactobacillus plantarum, Bacillus subtilis and Bacillus licheniformis at deep cultivation in the monoculture and coculture}; Khil'ko TV et al.; Active growth of some representatives of bacteria of Bacillus genus on earlier optimized nutrient medium for L . plantarum 8R-A3 is shown . Growth characteristics of the studied bacteria at mixed cultivation on this medium have been studied . The increase of their growth activity and accumulation of cell biomass has been determined . Taking into consideration the fact, that the process of obtaining of the studied strain mixed culture was determined by the type of ecological vital strategy of each separate species . B . licheniformis 5514 and L . plantarum 8R-A3 have been related to K-strategists and B . subtilis 5007--to r-strategists.

Mikrobiol Z, 2004 Mar-Apr, 66(2), 57 - 62
{Analysis of monosaccharides of bacterial glycocalix of Bacillus-Lactobacillus-Streptococcus group by gas-chromatography}; Sichkar SV; The glycocalix of some bacteria of the Bacillus--Lactobacillus--Streptococcus (Bacillus subtilis 3, B . subtilis 668, B . licheniformis 31, Lactobacillus plantarum 11/16, Enterococcus faecium K-50) group were isolated and composition of neutral monosaccharides was investigated . Such sugars as rhamnose, ribose, fucose, arabinose, xylose, mannose, galactose, and glucose were found in the investigated representatives of genus Bacillus--Lactobacillus--Streptococcus . Presence of significant quantity of glucose and galactose did not depend on cultivation conditions . This fact testifies to its conservatism and a possibility to use it as phylogenetic marker.

J Ind Microbiol Biotechnol, 2004 Jun, 31(5), 209 - 15 Epub 2004 Jun 16.
Nicotinic acid controls lactate production by K1-LDH: a Saccharomyces cerevisiae strain expressing a bacterial LDH gene; Colombie S et al.; Industrial applications for lactate, such as the production of chemicals, has led to interest in producing this organic acid by metabolically engineered a yeast such as Saccharomyces cerevisiae, which is more acid tolerant than lactic acid bacteria . This paper deals with lactate production by S . cerevisiae K1-LDH, in which the Lactobacillus plantarum lactate dehydrogenase (LDH) gene is integrated into the genome of the wine yeast strain K1 . We show that a vitamin, nicotinic acid (NiA), was the limiting factor for lactate production during fermentation with the K1-LDH strain . Increasing the NiA concentration in batch conditions or in the medium used to feed chemostats affected the lactate yield . Moreover, the addition of pulses of NiA or the exponential addition of NiA made it possible to control the lactate production kinetics throughout the fermentation process . The results point to the role of NiA in the regulation of metabolic pathways, but the physiological mechanisms remain poorly understood.

Digestion, 2004, 69(4), 225 - 8 Epub 2004 Jun 16.
Lactobacillus acidophilus protects tight junctions from aspirin damage in HT-29 cells; Montalto M et al.; BACKGROUND/AIMS: Non-steroidal anti-inflammatory drugs cause enterocyte damage inducing an increase of intestinal permeability . Tight junctions are the key structures in the permeability of the intestinal mucosa . ZO-1 is a tight junction associated protein considered a good marker of their integrity . It has been suggested that probiotics could play a protective role in the intestinal barrier function . We determined, in vitro, whether the heat-killed Lactobacillus acidophilus strain LB (LaLB) with its spent culture supernatant protects tight junctions of HT-29 cells from aspirin (ASA) damage . METHODS: HT-29 cells were treated with ASA alone or ASA and LaLB with its spent culture supernatant together . Morphological alterations of tight junctions were evaluated by immunofluorescence using an anti-ZO-1 antibody . Moreover, a semiquantitative assay for ZO-1 was performed by Western blot . RESULTS: Immunofluorescence analysis showed a fragmented and granulous ZO-1 staining, after ASA treatment . Using both ASA and LaLB with its spent culture supernatant together, we found a fine continuous linear web at cell-cell contacts similarly to control . Western blot revealed that ASA inhibited ZO-1 expression and LaLB with its spent culture supernatant counteracted this effect . CONCLUSIONS: This pilot study shows, for the first time, the protective effect of LaLB with its spent culture supernatant on tight junctions from ASA damage . These results suggest that probiotics could play a role in the prevention of ASA-induced alterations of intestinal permeability.

Eur J Paediatr Dent, 2004 Jun, 5(2), 107 - 9
High levels of salivary lactobacilli in Estonian schoolchildren; Koll-Klais P et al.; AIM: This was to assess oral salivary lactobacilli levels compared with oral health in a group of 12-year-old schoolchildren in Tartu, Estonia . METHODS: Whole saliva samples were collected and transferred to dip-slides (Dentocult LB) and incubated at 37 degrees C for 3 days . Dental caries, dental plaque, and data concerning the general health, dental habits and eating patterns were recorded . RESULTS: Salivary lactobacilli were found in all children, with a quarter of them having high or very high lactobacilli counts . Caries prevalence of 75% and 2.6 DMFT were recorded . A positive correlation was found between the DMFS counts and the lactobacilli counts . CONCLUSION: High levels of salivary lactobacilli were found in Estonian schoolchildren . Caries indicators of these children were slightly higher than in the same age group in Nordic countries.

Mikrobiologiia, 2004 Mar-Apr, 73(2), 211 - 7
{Expression vector pLF22 for the lactic acid bacteria}; Tarakanov BV et al.; The construction of the expression vector pLF22 for lactic acid bacteria is described . The vector contains a replicon of the cryptic plasmid pLF1311 from Lactobacillus fermentum and a multiple cloning site of the lacZ' gene integrated with the plasmid rep operon . Such a construction of the vector provides for the constitutive transcription of the cloned sequences lacking the terminators of transcription in all the strains that maintain the replication of the vector . The vector is suitable for a wide range of gram-positive and gram-negative bacteria, including probiotic strains . The efficiency of the vector was verified by expressing the beta-galactosidase gene in a laboratory Escherichia coli strain and the synthetic gene of somatotropin releasing factor (SRF) in the probiotic strains of lactobacilli and enterococci . A recombinant strain with the SRF gene included in the diet of laboratory animals exerted an effect on their physiological and anthropometric parameters and on the histological characteristics of animal tissues.

Minerva Urol Nefrol, 2004 Mar, 56(1), 65 - 72
Guidelines on diagnosis and treatment of superficial bladder cancer; Oosterlinck W; This manuscript reviews the guidelines of the European Association of Urology (EAU) on superficial bladder tumors and adds new data which has come available since 2001 . It emphasises the data which are evidenced based and clearly explained where still insufficient research is available to make clear recommendations . Intravenous urethrography (IVU) is only necessary in grade 3 tumors . A good transurethral resection (TUR), with muscle in the specimen is essential . Random biopsies are only necessary when there is positive urinary cytology or when tumor in situ (TIS) is suspected . The variability in pathology interpretation remains a problem which seems not to have been solved by the new WHO 1998 classification . A review of pathology seems indicated when aggressive therapy is planned or there is a discrepancy between the visual findings and pathology . The visual judgement of urologists in superficial bladder tumors is very good . Second resection is indicated whenever insufficient material is delivered and in any T1 G3 tumor . In the last infiltrative tumors are regularly found . The treatment largely depends on prognostic parameters . For recurrence rate multiplicity of the tumor is most important, followed by recurrence rate, volume of the tumor, grade and T category . For progression the most important tumor is the anaplasia grade and the T category . Up to 50% of T1 G3 tumors and TIS evaluate to invasive tumors . Even low risk tumors still have an important recurrence rate of at least 20%/year in the first years after diagnosis . One chemo instillation immediately after TUR is indicated in low and intermediate risk superficial bladder tumors . Intravesical chemotherapy prevents recurrence but not progression . Ideal dosage and schedule of instillation is not clearly defined . Longterm therapy is not worthwhile . Bacille Calmette-Guerin (BCG) therapy is indicated in all tumors at high risk for progression . In tumors at high risk for recurrence it is also superior to intravesical chemotherapy, but its side-effects are more pronounced . Local or systemic side-effects are not related to efficacy and side-effects do not increase over time . The ideal schedule for BCG has not yet been found . It is however clear that some kind of maintenance therapy is necessary to obtain good results . BCG failure is probably any tumor which recurs at 3 and 6 months under BCG therapy . One third dose seems as sufficient as a full dose BCG . That BCG can spare the bladder in T1g3 tumors is largely documented but the chance to save the bladder when the tumor is still present after 2 cycles of BCG is very low . Cystectomy is indicated in these BCG failures . Vitamin E, A, and Lactobacillus Casei are probably effective in the prevention of the disease . Stopping smoking is advocated . Cystoscopy is still the gold standard in follow-up . It is advocated at 3 months and thereafter according to the prognostic parameters . High grade tumors are at risk life long . Follow-up of 5 years for low risk tumors seems reasonable.

Int J Food Microbiol, 2004 Jul 15, 94(2), 123 - 35
Interactions between high pressure homogenization and antimicrobial activity of lysozyme and lactoperoxidase; Vannini L et al.; It was the objective of this work to evaluate the effect of high pressure homogenization on the activity of antimicrobial enzymes such as lysozyme and lactoperoxidase against a selected group of Gram positive and Gram negative species inoculated in skim milk . Lactobacillus helveticus, Lactobacillus plantarum and Listeria monocytogenes were the most pressure resistant species while Bacillus subtilis, Pseudomonas putida, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Salmonella enteritidis were found to be very sensitive to the hyperbaric treatment . The enzyme addition enhanced the instantaneous pressure efficacy on almost all the considered species as indicated by their instantaneous viability loss following the treatment . Moreover, the combination of the enzyme and high pressure homogenization significantly affected the recovery and growth dynamics of several of the considered species . Although L . monocytogenes was slightly sensitive to pressure, the combination of the two stress factors induced a significant viability loss within 3 h and an extension of lag phases in skim milk during incubation at 37 degrees C . The hypothesis formulated in this work is that the interaction of high pressure homogenization and lysozyme or lactoperoxidase is associated to conformational modifications of the two proteins with a consequent enhancement of their activity . This hypothesis is supported by the experimental results also regarding the increased antimicrobial activity against L . plantarum of the previously pressurised lysozyme with respect to that of the native enzyme.

J Dent, 2004 Jul, 32(5), 385 - 9
Efficacy of three surface disinfectants for dental radiographic films and gloves; Coogan MM et al.; Contaminated radiographic films and gloves may transmit infectious diseases . Objective . To determine whether Pre Sept (NaDC), a sodium dichloroisocyanurate based disinfectant; Bronocide SP (AIP) an alcohol-phenol-iodine disinfectant and polyvinyl pyrrolidine iodine (PVPI) can be used to disinfect radiographic films and gloves . Study design . Radiographic films were contaminated with Candida albicans, Streptococcus mutans, Lactobacilli or saliva and placed in either 0.1, 0.25 or 0.5 NaDC for 1, 2 or 5 min; PVPI for 5 min or sprayed with AIP . Gloves contaminated with C . albicans, S . mutans or Lactobacilli were sprayed with AIP . After treatment the films and gloves were tested for viable microorganisms . Results . A 0.5% solution of NaDC killed all microorganisms after one-minute exposure . PVPI killed 99.8% and AIP spray between 95.8 and 99% of microorganisms . Conclusions . NaDC was the most successful disinfectant in the laboratory and clinical setting followed by PVPI and AIP.

J Dairy Res, 2004 May, 71(2), 222 - 30
Lactobacilli isolated from kefir grains: evidence of the presence of S-layer proteins; Garrote GL et al.; In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains . Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66-71 kDa as measured by SDS-PAGE . The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl . The above mentioned proteins were recovered in the LiCl extracts . After dialysis and concentration, the proteins extracted were able to reassemble in a regular array . Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen . Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment . The production of this surface structure under different culture conditions was also evaluated . Finally, the relationship between the presence of S-layer proteins and surface properties (e.g . adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.

Lett Appl Microbiol, 2004, 39(1), 19 - 24
The expression of COX2 protein induced by Lactobacillus rhamnosus GG, endotoxin and lipoteichoic acid in T84 epithelial cells; Korhonen R et al.; AIMS: Lactobacillus rhamnosus GG (L . rhamnosus GG) possess immunomodulatory effects in the host . In the present study, the effect of L . rhamnosus GG on cyclooxygenase 2 (COX2) expression and its pharmacological control was investigated in human T84 colon epithelial cells . METHODS AND RESULTS: T84 cells were exposed to freeze-dried L . rhamnosus GG in vitro, and the expression of COX2 was detected by Western blot . CONCLUSIONS: L . rhamnosus GG induced COX2 expression in a time- and concentration-dependent manner in T84 cells . COX2 expression was inhibited by tyrosine kinase inhibitor genistein (100 micromol l(-1)), p38 mitogen-activated protein kinase (MAP kinase) inhibitor (SB203580; 1 micromol l(-1)) and dexamethasone (10 micromol l(-1)), whereas inhibitors of p42/44 MAP kinase (PD98095; 10 micromol l(-1)), protein kinase C (Ro 31-8220; 1 micromol l(-1)) and transcription factor nuclear factor kappa B (NF-kappaB) {pyrrolidinedithiocarbamate (PDTC) 100 micromol l(-1)} had practically no effect . SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide novel information on the cellular mechanisms involved in the interaction between L . rhamnosus GG and colon epithelial cells.

J Appl Microbiol, 2004, 97(1), 104 - 13
Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua; Fitzgerald DJ et al.; AIMS: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua . METHODS AND RESULTS: In laboratory media, MICs of 15, 75 and 35 mmol l(-1) vanillin were established for E . coli, Lact . plantarum and L . innocua, respectively . The observed inhibition was found to be bacteriostatic . Exposure to 10-40 mmol l(-1) vanillin inhibited respiration of E . coli and L . innocua . Addition of 50-70 mmol l(-1) vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact . Exposure to 50 mmol l(-1) vanillin completely dissipated potassium ion gradients in cultures of Lact . plantarum within 40 min, while partial potassium gradients remained in cultures of E . coli and L . innocua . Furthermore, the addition of 100 mmol l(-1) vanillin to cultures of Lact . plantarum resulted in the loss of pH homeostasis . However, intracellular ATP pools were largely unaffected in E . coli and L . innocua cultures upon exposure to 50 mmol l(-1) vanillin, while ATP production was stimulated in Lact . plantarum cultures . In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe . CONCLUSIONS: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific . These effects initially do not halt the production of ATP . SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.

Pediatr Dent, 2004 May-Jun, 26(3), 231 - 9
Microbiological screening for cariogenic bacteria in children 9 to 36 months of age; Barsamian-Wunsch P et al.; PURPOSE: The objective of this study was to evaluate sampling methods for recovery of mutans streptococci (MS) and lactobacilli (LB) in children 9 to 36 months of age . METHODS: Tongue and plaque specimens collected on cotton swabs and stimulated saliva were diluted and plated on selective and nonselective media . Tongue specimens on a swab and mouth mirror were inoculated directly on selective agar media (MS only) . Sampling methods were compared by frequency of recovery of MS or LB, correlation of microbial counts with dmfs scores, and potential of specific microbial counts to predict caries presence or absence . RESULTS: The mean dmfs score of 87 subjects was 6.3; 48 subjects were caries free . Levels of MS and LB were consistently higher in plaque than in other sampling techniques (P<.001), and frequencies of recovery of MS were highest in plaque (P<.041) and tongue (P<.006) . Frequency of LB recovery did not differ significantly between sampling methods . Counts of MS or LB in total subjects and subjects aged 9 to 24 months correlated positively with dmfs scores (P<.028) . Threshold levels of MS which were predictive of presence of caries were: (1) plaque=>2x10(5); (2) tongue=>10(4); (3) saliva=>10(5); (4) mirror=>50; and (5) swab=>50 . Comparable levels of LB were: plaque, >10(3); tongue, >10(2) and saliva, >10(3) . Specificities associated with these predictions were higher than sensitivities for all sampling methods . CONCLUSIONS: (1) All sampling methods were adequate for microbial risk assessment tests in children under 3 years of age; (2) MS was a stronger indicator of caries status than LB.

Appl Environ Microbiol, 2004 Jun, 70(6), 3673 - 80
Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus; Turner MS et al.; Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep . The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced . Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum . The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii . The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins . We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases . Sep was investigated as a heterologous peptide expression vector in L . fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363 . Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L . fermentum, L . rhamnosus, and L . lactis hosts . Sep as well as the previously described surface protein BspA were used to express and secrete in L . fermentum or L . rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes . This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.

Appl Environ Microbiol, 2004 Jun, 70(6), 3239 - 45
Metabolism of zearalenone by genetically modified organisms expressing the detoxification gene from Clonostachys rosea; Takahashi-Ando N et al.; Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101 . An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli . Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized . Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45 degrees C and pH 10.5 (k(cat) at 30 degrees C, 0.51 s(-1)) . The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors . ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k(cat) was highest with zearalanone (1.5 s(-1)) and lowest with beta-zearalenol (0.075 s(-1)) . EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity . Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions . We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN . While recombinant E . coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, beta-zearalenol, by an as-yet-unidentified reductase and remained in the medium . Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H . El-Nezami, N . Polychronaki, S . Salminen, and H . Mykkuane, Appl . Environ . Microbiol . 68:3545-3549, 2002) . An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.

Appl Environ Microbiol, 2004 Jun, 70(6), 3213 - 21
Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96; Choi YJ et al.; A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp . The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P(mxaF)), and alcohol oxidase (AOX1) promoters, respectively . The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558 . E . coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S . tag peptide . The recombinant L . casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose . The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C, respectively . Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 377 - 84
Transcriptional analysis of bacteriocin production by malt isolate Lactobacillus sakei 5; Vaughan A et al.; Malt isolate Lactobacillus sakei 5 produces three bacteriocins, sakacin P, sakacin T and sakacin X . The structural and regulatory genes for sakacin T and sakacin X are part of the sakacin TX locus, which consists of two adjacent, but divergently oriented gene clusters . Primer extension transcriptional analysis pointed to the existence of three distinct promoters within the sakacin TX locus, indicating that the three-component regulatory system in this locus is atypical in the sense that it is divided into a pheromone-specifying operon and an operon containing the genes for the histidine protein kinase and response regulator . Quantitative real-time PCR analyses showed that a transient increase in the expression of these two regulatory operons precedes transcription of the bacteriocin genes and appearance of bacteriocins in the culture medium . The identified promoters of the sakacin TX locus contain putative regulatory sequences (direct repeats) at corresponding positions in front of their -10 regions, which are likely to play a role in gene regulation.

Bioresour Technol, 2004 Sep, 94(3), 331 - 7
Kinetic growth parameters of different amylolytic and non-amylolytic Lactobacillus strains under various salt and pH conditions; Rao MS et al.; Four Lactobacillus species were studied for their ability to grow at high NaCl concentrations and different initial pH values . Lactobacillus plantarum 541 and A6 could ferment glucose and produce lactic acid in the presence of 8% salt in the medium . For strain 541, the specific rate of lactate production (q(lac)) and the yield of lactic acid relative to substrate (Y(p/s)) remained constant, whereas the yield of biomass relative to substrate (Y(x/s)) decreased up to 6% salt . In contrast, for strain A6, Y(p/s) decreased up to 6% salt whereas Y(x/s) did not vary markedly . Combined effects of salt and pH studied through a factorial design did not show significant interaction between salt and pH . The pH was the dominant factor in glucose fermentation for both the strains . Considering overall performance, 4% salt and pH between 6.0 and 6.6 can be taken as appropriate conditions, for the use of both strains as starters in processes where higher salt concentrations are required.

Biotechnol Prog, 2004 May-Jun, 20(3), 975 - 8
Optimization of enzymatic gas-phase reactions by increasing the long-term stability of the catalyst; Ferloni C et al.; Enzymatic gas-phase reactions are usually performed in continuous reactors, and thus very stable and active catalysts are required to perform such transformations on cost-effective levels . The present work is concerned with the reduction of gaseous acetophenone to enantiomerically pure (R)-1-phenylethanol catalyzed by solid alcohol dehydrogenase from Lactobacillus brevis (LBADH), immobilized onto glass beads . Initially, the catalyst preparation displayed a half-life of 1 day under reaction conditions at 40 degrees C and at a water activity of 0.5 . It was shown that the observed decrease in activity is due to a degradation of the enzyme itself (LBADH) and not of the co-immobilized cofactor NADP . By the addition of sucrose to the cell extract before immobilization of the enzyme, the half-life of the catalyst preparation (at 40 degrees C) was increased 40 times . The stabilized catalyst preparation was employed in a continuous gas-phase reactor at different temperatures (25-60 degrees C) . At 50 degrees C, a space-time yield of 107 g/L/d was achieved within the first 80 h of continuous reaction.

J Hum Hypertens, 2004 Nov, 18(11), 795 - 802
Effect of ingesting sour milk fermented using Lactobacillus helveticus bacteria producing tripeptides on blood pressure in subjects with mild hypertension; Tuomilehto J et al.; Angiotensin-converting enzyme (ACE) is important in the regulation of blood pressure (BP) . Two tripeptides that inhibit ACE, isoleucyl-prolyl-proline (Ile-Pro-Pro) and valyl-prolyl-proline (Val-Pro-Pro), have been isolated from certain sour milks . The aim of the study reported was to evaluate the effect on BP in subjects with mild hypertension of a new sour milk containing tripeptides . The initial number of subjects was 60 (36 men, 24 women) . Among the criteria for inclusion in the study were systolic BP (SBP) between 140 and 180 mmHg and/or diastolic BP (DPB) between 90 and 110 mmHg, without antihypertensive drug therapy . There were two study periods with a washout period between . All subjects were given 1.5 dl per day of a placebo (regular sour milk) or of the active product, a milk that had been fermented with Lactobacillus helveticus bacteria and contained 2.4-2.7 mg of Ile-Pro-Pro and 2.4-2.7 mg of Val-Pro-Pro per 1.5 dl . In the first phase, SBP fell 16 mmHg from baseline in the active group, 2 mmHg more than in the placebo group (P=0.0668) and no difference in DBP (P=0.92) . There was a statistically significant downward trend both in SBP and DBP (P=0.0001) . During the second phase, SBP fell 11 mmHg in the active group (P=0.008) . The reduction in SBP was significantly larger in active than placebo group (P=0.012) . In the crossover analysis combining both phases, SBP fell on average 2.6+/-15.9 mmHg more on the active product compared with the placebo product, but this difference was not statistically significant (P=0.3111) . The difference in DBP, 1.0+/-8.3 mmHg between the two test products was not significant either (P=0.4431) . In conclusion, the ingestion of sour milk fermented by L . helveticus bacteria and that containing ACE inhibitory tripeptides seems to lower BP modestly.

J Bacteriol, 2004 Jun, 186(12), 3749 - 59
Characterization and functional analysis of the poxB gene, which encodes pyruvate oxidase in Lactobacillus plantarum; Lorquet F et al.; The pyruvate oxidase gene (poxB) from Lactobacillus plantarum Lp80 was cloned and characterized . Northern blot and primer extension analyses revealed that transcription of poxB is monocistronic and under the control of a vegetative promoter . poxB mRNA expression was strongly induced by aeration and was repressed by glucose . Moreover, Northern blotting performed at different stages of growth showed that poxB expression is maximal in the early stationary phase when glucose is exhausted . Primer extension and in vivo footprint analyses revealed that glucose repression of poxB is mediated by CcpA binding to the cre site identified in the promoter region . The functional role of the PoxB enzyme was studied by using gene overexpression and knockout in order to evaluate its implications for acetate production . Constitutive overproduction of PoxB in L . plantarum revealed the predominant role of pyruvate oxidase in the control of acetate production under aerobic conditions . The DeltapoxB mutant strain exhibited a moderate (20 to 25%) decrease in acetate production when it was grown on glucose as the carbon source, and residual pyruvate oxidase activity that was between 20 and 85% of the wild-type activity was observed with glucose limitation (0.2% glucose) . In contrast, when the organism was grown on maltose, the poxB mutation resulted in a large (60 to 80%) decrease in acetate production . In agreement with the latter observation, the level of residual pyruvate oxidase activity with maltose limitation (0.2% maltose) was less than 10% of the wild-type level of activity.

Biosci Biotechnol Biochem, 2004 May, 68(5), 1004 - 10
A new assay using surface plasmon resonance (SPR) to determine binding of the Lactobacillus acidophilus group to human colonic mucin; Uchida H et al.; A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000 . Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen {(GalNAcalpha1-3(Fucalpha1-2)Gal)-} or human colonic mucin of blood type A (HCM-A) as ligands . In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L . acidophilus OLL2769, L . crispatus JCM8778, LA205 and LA206 . In the second screening, which used HCM-A, only L . acidophilus OLL2769 and L . crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen . The results indicated that some strains of the L . acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.

Shokuhin Eiseigaku Zasshi, 2004 Feb, 45(1), 25 - 8
Detection of Leuconostoc strains at a meat processing plant using polymerase chain reaction; Goto S et al.; To simplify the labor-intensive conventional routine testing of samples to detect Leuconostoc at a meat processing plant, we developed polymerase chain reaction (PCR) primers specific for Leuconostoc from 16S rRNA gene sequences . These primers did not detect other common lactic acid bacteria such as Lactobacillus plantarum, Lact . sake, Lact . fermentum, Lact . acidophilus and Weissella viridescens . PCR with this primer detected all Leuconostoc species tested (Leu . mesenteroides subsp . mesenteroides, Leu . pseudomesenteroides, Leu . carnosum, Leu . lactic, Leu . citreum, Leu . amelibiosum, Leu . gelidum), except for Leu . fallax, and no other lactic acid bacteria on agarose gel electrophoresis . The method could identify areas contaminated with Leuconostoc in a large-scale industrial meat processing plant . Of 69 samples analyzed, 34 were positive for Leuconostoc according to the conventional culture method (isolation of LAB producing dextran) and PCR, whereas 29 were negative according to both . Six samples were culture-negative but positive by PCR . No false negative results were generated by PCR . The method is rapid and simple, is useful for routinely monitoring areas contaminated with Leuconostoc in meat processing plants, and could help to prevent the spoilage of meat products.

Int J Food Microbiol, 2004 Jun 15, 93(3), 319 - 23
Investigation of the effectiveness of Ascopyrone P as a food preservative; Thomas LV et al.; Ascopyrone P (APP), a novel antibacterial from fungi, was evaluated as a food preservative . Efficacy was generally assessed by comparing the time taken for test strains to grow to 10(6) CFU/g in food +/- APP . In chilled chicken soup, 2000 mg kg-1 APP prevented Bacillus cereus, Listeria monocytogenes, Pseudomonas fluorescens, Salmonella and Escherichia coli reaching this threshold for >60 days . Good activity was also observed at 500-1000 mg kg-1 but not against L . monocytogenes . No activity was observed against Saccharomyces cerevisiae . Activity was reduced at 20 degrees C, although 2000 mg kg-1 was still effective against B . cereus and P . fluorescens . APP was less effective in chilled cooked meat systems and ineffective in raw meat . In a cooked meat system at 8 degrees C, bacteriostatic effect was generally observed at 2000 mg kg-1 against Salmonella typhimurium, E . coli and P . fluorescens but not against L . monocytogenes or Lactobacillus sake . Activity against Gram-negative enteric bacteria was enhanced by low temperature . In milk, 2000 mg l-1 was effective against P . fluorescens at chilled but not ambient temperature . APP was ineffective against yeasts and the mould Byssochlamys in apple juice . A minimum of 2000 mg kg-1 APP would appear to be necessary for antibacterial efficacy in food, although low-temperature storage may help . Observed variations in sensitivity may be related to APP stability, which decreases >pH 5.5 . Toxicology testing is needed before consideration of APP for food use.

J Clin Pediatr Dent, 2004 Spring, 28(3), 239 - 48
Salivary characteristics of children and its relation to oral microorganism and lip mucosa dryness; Alamoudi N et al.; The aim of this paper was to present baseline data on various saliva properties among a group of Saudi children aged 5 to 11 years and to study the relationship of these properties to some oral micro-organisms as well as to lip and oral mucosa dryness . The results showed a mean of resting and stimulated flow rate of 0.54 +/- 0.40 and 1.23 +/- 0.59 respectively and mean pH value of 7.27 +/- 0.38 and 7.5 +/- .035 respectively . Fluoride concentration was estimated to be 0.151 +/- 0.07 and 0.145 +/- 0.06 in resting and stimulated saliva respectively . Children with dry lip represented 33.9% of the sample population, whereas, those with dry mucosa represented only 0.8% . No significant sex difference was evident in all parameters . 59.1% of children showed medium buffering capacity in the resting saliva, whereas, the majority of children (73.7%) showed high stimulated buffering capacity . Children showed generally high Lactobacillus counts (Lb) in the resting and stimulated saliva (57.9% and 60.5% of children) . The presence of yeast also in resting and stimulated saliva seemed high in general (40% and 53% of children had high count) . However, Streptococcus mutans (S . mutans) counts showed no discriminating trend in both types of saliva . The data showed no significant association between flow rate and Lb counts in both resting and stimulated saliva although there was a trend toward higher counts associated with low flow rate . The same trend was observed in resting saliva although not significant . Similarly, low resting buffering capacity was associated with high counts of Lb among a high proportion of children (68.6% of children) although not significant . A significant reverse relation was evident between S . mutans counts and stimulated flow rate (p=0.049) . The majority of children with normal level of saliva pH showed no yeast colonization (62.1%) . The association was significant (p=.024) . Similarly, the same association was observed in the medium and high buffering group (66.2%) (p=.040) . It was concluded that salivary Lb count seems to be primarily affected by some local factors other than salivary properties, such as diet . Significant inverse relationship was found between S . mutans and stimulated salivary flow . Children in general showed high percentage of yeast reflecting the affect of poor diet among the studied population group . Buffering capacity and pH had an important role in yeast colonization.

Microbiol Res, 2004, 159(1), 35 - 42
Effect of preculturing conditions on growth of Lactobacillus rhamnosus on medium containing glucose and citrate; Jyoti BD et al.; Lactobacillus rhamnosus can metabolize citrate through a citrate inducible transport system . The growth curves of L . rhamnosus on medium containing glucose and citrate was found to be highly dependent on preculturing conditions . It exhibited diauxic growth when precultured on glucose, but demonstrated simultaneous consumption when cultured on citrate . The maximum specific growth rate for cells growing on glucose + citrate was 0.38 h-1, which was higher than the growth rate on individual substrates (0.28 h-1) . Simultaneous consumption also yielded higher net flavour compounds, diacetyl and acetoin . Flux analysis indicated that L . rhamnosus requires oxygen for balancing excess NADH through NADH oxidase . The flux analysis provided insights into the metabolic network of L . rhamnosus.

Methods Mol Biol, 2004, 268, 491 - 502
Microflora of the gastrointestinal tract: a review; Hao WL et al.; The mucosal surface of the human gastrointestinal (GI) tract is about 200-300 m2 and is colonized by 1013-14 bacteria of 400 different species and subspecies . Savage has defined and categorized the gastrointestinal microflora into two types, autochthonous flora (indigenous flora) and allochthonous flora (transient flora) . Autochthonous microorganisms colonize particular habitats, i.e., physical spaces in the GI tract, whereas allochthonous microorganisms cannot colonize particular habitats except under abnormal conditions . Most pathogens are allochthonous microorganisms; nevertheless, some pathogens can be autochthonous to the ecosystem and normally live in harmony with the host, except when the system is disturbed.The prevalence of bacteria in different parts of the GI tract appears to be dependent on several factors, such as pH, peristalsis, redox potential, bacterial adhesion, bacterial cooperation, mucin secretion, nutrient availability, diet, and bacterial antagonism . Because of the low pH of the stomach and the relatively swift peristalsis through the stomach and the small bowel, the stomach and the upper two-thirds of the small intestine (duodenum and jejunum) contain only low numbers of microorganisms, which range from 103 to 104 bacteria/mL of the gastric or intestinal contents, mainly acid-tolerant lactobacilli and streptococci . In the distal small intestine (ileum), the microflora begin to resemble those of the colon, with around 107-108 bacteria/mL of the intestinal contents . With decreased peristalsis, acidity, and lower oxidation-reduction potentials, the ileum maintains a more diverse microflora and a higher bacterial population . Probably because of slow intestinal motility and very low oxidation-reduction potentials, the colon is the primary site of microbial colonization in humans . The colon harbors tremendous numbers and species of bacteria . However, 99.9% of colonic microflora are obligate anaerobes.

Methods Mol Biol, 2004, 268, 471 - 4
Meat-model system development for proteolytic activity determination; Vignolo G et al.; Many technological parameters that affect the nature and functional properties of proteins are involved in the preparation of meat products . The dry-curing process is quite complex because of the coexistence of enzymes from endogenous and bacterial origins . The protein breakdown that takes place during the ripening of dry fermented sausages leads to an increase in the concentration of peptides and free amino acids . The proteolytic events have been thoroughly investigated not only because of their physiological significance but also for their technological connotations in terms of texture and flavor development . Lactic acid bacteria and Staphylococcus or Kocuria are used as starter cultures in fermented meat products . In recent years, the proteolytic system of lactobacilli involved in meat fermentation became the focus of an increasing number of studies because of the technological roles of these organisms . Although results obtained from broth systems show proteolytic activity, studies involving food systems must be done to confirm their effectiveness . A simplified soluble muscle extract to determine the capacity to degrade meat proteins by lactic acid bacteria was developed using a spectrophotometric method based on the reaction of the alpha-amino groups released by hydrolysis with o-phtaldialdehyde and beta-mercaptoethanol to form an adduct (1-thioalkyl-2-alkylisoindoles) that is enhanced at basic pH and absorbs strongly at 360 nm.

Methods Mol Biol, 2004, 268, 447 - 52
Hydroxylapatite beads as an experimental model to study the adhesion of lactic acid bacteria from the oral cavity to hard tissues; Ostengo Mdel C et al.; The oral environment contains many different types of microorganisms, including both Gram-negative and Gram-positive cocci, bacilli, and spirochetes . From the ecological point of view, the oral cavity is a perfect niche for certain bacteria such as lactobacilli because they interact, forming different types of communities . Lactobacilli have been associated with the generation of caries in some reports, secondary to the cariogenic Streptococcus.In previous papers, the isolation and identification of 145 strains from healthy subjects and from subjects with active caries were performed . Strains were characterized by their surface properties and also by the production of inhibitory substances . From all the strains, one isolated from the teeth of healthy patients and another from a patient with caries, sharing some surface properties, were selected for further study of their adhesion properties in an experimental model by using hydroxylapatite beads.Adhesion is the first step in the association of microorganisms with surfaces or mucous membranes . The first approach is a nonspecific interaction of both surfaces; later some other types of interactions can occur, involving more specific mediators of adhesions.Many different assays are available to study the adhesion phenomenon; some of them use predictive characteristics, and others use experimental models resembling the in vivo situation . Two model systems predominate . The most widely used has been saliva-coated hydroxylapatite or hydroxylapatite coated with buffers, proteins, and other substances.In an attempt to increase knowledge of the mechanisms of adhesion of oral lactobacilli with hard surfaces, this chapter describes an experimental model for studing the adhesion between lactobacilli and hard tissues represented by hydroxylapatite, the component most abundant in the teeth.The following steps were performed: 1 . Obtaining the microorganisms . 2 . Preparation of the hydroxylapatite beads . 3 . Adhesion assay.

Methods Mol Biol, 2004, 268, 441 - 5
Adhesion ability of Lactobacillus to vaginal epithelial cells: study by microbiological methods; Ocana VS et al.; Adhesion of lactobacilli to the epithelium has been described as the first step in the formation of a barrier to prevent undesirable microbial colonization; consequently, it has been defined as a characteristic of interest for selecting probiotic strains.Several methods have been described to predict the adhesion ability of Lactobacillus . Early studies were phenomenological: it was useful to determine whether a particular bacterium could hemagglutinate, or bind to coated particles . Later, studies based on the adhesion of bacteria to epithelial cells in vitro were developed . These last assays were based on the numbers of bacteria attached to epithelial cells, which were determined by counting stained microorganisms under light microscopy or by measuring the radioactivity of previously radiolabeled bacteria . Because the microscope technique, had some disadvantages, a modification was developed in our laboratory . The technique described in this chapter is a modification of the Mardh and Westron method . The method allows the study of the adhesion ability of bacteria, even if they are aggregating or if they are high-adherent bacteria covering a large area of the epithelial cell surface . Determination of the number of adherent bacteria by counting colonies grown in a selective media avoids the time-consuming, tedious, and hazardous counting under the light microscope and the use of radioactive methods.

Methods Mol Biol, 2004, 268, 435 - 40
Bacterial surface characteristics applied to selection of probiotic microorganisms; Otero MC et al.; For the study of probiotic microorganisms, the in vitro selection tests need to be based on a solid scientific foundation . Surface characteristics, one of the in vitro properties are used to evaluate the potentially probiotic strains of lactobacilli.Bacterial surface properties have been associated with attachment to a variety of substrata . Bacterial adhesion to tissues is considered the first step, and such adhesion can also determine the colonization capability of a microorganism . Through adhesion ability and colonization of tissues, probiotic microorganisms can prevent pathogen access by steric interactions or specific blockage on cell receptors.One of the main characteristics studied is the hydrophobic nature of the bacterial cell surface . To test this property, Rosenberg and Doyle divided microbial cell hydrophobicity assays into two categories . The first includes contact angle measurements (CAMs), partitioning of cells into one or another liquid phase (TTP), and adsorption of individual hydrophobic molecular probes at the cell surface . The second category includes microbial adhesion to hydrocarbons (MATH), hydrophobic interaction chromatography (HIC), and adhesion to polystyrene and other hydrophobic solid surfaces . The tests included in the first category measure hydrophobic properties of the outer cell surface as a whole; those in the second measure hydrophobicity in terms of adhesion . Finally, those bacterium classified as hydrophobic can be considered as able to mediate adhesion.The objective of this chapter is to describe three different methods applied in our laboratory for the study of bacterial surface properties . They can be used to screen characteristics of lactobacillus strains for probiotic purposes . They are: Microbial adhesion to hydrocarbons (MATH); Salt aggregation test (SAT); Hemagglutination (HA) reaction.

Methods Mol Biol, 2004, 268, 417 - 22
Animal model for in vivo evaluation of cholesterol reduction by lactic Acid bacteria; Taranto MP et al.; For many years, it has been recognized that elevated serum cholesterol is a risk factor associated with atherosclerosis and coronary heart disease, the latter being a major cause of death in Western countries . Numerous drugs that lower cholesterol have been used to treat hypocholesterolemic individuals . However, the undesirable side effects of these compounds have caused concerns about their therapeutic use . Ingestion of probiotic (beneficial for health) lactic acid bacteria (LAB) would possibly be a more natural method to decrease serum cholesterol in humans, as has been was reported.Previous studies have demonstrated that Lactobacillus reuteri administered in low doses has a hypocholesterolemic effect both therapeutically and preventively . One of the key studies in the development of a probiotic is to determine the minimal effective dose of live microorganisms that might be ingested without producing adverse effects (i.e., translocation) in the host.In this chapter, we describe an animal model that allows us to evaluate reduction in hypercholesterolemia by LAB and, also to determine the minimal dose of the microorganism, a critical step in the development of a safe probiotic product.

Methods Mol Biol, 2004, 268, 401 - 10
Effect of lactobacilli administration in the vaginal tract of mice: evaluation of side effects and local immune response by local administration of selected strains; Vintini E et al.; Lactobacilli are the predominant microorganisms in the vaginal tract of human and some homeothermic animals . They can maintain the ecological equilibrium of the tract by protecting against pathogenic microorganisms . In the last few years, there has been an increased tendency to use probiotic microorganisms to restore the ecological equilibrium and to protect against infections . This principle has been widely applied to the gastrointestinal tract . More recently, some other studies have reported the application of probiotics in different tracts, for example, the urogenital or respiratory tract.One of the objectives of our group is to design probiotic products for the urogenital tract . With this purpose, lactobacilli were isolated from the human vagina, and later some of them were selected for their probiotic characteristics (production of antagonistic substances or adhesion capability).The application of probiotic products in the vaginal tract has been approached empirically; some pharmaceuticals containing these microorganisms are available in the United States or Europe or are protected under the patent process or intellectual property rights . There are not enough studies in humans or animals to determine whether their administration can produce some type of collateral or adverse effect.Using Balb/c mice as the experimental model, the object of the present work was to study (1) whether intravaginal administration of human lactobacilli can produce colonization of the tract; (2) whether such administration produces some type of adverse or collateral effect; and (3) whether probiotics are able to stimulate the local immune system . Keeping in mind that hormones can affect the colonization or persistence ability of microorganisms, and with the purpose of having all animals at the same point in the sexual cycle, animals were cycled with estradiol 48 h before inoculation with lactobacilli . They were then inoculated im with hormones 48 h before beginning microorganism inoculations . Later they were intravaginally inoculated with the appropriate dose of each Lactobacillus strains . The animals were sacrificed on different days after inoculation to perform the following studies: 1 . Microbiological assays: To determine the number of lactobacilli in the tract (in vaginal washes or in organ homogenates), by plating the samples in selective media containing antibiotic (to differentiate the resident flora from those administered experimentally).

Methods Mol Biol, 2004, 268, 387 - 399
Effects of estrogen administration on the colonization capability of lactobacilli and Escherichia coli in the urinary tracts of mice; Silva C et al.; The use of probiotic microorganisms has been widely promoted in the last 20 yr . They have been used in the gastrointestinal tract as capsules or as fermented milks . The characteristics of the strains proposed as probiotics have been published or patented under an elaboration process . The first step in designing a probiotic product is to isolate and characterize strains with some beneficial properties . The second step is to determine the optimal conditions to obtain the highest amount of viable microorganisms, together with the study of the best conditions to produce antagonistic substances.Urinary tract infections (UTIs) constitute a common cause of illness in pre- and postmenopausal women . It was estimated that 40-50% of adult women suffer a cystitis during their life . Ninety percent of acquired ambulatory UTIs and 30% of nosocomial infections are produced by Escherichia coli . The healthy human urinary tract is free of microorganisms, except for the anterior urethra, which is colonized by indigenous microbiota . The vaginal environment is a dynamic and complex ecological system with a highly heterogeneous microflora; thus favorable conditions exist for the colonization process, which is also affected by factors external to the tissues . The distal urethra and periurethral areas are separated ecological niches, both covered by the vaginal secretions that contain approx 109 microorganisms/mL . In these secretions, members of the genus Lactobacillus are predominant . Bacterial colonization does not increase because of the urinary flux, which clears the bacterial cells from the outer surfaces, as well as other factors such as pH, osmolarity, and urea concentration.

Methods Mol Biol, 2004, 268, 373 - 85
Colonization capability of lactobacilli and pathogens in the respiratory tract of mice: microbiological, cytological, structural, and ultrastructural studies; Cangemi de Gutierrez R et al.; Respiratory tract infections are among the bacterial infections that affect humans with higher frequency . Those produced by Streptococcus pneumoniae are reported to have the highest incidence in the world, affecting both children and old people . As a 2001 report from the World Health Organization expressed it, the basic fight of children under 5 yr old is to survive . Five different conditions (acute respiratory infections, diarrhea, measles, palludism, and undernutrition) directly produce more than 50% of the deaths in this age group . Respiratory tract infections in the developing countries in the Americas are among the first three causes of death in children under 1 yr and between the first and second cause in children between 1 and 4 yr old . Pneumonia is responsible for 85 and 90% of deaths in children under 5 yr old (approx 150,000 annually), 95% of them occurring in the developing countries in the Americas.There is an increased worldwide tendency to use preventive measures and to consume products that help to maintain the health status of the individual . Thus the use of probiotics has increased systematically during the last decade, and the scientific literature trying to demonstrate the positive effect of such preparations has also increased . The term probiotic has been applied to products that (1) contain live microorganisms, freeze-dried or included in fermented products or (2) improve the health status of humans and animals, exerting effects in the mouth or gastrointestinal tract (included in foods or capsules), in the respiratory tract (as aerosols), or in the urogenital tract (by local application)Having in mind the high incidence of respiratory tract infections, and looking for preventive measures as well as the possible applications of probiotics, the aim of this chapter was to use mice as experimental models to determine whether members of the genus Lactobacillus were able to colonize and give protection from infections after inoculation by the intranasal route . To this end, the following procedures were carried out: 1 . Screening of the predominant bacterial species in respiratory organs . 2 . Study of the kinetics of colonization of the different groups of microorganisms from 15 d up to adult (2 mo) . 3 . Screening of the probiotic characteristics of all the isolated strains.

Methods Mol Biol, 2004, 268, 355 - 65
Statistical models to optimize production of probiotic characteristics; Tomas MS et al.; The use of probiotic microorganisms has been widely promoted in the last 20 years . Probiotics have applied to the gastrointestinal tract as capsules or as fermented milks . The characteristics of the strains proposed as probiotics have been published or patented after elaboration of the product.The first step in designing a probiotic product is to isolate and characterize strains with beneficial properties . Our group has reported the isolation and characterization of 134 strains of lactobacilli from the vagina of women from our city . Surface properties and the production of antagonistic substances has also been published . From these studies, certain strains were selected because they shared probiotic characteristics.The second step in the design of a pharmaceutical product is to determine the optimal conditions for obtaining the highest amount of viable microorganisms, as well as the best conditions to produce antagonistic substances . To perform these types of experiments, laboratory assays can be carried out to determine the optimum conditions in which the microorganisms can be cultured . Growth parameters and optimal growth conditions can be studied by the classical methodology or by the application of alternative methodologies . Alternative methods have been widely used in the food industry, including experimental designs to test multiple conditions at the same time, complemented by the use of statistical models to evaluate and compare all the conditions tested.The object of this chapter is to describe the experimental designs used to study the technological characteristics of vaginal probiotic lactobacilli as well as the application of statistical models for evaluation of the different conditions tested for the production of biomass and antagonistic substances in these bacteria . With this objective in mind, in the present chapter, the following areas are explored: 1 . Growth experiments performed to study the effect of temperature, pH, agitation, and culture media on the growth and production of antagonistic substances by vaginal lactobacilli . 2 . Fractional and complete factorial experimental designs used to perform the growth experiments . 3 . Statistical models applied to evaluate the growth parameters and their modifications under the conditions assayed.

Methods Mol Biol, 2004, 268, 347 - 53
Production of antimicrobial substances by lactic acid bacteria II: screening bacteriocin-producing strains with probiotic purposes and characterization of a Lactobacillus bacteriocin; Ocana VS et al.; Bacteriocins have been defined as proteinaceous, bactericidal substances synthesized by bacteria, which usually have a narrow spectrum of activity, only inhibiting strains of the same or closely related species . The term bacteriocin-like substance is applied to antagonistic substances that are not completely defined or do not fit the typical criteria of bacteriocins . They have been reported to inhibit a wide range of both Gram-positive and Gram-negative bacteria as well as fungi.Lactobacillus species are the dominant microorganisms isolated from the vagina of healthy premenopausal women . In this environment, they exert a protective effect against pathogenic microorganisms by different mechanisms such as production of antimicrobial agents, which include organic acids, hydrogen peroxide, and probably bacteriocins . The production of bacteriocins by vaginal lactobacilli has been demonstrated in vitro; however, it is not yet well established whether they are produced in vivo as another antagonistic mechanism exerted by the normal microflora.Bacteriocin-producing bacteria as well as bacteriocins per se are of growing interest as biological controls in the manufacture of beverages and fermented products, mainly in the area of dairy products . These bacteria have also been proposed as probiotic candidates for human or animal use.The objectives of the present chapter are to describe the methods employed for: 1 . Detection of production of bacteriocins among vaginal Lactobacillus strains . 2 . Characterization of the bacteriocin or bacteriocin-like substances . 3 . Study of the kinetics of production and mode of action of bacteriocins . 4 . Determination of the inhibition of pathogenic microorganisms by bacteriocin-producing strains in mixed cultures.

Methods Mol Biol, 2004, 268, 337 - 46
Production of antimicrobial substances by lactic acid bacteria I: determination of hydrogen peroxide; Tomas MS et al.; Restoration of the balance of different ecological niches has been proposed as a way to control the income of pathogenic microorganisms . The genus Lactobacillus has been used in different human and animal tracts as probiotic microorganisms with this objective in mind . The characteristics of the strains proposed as probiotics have been published or patented under the process of elaboration of different types of products . One of the mechanisms suggested to control the vaginal ecosystem is the production of antagonistic substances (lactic acid, bacteriocins, or H2O2) . The H2O2-producing microorganisms present in the vagina of healthy women have been suggested as some of the bacteria responsible for maintenance of ecological balance, mainly in pregnant women . The absence of these microorganisms is related to a higher risk of: bacterial vaginosis, recurrent urinary tract infections by Escherichia coli, and acquisition of human immunodeficiency virus type 1 (HIV-1) . Bauer has proposed that H2O2-producing lactobacilli also might exert control over vaginal cancer through specific interactions of reactive oxygen species, such as superoxide anion, hydroxyl radicals, and hypochlorous acid . The conversion of H2O2 into more toxic compounds during the oxidative process is potentiated by peroxidase and halures . This enzyme and some halures, such as chloride and bromide, are present in vaginal washes in sufficient amounts to allow an optimal environment for successful inhibition of pathogens.In vitro tests provide an approach for determining the ability of lactobacilli to produce H2O2 . The H2O2 amounts produced in such systems are probably not a direct reflection of what happens in the vaginal tract of women or animals, which is not yet know . However, there is a registered patent with an H2O2-generating L . crispatus strain, also supporting the use of H2O2-producing lactobacilli to restore the vaginal ecosystem.

Infect Immun, 2004 Jun, 72(6), 3299 - 309
Bacterial probiotic modulation of dendritic cells; Drakes M et al.; Intestinal dendritic cells are continually exposed to ingested microorganisms and high concentrations of endogenous bacterial flora . These cells can be activated by infectious agents and other stimuli to induce T-cell responses and to produce chemokines which recruit other cells to the local environment . Bacterial probiotics are of increasing use against intestinal disorders such as inflammatory bowel disease . They act as nonpathogenic stimuli within the gut to regain immunologic quiescence . This study was designed to determine the ability of a bacterial probiotic cocktail VSL#3 to alter cell surface antigen expression and cytokine production in bone marrow-derived dendritic cell-enriched populations . Cell surface phenotype was monitored by monoclonal fluorescent antibody staining, and cytokine levels were quantitated by enzyme-linked immunosorbent assay . High-dose probiotic upregulated the expression of C80, CD86, CD40, and major histocompatibility complex class II I-Ad . Neither B7-DC or B7RP-1 was augmented after low-dose probiotic or Lactobacillus casei treatment, but B7RP-1 showed increased expression on dendritic cells stimulated with the gram-negative bacterium Escherichia coli . Functional studies showed that probiotic did not enhance the ability of dendritic cells to induce allogeneic T-cell proliferation, as was observed for E . coli . Substantial enhancement of interleukin-10 release was observed in dendritic cell-enriched culture supernatants after 3 days of probiotic stimulation . These results demonstrate that probiotics possess the ability to modulate dendritic cell surface phenotype and cytokine release in granulocyte-macrophage colony-stimulating factor-stimulated bone marrow-derived dendritic cells . Regulation of dendritic cell cytokines by probiotics may contribute to the benefit of these molecules in treatment of intestinal diseases.

Biotechnol Appl Biochem, 2004 Jun, 39(Pt 3), 347 - 54
Modified alginate and chitosan for lactic acid bacteria immobilization; Le-Tien C et al.; Beads with enhanced-stability acid media, which were based on alginate and chitosan functionalized by succinylation (increasing the anionic charges able to retain protons) or by acylation (improving matrix hydrophobicity), were developed for immobilization of bacterial cells . Beads (3 mm diameter) formed by ionotropic gelation with CaCl(2) presented good mechanical characteristics . After 30 min incubation of viable free Lactobacillus rhamnosus cells in simulated gastric fluid (pH 1.5), we noticed that the level of viable bacteria was undetectable . Bacterial immobilization in native-alginate-based beads generated a viable-cell count of 22-26%, whereas, when entrapped in succinylated alginate and chitosan beads, the percentage of viable cells was of 60 and 66%, respectively . Best viability (87%) was found for bacteria immobilized in N -palmitoylaminoethyl alginate, which affords a high protective effect, probably due to long alkyl pendants that improve the beads' hydrophobicity, limiting hydration in the acidic environment.

J Econ Entomol, 2004 Apr, 97(2), 235 - 50
Influence of antimicrobial agents on the spoilage of a meat-based entomophage diet; Inglis GD et al.; The microbial decomposition of a meat-based entomophage diet presented in Parafilm packets was investigated . Considerable bacteria but not fungi were associated with components used to prepare the diet (i.e., hens' eggs, liver, and ground beef) . At the initial sampling time, there were no differences among diet treatments in the size of bacterial or fungal populations . Bacterial populations in diets not containing antibacterial agents rapidly increased and reached an asymptote by 24 h (approximately 10(10) colony-forming units per gram) . Bacterial populations also increased in diets containing antibacterial agents, but they were significantly smaller than in diets not containing antibacterial agents . The most prevalent bacteria isolated were Carnobacterium piscicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides, and Enterococcus spp., regardless of the antibacterial treatment used . The proliferation of fungi was delayed relative to bacteria, but significant differences were observed among the diet treatments . Fungi were most inhibited by sorbic acid and propionic acid in the absence of antibacterial agents . The most common fungi isolated were the yeasts Candida zeylanoides, Torulaspora globosa, and Yarrowia lipolytica . The pH of diets not containing antibacterial agents decreased rapidly and was highly correlated with increases in bacteria but not fungi . The results of this study demonstrate that antimicrobial agents significantly inhibit spoilage microorganisms in a meat-based diet and that alternative management strategies to delay the decomposition of such diets presented in Parafilm packets should target lactic acid spoilage bacteria, particularly Carnobacterium and Lactobacillus species.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 809 - 12
Reclassification of Lactobacillus cellobiosus Rogosa et al . 1953 as a later synonym of Lactobacillus fermentum Beijerinck 1901; Dellaglio F et al.; The name Lactobacillus cellobiosus is validly published, but the species is often neglected in taxonomic studies, due to its high similarity to Lactobacillus fermentum . In the present paper, literature data concerning the two species were reviewed . Phylogenetic placement of L . cellobiosus was obtained based on 16S rDNA sequences, and genetic similarity was further investigated by comparing partial recA gene sequences for the type strains of L . cellobiosus and L . fermentum . Based on the high identity values for 16S rDNA (99 %) and recA gene (98 %) sequences, the results of DNA-DNA hybridization assays and phenotypic traits available from the literature, it is proposed that L . cellobiosus be reclassified and, as a rule of priority, renamed as L . fermentum, the first described species.

Poult Sci, 2004 May, 83(5), 788 - 95
Effects of Lactobacilli and an acidophilic fungus on the production performance and immune responses in broiler chickens; Huang MK et al.; Accumulated lines of evidence indicate that inactivated probiotics could have beneficial effects similar to those of live probiotics . Two strains of disrupted, cobalt-enriched, lactic acid bacteria (Lactobacillus acidophilus and Lactobacillus casei) and a disrupted fungal mycelium (Scytalidium acidophilum) were spray-mixed onto a mash basal feed, in 2 concentrations, prior to pelleting . The effects of these probiotics on production performance and immune response in broiler chickens were investigated . The production parameters, including BW, feed intake (FI), BW gain (BWG), and feed conversion ratio (FCR), were monitored weekly during a 6-wk trial . The immune response was evaluated by immunizing the birds with the antigen keyhole limpet hemocyanin (KLH) followed by a serological assay to measure blood IgA and IgG titers . Some of the production parameters were significantly improved by low L . casei (LCL; for BW and BWG), high L . acidophilus (LAH; for BW and BWG), and high fungal (FH; for BW, BWG, and FI) in comparison with the nonadditive control (NC-) . However, these 3 treatments (LCL, LAH, and FH) did not enhance the measured immune responses . Instead, the titers of serum KLH-specific IgA in high L . casei (LCH) and low L . acidophilus (LAL) were significantly higher than those of NC-, 10 d after immunization . None of the probiotic treatments increased the titer of KLH-specific IgG in blood . Our results indicate that disrupted and cobalt-enriched L . acidophilus or L . casei was able to enhance production performance of broiler chickens . The fungal mycelium, S . acidophilum, when used at a high concentration, also demonstrated its potential for the first time to be used as a probiotic . In addition, the optimal concentration for administering probiotics is strain dependent . A higher dose does not always result in a better performance.

Int J Paediatr Dent, 2004 May, 14(3), 182 - 91
Comparison of the efficacy of chemomechanical caries removal (Carisolv) with that of conventional excavation in reducing the cariogenic flora; Azrak B et al.; OBJECTIVES: The usage of minimally invasive procedures and attention to patient comfort are of great importance, especially for dental treatment in small children . This has led to the development of chemomechanical methods for caries removal . The aim of this study was to compare the efficacy of chemomechanical caries removal with that of conventional excavation in reducing the count of the cariogenic flora . DESIGN AND SETTING: Subjects for this study were chosen from children admitted to dental clinic for restorative procedures under general anaesthesia . SAMPLES AND METHODS: Twenty-one children (mean age 43.5 +/- 12.0 months) with early childhood caries were included in this study . Two primary teeth with comparable degrees of carious destruction were chosen in each child (n = 42) for caries removal with Carisolv' or by means of rotary instruments . Samples from carious dentine were taken with a sterile scraping instrument, then all softened dentine was removed and a second sample was taken . All samples (n = 84) were serially diluted and plated on two different nutrient agar plates . RESULTS: After 24 h of incubation, colony forming units were determined for total bacterial counts and lactobacilli . Twelve per cent of the samples from carious dentine contained more than 10(6) bacteria, 23.8% contained more than 10(5) lactobacilli . Both methods of caries removal produced a statistically significant reduction in the bacterial counts (P = 0.0001) . In at least 90.5% of the samples taken after the removal, the total bacterial count was below 10(2), and in 95.2% lactobacilli fell below 10(2) . CONCLUSION: These results indicate that the efficacy of chemomechanical removal of carious dentine in children by means of Carisolv' is comparable to the results obtained by conventional methods, and thus might serve as a suitable alternative.

J Appl Microbiol, 2004, 96(6), 1324 - 32
Detection of resistance of lactic acid bacteria to a mixture of the hop analogue compounds tetrahydroiso-alpha-acids by noninvasive measurement of intracellular pH; Yansanjav A et al.; AIMS: To examine the resistance of beer isolates of lactic acid bacteria (LAB) towards a mixture of tetrahydroiso-alpha-acids (Tetra) by growth experiments as well as by measurement of intracellular pH . METHODS AND RESULTS: Beer LAB isolates were identified to species level by SDS-PAGE of whole-cell proteins . Beer isolates of Lactobacillus brevis showed better ability for growth in the presence of Tetra than nonbeer isolates of the L . brevis or other species of LAB including beer and nonbeer isolates . The antimicrobial effect of Tetra was also examined by noninvasive measurement of intracellular pH by fluorescence ratio imaging microscopy for selected beer isolates of L . brevis and Pediococcus inopinatus . Strains of L . brevis showing limited decrease of intracellular pH during exposure to Tetra also showed better ability for growth in the presence of these compounds as well as in commercial beer products . CONCLUSIONS: It was possible to apply a method for noninvasive measurement of intracellular pH to predict the resistance of beer spoilage LAB towards the Tetra hop analogue compounds . SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the usability of a new rapid method for detecting hop-resistant variants of known beer spoilage LAB species.

Pharm Res, 2004 Apr, 21(4), 585 - 91
A dynamic artificial gastrointestinal system for studying the behavior of orally administered drug dosage forms under various physiological conditions; Blanquet S et al.; PURPOSE: The purpose of this study was to demonstrate the potential of a dynamic, multicompartmental in vitro system simulating the human stomach and small intestine (TIM-1) for studying the behavior of oral drug dosage forms under various physiological gastrointestinal conditions . METHODS: Two model drug compounds were studied in TIM-1: a lyophilized Lactobacillus strain and paracetamol (acetaminophen) . The Lactobacillus survival rate was determined by bacterial counting in the gastric and ileal effluents while simulating the conditions of the gastrointestinal tract of infants or adults . The availability for absorption of paracetamol from two oral dosage forms was investigated by measuring the drug concentration in jejunal dialysis fluid . The effect of gastrointestinal passage time and food intake on paracetamol absorption was also studied . RESULTS: The Lactobacillus survival rate in both gastric and ileal effluents was higher during simulation of the infant compared to adult conditions . We also showed that (i) paracetamol absorption was faster when it was administered as a free powder than in sustained-release tablet form, (ii) a slow passage time resulted in a delay in the absorption of paracetamol, and (iii) there was a lower rate of absorption when paracetamol was ingested with a standard breakfast as opposed to water . The in vitro results were consistent with in vivo data, showing the predictive value of TIM-1 . CONCLUSIONS: TIM-1 is a powerful tool for supplying valuable information about the effects of various gastrointestinal conditions on biopharmaceutical behavior and efficacy of drug delivery systems in the development of oral formulations.

Int J Food Microbiol, 2004 Jun 1, 93(2), 219 - 30
Contribution of starter cultures to the proteolytic process of a fermented non-dried whole muscle ham product; Scannell AG et al.; Porcine longissimus dorsi muscles were cured by brine injection . Curing brine containing 15% (w/v) NaCl, 1.33% (w/v) glucose, 750 ppm sodium nitrite, and appropriate levels of either Lactobacillus sakei LAD, L . sakei LAD plus Kocuria varians FT4 (formally Micrococcus varians), L . sakei LAD plus papain and GDL (glucono-delta-lactone) plus K . varians FT4, was injected to the muscle at a pumping rate 15% w/v . The effect of these treatments on the proteolysis in the ham system was compared to a control ham, produced without starter culture and containing GDL acidulant to control pH and antibiotics to reduce the contribution of background microflora . Hydrolysis of sarcoplasmic and myofibrillar protein fractions was evaluated by SDS-PAGE and reverse phase-HPLC . Hams with different treatments were also investigated for differences in amino acid profile, protein and non-protein nitrogen level, colour, pH, water activity and moisture and microbiological evolution . There was no significant difference in the gross compositional analysis of any of the treatments compared to the control . There was no significant difference (p>0.05) in the protein content, non-protein nitrogen level, SDS-PAGE and free amino acid analysis between the control ham and ham inoculated with proteolytic starter culture . However, it was observed that hams containing starter cultures exhibited decreases in certain peptide fractions and corresponding increases in some free amino acids compared to the uninoculated control . It can be concluded that, while the principle mechanisms resulting in the proteolysis of this non-dried ham product involve the activity of endogeneous cathepsins, the addition of proteolytic starter cultures influence the amino acid profile thereby potentially enhancing the sensorial attributes of the ham .

Int J Food Microbiol, 2004 May 15, 93(1), 115 - 21
Souring and breakdown of cyanogenic glucosides during the processing of cassava into akyeke; Obilie EM et al.; The population and composition of the lactic acid bacteria microbiota as well as the content of cyanogenic glucosides occurring at various stages of fermentation and subsequent processing of cassava roots into akyeke, a steamed sour cassava meal, were investigated . The number of lactic acid bacteria and percentage titratable acidity increased during 5 days of fermentation, but decreases were observed in the subsequent operations of 'washing' the dough with water followed by partial drying and steaming . In field and laboratory samples, Lactobacillus plantarum accounted for 59.3% and 52.3%, Lactobacillus brevis 23.3% and 22.8% and Leuconostoc mesenteroides subsp . cremoris 14.5% and 15.8%, respectively, of all lactic acid bacteria isolated at various stages of fermentation and processing . A reduction of about 98% occurred in the total cyanogens (CN) content of cassava roots during processing, from 69.3 to 1.4 and 110.3 to 2.8 mg CN equivalent/kg dry weight for laboratory and field samples of akyeke, respectively .

Microbiology, 2004 May, 150(Pt 5), 1339 - 51
Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance; Len AC et al.; Streptococcus mutans is an important pathogen in the initiation of dental caries as the bacterium remains metabolically active when the environment becomes acidic . The mechanisms underlying this ability to survive and proliferate at low pH remain an area of intense investigation . Differential two-dimensional electrophoretic proteome analysis of S . mutans grown at steady state in continuous culture at pH 7.0 or pH 5.0 enabled the resolution of 199 cellular and extracellular protein spots with altered levels of expression . Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 167 of these protein spots . Sixty-one were associated with stress-responsive pathways involved in DNA replication, transcription, translation, protein folding and proteolysis . The 61 protein spots represented isoforms or cleavage products of 30 different proteins, of which 25 were either upregulated or uniquely expressed during acid-tolerant growth at pH 5.0 . Among the unique and upregulated proteins were five that have not been previously identified as being associated with acid tolerance in S . mutans and/or which have not been studied in any detail in oral streptococci . These were the single-stranded DNA-binding protein, Ssb, the transcription elongation factor, GreA, the RNA exonuclease, polyribonucleotide nucleotidyltransferase (PnpA), and two proteinases, the ATP-binding subunit, ClpL, of the Clp family of proteinases and a proteinase encoded by the pep gene family with properties similar to the dipeptidase, PepD, of Lactobacillus helveticus . The identification of these and other differentially expressed proteins associated with an acid-tolerant-growth phenotype provides new information on targets for mutagenic studies that will allow the future assessment of their physiological significance in the survival and proliferation of S . mutans in low pH environments.

Lett Appl Microbiol, 2004, 38(6), 499 - 504
Thermal and chemical resistance of Lactobacillus casei and Lactobacillus paracasei bacteriophages; Capra ML et al.; AIMS: The survival of two collection Lactobacillus casei and L . paracasei bacteriophages when subjected to thermal and chemical treatments was investigated . METHODS AND RESULTS: Thermal resistance was evaluated by heating phage suspensions at 63, 72 and 90 degrees C in three different media {Tris-magnesium gelatin (TMG) buffer: 10 mmol l(-1) Tris-Cl, 10 mmol l(-1) MgSO(4) and 0.1% w/v gelatin; Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)} . A marked heat sensitivity was evident in both phages, as 15 min at 72 degrees C was enough to completely inactivate (6 log(10) reduction) them . No clear influence was demonstrated by the suspension media . The phages also showed similar resistance to biocides . Peracetic acid and sodium hypochlorite (800 ppm) were the most effective ones, destroying the phages within 5 min . Concentrations of 75 and 100% ethanol were not suitable to inactivate phage particles even after 45 min . Isopropanol did not show an effect on phage viability . CONCLUSIONS: The data obtained in this work are important to design more effective control procedures in order to inactivate phages in dairy plants and laboratories . SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute to enhance the background knowledge about phages of probiotic bacteria.

Lett Appl Microbiol, 2004, 38(6), 459 - 63
Characterisation of autolytic enzymes in Lactobacillus pentosus; Cibik R et al.; AIMS: To characterize autolysis and autolytic system of the lactic acid bacterium Lactobacillus pentosus . METHODS AND RESULTS: Autolysis of nine Lact . pentosus strains was evaluated in buffer solution . Their peptidoglycan hydrolase profiles were examined by renaturing SDS-PAGE and revealed two major activity bands at 58 and 112 kDa . Specificity analysis indicated the presence of at least two different types of peptidoglycan hydrolase activities in Lact . pentosus 1091 . CONCLUSIONS: Autolysis of Lact . pentosus was shown to be strain dependent and involvement of at least two different autolysins was evidenced . SIGNIFICANCE AND IMPACT OF THE STUDY: The autolytic system of Lact . pentosus was characterized for the first time and the data obtained could be used in the selection of strains of technological interest.

Appl Environ Microbiol, 2004 May, 70(5), 3146 - 8
Transformation of folate-consuming Lactobacillus gasseri into a folate producer; Wegkamp A et al.; Five genes essential for folate biosynthesis in Lactococcus lactis were cloned on a broad-host-range lactococcal vector and were transferred to the folate auxotroph Lactobacillus gasseri . As a result L . gasseri changed from a folate consumer to a folate producer . This principle can be used to increase folate levels in many fermented food products.

Appl Environ Microbiol, 2004 May, 70(5), 2906 - 11
Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli; Kawai Y et al.; Lactobacillus gasseri LA39 and L . reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences . However, reutericin 6 has a narrower spectrum than gassericin A . In this study, gassericin A inhibited the growth of L . reuteri LA6, but reutericin 6 did not inhibit the growth of L . gasseri LA39 . Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different . Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of alpha-helices, the spectra of the bacteriocins did not coincide . The results of D- and L-amino acid composition analysis showed that two residues and one residue of D-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively . These findings suggest that the different D-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures . This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in D-amino acid contents in the molecules.

Appl Biochem Biotechnol, 2004 Apr, 117(1), 33 - 48
Characterization and comparison of metal accumulation in two Escherichia coli strains expressing either CopA or MntA, heavy metal-transporting bacterial P-type adenosine triphosphatases; Zagorski N et al.; MntA from Lactobacillus plantarum and copA from Enterococcus hirae both encode membrane proteins that are members of the P-type family of adenosine triphosphatases (ATPases) . Both transporters act as metal importers to take up nutritionally required substrates; MntA translocates Mn(II) and CopA translocates Cu(I) . Both ATPases can also translocate secondary substrates, Cd(II) and Ag(I), respectively . Although functionally and sequentially similar, these ATPases differ in several key residues and in their membrane topologies . The bioaccumulation properties of these two proteins were examined by coexpressing the transporters with overexpressed metallothionein in Escherichia coli cells, a system that has previously shown high levels of substrate-specific uptake . Both strains exhibited rapid metal accumulation, both saturated at around 50 microM metal, and both displayed temperature-sensitive uptake . However, the transporters responded differently when external conditions were varied; MntA displayed increased sensitivity to ionic strength, while CopA was more pH sensitive and more inhibited by chelating agents . The differences in accumulation are likely owing to structural differences in the transmembrane region of these two ATPases .

J Biomed Biotechnol, 2004, 2004(1), 61 - 69
Microencapsulated Genetically Engineered Lactobacillus plantarum 80 (pCBH1) for Bile Acid Deconjugation and Its Implication in Lowering Cholesterol; Jones ML et al.; Cholesterol is known to be a major risk factor for coronary heart disease (CHD) . Current treatments for elevated blood cholesterol include dietary management, regular exercise, and drug therapy with fibrates, bile acid sequestrants, and statins . Such therapies, however, are often suboptimal and carry a risk for serious side effects . This study shows that microencapsulated Lactobacillus plantarum 80 (pCBH1) cells can efficiently break down and remove bile acids, and establishes a basis for their use in lowering blood serum cholesterol . Results show that microencapsulated LP80 (pCBH1) is able to effectively break down the conjugated bile acids glycodeoxycholic acid (GDCA) and taurodeoxycholic acid (TDCA) with bile salt hydrolase (BSH) activities of 0.19 and 0.08 $\mu$ mol DCA/mg CDW/h respectively . This article also summarizes the physiological interrelationship between bile acids and cholesterol and predicts the oral doses of microencapsulated Lactobacillus plantarum 80 (pCBH1) cells required for lowering cholesterol.

J Antimicrob Chemother, 2004 May, 53(5), 772 - 7 Epub 2004 Apr 07.
Selection for high-level resistance by chronic triclosan exposure is not universal; McBain AJ et al.; OBJECTIVES: To investigate the effect of triclosan exposure on the antimicrobial susceptibilities of numerically important dental bacteria . METHODS: A gradient plate technique was used to expose Fusobacterium nucleatum, Lactobacillus rhamnosus, Neisseria subflava, Porphyromonas gingivalis, Actinomyces naeslundii, Prevotella nigrescens, Streptococcus oralis, Streptococcus sanguis, Streptococcus mutans and Veillonella dispar repeatedly to escalating, sublethal concentrations of triclosan . Escherichia coli ATCC 8739 was included as an organism showing the triclosan resistance development trait . MIC values towards chlorhexidine, metronidazole and tetracycline were determined before and after biocide exposure . RESULTS: N . subflava, Pr . nigrescens Po . gingivalis and E . coli were highly susceptible to triclosan (MIC range 0.1-3.9 mg/L), whereas the lactobacillus and S . mutans were less susceptible (MIC range 15.6-20.8 mg/L) . Triclosan exposure resulted in a highly significant ( approximately 400-fold) reduction in triclosan susceptibility (P < 0.01) for the positive control E . coli, although its MICs towards chlorhexidine, metronidazole and tetracycline were not significantly altered . Minor ( approximately two-fold) decreases in triclosan susceptibility (MIC) occurred for Pr . nigrescens and in S . sanguis and S . oralis (MBC) . Mean changes in susceptibilities (MIC and MBC) of the oral species to chlorhexidine, metronidazole and tetracycline did not exceed two-fold, although chlorhexidine MBCs for S . sanguis were markedly, but transiently, increased . CONCLUSIONS: These data fail to demonstrate biologically significant drug resistance in triclosan-exposed bacteria and suggest that markedly decreased triclosan susceptibility, although confirmed for E . coli, is not a universal phenomenon . Other bacteria possibly possess more susceptible targets than FabI that are highly conserved, which may govern triclosan activity.

J Med Food, 2004 Spring, 7(1), 38 - 44
The depletion of sodium nitrite by lactic acid bacteria isolated from kimchi; Oh CK et al.; Nitrites, whether added or naturally occurring in foods, are potential carcinogens, and controlling their concentrations is important for maintaining a safe food supply . In this study we investigated the depletion of sodium nitrite (150 microg/mL) during the fermentation in Lactobacilli MRS broth at 5, 10, 15, 20, 25, 30, and 36 degrees C by lactic acid bacteria (LAB-A, -B, -C, and -D) isolated from kimchi and Leuconostoc mesenteroides strain KCTC3100 . The four species of lactic acid bacteria isolated from kimchi were identified as L . mesenteroides, and all produced depletion of less than 20% of sodium nitrite after 10 days of incubation at 5 degrees C . There was less than 40% depletion after 9 days at 10 degrees C, 86.4-92.8% after 7 days at 15 degrees C, 81.4-87.8% after 4 days and more than 90.0% after 5 days at 20 degrees C, 76.3-85.7% after 3 days and more than 90.0% after 5 days at 25 degrees C, and more than 90.0% after 2 days at 30 and 36 degrees C . The depletion by LAB isolates was similar or higher than that by L . mesenteroides strain KCTC3100, and in particular, the LAB-D strain showed the highest depletion effect of all the strains tested, up to 15 degrees C . From these results, the strains isolated from kimchi were very effective for the depletion of sodium nitrite at high temperature, and all sodium nitrite was depleted at the initial period of incubation (1-2 days) at 30 and 36 degrees C . But as the temperature was lowered, the depletion effect of sodium nitrite was decreased in all the strains tested from kimchi . This illustrates that the depletion of nitrite by each strain is subject to the influence of temperatures.

Can Fam Physician, 2004 Apr, 50, 583 - 7
Caveat emptor . "Probiotics" might not be what they seem; Huff BA; OBJECTIVE: To assess whether commercially prepared probiotic products contain viable organisms, as claimed by their manufacturers, particularly whether products labeled as containing Lactobacillus did so . To identify and quantify as many species as feasible and to compare them with the contents listed on labels . DESIGN: Randomized, double-blind trial . SETTING: Community hospital in Chilliwack, BC . MAIN OUTCOME MEASURES: Trial of 10 randomly chosen brands of probiotic preparations bought over-the-counter in British Columbia's lower mainland . Only products claiming to contain lactobacillus were included in this study . Viable organisms in each probiotic brand and quantities of Lactobacillus in each product . RESULTS: None of the 10 products tested matched their labeled microbiologic specifications . Two brands grew nothing aerobically or anaerobically . No Lactobacillus grew in five brands, although their labels stated that this was the main species . Eight brands contained viable cells, but only 10% of the number stated by their manufacturers . CONCLUSION: Most product labels did not adequately identify or quantify microbes . Use of probiotics should not be recommended at this time.

Chem Commun (Camb), 2004 May 7, (9), 1084 - 5 Epub 2004 Mar 30.
Use of an ionic liquid in a two-phase system to improve an alcohol dehydrogenase catalysed reduction; Eckstein M et al.; Due to favourable partition coefficients the highly enantioselective reduction of 2-octanone, catalysed by an alcohol dehydrogenase from Lactobacillus brevis, is faster in a biphasic system containing buffer and the ionic liquid {BMIM}{(CF(3)SO(2))(2)N} compared to the reduction in a biphasic system containing buffer and methyl tert-butyl ether.

J Nutr, 2004 May, 134(5), 1120 - 3
Lactobacillus casei alters hPEPT1-mediated glycylsarcosine uptake in Caco-2 cells; Neudeck BL et al.; Augmentation of the normal flora of the gastrointestinal tract with probiotic bacteria is currently under investigation as a therapeutic tool for several diseases . However, it is unknown whether probiotic bacteria such as Lactobacillus casei alter the expression and function of intestinal transport proteins such as hPEPT1 . The effects of 24 and 48 h incubation of Caco-2 cells with 10(8)/L L . casei on the hPEPT1-mediated uptake rate of 20 micro mol/L {(3)H}glycylsarcosine were examined . Dipeptide uptake did not differ from the control at 24 h (15.9 +/- 2.4 vs . 11.5 +/- 1.4 cm.s(-1).mg protein(-1)); however, a significant increase in uptake occurred after 48 h of L . casei treatment (23.7 +/- 1.5 vs . 12.0 +/- 1.9 cm.s(-1).mg protein(-1); P = 0.005) . hPEPT1 involvement was confirmed in experiments using excess substrate . Increased uptake of {(3)H}glycylsarcosine appeared to be the result of the direct interaction of the bacteria with Caco-2 cells because conditioned medium had no effect on dipeptide uptake . hPEPT1 mRNA levels did not differ at any time point . These results show that prolonged incubation of Caco-2 cells leads to increased hPEPT1 activity and that this occurs by a mechanism distinct from increased gene expression.

J Virol, 2004 May, 78(10), 5147 - 56
Theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry; Yasin B et al.; We tested the ability of 20 synthetic theta defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively) . The peptides included rhesus theta defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human theta-defensin (DEFT) pseudogenes . We also tested 14 retrocyclin analogues, including the retro, enantio, and retroenantio forms of retrocyclin 1 . Retrocyclins 1 and 2 and RTD 3 protected cervical epithelial cells from infection by both HSV serotypes, but only retrocyclin 2 did so without causing cytotoxicity or requiring preincubation with the virus . Surface plasmon resonance studies revealed that retrocyclin 2 bound to immobilized HSV-2 glycoprotein B (gB2) with high affinity (K(d), 13.3 nM) and that it did not bind to enzymatically deglycosylated gB2 . Temperature shift experiments indicated that retrocyclin 2 and human alpha defensins human neutrophil peptide 1 (HNP 1) to HNP 3 protected human cells from HSV-2 by different mechanisms . Retrocyclin 2 blocked viral attachment, and its addition during the binding or penetration phases of HSV-2 infection markedly diminished nuclear translocation of VP16 and expression of ICP4 . In contrast, HNPs 1 to 3 had little effect on binding but reduced both VP16 transport and ICP4 expression if added during the postbinding (penetration) period . We recently reported that theta defensins are miniature lectins that bind gp120 of human immunodeficiency virus type 1 (HIV-1) with high affinity and inhibit the entry of R5 and X4 isolates of HIV-1 . Given its small size (18 residues), minimal cytotoxicity, lack of activity against vaginal lactobacilli, and effectiveness against both HSV-2 and HIV-1, retrocyclin 2 provides an intriguing prototype for future topical microbicide development.

BMC Gastroenterol . 2004 Mar 15;4(1):5.
Lactobacillus GG in inducing and maintaining remission of Crohn's disease; Schultz M et al.; BACKGROUND: Experimental studies have shown that luminal antigens are involved in chronic intestinal inflammatory disorders such as Crohn's disease and ulcerative colitis . Alteration of the intestinal microflora by antibiotic or probiotic therapy may induce and maintain remission . The aim of this randomized, placebo-controlled trial was to determine the effect of oral Lactobacillus GG (L . GG) to induce or maintain medically induced remission . METHODS: Eleven patients with moderate to active Crohn's disease were enrolled in this trial to receive either L . GG (2 x 10(9) CFU/day) or placebo for six months . All patients were started on a tapering steroid regime and received antibiotics for the week before the probiotic/placebo medication was initiated . The primary end point was sustained remission, defined as freedom from relapse at the 6 months follow-up visit . Relapse was defined as an increase in CDAI of >100 points . RESULTS: 5/11 patients finished the study, with 2 patients in each group in sustained remission . The median time to relapse was 16 +/- 4 weeks in the L . GG group and 12 +/- 4.3 weeks in the placebo group (p = 0.5) . CONCLUSION: In this study we could not demonstrate a benefit of L . GG in inducing or maintaining medically induced remission in CD.

Int J Food Microbiol, 2004 Apr 15, 92(2), 153 - 60
Characterization of thoeniicin 447, a bacteriocin isolated from Propionibacterium thoenii strain 447; Van der Merwe IR et al.; Fifteen strains of propionibacteria, isolated from dairy products, were screened for the production of bacteriocins . Propionibacterium thoenii 447 produced an antimicrobial peptide, thoeniicin 447, which acted bactericidal against Lactobacillus delbrueckii subsp . bulgaricus and bacteriostatic against Propionibacterium acnes . Thoeniicin 447 remained active after 15 min at 100 degrees C and after 30 min of incubation at pH 1-10 . The peptide was inactivated when treated with pepsin, pronase, alpha-chymotrypsin, trypsin and proteinase K . Optimal bacteriocin production was detected during late exponential growth . The peptide was partially purified by ammonium sulfate precipitation, followed by SP-Sepharose cation exchange chromatography . The estimated size of thoeniicin 447, according to tricine-SDS-PAGE, is 6 kDa . Based on DNA sequencing, the mature peptide is 7130.20 Da in size and homologous to propionicin T1, produced by P . thoenii strain 419 (=NCFB 568(T)) . Strain 447 is phenotypically different from strain 419 and belongs to a separate ribotype cluster . To our knowledge, this is the first report of a bacteriocin from a Propionibacterium species active against P . acnes.

Dis Colon Rectum, 2004 Jun, 47(6), 876 - 84 Epub 2004 Apr 19.
Delay of the first onset of pouchitis by oral intake of the probiotic strain Lactobacillus rhamnosus GG; Gosselink MP et al.; PURPOSE: Proctocolectomy with ileal pouch-anal anastomosis is the operation of choice for patients with refractory or fulminant ulcerative colitis . The most common long-term complication in these patients is pouchitis . This study was designed to investigate the efficacy of probiotic Lactobacillus rhamnosus GG in long-term delaying the first onset of pouchitis . METHODS: Between 1989 and 2001, a consecutive series of 127 patients presenting with ulcerative colitis underwent an ileal pouch-anal anastomosis at the Erasmus Medical Center in Rotterdam . Histopathologic investigation of the resected specimens revealed unsuspected Crohn's disease in five patients . Postoperative complications resulted in pouch excision in five patients . The remaining 117 patients were included in this study . All episodes of pouchitis occurring in this group were analyzed . Pouchitis was diagnosed on the basis of clinical symptoms and endoscopic and histologic features . The 39 patients, who underwent an ileal pouch-anal anastomosis between 1996 and 2001, started immediately after the operation with the daily intake of L . rhamnosus GG in a fermented product . The 78 patients, in whom an ileal pouch-anal anastomosis was performed between 1989 and 1996, received no L . rhamnosus GG . RESULTS: Except for the duration of follow-up, the patient characteristics, indications for proctocolectomy, number of postoperative complications, and functional outcome were similar in both groups . First episodes of pouchitis were observed less frequently in patients with a daily intake of L . rhamnosus GG (cumulative risk at 3 years: 7 vs . 29 percent; P = 0.011) . CONCLUSIONS: Daily intake of fermented products containing L . rhamnosus GG provides significant clinical benefit, without side effects . Based on the results of this study, we recommend a daily intake of Lactobacillus rhamnosus GG (dose 1-2 x 10(10) bacteria) to delay the first onset of pouchitis.

J Endod, 2004 May, 30(5), 352 - 8
Effect of dentin on the antibacterial activity of dentin bonding agents; Schmalz G et al.; Dentin bonding agents with antibacterial effects may inhibit secondary caries formation and pulp inflammation by eliminating residual bacteria in and on dentin . Therefore, the antibacterial effects of Prime & Bond NT (PB), Prime & Bond NT without fluoride (PBNF), Gluma Comfort Bond (GL), ABF, Xeno CF II (XE), 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEG-DMA), and 0.2% chlorhexidine were tested against Streptococcus mutans, S . sobrinus, and Lactobacillus acidophilus using the agar-diffusion method with and without bovine-dentin disks (200 microm and 500 microm thickness) placed between the bacteria and the test substances . Without dentin, ABF Primer showed growth inhibition for all bacterial strains . XE inhibited S . mutans and S . sobrinus, and PB S . sobrinus . ABF Bonding inhibited L . acidophilus . PBNF, HEMA, and TEGDMA did not have any antibacterial effects . Dentin disks of 500 microm thickness reduced the inhibitory effect of chlorhexidine to 23% to 54% compared with direct application . ABF Primer (nonpolymerized) produced inhibition zones against all tester strains regardless of dentin disks interposed or not . XE (against S . mutans and S . sobrinus) and PB (against S . sobrinus) did not produce any inhibition zones on 200 microm thick dentin . After polymerization, the ABF system did not inhibit bacterial growth on 200 microm thick dentin disks . A dentin barrier reduces significantly the antibacterial activity of chlorhexidine and dentin bonding agents.

J Endod, 2004 May, 30(5), 333 - 8
Comparison of type 1 and type 2 cytokine production by mononuclear cells cultured with streptococcus mutans and selected other caries bacteria; Hahn CL et al.; A feature of pulpal immune responses is the predominance of type 1 cytokine mRNA under shallow caries and a mixed (type 1/type 2) profile under deep caries . These results prompted an examination of the cytokine profiles induced by bacteria in shallow caries (Streptococcus mutans and Actinomyces viscosus) and deep caries (Lactobacillus casei, Pseudoramibacter alactolyticus, and Prevotella intermedia) . All isolates induced interferon-gamma and interleukin-10, whereas interleukin-4 and interleukin-2 titers were low to undetectable . S . mutans was the most potent and persistent interferon-gamma inducer . Differences in interleukin-10 were apparent at low doses but were less dramatic, with L . casei the dominant producer . S . mutans induced substantially more interferon-gamma than interleukin-10 over all doses and time points, suggesting strong type 1 polarization . P . alactolyticus induced significantly more interleukin-10 than interferon-gamma at higher concentrations, suggesting polarization toward type 2 . A similar amount of interferon-gamma and interleukin-10 induced by L . casei, A . viscosus, and P . intermedia reflected a mixed profile . A better understanding of pulpal immune response to caries bacteria may enable us to develop an immune system-based pulp therapy in the future.

Infect Immun, 2004 May, 72(5), 2731 - 7
Enhanced mucosal delivery of antigen with cell wall mutants of lactic acid bacteria; Grangette C et al.; The potential of recombinant lactic acid bacteria (LAB) to deliver heterologous antigens to the immune system and to induce protective immunity has been best demonstrated by using the C subunit of tetanus toxin (TTFC) as a model antigen . Two types of LAB carriers have mainly been used, Lactobacillus plantarum and Lactococcus lactis, which differ substantially in their abilities to resist passage through the stomach and to persist in the mouse gastrointestinal tract . Here we analyzed the effect of a deficiency in alanine racemase, an enzyme that participates in cell wall synthesis, in each of these bacterial carriers . Recombinant wild-type and mutant strains of L . plantarum NCIMB8826 and L . lactis MG1363 producing TTFC intracellularly were constructed and used in mouse immunization experiments . Remarkably, we observed that the two cell wall mutant strains were far more immunogenic than their wild-type counterparts when the intragastric route was used . However, intestinal TTFC-specific immunoglobulin A was induced only after immunization with the recombinant L . plantarum mutant strain . Moreover, the alanine racemase mutant of either LAB strain allowed induction of a much stronger serum TTFC-specific immune response after immunization via the vagina, which is a quite different ecosystem than the gastrointestinal tract . The design and use of these mutants thus resulted in a major improvement in the mucosal delivery of antigens exhibiting vaccine properties.

Korean J Gastroenterol, 2004 Apr, 43(4), 234 - 45
{Prophylactic effect of Lactobacillus GG in animal colitis and its effect on cytokine secretion and mucin gene expressions}; Moon G et al.; BACKGROUND/AIMS: Lactobacillus rhamnosus GG (LGG) has been used in acute colitis treatment . However, it is unclear whether the LGG prevents chronic colitis . The aim of this study was to examine the prophylactic effect of LGG on animal colitis, cytokine secretion, and mucin gene expression . METHODS: BALB/c mice (n=64) were exposed to 5% dextran sulfate sodium (DSS) for 7 days followed by 10 days recovery period and repeatedly exposed for 4 days . Then, the mice were devided into three group; group of oral LGG adminstration throughout the recovery and repeated colitis period; PBS group of PBS administration; control group . Colon length, histologic score, tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) levels, mucin gene expressions were determined at each period . RESULTS: In acute colitis period, the LGG group showed higher levels of disease activity index (DAI), histologic score, TNF-alpha, IL-10, but shorter colon length, lower levels of mucin gene expressions than the control group . However, in repeated colitis period, the LGG group showed markedly lower levels of DAI and IL-10 but significantly longer colon length than PBS group (p<0.05) . There was no difference in the mucin gene expression . CONCLUSIONS: These results suggest that LGG prevents chronic murine colitis . It may be associated with cytokine modulation and competitive inhibition of pathogenic bacteria . However, it may not be related with gene expression.

J Med Microbiol, 2004 May, 53(Pt 5), 451 - 3
Case of aortic endocarditis caused by Lactobacillus casei; Ze-Ze L et al.; A case of Lactobacillus aortic valve endocarditis in a 53-year-old immunocompetent patient with past history of rheumatic fever is reported . Clinical symptoms began after a dental extraction and the patient's diet included several yogurts per day . Blood, bone marrow cultures and the replaced aortic valve were positive for Lactobacillus: The clinical isolate was identified as Lactobacillus casei by 16S rDNA sequencing.

Curr Opin Infect Dis, 2004 Feb, 17(1), 41 - 4
Bacterial vaginosis and miscarriage; Hay PE; PURPOSE OF REVIEW: This review aims to summarize current knowledge about the relationship between bacterial vaginosis and miscarriage . RECENT FINDINGS: Studies investigating the relationship between bacterial vaginosis and infertility, implantation and early pregnancy loss have produced conflicting results . One study demonstrated a beneficial effect of colonization with hydrogen peroxide producing lactobacilli . A community based study found no association between bacterial vaginosis and first trimester loss, but a positive association with early second trimester loss . In a randomized controlled trial, treatment of bacterial vaginosis with oral clindamycin was associated with a fivefold reduction in the incidence of late miscarriage . SUMMARY: The relationship between bacterial vaginosis and infertility and first trimester loss needs further elucidation . Measures to support a healthy lactobacillus flora such as probiotic therapy warrant study . The randomized controlled trials of clindamycin treatment need to be replicated in different settings.

Dis Colon Rectum . 2004 Apr 19; {Epub ahead of print}
Delay of the First Onset of Pouchitis by Oral Intake of the Probiotic Strain Lactobacillus rhamnosus GG; Gosselink MP et al.; PURPOSE: Proctocolectomy with ileal pouch-anal anastomosis is the operation of choice for patients with refractory or fulminant ulcerative colitis . The most common long-term complication in these patients is pouchitis . This study was designed to investigate the efficacy of probiotic Lactobacillus rhamnosus GG in long-term delaying the first onset of pouchitis.METHODS: Between 1989 and 2001, a consecutive series of 127 patients presenting with ulcerative colitis underwent an ileal pouch-anal anastomosis at the Erasmus Medical Center in Rotterdam . Histopathologic investigation of the resected specimens revealed unsuspected Crohn's disease in five patients . Postoperative complications resulted in pouch excision in five patients . The remaining 117 patients were included in this study . All episodes of pouchitis occurring in this group were analyzed . Pouchitis was diagnosed on the basis of clinical symptoms and endoscopic and histologic features . The 39 patients, who underwent an ileal pouch-anal anastomosis between 1996 and 2001, started immediately after the operation with the daily intake of L . rhamnosus GG in a fermented product . The 78 patients, in whom an ileal pouch-anal anastomosis was performed between 1989 and 1996, received no L . rhamnosus GG.RESULTS: Except for the duration of follow-up, the patient characteristics, indications for proctocolectomy, number of postoperative complications, and functional outcome were similar in both groups . First episodes of pouchitis were observed less frequently in patients with a daily intake of L . rhamnosus GG (cumulative risk at 3 years: 7 vs . 29 percent; P = 0.011).CONCLUSIONS: Daily intake of fermented products containing L . rhamnosus GG provides significant clinical benefit, without side effects . Based on the results of this study, we recommend a daily intake of Lactobacillus rhamnosus GG (dose 1-2 x 10(10) bacteria) to delay the first onset of pouchitis.

J Med Chem, 2004 Apr 22, 47(9), 2264 - 75
Heterocyclic bis-cations as starting hits for design of inhibitors of the bifunctional enzyme histidine-containing protein kinase/phosphatase from Bacillus subtilis; Ramstrom H et al.; The main mechanism of carbon catabolite repression/activation in low-guanine and low-cytosine Gram-positive bacteria seems to involve phosphorylation of HPr (histidine-containing protein) at Ser-46 by the ATP-dependent HPr kinase, which in Bacillus subtilis, Lactobacillus casei, and Staphylococcus xylosus also exhibits phosphatase activity and is thus a bifunctional enzyme (HPrK/P) . Since deficiency of HPrK/P in S . xylosus, L . casei, and B . subtilis mutants leads to severe growth defects, inhibitors of the enzyme could form a new family of antibiotic drugs . The aim of the study was to screen an in-house chemical library for identification of hits as inhibitors of HPrK/P in B . subtilis and to further extract additional information of structural features from hit optimization using a radioactive in vitro assay . A symmetrical bis-cationic compound LPS 02-10-L-D09 (2a) with a 12-carbon alkyl linker bridging the two 2-aminobenzimidazole moieties was identified as a non-ATP mimetic compound exhibiting an EC(50) value of 10 microM in a kinase assay with HPr as substrate . The substance also inhibited the phosphatase activity of HPrK/P triggered by the addition of inorganic phosphate . Similar results were obtained with 2a and catabolite repression HPr, which, like HPr, can be phosphorylated at Ser-46 by HPrK/P and is involved in catabolite repression . Structure-activity relationship analysis indicated the importance in its structure of a substituted 2-aminobenzimidazole . This typical heterocycle is linked through a C12 alkyl chain to a second scaffold that can bear a cationic or a noncationic moiety but in all cases should present an aromatic ring in its vicinity.

Eur J Nutr, 2004 Apr, 43(2), 61 - 8 Epub 2004 Jan 06.
The effect of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women; Narva M et al.; BACKGROUND: Milk fermented with Lactobacillus helveticus ( L . helveticus) has been shown to lower blood pressure and to increase bone mineral content in spontaneously hypertensive rats . The effect of L.helveticus may be due to better calcium availability . AIM OF THE STUDY: In the present study the effect of milk fermented with L . helveticus on acute changes in calcium metabolism and bone resorption in postmenopausal women was studied . METHODS: The study was performed as a randomised double-blind crossover study of 20 postmenopausal women (mean age 65, range 50-78) . The study was carried out in two parts . Firstly, L . helveticus fermented milk was compared to a control milk . Secondly, juice containing peptides formed with L . helveticus bacteria was compared to a control juice . The acute effect on calcium metabolism was measured during the study day by serum ionised calcium (iCa), parathyroid hormone (PTH), calcium (Ca), phosphate (P), and urinary calcium . A direct marker of bone turnover, carboxyterminal telopeptide of type I collagen (ICTP), was measured from the serum . RESULTS: L . helveticus fermented milk reduced serum PTH (405.3 +/- 37 ng/l vs . 454.9 +/- 37, p = 0.012) and increased serum calcium (19.1 +/- 0.2 mmol/l vs . 18.8 +/- 0.2, p = 0.031) compared to the control milk . L . helveticus derived peptides had no significant acute effect on calcium metabolism, in fact, ionised calcium was lower and PTH higher after the juice containing peptides compared to the control juice . CONCLUSIONS: Fermentation of milk with Lactobacillus helveticus had a positive acute effect on calcium metabolism . This effect was not explained by the small peptides formed by L . helveticus.

Gut, 2004 May, 53(5), 694 - 700
Is the mucosal route of administration essential for probiotic function? Subcutaneous administration is associated with attenuation of murine colitis and arthritis; Sheil B et al.; BACKGROUND: We and others have reported the prophylactic efficacy of oral consumption of probiotic lactobacilli in the interleukin 10 knockout (IL-10 KO) model of colitis . It has not been demonstrated that the oral route is essential for probiotic efficacy . AIMS: (i) To determine the effect of parenteral administration (subcutaneous) of Lactobacillus salivarius 118 on colitis of IL-10 KO mice; and (ii) to determine if observed responses are disease specific . METHODS: (i) IL-10 KO mice were injected subcutaneously with L salivarius 118 or saline over 19 weeks . At sacrifice, the bowels were histologically scored . Isolated splenocytes were cultured in vitro and cytokine levels measured . (ii) In the collagen induced arthritis model, DBA/1 mice were injected subcutaneously with the probiotic or saline . At sacrifice, paw thickness was measured and joints were histologically scored . RESULTS: (i) Colonic inflammatory scores were significantly decreased in IL-10 KO mice injected with L salivarius 118 compared with controls (p<0.05) . Proinflammatory cytokine production from stimulated splenocytes was significantly lower for the probiotic group whereas stimulated transforming growth factor beta (TGF-beta) levels were significantly increased (p<0.05) . (ii) Scoring of arthritis and paw thickness were significantly improved in the group of mice injected with L salivarius 118 compared with controls . CONCLUSIONS: (1) Subcutaneous administration of L salivarius 118 significantly attenuated colitis in the IL-10 KO model and suppressed collagen induced arthritis, suggesting that the oral route may not be essential for probiotic anti-inflammatory effects and that responses are not disease specific . (2) The probiotic effect was associated with reduced production of proinflammatory (T helper 1) cytokines and maintained production of anti-TGF-beta.

J Appl Microbiol, 2004, 96(5), 1082 - 9
Characterization and purification of acidocin CH5, a bacteriocin produced by Lactobacillus acidophilus CH5; Chumchalova J et al.; AIMS: To characterize and to purify a bacteriocin produced by Lactobacillus acidophilus strain with its activity restricted to Gram-positive bacteria . METHODS AND RESULTS: Native acidocin CH5, a bacteriocin produced by L . acidophilus CH5 an isolate from a dairy starter culture forms in MRS (Oxoid, Basingstoke, UK) broth high-molecular weight aggregates which can dissociate into smaller units (retained by 5 kDa membrane) with higher activity . Acidocin CH5 was purified using combinations of chromatographic methods based on hydrophobic and cation exchange principles and the N-terminal region was sequenced . CONCLUSIONS: Based on our results it is evident that acidocin CH5 belongs, according to bacteriocin classification, to the class II bacteriocins with identical N-terminal amino acid sequence described in the literature previously . SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on bacteriocin acidocin CH5 from class II with wide spectrum of antimicrobial activity atypical for bacteriocins produced by L . acidophilus sharing the same homology.

J Appl Microbiol, 2004, 96(5), 1024 - 39
Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances; Corcoran BM et al.; AIMS: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products . METHODS AND RESULTS: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C . Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively . Stationary phase Lact . rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C . Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45% . To compare different probiotic lactobacilli during spray-drying, stationary phase Lact . rhamnosus E800 and Lact . salivarius UCC 500 were spray-dried using the same parameters as for Lact . rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v) . Lact . rhamnosus E800 experienced approx . 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact . salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media . In addition to the superior survival of Lact . rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact . rhamnosus GG . CONCLUSIONS: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible . The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage . SIGNIFICANCE AND IMPACT OF THE STUDY: High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.

J Appl Microbiol, 2004, 96(5), 946 - 53
Genetic characterization of non-spoilage variant isolated from beer-spoilage Lactobacillus brevis ABBC45; Suzuki K et al.; AIMS: To characterize the non-spoilage variant obtained from beer-spoilage Lactobacillus brevis ABBC45C and to identify a potential genetic marker capable of discriminating beer-spoilage L . brevis strains from non-spoilers . METHODS AND RESULTS: A non-spoilage variant was obtained from beer-spoilage L . brevis ABBC45C by repeatedly subculturing the strain at 37 degrees C . Genetic characterization of the variant revealed that 12,605 bp portion of one plasmid, designated pRH45II, was lost in the variant . The sequence analysis indicates the presence of 12 ORFs in the deleted region of pRH45II . The PCR and Southern hybridization study revealed that the homologues of ORF5 found in the deleted region were present in all of the beer-spoilage L . brevis strains examined in this study . In contrast, the homlogues appeared to be absent in non-spoilage L . brevis strains . CONCLUSIONS: The presence or absence of ORF5 homologues was found to be highly correlated with the beer-spoilage ability of L . brevis strains, indicating this ORF is potentially a useful genetic marker capable of differentiating beer-spoilage strains among L . brevis . SIGNIFICANCE AND IMPACT OF THE STUDY: A non-spoilage variant was successfully isolated from beer-spoilage L . brevis ABBC45C . This study could facilitate the understanding of mechanisms underlying beer-spoilage ability of L . brevis.

Protein Pept Lett, 2004 Apr, 11(2), 189 - 94
Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels; Silva J et al.; Protein extraction and the proteome of Lactobacillus delbrueckii subsp . bulgaricus were studied using different stains . The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain . Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.

Community Dent Health, 2004 Mar, 21(1 Suppl), 121 - 30
International comparisons of health inequalities in childhood dental caries; Pine CM et al.; OBJECTIVE: To undertake formative studies investigating how the experience of dental caries in young children living in diverse settings relates to familial and cultural perceptions and beliefs, oral health-related behaviour and oral microflora . PARTICIPANTS: The scientific consortium came from 27 sites in 17 countries, each site followed a common protocol . Each aimed to recruit 100 families with children aged 3 or 4 years, half from deprived backgrounds, and within deprived and non-deprived groups, half to be "caries-free" and half to have at least 3 decayed teeth . OUTCOME MEASURES: Parents completed a questionnaire, developed using psychological models, on their beliefs, attitudes and behaviours related to their child's oral health . 10% of children had plaque sampled . RESULTS: 2,822 children and families were recruited . In multivariate analyses, reported toothbrushing behaviours that doubled the odds of being caries-free were a combination of brushing before age 1, brushing twice a day and adult involvement in brushing . Analyses combining beliefs, attitudes and behaviours found that parents' perceived ability to implement regular toothbrushing into their child's daily routine was the most important predictor of whether children had caries and this factor persisted in children from disadvantaged communities . 90% of children with lactobacillus had caries . CONCLUSIONS: Parental beliefs and attitudes play a key role in moderating oral health related behaviour in young children and in determining whether they develop caries . Further research is indicated to determine whether supporting the development of parenting skills would reduce dental caries in children from disadvantaged communities independent of ethnic origin.

Community Dent Health, 2004 Mar, 21(1 Suppl), 96 - 101
A multi-country comparison of caries-associated microflora in demographically diverse children; Beighton D et al.; OBJECTIVE: The aim of this formative international collaborative research on childhood dental caries was to undertake an initial investigation comparing the dental plaque of young children from diverse ethnic and socioeconomic backgrounds with and without dental caries . BASIC RESEARCH DESIGN: The following four null hypotheses were investigated . There were no differences in numbers of individual taxa when comparing plaque samples from: 1) caries-free children from deprived and non-deprived backgrounds; 2) children from deprived and non-deprived backgrounds with at least 3 decayed teeth; 3) children from non-deprived backgrounds who are caries free with those from similar backgrounds with at least 3 decayed teeth; and, 4) children from deprived backgrounds who are caries free with those from similar backgrounds with at least 3 decayed teeth . PARTICIPANTS: 277 children aged 3-4 years from 5 countries . MAIN OUTCOME MEASURES: A sample of interproximal plaque from anterior teeth was collected using sterile dental floss, and cultured according to accepted international standards . RESULTS: Analysis of the data found that the first null hypothesis was accepted and that the fourth was rejected . Unexpectedly, the second null hypothesis was rejected as the children with caries from deprived and non-deprived backgrounds had a different caries-associated flora . In particular, children living in deprivation harbored more caries-associated bacteria {mutans streptococci and lactobacilli} . This greater microbial challenge was associated with a higher level of cavitated carious lesions and with more frequent consumption of confectionery . Conclusions Children from deprived backgrounds with caries may be further disadvantaged by having higher levels of caries-associated microflora.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2004 Mar, 35(2), 217 - 9
{Amplification of Lactobacillus bulgaricus beta-galactosidase gene and optimization of the reaction condition}; Wang C et al.; OBJECTIVE: To obtain the gene which encode high activity beta-galactosidase from Lactobacillus bulgaricus and study the influencing factors of amplification . METHODS: Lysozyme and freeze-thaw cycles were applied to obtain Lactobacillus bulgaricus DNA; different template concentration and annealing temperature were selected for the amplification . RESULTS: 60 ng/microliter template concentration and 66 degrees C annealing temperature were the best reaction conditions of amplifying the gene . CONCLUSION: This study optimized the template concentration and anneal temperature conditions of amplifying Lactobacillus bulgaricus beta-galactosidase gene, thus providing a foundation for further studies.

Shanghai Kou Qiang Yi Xue, 1998 Sep, 7(3), 133 - 5
{The influences of lanthanum,cerium and fluoride on the activity of glucosyltransferase(GTF) and adherence of oral cariogenic bacteria in vitro}; Li MY et al.; OBJECTIVE:Study of the influences of lanthanum,cerium and fluoride on the activity of flucosyltransferase(GTF) and adherence of oral cariogenic bacteria . METHODS: The influences of lanthanum,certium and fluoride on the acitivity of GTF and adherences of Steptococcus sobrinus 6715,Lactobacillus 4356 and Actinomycetes viscosus 19246 in vitro were investigated . RESULTS: Lanthanum and cerium can efficiently inhibit the acitivity of GTF than fluoride . CONCLUSION: The result was given a data that lanthanum and cerium could be used as a cariostatic agent in future.At the same time the inhibition of the bacterial adherence by these trace elements was also revealed.

Shanghai Kou Qiang Yi Xue, 1998 Dec, 7(4), 226 - 7
{The measurement of growth curve and generation time of lactobacillus}; Gu SP et al.; OBJECTIVE:The purpose of this test was to understand the growth pattern of lactobacillus for the research of its cariogenicity . METHODS: The growth quantity of lactobacillus which was culture in a constant condition was measured periodically by spectrophotometry and flora counting,and its growth curve and generation time were measured . RESULTS: It was found that the logarithmic phase of lactobacillus was 6-16 hours after it was cultured.And its generatin time was 54 minutes . CONCLUSIN: The growth curve of lactobacillus was in accordance with streptococcus mutan.

Shanghai Kou Qiang Yi Xue, 1998 Dec, 7(4), 224 - 5
{The effect of PH on the growth of lactobacillus and Streptococcus mutan}; Gu SP et al.; OBJECTIVE:The purpose of this study was to understand the effect of pH of culture media on the growth of lactobacillus and streptococcus mutan.METHODS: In this study the growth of two bacteria in differential pH culture media was measured by spectrophotometry . RESULTS: It was found that the growth of the bacteria was equal when the pH of the culture media was 6.2 . CONCLUSION: This pH should be selected for the culture media which is used for the analysis of the cariogenicity of the two bacteria.

Helicobacter, 2004 Apr, 9(2), 165 - 72
The influence of Lactobacillus brevis on ornithine decarboxylase activity and polyamine profiles in Helicobacter pylori-infected gastric mucosa; Linsalata M et al.; BACKGROUND: Functional probiotics may prevent Helicobacter pylori infection, and some evidence suggests that they also possess antitumor properties . Lactobacillus brevis (CD2) is a functional Lactobacillus strain with peculiar biochemical features, essentially related to the activity of arginine deiminase . This enzyme catalyzes the catabolism of arginine and affects the biosynthesis of polyamines (putrescine, spermidine, and spermine) . Polyamines are polycations found in high concentrations in both normal and neoplastic cells . Our aims were: 1, to assess whether oral administration of L . brevis (CD2) affects H . pylori survival in the human gastric mucosa; 2, to evaluate the effects of L . brevis (CD2) on polyamine biosynthesis in gastric biopsies from H . pylori-positive patients . MATERIALS AND METHODS: For 3 weeks before endoscopy, 22 H . pylori-positive dyspeptic patients randomly received (ratio 1 : 1) high oral doses of L . brevis (CD2) or placebo . Before and after treatment, H . pylori infection was determined by urea breath test (UBT) . In gastric biopsies, ornithine decarboxylase activity and polyamine levels were, respectively, evaluated by a radiometric technique and high-pressure liquid chromatography (HPLC) . RESULTS: L . brevis (CD2) treatment did not eradicate H . pylori . However, a reduction in the UBT delta values occurred, suggesting a decrease in intragastric bacterial load . Significantly, L . brevis (CD2) induced a decrease in gastric ornithine decarboxylase activity and polyamine levels . CONCLUSIONS: Our data support the hypothesis that L . brevis (CD2) treatment decreases H . pylori colonization, thus reducing polyamine biosynthesis . Alternatively, the arginine deiminase activity following L . brevis (CD2) administration might cause arginine deficiency, preventing polyamine generation from gastric cells.

J Dairy Res, 2004 Feb, 71(1), 107 - 15
Texture, proteolysis and viable lactic acid bacteria in commercial cheddar cheeses treated with high pressure; Wick C et al.; High pressure processing was investigated for controlling Cheddar cheese ripening . One-month-or 4-month-old Cheddar cheeses were subjected to pressures ranging from 200 to 800 MPa for 5 min at 25 C . The number of viable Lactococcus lactis (starter) and Lactobacillus (nonstarter) cells decreased as pressure increased . Subsequent storage of the control and pressure-treated cheeses at 10 degrees C caused viable cell counts to change in some cases . Free amino acid content was monitored as an indicator of proteolysis . Cheeses treated with pressures > or = 400 MPa evolved free amino acids at significantly lower rates than the control . No acceleration in free amino acid development was observed at lower pressures . Pressure treatment did not accelerate the rate of textural breakdown compared with the non-pressure treated control . On the contrary, pressure treatment at 800 MPa reduced the time-dependent texture changes . Results indicate that high pressure may be useful in arresting Cheddar cheese ripening.

Appl Environ Microbiol, 2004 Apr, 70(4), 2367 - 72
Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus; Wang X et al.; Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research . FA in cereals exists mainly as feruloylated sugar ester . To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73) . In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time . The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography . The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa . It has optimum pH and temperature characteristics (5.6 and 37 degrees C, respectively) . The metal ions Cu(2+) and Fe(3+) (at a concentration of 5 mmol liter(-1)) inhibited FAE activity by 97.25 and 94.80%, respectively . Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K(m) of 0.0953 mmol liter(-1) and a V(max) of 86.27 mmol liter(-1) min(-1) mg(-1) of protein . Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T . The FAE released FA from O-(5-O-feruloyl-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran . Moreover, it released two times more FA from FAXX in the presence of added xylanase.

Appl Environ Microbiol, 2004 Apr, 70(4), 2271 - 8
Sodium chloride reduces production of curvacin A, a bacteriocin produced by Lactobacillus curvatus strain LTH 1174, originating from fermented sausage; Verluyten J et al.; Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A . Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium . A model was set up to describe the effects of different NaCl concentrations on microbial behavior . Both cell growth and bacteriocin activity were affected by changes in the salt concentration . Sodium chloride clearly slowed down the growth of L . curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k(B)) and hence on overall bacteriocin activity . Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration . The inhibitory effect of NaCl was mainly due to its role as an a(w)-lowering agent . Further, it was clear that salt interfered with bacteriocin induction . Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X(B)) was 0.90 g (cell dry mass) per liter . Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X(B), an increase in k(B), and hence an increase in the maximum attainable bacteriocin activity.

Appl Environ Microbiol, 2004 Apr, 70(4), 2146 - 53
Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum; Gury J et al.; Lactobacillus plantarum displays a substrate-inducible padA gene encoding a phenolic acid decarboxylase enzyme (PadA) that is considered a specific chemical stress response to the inducing substrate . The putative regulator of padA was located in the padA locus based on its 52% identity with PadR, the padA gene transcriptional regulator of Pediococcus pentosaceus (L . Barthelmebs, B . Lecomte, C . Divies, and J.-F . Cavin, J . Bacteriol . 182:6724-6731, 2000) . Deletion of the L . plantarum padR gene clearly demonstrates that the protein it encodes is the transcriptional repressor of divergently oriented padA . The padR gene is cotranscribed with a downstream open reading frame (ORF1), the product of which may belong to a group of universal stress proteins (Usp) . The padR deletion mutant overexpressed padA constitutively, and the padA promoter appears to be tightly regulated in this bacterium . Gel mobility shift assays using the padA gene promoter region and purified PadR expressed in Escherichia coli indicated that operator DNA binding by PadR was not eliminated by addition of p-coumarate . Gel mobility shift assays using partially purified extracts of native PadR protein from both phenolic acid-induced and noninduced L . plantarum cells demonstrate that inactivation of PadR by phenolic acids requires the integrity of L . plantarum and mediation by a specific protein absent in E . coli.

Appl Environ Microbiol, 2004 Apr, 70(4), 1883 - 94
Study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods; Cocolin L et al.; In this study, a polyphasic approach was used to study the ecology of fresh sausages and to characterize populations of lactic acid bacteria (LAB) . The microbial profile of fresh sausages was monitored from the production day to the 10th day of storage at 4 degrees C . Samples were collected on days 0, 3, 6, and 10, and culture-dependent and -independent methods of detection and identification were applied . Traditional plating and isolation of LAB strains, which were subsequently identified by molecular methods, and the application of PCR-denaturing gradient gel electrophoresis (DGGE) to DNA and RNA extracted directly from the fresh sausage samples allowed the study in detail of the changes in the bacterial and yeast populations during storage . Brochothrix thermosphacta and Lactobacillus sakei were the main populations present . In particular, B . thermosphacta was present throughout the process, as determined by both DNA and RNA analysis . Other bacterial species, mainly Staphylococcus xylosus, Leuconostoc mesenteroides, and L . curvatus, were detected by DGGE . Moreover, an uncultured bacterium and an uncultured Staphylococcus sp . were present, too . LAB strains isolated at day 0 were identified as Lactococcus lactis subsp . lactis, L . casei, and Enterococcus casseliflavus, and on day 3 a strain of Leuconostoc mesenteroides was identified . The remaining strains isolated belonged to L . sakei . Concerning the yeast ecology, only Debaryomyces hansenii was established in the fresh sausages . Capronia mansonii was initially present, but it was not detected after the first 3 days . At last, L . sakei isolates were characterized by randomly amplified polymorphic DNA PCR and repetitive DNA element PCR . The results obtained underlined how different populations took over at different steps of the process . This is believed to be the result of the selection of the particular population, possibly due to the low storage temperature employed.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 289 - 95
Production of phenyllactic acid by lactic acid bacteria: an approach to the selection of strains contributing to food quality and preservation; Valerio F et al.; The ability of lactic acid bacteria (LAB) to produce phenyllactic (PLA) and 4-hydroxy-phenyllactic (OH-PLA) acids, metabolites involved in food quality and preservation, has been evaluated by HPLC analysis in 29 LAB strains belonging to 12 species widely used in the production of fermented foods . Metabolite production was demonstrated for all strains of the species Lactobacillus plantarum, Lactobacillus alimentarius, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, Lactobacillus hilgardii, Leuconostoc citreum, and for some strains of Lactobacillus brevis, Lactobacillus acidophilus and Leuconostoc mesenteroides subsp . mesenteroides . Strains were distinguished by analysis of variance in three groups including 15 strains that produced both metabolites (0.16-0.46 mM PLA and 0.07-0.29 mM OH-PLA), five strains accumulating in culture only PLA (0.17-0.57 mM) and nine non-producer strains (< or = 0.10 mM PLA and < or = 0.02 mM OH-PLA) . Improvement of phenyllactic acid production was obtained in a selected L . plantarum strain by increasing the concentration of phenylalanine in culture and using low amounts of tyrosine.

J Microbiol Methods, 2004 May, 57(2), 181 - 6
Resin straw as an alternative system to securely store frozen microorganisms; Thammavongs B et al.; Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms . In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections . Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce . In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes . Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp . cremoris and Lactobacillus delbrueckii subsp . bulgaricus and a deuteromycete fungus, Geotrichum candidum . The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms . Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes . However, conservation in the resin straws lead to a slight increase in the survival of L . cremoris and G . candidum compared to microtubes . In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.

Pediatr Allergy Immunol, 2004 Apr, 15(2), 152 - 8
Treatment of perennial allergic rhinitis with lactic acid bacteria; Wang MF et al.; Probiotics are perceived to exert beneficial effects in the prevention and treatment of allergic diseases via modifying the gut ecosystem . The aim of this study was to assess whether ingestion of fermented milk containing Lactobacillus paracasei-33 (LP-33), a strain newly isolated from the human intestinal tract, can improve the quality of life of patients with perennial allergic rhinitis . In a randomized, double-blind, placebo-controlled trial, we gave patients fermented milk with (n = 60) or without (n = 20) the addition of LP-33 (2 x 10(9) colony-forming units per bottle) for 30 days . A modified questionnaire concerning pediatric rhinoconjunctivitis quality of life was administered to all subjects or their parents at each clinical visit . Scores for the overall quality of life significantly decreased in the LP-33 group as compared with the placebo group, in both frequency (-16.02 +/- 2.14 vs . -7.27 +/- 3.55, respectively; p = 0.037) and level of bother (-16.35 +/- 2.33 vs . -6.20 +/- 3.13, respectively; p = 0.022) after the 30-day treatment . Subjects reported no severe adverse effects such as fever, abdominal pain, or diarrhea . The results suggest that ingestion of LP-33-fortified fermented milk for 30 days can effectively and safely improve the quality of life of patients with allergic rhinitis, and may serve as an alternative treatment for allergic rhinitis.

Biotechnol Prog, 2004 Mar-Apr, 20(2), 537 - 42
Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius; Saha BC; Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol . Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol . The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography . The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500 . The isoelectric point of the enzyme was at pH 4.7 . Both subunits had the same N-terminal amino acid sequence . The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C . The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose . The optimum pH for the oxidation of mannitol to fructose was 7.0 . The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C . The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively . It did not have any reductive activity on glucose, xylose, and arabinose . The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor . This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB . Comparative properties of the enzyme with other microbial MDHs are presented.

Biotechnol Lett, 2004 Feb, 26(4), 317 - 20
Antagonistic activity of lactic acid bacteria as probiotics against selected bacteria of the Enterobaceriacae family in the presence of polyols and their galactosyl derivatives; Klewicki R et al.; Probiotic lactic acid bacteria were grown on erythritol, xylitol, sorbitol or lactitol and produced various derivatives: gal-erythritol, gal-xylitol, and gal-sorbitol as prebiotics . Galactosyl derivatives of erythritol, xylitol and sorbitol were metabolised by Lactobacillus spp . This resulted in their antagonistic activity against the test microflora . No activity was observed in the presence of xylitol and erythritol . Gal-sorbitol obtained by enzymatic transglycosylation from lactose had the same abilities of inducing the antagonistic activity of lactic acid bacteria that lactitol had.

J Agric Food Chem, 2004 Apr 7, 52(7), 1861 - 6
Resistance screening essay of wine lactic acid bacteria on lysozyme: efficacy of lysozyme in unclarified grape musts; Delfini C et al.; In wine making, the bacteriolytic activity of lysozyme has primarily been used to control the malolactic fermentation in wines . The use of lysozyme in musts before settling and the beginning of the alcoholic fermentation to inhibit the growth of lactic acid bacteria could be very beneficial . In a resistance test carried out in MT/b broth, lysozyme had greater antimicrobial activity toward Oenococcus oeni than Lactobacillus species . Several strains of wine bacteria belonging to Oenococcus proved sensitive to the bacteriolytic activity of lysozyme at low concentrations in both synthetic medium (MT/b) (50 mg/L), white must, or red must made with or without the skins (100 mg/L) . Lactobacillus and Pediococcus strains survived at lysozyme concentrations of 200-500 and 500 mg/L, respectively, in MT/b and musts . Suspended solids in unclarified musts may strongly bind to lysozyme thereby causing its removal by filtration or centrifugation . One hour after lysozyme was added to musts, it was quantified by HPLC and found after centrifugation to be 40-50% and only 10% in musts made with or without the skins, respectively . Although appreciable amounts of lysozyme were bound to wine components, this did not appear to be a serious hindrance to lysozyme activity.

Nahrung, 2004 Feb, 48(1), 73 - 9
Adjunct starter properties affect characteristic features of Swiss-type cheeses; Weinrichter B et al.; A large number of microorganisms, both starter microorganisms and non-starter lactic acid bacteria originating from the base milk, or from various contamination sources during cheese manufacture, is associated with cheese ripening and the formation of flavour, texture and aroma . Under controlled conditions, Emmental and Bergkase, a Gruyere-type cheese variety, were produced from pasteurised milk with standard starters and defined strains of facultatively heterofermentative lactobacilli (FHL), and partly with addition of a defined mixture of enterococci . Lactobacillus casei subsp . casei and L . rhamnosus (two strains each) were selected with respect to their potential for the utilisation of citric acid and ribose as sole energy source . The FHL developed up to 10(8) cfu/g within the first weeks of ripening, and viable counts in mature cheeses were 10(7) cfu/g, independent of the cheese variety . Bergkase made with addition of L . rhamnosus strains showed a more pronounced proteolysis, resulting in reduced firmness and elasticity values of the cheese body, and FHL strains able to utilise citric acid improved the appearance of the cheeses by increasing the number of small eyes to the desired level . In Emmental cheese, the citric acid (+) strains reduced the intensity of propionic acid formation as the FHL apparently competed with the propionibacteria, and enterococci disappeared completely during maturation . Although further work is needed the study shows that, depending on the cheese variety, particular properties of FHL adjunct starters significantly affect important quality attributes of the resulting cheeses.

Syst Appl Microbiol, 2004 Feb, 27(1), 109 - 17
Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods; Nishitani Y et al.; A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production . With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L . paraplantarum, or L . pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species . Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species . Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L . acidophilus, one as Pediococcus acidilactici, one as P . pentosaceus, and two remained unidentifiable . The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family . Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L . plantarum predominant clusters, one L . paraplantarum predominant, and one L . pentosus predominant . The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L . pentosus, were <40% similarity to those belonging to the 4 clusters . A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.

Can J Microbiol, 2001 Jan, 47(1), 18 - 24
The effect of the addition of proteases and glucanases during yeast autolysis on the production and properties of yeast extracts; Conway J et al.; Yeast extracts (YE) were produced with the addition of proteases or glucanases during bakers' yeast (Saccharomyces cerevisiae) autolysis . Chemical composition, physical properties, and biological value of the YE were examined . Proteases had the highest impact on the turbidity and filterability of YE . All 11 proteases and two glucanases increased YE yields (% yeast solids solubilized) obtained from heated (80 degrees C/15 min) bakers' yeast creams (BYC) . However, when proteases were added to native (unheated) BYC during autolysis, few increased YE yields, with papain being the most effective . The increased yields were generally related to increased levels of total nitrogen (TN) and alpha-amino nitrogen (alpha-AN) in the YE . Media were supplemented with the various yeast extracts, and the highest growth rates (mumax) and biomass values (ODmax) of Lactobacillus acidophilus were noted . The best growth was obtained with YE produced with native BYC treated with a fungal protease, and results of this study show that some enzymes could be used to produce improved YE for microbiological media.

J Surg Res, 2004 Apr, 117(2), 262 - 71
Probiotics partly reverse increased bacterial translocation after simultaneous liver resection and colonic anastomosis in rats; Seehofer D et al.; BACKGROUND: Bacterial translocation is one important cause of nosocomial infections following major abdominal surgery . Oral administration of probiotics has been proposed to diminish bacterial translocation . MATERIAL AND METHODS: In total 68 rats were divided into seven groups: five of the groups received standard rat chow and were subjected to either sham-operation, 70% liver resection, colonic anastomosis, or a combination of 30 or 70% liver resection with synchronous colonic anastomosis, respectively . In two additional groups with synchronous operation, a combination of four different lactic acid bacteria and four fibers was administered two times daily pre- and postoperatively . Bacterial concentrations in cecum, mesenteric lymph nodes, liver, and spleen were analyzed and blood cultures were taken 48 h after operation . Furthermore, the following parameters were assessed: histological changes in the intestine, intestinal paracellular permeability (Ussing chamber), bursting pressure of the colonic anastomosis, and mitosis rate of the remnant liver . RESULTS: Bacterial translocation was observed in all rats, except in the sham group . Following liver resection, the highest bacterial concentrations were seen in liver and spleen, following colon anastomosis in the mesenteric lymph nodes . Bacterial translocation was increased in the animals with combined operation, in parallel to the extent of liver resection . In rats with colon anastomosis, bacterial concentration in the cecum was also higher than in the sham group . Application of probiotics significantly decreased bacterial concentration in the lymph nodes . In addition, animals with a high cecal concentration of lactobacilli had less translocation than the others . No histological changes were observed in the intestine . Paracellular permeability for ions, but not for the larger molecule lactulose, was increased in the colon in all groups with colon anastomosis . The bursting pressure of the colon anastomosis was not significantly different between the groups . Seventy percent liver resection led to a high rate of hepatocyte mitosis, whereas combination with colon anastomosis impaired the regeneration process . CONCLUSION: Synchronous liver resection and colon anastomosis led to increased bacterial translocation compared to the single operations in the rat model . It is possible to diminish this process by oral administration of probiotics . Bacterial overgrowth in the cecum and impaired hepatic regeneration, but not histological changes or alterations of paracellular permeability, are potential pathogenic mechanisms for translocation in this setting.

BMC Fam Pract . 2004 Mar 28;5(1):5.
The PAV trial: does lactobacillus prevent post-antibiotic vulvovaginal candidiasis? Protocol of a randomised controlled trial {ISRCTN24141277}; Pirotta M et al.; BACKGROUND: Complementary and alternative medicines are used by many consumers, and increasingly are being incorporated into the general practitioner's armamentarium . Despite widespread usage, the evidence base for most complementary therapies is weak or non-existent . Post-antibiotic vulvovaginitis is a common problem in general practice, for which complementary therapies are often used . A recent study in Melbourne, Australia, found that 40% of women with a past history of vulvovaginitis had used probiotic Lactobacillus species to prevent or treat post-antibiotic vulvovaginitis . There is no evidence that this therapy is effective . This study aims to test whether oral or vaginal lactobacillus is effective in the prevention of post-antibiotic vulvovaginitis . METHODS/DESIGN: A randomised placebo-controlled blinded 2 x 2 factorial design is being used . General practitioners or pharmacists approach non-pregnant women, aged 18-50 years, who present with a non-genital infection requiring a short course of oral antibiotics, to participate in the study . Participants are randomised in a four group factorial design either to oral lactobacillus powder or placebo and either vaginal lactobacillus pessaries or placebo . These interventions are taken while on antibiotics and for four days afterwards or until symptoms of vaginitis develop . Women self collect a vaginal swab for culture of Candida species and complete a survey at baseline and again four days after completing their study medications . The sample size (a total of 496--124 in each factorial group) is calculated to identify a reduction of half in post-antibiotic vulvovaginitis from 23%, while allowing for a 25% drop-out . An independent Data Monitoring Committee is supervising the trial . Analysis will be intention-to-treat, with two pre-specified main comparisons: (i) oral lactobacillus versus placebo and (ii) vaginal lactobacillus versus placebo.

Berl Munch Tierarztl Wochenschr, 2004 Mar-Apr, 117(3-4), 145 - 7
Testing two Lactobacillus plantarum and Lactobacillus acidophilus strains for their suitability as a lipoid probiotic; Marsalkova S et al.; Two strains of lactobacilli (Lactobacillus acidophilus T-135 and Lactobacillus plantarum 4/97) were selected in order to study their inhibitory properties against frequent udder pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Salmonella enteritidis and Bacillus pumilus), their production of organic acids as well as their ability to survive on the teat skin, the teat duct mucosa and in a lipoid emulsion . Both strains inhibited the tested pathogenic microbes and survived on the investigated surfaces and in an emulsion for more than 6 hours and 11 days, respectively.

Syst Appl Microbiol, 2004 Mar, 27(2), 229 - 37
A mixed culture of Propionibacterium jensenii and Lactobacillus paracasei subsp . paracasei inhibits food spoilage yeasts; Schwenninger SM et al.; Screening for antimicrobial features of 197 propionibacteria and tests with several antifungal lactobacilli led to the development of three protective cultures containing Propionibacterium jensenii SM11 and Lactobacillus paracasei subsp . paracasei strain SM20, SM29 or SM63 . These cultures showed inhibitory activities (up to 5 orders of magnitude) against yeasts in dairy products such as yoghurt or cheese surface at refrigerator temperatures (6 degrees C) without an influence on the quality properties of the food . Initial cell numbers of 5 x 10(7) cells/g of propionibacteria and 1 x 10(8) cells/g of lactobacilli were the optimal concentrations to yield a total inhibition of the spoilage yeasts (Candida pulcherrima, Candida magnoliae, Candida parapsilosis and Zygosaccharomyces bailii).

Syst Appl Microbiol, 2004 Mar, 27(2), 186 - 91
Whole-organism fingerprinting of the genus Carnobacterium using Fourier transform infrared spectroscopy (FT-IR); Lai S et al.; Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy . The effects of culture age and reproducibility over a six month period were also investigated . The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies . Taxonomic correlations were observed between the FT-IR data and these studies . Culture age was observed to have little effect on the spectra obtained . The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period . It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.

Syst Appl Microbiol, 2004 Mar, 27(2), 151 - 9
Characterisation of the microbiota of rice sourdoughs and description of Lactobacillus spicheri sp . nov; Meroth CB et al.; The microbiota of two industrially processed rice sourdoughs was characterised by bacteriological culture in combination with PCR-denaturing gradient gel electrophoresis (DGGE) and 16S/28S rDNA sequence analysis . Rice sourdough I was continuously propagated for several years by back-slopping every week, whereas sourdough II was processed by using a commercial starter culture and back-slopping daily for three days . In rice sourdough II Candida krusei and Saccharomyces cerevisiae as well as Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus kimchii, Lactobacillus plantarum, and Lactobacillus pontis dominated at the first day of fermentation . RAPD analysis of lactobacilli revealed identical profiles for each of the species except for L . fermentum and L . pontis indicating the presence of different strains . Fluctuations within the LAB community during fermentation were monitored by PCR-DGGE . L . pontis decreased in numbers over time and L . curvatus became dominant after 3 days of fermentation . Rice sourdough I contained S . cerevisiae, Lactobacillus paracasei (present with three different RAPD types), Lactobacillus paralimentarius, and a Lactobacillus strain which could not be allotted to any valid species . Phylogenetic analysis based on 16S rDNA sequences revealed Lactobacillus brevis as the closest relative (97.3% sequence similarity) . Differences in some phenotypic characteristics and DNA-DNA relatedness indicated that the strain represents a new Lactobacillus species, for which the name Lactobacillus spicheri is proposed.

J Mol Microbiol Biotechnol, 2003, 6(2), 76 - 87
CbsT2 from Lactobacillus johnsonii 100-100 is a transport protein of the major facilitator superfamily that facilitates bile acid antiport; Elkins CA et al.; We previously identified two conjugated bile acid transporters, CbsT1 and CbsT2, in Lactobacillus johnsonii 100-100 and Lactobacillus acidophilus KS-13 that are gene duplicates encoded in tandem with a conjugated bile salt hydrolase (BSH) {Elkins and Savage, J . Bacteriol . 180:4344-4349, 1998; Elkins et al., Microbiology 147: 3403-3412, 2001} . CbsT2 from 100-100 was shown to increase taurocholic acid (TCA) uptake in Escherichia coli; however, higher levels were achieved when an extracellular factor (EF) from 100-100 was present in the assay medium (spent medium from 100-100, pH 4.2) . We continued this study here to determine the role of EF in this transport system . Kinetic studies revealed that the previously observed CbsT2- and EF-mediated TCA accumulation is rapid (< 15 s) but not saturable, suggesting that EF is limiting . In addition, uptake of TCA by E . coli expressing CbsT2 was insensitive to ionophores, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, and thus, is independent of the proton motive force . Since BSH converts {24-(14)C}TCA to {24-(14)C}cholic acid (CA), we measured net radiolabel uptake in E . coli cells expressing transporter(s) and BSH . Interestingly, such cells accumulated less 14C radiolabel (by approximately half) than cells expressing CbsT2 alone . These data can be explained if CA diffuses out of E . coli through the transporter(s) . We, therefore, added exogenous, unlabeled CA to EF-spent media, which under our assay conditions, performed similarly to EF+ culture supernatant in TCA and CA uptake assays . Thus, unlabeled CA (a protonated, neutral lipophile) can partition directly into E . coli cells especially at low pH . These findings were validated in uptake assays with {3H}TCA, which yields {3H}taurine (a hydrophilic moiety) upon hydrolysis by the BSH . Amounts of cell-associated 3H radiolabel remained similar in cells expressing CbsT2 and BSH versus cells expressing only CbsT2, both of which were higher than in cells expressing BSH alone . Our data support a hypothesis that these transporters, which comprise a new subfamily of the major facilitator superfamily, facilitate antiport of TCA and CA .

FEMS Microbiol Lett, 2004 Apr 1, 233(1), 131 - 8
Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation; Palumbo E et al.; A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps . When the mutant was supplemented with D-alanine, growth and viability were unaffected . Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis . Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h . Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells . Prolonged d-alanine starvation resulted in cells with an aberrant morphology . Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region . We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.

Ceska Gynekol, 2003 Nov, 68(6), 439 - 42
{Treatment of aerobic vaginitis and clinically uncertain causes of vulvovaginal discomfort}; Cepicky P et al.; OBJECTIVE: The treatment of clinically uncertain conditions of vaginal discomforts with a mixed preparation of nifuratel + nystatin (Macmiror complex) and the relation of uncertain conditions to aerobic vaginitis . DESIGN: A prospective study . SETTING: Gynecology-Obstetrics Outpatient Department LEVRET Ltd., AescuLab Ltd., Laboratory of Microbiology, Prague . METHODS: 50 women with vaginal discomfort, causes of which had not been clarified by gynecological examination, determination of pH and the amine test, were examined by vaginal smears using microscopy . The results were evaluated in relation to aerobic vaginitis in a pure form or in combination with other nosological units . The authors also evaluated results of therapy by oral nifuratel (Macmiror tbl) 3 x 200 mg daily and a vaginal combined preparation containing nifuratel 500 mg + nystatin 200 kIU (Macmiror complex 500 glo vag) for the period of 7 days . RESULTS: In 50 women candida was demonstrated 24 times, presence of key cells 11 times, lactobacillus nine times with more than 50 in the field, six women were affected by aerobic vaginitis . In all these cases the pH was 4.8 or higher, leukocytes were significantly represented in all cases (> 15 in the field), as well as gram-negative bacteria and/or cocci (> 30 in the field), indicating a combined picture of mycosis, anaerobic vaginosis or lactobacillosis with aerobic vaginitis . The therapy was successful in all cases, the relapse of complaints during one month occurred in three cases . CONCLUSIONS: Aerobic vaginitis in a pure form or with anaerobic vaginosis, mycosis or lactobacillosis is frequently concealed under clinically uncertain pictures of vulvo-vaginal discomfort . The therapy by a combination of nifurated 3 x 200 mg orally together with the combined vaginal preparation nifuratel 500 mg + nystatin 200 kIU for the period of 7 days exerts high effect and a low number of relapses.

Infect Immun, 2004 Apr, 72(4), 2160 - 9
Cell surface-associated elongation factor Tu mediates the attachment of Lactobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins; Granato D et al.; The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins . Incubation of Caco-2 intestinal epithelial cells with an L . johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor . The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria . Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule . Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent . Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity . In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14 . Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.

Infect Immun, 2004 Apr, 72(4), 1866 - 73
Effect of Escherichia coli and Lactobacillus rhamnosus on macrophage inflammatory protein 3 alpha, tumor necrosis factor alpha, and transforming growth factor beta release by polarized rat uterine epithelial cells in culture; Crane-Godreau MA et al.; Entry of bacteria from the vagina into the uterus raises the question of uterine epithelial cell (UEC) signaling in response to the presence of bacteria . Our model system helps to define microbially elicited UEC basolateral cytokine release, important in regulating underlying stromal immune cell protection . UECs from adult rats were grown in cell culture inserts to establish a confluent polarized monolayer as was determined by transepithelial resistance (TER) . Polarized epithelial cell cultures were treated apically with live or heat-killed Escherichia coli or Lactobacillus rhamnosus prior to collection of basolateral media after 24 h of incubation . Coculture of polarized UECs with live E . coli had no effect on epithelial cell TER . In response to exposure to live E . coli, epithelial cell basolateral release of macrophage inflammatory protein 3 alpha (MIP3 alpha) and tumor necrosis factor alpha (TNF-alpha) increased at a time when basolateral release of biologically active transforming growth factor beta (TGF-beta) decreased . Incubation of UECs with heat-killed E . coli resulted in an increased basolateral release of MIP3 alpha and TNF-alpha, without affecting TER or TGF-beta . In contrast to E . coli, live or heat-killed L . rhamnosus had no effect on TER or cytokine release . These studies indicate that polarized rat UECs respond to gram-negative E . coli by releasing the cytokines MIP3 alpha and TNF-alpha, signals important to both the innate and adaptive immune systems . These findings suggest that UEC responses to bacteria are selective and important in initiating and regulating immune protection in the female reproductive tract.

J Am Chem Soc, 2004 Mar 31, 126(12), 3755 - 61
Control of bacteria adhesion by cell-wall engineering; Sadamoto R et al.; UDP-MurNAc-pentapeptide derivative bacterial cell-wall precursors were synthesized as effective tools for surface display on living bacteria . Lactobacilli were incubated in the ketone-modified precursor-containing medium, and the ketone moiety was displayed on the bacterial surface through cell-wall biosynthesis . Oligomannose was coupled with the ketone moiety on the bacterial surface via a aminooxyl linker, thereby displaying this oligosaccharide on the surface of the bacteria . The increase in the adhesion of the sugar-displaying bacteria onto a concanavalin A-attached film compared to that of native bacteria was confirmed by microscopic observation and surface plasmon resonance measurement . The incorporation of the artificial cell-wall precursors was enhanced when incubated with fosfomycin, an inhibitor of cell-wall precursor biosynthesis.

J Food Prot, 2004 Mar, 67(3), 610 - 5
A PCR assay for detection of acetic acid-tolerant lactic acid bacteria in acidic food products; Nakano S et al.; A PCR assay for the detection of acetic acid-tolerant lactic acid bacteria in the genera of Lactobacillus and Pediococcus was developed in this study . Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay . To determine the specificity of the assay, 56 different bacterial strains (of 33 genera), 2 fungi, 3 animals, and 4 plants were tested . Results were positive for most tested bacterial members of 16S rRNA gene-based phylogenetic groups (classified in the Lactobacillus casei and Pediococcus group), including Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus plantarum, and Lactobacillus paracasei . For all other bacterial strains and eukaryote tested, results were negative . Bacterial DNA for PCR was prepared with a simple procedure with the use of Chelex 100 resin from culture after growth in deMan Rogosa Sharpe broth (pH 6.0) . To test this PCR assay for the monitoring of the acetic acid-tolerant lactic acid bacteria, L . fructivorans was inoculated into several acidic food as an indicator . Before the PCR, the inoculation of 10 to 50 CFU of bacteria per g of food was followed by a 28-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells . Including the enrichment culture step, the entire PCR detection process can be completed within 30 h.

Biotechnol Appl Biochem, 2004 Apr, 39(Pt 2), 189 - 98
Modified alginate matrices for the immobilization of bioactive agents; Le-Tien C et al.; Bioactive agents (catalase - an enzyme, and nisin - a bacteriocin) were covalently immobilized on alginate activated with sodium periodate (oxidatively converting 2,3-dihydroxy groups into dialdehyde residues), followed or preceded by ionotropic gelation . For the same protein coupling yield, the retained enzyme activity of the immobilized enzyme (ImE) can be markedly increased by diminishing the bead diameter, a phenomenon that illustrates the role of substrate/product diffusion through the bead gel layer . When the amount of enzyme introduced for coupling was about 15 mg/100 mg of support and the bead diameter was about 100 microm, a high retained specific activity (95-98%) was obtained . Diffusion phenomena can be markedly decreased by enzyme immobilization on the surface of microbeads (obtained by gelation of activated alginate prior to immobilization) . In this case, the retained activity was approx . 75% of that of the free enzyme . A slightly higher K (m) value of ImE suggested that the enzyme-substrate affinity was almost maintained . The profiles of ImE activities at various pH values, at various temperatures and when undergoing proteolysis showed a overall higher stability for the immbolized than that for the free enzyme . Nisin immobilized on the microbead surface, when submitted to proteolysis, conserved its bacteriocin activity, strongly inhibiting the growth of Lactobacillus sake when subjected to an agar spot test, whereas free nisin totally lost its activity.

Antonie Van Leeuwenhoek, 2004 Apr, 85(3), 191 - 8
Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442; Mataragas M et al.; The aim of this study was to investigate the effect of complex nutrients on microbial growth and bacteriocin production, in order to improve bacteriocin synthesis during the growth cycle of Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442 . The fermentations were conducted at the optimum pH and temperature for bacteriocin production (pH 5.5+/-0.1 and temperature 25+/-0.1 degrees C) . Because of their association with the final biomass, conditions favouring the increase of the produced biomass resulted in the increase of bacteriocin activity in the growth medium . Since the produced final biomass and the final concentration of the bacteriocins were associated with the amount of the carbon (glucose) and nitrogen source, better growth of the lactic acid bacterial strains favoured the increase of the specific bacteriocin production . Additionally, the bacteriocin production was influenced by carbon/nitrogen ratio.

Antonie Van Leeuwenhoek, 2004 Apr, 85(3), 209 - 16
Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins; Carl GD et al.; Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed . They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus . Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT) . Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers . The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results . No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found . The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.

J Antimicrob Chemother, 2004 May, 53(5), 797 - 803 Epub 2004 Mar 17.
Comparative in vitro activity of telavancin (TD-6424), a rapidly bactericidal, concentration-dependent anti-infective with multiple mechanisms of action against Gram-positive bacteria; King A et al.; OBJECTIVE: We compared the in vitro activity of telavancin with that of vancomycin, teicoplanin, linezolid, quinupristin/dalfopristin, moxifloxacin and ampicillin, penicillin or oxacillin as appropriate, by the NCCLS/EUCAST and BSAC methods . METHODS: The organisms (n = 401) included in the study were patient isolates from St Thomas' Hospital and were selected to include representatives of the clinically important Gram-positive aerobic species . Susceptibility testing was performed by agar dilution methods on Mueller-Hinton agar according to the NCCLS/EUCAST guidelines, in comparison with Iso-Sensitest agar according to the BSAC guidelines . RESULTS: Telavancin was active against all the Gram-positive species tested and nearly 90% of isolates included in the study had telavancin MICs </= 1 mg/L . Vancomycin-resistant enterococci and lactobacilli isolates with vancomycin MICs > 64 mg/L had telavancin MIC ranges of 0.5-8 and 2-16 mg/L, respectively . There was no evidence of cross-resistance with other comparator drugs . The results for telavancin for the two susceptibility testing methods were mostly either the same or within one doubling dilution . CONCLUSION: The susceptibility breakpoints for telavancin have yet to be established, but it would appear that telavancin has superior potency to the other tested glycopeptides, and on a weight-for-weight basis displays activity that is comparable to, or better than, that of the other agents tested.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2004, 39(2), 515 - 33
Ensilage of cardboard and date palm leaves; Baroon Z et al.; Eight treatments of cardboard (CB) and date palm leaves (DPL) with or without alkali treatments plus molasses, dates, and ammonium sulfate were ensiled in laboratory silos . Latobacillus plantarum and Saccharomyces cerevisiae inoculants were added at an application rate of 10(5) cfu/g of silage to study their possible influence on the fermentation process . Silos were sealed and incubated at 30 degrees C . The anaerobic ensiling was evaluated for chemical and microbiological variables by opening the silos after 30, 60, and 90 days . After 90 days, significantly low pH values (5.2-3.5) were obtained . The percentages of hemicellulose, cellulose, lignin, and ash were determined . Concentrations of reducing sugars, ether extract, and crude protein tended to be acceptable among treatments . Silages showed a significantly high lactic acid concentration (up to 4.3%) and only traces of butyric acid (below 1%) . Also changes in acetic and propionic acids concentrations were determined . Lactobacillus populations remained almost static while yeast and molds populations tended to be lower after 90 days of ensilage . Neither alkali treatment nor microbial inoculants showed a relationship with the ensiling process . Thus, overall, it was concluded that CB and DPL can be effectively ensiled and the resultant silages are acceptable to merit their use as ruminant feed in arid regions.

Artif Cells Blood Substit Immobil Biotechnol, 2004 Feb, 32(1), 55 - 65
Encapsulated lactic acid bacteria for control of malolactic fermentation in wine; Kosseva MR et al.; The kinetics of both malolactic fermentation in Chardonnay wine by encapsulating Lactobacillus casei cells in pectate gel and lyophilized Oenococcus oeni culture has been carried out . The influence of acidity, sulfur dioxide content, and organic acid content on the malolactic activity of the bacteria has been controlled . Encapsulated bacteria degraded 30%, of malic acid in white wine, deacidifying it from pH 3.15 to 3.40, whereas the lyophilized culture degraded 48% of malic acid, deacidifying from pH 3.15 to 3.60 . The degree of conversion of malic acid in wine by the encapsulated cells was twice as high as that obtained by the free Lactobacillus casei cells . The operational stability of calcium pectate gel capsules was 6 months . It has been proved that the encapsulated biocatalyst increases the rate of fermentation, and induces the fermentation to take place at high ethanol concentrations . The proposed encapsulated biocatalyst is an attractive material for industrial applications in continuous winemaking processes.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 551 - 6
Reclassification of Lactobacillus kefirgranum Takizawa et al . 1994 as Lactobacillus kefiranofaciens subsp . kefirgranum subsp . nov . and emended description of L . kefiranofaciens Fujisawa et al . 1988; Vancanneyt M et al.; Fourteen homofermentative lactic acid bacteria that were isolated from kefir grains and kefir fermented milks were assigned to either Lactobacillus kefiranofaciens or Lactobacillus kefirgranum, based on their characteristic morphotypes, phenotypic features and SDS-PAGE profiles of whole-cell proteins . Further genotypic analyses on representative strains from both taxa demonstrated that L . kefiranofaciens and L . kefirgranum share 100 % 16S rDNA sequence similarity and belong phylogenetically to the Lactobacillus acidophilus species group . DNA-DNA binding values of >79 % and analogous DNA G+C contents of 37-38 mol% showed that the strains studied belonged to one species: L . kefirgranum is a later synonym of L . kefiranofaciens . An emended description is proposed for L . kefiranofaciens . Due to the specific morphological and biochemical characteristics of these taxa in kefir grain formation, it is proposed that L . kefirgranum should be reclassified as L . kefiranofaciens subsp . kefirgranum subsp . nov.

Infect Dis Obstet Gynecol, 2003, 11(3), 147 - 56
Inhibition of vaginal lactobacilli by a bacteriocin-like inhibitor produced by Enterococcus faecium 62-6: potential significance for bacterial vaginosis; Kelly MC et al.; OBJECTIVE: Bacterial vaginosis (BV) is characterized by a shift in vaginal tract ecology, which includes a decrease in the concentration and/or prevalence of facultative lactobacilli . Currently, mechanisms which could account for the disappearance of lactobacilli are not well understood . The objective of this study was to determine whether vaginal streptococci/enterococci can produce bacteriocin-like inhibitors antagonistic to vaginal lactobacilli . METHODS: Seventy strains of vaginal streptococci or enterococci were tested for antagonistic activities against vaginal lactobacilli using the deferred antagonism technique . RESULTS: One strain, Enterococcus faecium 62-6, which strongly inhibited growth of lactobacilli was selected for further characterization . The spectrum of inhibitory activity of strain 62-6 included Gram-positive organisms from the vaginal environment, although native lactobacilli from the same host were resistant to inhibitor action . Following growth in MRS broth the strain 62-6 inhibitor was shown to be heat- (100 degrees C, 30 minutes), cold- (4 degrees C, less than 114 days) and pH- (4-7) stable . The sensitivity of inhibitor-containing supernatants to pepsin and alpha-chymotrypsin suggested an essential proteinaceous component . The inhibitor was sensitive to lipase but resistant to lysozyme . Dialysis of inhibitor-containing culture supernatants suggested a molecular mass greater than 12,000 Da . All physicochemical properties were consistent with its classification as a bacteriocin-like inhibitor . Kinetic assays demonstrated a sharp onset of inhibitor production coinciding with a concentration of 62-6 of 10(7) cfu/ml, suggesting that production may be regulated by quorum sensing . CONCLUSIONS: These results may have clinical significance as a novel mechanism to account for the decline of vaginal Lactobacillus populations and contribute to both the establishment and recurrence of BV.

Peptides, 2003 Nov, 24(11), 1733 - 40
Two different propionicins produced by Propionibacterium thoenii P-127; Ben-Shushan G et al.; The bacteriocin GBZ-1 was purified from the growth media of Propionibacterium thoenii P-127 and was found to have a molecular weight of 6000Da . P . thoenii P-127 also known as the producer of the bacteriocin PLG-1 (MW 10kDa) . Under specific growth conditions, on semi-solid media, P . thoenii P-127 produced both PLG-1 and GBZ-1 . The N-terminal of GBZ-1 was microsequenced, the gene was cloned and the DNA sequence was determined and identified . GBZ-1 is highly homologous to a protease-activated antimicrobial peptide (PAMP) . In contrast to PAMP, it was purified in its active form and no protease digestion was required for its activation . The survival curve of indicator bacteria Lactobacillus delbrueckii subsp . lactic ATCC 4797 showed two phases . The fast phase of 20min was followed by a slow phase . While bacterial survival was reduced by 2logs during the fast phase, bacterial survival was reduced by additional 3logs up to 200min during the slow phase . GBZ-1 activity was affected by magnesium and its activity was completely abolished at 50mM magnesium chloride . Other divalent cations had no effect on GBZ-1 activity of GBZ-1 . To the best of our knowledge this is the first report of a bacterium producing two different bacteriocins under different growth conditions.

BMC Infect Dis . 2004 Feb 13;4(1):5.
Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis; Ferris MJ et al.; BACKGROUND: Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species . The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes . The bacterium Atopobium vaginae has been recognized only recently . It is not readily identified by commercial diagnostic kits . Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess . METHODS: Nucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp . predominant; Nugent score < or = 3) and abnormal flora (Nugent score > or = 4) . These women ranged in age from 14 to 48 and 82% were African American . RESULTS: The DGGE banding patterns of normal and BV-positive patients were recognizably distinct . Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane . More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients . (P < 0.001) Two A . vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism . Two A . vaginae 16S rRNA gene sequences were identified among the clinical isolates . The same two sequences were obtained from DGGE bands of the corresponding vaginal flora . The sequences differed by one nucleotide over the short (approximately 300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels . Both isolates were strict anaerobes and highly metronidazole resistant . CONCLUSION: The results suggest that A . vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora . This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant.

Shanghai Kou Qiang Yi Xue, 2000 Sep, 9(3), 153 - 5
{An analysis of metablic organic Acid of anaerobes by gas chromatography}; Gu SP et al.; OBJECTIVE:The purpose of this study was to test the reliability of measurement of metablic organic acid of anaerobic bacteria by gas chromatography for the identification of anaerobes.METHODS:The metablic organic acid of 60 strains of 6 species of oral residential anaerobes were tested by gas chromatography qualitatively and quantitatively . RESULTS:It was found that the types and quantities of metablic organic acid of all anaerobes but two strains of lactobacillus were coincident with the standard of identification of anaerobes,and the total coincidence was 97.67%.CONCLUSION:The anaerobes could be identificated accurately and quickly by gas chromatography.

Clin Diagn Lab Immunol, 2004 Mar, 11(2), 266 - 71
Stimulation of interleukin-10 production by acidic beta-lactoglobulin-derived peptides hydrolyzed with Lactobacillus paracasei NCC2461 peptidases; Prioult G et al.; We have previously demonstrated that Lactobacillus paracasei NCC2461 may help to prevent cow's milk allergy in mice by inducing oral tolerance to beta-lactoglobulin (BLG) . To investigate the mechanisms involved in this beneficial effect, we examined the possibility that L . paracasei induces tolerance by hydrolyzing BLG-derived peptides and liberating peptides that stimulate interleukin-10 (IL-10) production . L . paracasei peptidases have been shown to hydrolyze tryptic-chymotryptic peptides from BLG, releasing numerous small peptides with immunomodulating properties . We have now shown that acidic tryptic-chymotryptic peptides stimulate splenocyte proliferation and gamma interferon (IFN-gamma) production in vitro . Hydrolysis of these peptides with L . paracasei peptidases repressed the lymphocyte stimulation, up-regulated IL-10 production, and down-regulated IFN-gamma and IL-4 secretion . L . paracasei NCC2461 may therefore induce oral tolerance to BLG in vivo by degrading acidic peptides and releasing immunomodulatory peptides stimulating regulatory T cells, which function as major immunosuppressive agents by secreting IL-10.

J Appl Microbiol, 2004, 96(4), 819 - 27
Use of bacteriocin promoters for gene expression in Lactobacillus plantarum C11; Mathiesen G et al.; AIMS: To exploit promoters involved in production of the bacteriocin sakacin P for regulated overexpression of genes in Lactobacillus plantarum C11 . METHODS AND RESULTS: Production of sakacin P by Lact . sakei LTH673 is controlled by a peptide-based quorum sensing system that drives strong, regulated promoters . One of these promoters (PorfX) was used to establish regulated overexpression of genes encoding chloramphenicol acetyltransferase from Bacillus pumilus, aminopeptidase N from Lactococcus lactis or chitinase B from Serratia marcescens in Lact . plantarum C11, a strain that naturally possesses the regulatory machinery that is necessary for promoter activation . The expression levels obtained were highly dependent on which gene was used and on how the promoter was coupled to this gene . The highest expression levels (14% of total cellular protein) were obtained with the aminopeptidase N gene translationally fused to the regulated promoter . CONCLUSIONS: Sakacin promoters permit regulated expression of a variety of genes in Lact . plantarum C11 . SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the usefulness of regulated bacteriocin promoters for developing new gene expression systems for lactic acid bacteria, in particular lactobacilli.

J Appl Microbiol, 2004, 96(4), 677 - 83
Genetic characterization and specific detection of beer-spoilage Lactobacillus sp . LA2 and related strains; Suzuki K et al.; AIMS: Lactobacillus sp . LA2 (DSM15502) and related strains (LA2 group) possess strong beer-spoilage ability . The 16S rDNA sequence of LA2 strain is virtually indistinguishable from that of L . collinoides, generally considered to be nonbeer-spoilage bacteria . The aim of this study was to identify the genetic marker to distinguish between Lactobacillus sp . LA2 group and L . collinoides and to provide a rapid means of identifying beer-spoilage strains belonging to Lactobacillus sp . LA2 group . METHODS AND RESULTS: The 16-23S rDNA intergenic spacer (ITS) regions of Lactobacillus sp . LA2 and L . collinoides JCM1123T were sequenced to identify a genetic marker to distinguish between the two groups . As a result, 300 and 500 bp ITS regions of Lactobacillus sp . LA2 were found to be almost identical with those of L . collinoides JCM1123T . Sequence comparison analysis between Lactobacillus sp . LA2 and L . collinoides JCM1123T revealed that the two contiguously located nucleotides are absent in both ITS regions of Lactobacillus sp . LA2 . Based on the sequence difference, we have designed specific PCR primers with a minor modification to the primer sequence that can differentiate between beer-spoilage Lactobacillus sp . LA2 group and nonbeer-spoilage L . collinoides . CONCLUSIONS: The PCR-based method has been developed to identify Lactobacillus sp . LA2 group, providing a rapid and sensitive means of determining the beer-spoilage ability of detected bacterial strains . SIGNIFICANCE AND IMPACT OF THE STUDY: The substitution of one nucleotide, located at the third position to the 3'-end in the primer sequence, enhanced the specificity of the PCR method while retaining sufficient sensitivity . The nucleotide gap identified in this study appeared to serve as a useful genetic marker that can differentiate 12 beer-spoilage Lactobacillus sp . LA2 group strains from its close relatives that exhibit no beer-spoilage ability.

Springer Semin Immunopathol, 2004 Feb, 25(3-4), 237 - 55 Epub 2003 Oct 08.
Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders; Rook GA et al.; In the rich, developed parts of the world there has been a steady and simultaneous increase in at least three groups of disease: (1) allergies, (2) inflammatory bowel diseases (IBD; e.g . Crohn's disease and ulcerative colitis) and (3) autoimmunity (e.g . type 1 diabetes and multiple sclerosis) . Because the medical world is so compartmentalised it was some time before the connection between these increases was noticed and understood . There is now evidence that the simultaneous increase in these diseases of immunodysregulation is at least partly attributable to malfunction of regulatory T cells (Treg) . This paper provides an overview of relevant work in each of these fields of medicine (though with emphasis on the allergic disorders), and concludes that the increasing failure of Treg is a consequence of diminished exposure to certain micro-organisms that are "old friends", because of their continuous presence throughout mammalian evolution . These organisms, which include saprophytic mycobacteria, helminths and lactobacilli, are recognised by the innate immune system as harmless, and as adjuvants for Treg induction . Polymorphisms of components of the innate immune system such as TLR2 and NOD2 appear to define subsets of the population that will develop immunoregulatory disorders when living in the modern environment . A further role of the "old friends" and of the Treg that they induce might be to maintain the levels of regulatory IL-10 secreting macrophages and antigen-presenting cells, which are depleted in asthma and Crohn's disease . These concepts are leading to novel therapies based on harmless organisms or their components . Phase I/II clinical trials have yielded some statistically significant results, and phase II trials are in progress.

Appl Environ Microbiol, 2004 Mar, 70(3), 1869 - 73
L-alanine auxotrophy of Lactobacillus johnsonii as demonstrated by physiological, genomic, and gene complementation approaches; van der Kaaij H et al.; Using a chemically defined medium without L-alanine, Lactobacillus johnsonii was demonstrated to be strictly auxotrophic for that amino acid . A comparative genetic analysis showed that all known genes involved in L-alanine biosynthesis are absent from the genome of L . johnsonii . This auxotrophy was complemented by heterologous expression of the Bacillus subtilis L-alanine dehydrogenase.

Appl Environ Microbiol, 2004 Mar, 70(3), 1522 - 7
Microbiological characterization of wet wheat distillers' grain, with focus on isolation of lactobacilli with potential as probiotics; Pedersen C et al.; Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective . After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol . The composition of lactobacilli in WWDG was simple, including primarily the species Lactobacillus amylolyticus, Lactobacillus panis, and Lactobacillus pontis, as determined by 16S rRNA gene sequencing . Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature . Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison . It was clear that all three species could grow at temperatures of 45 to 50 degrees C, with L . amylolyticus being able to grow at temperatures as high as 54 degrees C . This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms . Some strains of L . panis and L . pontis showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt . In addition, members of all three species were able to bind to immobilized mucus material in vitro . Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.

Appl Environ Microbiol, 2004 Mar, 70(3), 1336 - 46
Heat shock response in Lactobacillus plantarum; De Angelis M et al.; Heat stress resistance and response were studied in strains of Lactobacillus plantarum . Stationary-phase cells of L . plantarum DPC2739 had decimal reduction times (D values) (D value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75 degrees C, respectively . When mid-exponential-phase cells were used, the D values decreased . The temperature increases which caused a 10-fold reduction in the D value ranged from 9 to 20 degrees C, depending on the strain . Part of the cell population treated at 72 degrees C for 90 s recovered viability during incubation at 7 degrees C in sterile milk for 20 days . When mid-exponential- or stationary-phase cells of L . plantarum DPC2739 were adapted to 42 degrees C for 1 h, the heat resistance at 72 degrees C for 90 s increased ca . 3 and 2 log cycles, respectively . Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl . Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively . Twelve proteins were commonly induced . Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of L . plantarum DPC2739 were subjected to N-terminal sequencing . These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC . All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria . Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation . This study showed that the heat resistance of L . plantarum is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function . The physiological mechanisms of response to pasteurization in L . plantarum are fundamental for survival in cheese during manufacture.

Int J Paediatr Dent, 2004 Mar, 14(2), 118 - 26
Oral health and caries related microflora in children during the first three months following renal transplantation; Al Nowaiser A et al.; There is little information on the oral health of children undergoing renal transplantation during the early transplant period . METHODS: Twenty-four children undergoing renal transplantation aged 4-13.2 years and their matched controls were recruited . The dmfs, dmft, DMFS and DMFT, plaque, gingivitis and gingival enlargement scores were recorded . The oral microflora was sampled and cultured for S . mutans, Lactobacllus species and Candida species . RESULTS: There was a significantly lower mean dmfs (0.3 +/- 0.9; P = 0.03), dmft (0.3 +/- 0.9; P = 0.03), DMFS (2.3 +/- 5.3; P = 0.01) and DMFT (1.5 +/- 2.6; P = 0.02), respectively, in the transplant group . There was a significantly greater mean plaque score (14.7 +/- 11) for the permanent dentition, at baseline only, compared with 90 days post-transplantation (9.4 +/- 10.4; P = 0.02) . There was a significantly greater gingival enlargement score (1.8 +/- 1.4; P = 0.04) 90 days post-transplantation compared with baseline . The S . mutans and Lactobacillus counts were significantly lower both at baseline (P = 0.0001 and P = 0.004) and 90 days post-transplantation (P = 0.02; and P = 0.05), respectively, compared with the controls . CONCLUSIONS: The transplant children had less active dental disease than the controls although gingival enlargement needs careful monitoring.

J Med Invest, 2004 Feb, 51(1-2), 43 - 51
Role of unbalanced growth of gram-negative bacteria in ileal ulcer formation in rats treated with a nonsteroidal anti-inflammatory drug; Hagiwara M et al.; Nonsteroidal anti-inflammatory drugs (NSAIDs) induced formation of intestinal ulcers as side effects, in which an unbalanced increase in the number of gram-negative bacteria in the small intestine plays an important role . To clarify how intestinal microflora are influenced by NSAIDs, we examined the effects of 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl) thiophene (BFMeT), an NSAID, on intestinal motility and on the growth of Escherichia coli and Lactobacillus acidophilus . Transit index, a marker of peristalsis, was not different in BFMeT-treated and solvent-treated rats, indicating that BFMeT increased the number of gram-negative bacteria without suppression of peristalsis . The factors that affect the growth of intestinal bacteria were not found in intestinal contents of BFMeT-treated rats, because the growth of E . coli and that of L . acidophilus in the supernatants of small intestinal contents of BFMeT-treated rats and solvent-treated rats were not different . The mechanism of the increase in the number of gram-negative bacteria is still unclear, but heat-killed E . coli cells and their purified lipopolysaccharide (LPS) caused deterioration of BFMeT-induced ileal ulcers, while they could not cause the ulcers by themselves without the NSAID . Concentration of LPS and myeloperoxidase activity level were elevated correlatively in the intestinal mucosa of rats treated with LPS and BFMeT . These results suggest that an increase in the number of gram-negative bacteria and their LPS in the mucosa induces activation of neutrophils together with the help of NSAID action and causes ulcer formation.

J Bacteriol, 2004 Mar, 186(6), 1714 - 9
A -1 ribosomal frameshift in the transcript that encodes the major head protein of bacteriophage A2 mediates biosynthesis of a second essential component of the capsid; Garcia P et al.; The two major capsid proteins of Lactobacillus bacteriophage A2 share their amino termini . The smaller of these (gp5A) results from translation of orf5 and proteolytic processing after residue 123 . The larger form (gp5B) originates through a -1 ribosomal frameshift at the penultimate codon of orf5 mRNA, resulting in a product that is 85 amino acids longer than gp5A . Frameshifting needs two cis-acting elements: a slippery region with the sequence C CCA AAA (0 frame), and a stem-loop that begins 9 nucleotides after the end of the slippery sequence . Mutations introduced in the slippery sequence suppress the frameshift . Similarly, deletion of the second half of the stem-loop results in drastic reduction of frameshifting . Both gp5A and gp5B appear to be essential for phage viability, since lysogens harboring prophages that produce only one or the other protein become lysed upon induction with mitomycin C, though no viable phage progeny are observed.

Int J Food Microbiol, 2004 Mar 1, 91(2), 215 - 20
Fermentation of banana media by using kappa-carrageenan immobilized Lactobacillus acidophilus; Tsen JH et al.; Fermentation media were prepared by using banana as the raw material, and cell immobilization of Lactobacillus acidophilus by kappa-carrageenan entrapment was applied to enhance the fermentation efficiency . Gel beads of diameters around 3.0 mm were prepared for the immobilized cells, ripe bananas were used for preparation of media, and both free and immobilized cells were employed to carry out the fermentation for 80 h . Cells leaked out from the gel beads and proliferated in the medium solution during the fermentation of immobilized cells . The final viable cell number reached 10(5) CFU/ml in the medium suspension, over 10(8) CFU/(ml gel) in gel beads for the immobilized cell fermentation and around 10(6) CFU/ml for the free cell fermentation . Immobilized cells withstand the adverse conditions in banana media resulting in better fermentation efficiency compared to free cells . Variation of fructooligosaccharides (FOS) in banana media was not significant in immobilized cell fermentation compared to free cell . Immobilized L . acidophilus fermented banana medium was found to possess synbiotic properties.

Int J Food Microbiol, 2004 Mar 1, 91(2), 129 - 39
Genotypic and phenotypic diversity of Lactobacillus delbrueckii subsp . lactis strains of dairy origin; Giraffa G et al.; Eighty-nine strains of Lactobacillus delbrueckii subsp . lactis isolated from Italian hard and semi-hard cheeses and artisan starter cultures were characterised by phenotypic and genotypic methods . Phenotypic diversity was evaluated by studying biochemical characteristics (i.e . acidifying and peptidase activities) of technological interest . Genotypic diversity was evidenced by RAPD-PCR and pulsed field gel electrophoresis (PFGE) . Phenotypic characterisation indicated a wide variability of the acidifying activity within Lact . delbrueckii subsp . lactis . Although the data was variable, it allowed us to evidence groups of strains with different acidifying properties, especially in terms of acidification intensity . Concerning peptidase activity, Lact . delbrueckii subsp . lactis showed a homogeneously high x-prolil-dipeptidil-aminopeptidase activity and a considerable but more heterogeneous lysil-aminopeptidase activity . The increased resolution obtained by the use of two molecular typing techniques, i.e . RAPD-PCR and PFGE, allowed to widen the level of strain heterogeneity . Technological and ecological pressures are determinant in selecting Lact . delbrueckii subsp . lactis sub-populations which are more functional to the different cheese technologies.

J Agric Food Chem, 2004 Mar 10, 52(5), 1163 - 9
Fermentation of seeds of Teff (Eragrostis teff), grass-pea (Lathyrus sativus), and their mixtures: aspects of nutrition and food safety; Yigzaw Y et al.; Fermentation of pure teff (Eragrostis teff), pure grass-pea (Lathyrus sativus), and their mixtures, 9:1 and 8:2 (teff/grass-pea) has been done at two temperatures (room temperature and 35 degrees C) in duplicate using the strains of Lactobacillus plantarum, for bacterial fermentation, and Aspergillus oryzae and Rhizopus oligosporus in succession for solid-state fungal fermentation as inocula . In addition, the natural or spontaneous and back-slopping methods of bacterial fermentation have been done on the above four substrate groups . The pH and essential amino acid profiles of the different fermentation processes were compared . The back-slopping in teff at a temperature of 35 degrees C gave the sharpest pH drop . All fermentations done at 35 degrees C showed a steeper slope in their pH versus time plot compared to their room temperature counterpart . Fungal fermentation gave an improved amino acid profile for the essential ones in all of the substrate groups, except in pure grass-pea . Fermented teff/grass-pea (8:2) in this fungal fermentation has been found to be quite comparable in essential amino acid profile to an ideal reference protein recommended for children of 2-5 years of age . None of the bacterial fermentations produced a net change in their essential amino acid profile in any of the substrate groups investigated . Solid state fungal fermentation on pure grass-pea using the fungal strains R . oligosporous and A . oryzae in succession has shown that the neurotoxin beta-N-oxalyl-alpha,beta-diaminopropionic acid (beta-ODAP) in grass-pea has been removed by 80% on average for the high-toxin variety and by up to 97% for the low-toxin variety as determined by an improved chromatographic method with bioelectrochemical detection coupled on-line with refractive index detection.

Microbiology, 2004 Mar, 150(Pt 3), 621 - 30
Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri; van Hijum SA et al.; Lactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer {a fructan of the levan type, with beta(2-->6) linkages} from sucrose or raffinose . Purification of this FTF (a levansucrase), and identification of peptide amino acid sequences, allowed isolation of the first Lactobacillus levansucrase gene (lev), encoding a protein (Lev) consisting of 804 amino acids . Lev showed highest similarity with an inulosucrase of L . reuteri 121 {Inu; producing an inulin polymer with beta(2-->1)-linked fructosyl units} and with FTFs from streptococci . Expression of lev in Escherichia coli resulted in an active FTF (Lev Delta 773His) that produced the same levan polymer {with only 2-3 % beta(2-->1-->6) branching points} as L . reuteri 121 cells grown on raffinose . The low degree of branching of the L . reuteri levan is very different from bacterial levans known up to now, such as that of Streptococcus salivarius, having up to 30 % branches . Although Lev is unusual in showing a higher hydrolysis than transferase activity, significant amounts of levan polymer are produced both in vivo and in vitro . Lev is strongly dependent on Ca(2+) ions for activity . Unique properties of L . reuteri Lev together with Inu are: (i) the presence of a C-terminal cell-wall-anchoring motif causing similar expression problems in Escherichia coli, (ii) a relatively high optimum temperature for activity for FTF enzymes, and (iii) at 50 degrees C, kinetics that are best described by the Hill equation.

Microbiology, 2004 Mar, 150(Pt 3), 613 - 20
Complementation of a Delta ccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains; Esteban CD et al.; In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator . A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis . The replaced amino acids were located in the DNA- and cofactor (P-Ser-HPr)-binding domains . Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium . However, mutations in the cofactor-binding domain of L . casei CcpA had a different phenotype to that observed in the previous studies with B . megaterium . Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA . Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features.

Biochemistry, 2004 Mar 9, 43(9), 2384 - 93
Structures of purine 2'-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 A resolution; Anand R et al.; The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography . Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base . Both class I and class II enzymes are highly specific for deoxynucleosides . The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni {Armstrong, S . A., Cook, W . J., Short, S . A., and Ealick, S . E . (1996) Structure 4, 97-107} . The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme . In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot . The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides . The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond . The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.

Scand J Gastroenterol, 2004 Jan, 39(1), 37 - 45
Nitric oxide released by Lactobacillus farciminis improves TNBS-induced colitis in rats; Lamine F et al.; BACKGROUND: Beneficial effects of lactobacilli have been reported in experimental colitis . On the other hand, despite the controversial role of nitric oxide (NO) in the inflammatory gut process, a protective action of exogenous NO in inflammation has been suggested . Consequently, this study aimed to determine the effect of (i) sodium nitroprusside (SNP), a NO donor and (ii) treatment with Lactobacillus farciminis, which produces NO in vitro, on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats and to evaluate the role of exogenous NO in this effect . METHODS: Rats were divided into three groups receiving one of the following: (i) a continuous intracolonic (IC) infusion of SNP for 4 days, (ii) L . farciminis orally for 19 days, or (iii) saline . On day 1 and day 15, respectively, TNBS and saline were administrated IC, followed by a continuous IC infusion of saline or haemoglobin, a NO scavenger . At the end of treatments, the following parameters were evaluated: macroscopic damage of colonic mucosa, myeloperoxidase and nitric oxide synthase activities and colonic luminal NO production . RESULTS: In colitic rats, SNP and L . farciminis treatment significantly (P < 0.05) reduced macroscopic damage scores, myeloperoxidase and nitric oxide synthase activities compared to controls . Haemoglobin infusion abolished the anti-inflammatory effect of both NO donor treatments, but had no effect per se on colitis . CONCLUSION: NO released intraluminally by SNP infusion or by L . farciminis given orally improves TNBS-induced colitis in rats . These results indicate a protective role of NO donation in colonic inflammation and show for the first time a mechanism involving NO delivery by a bacterial strain reducing an experimental colitis.

FEBS Lett, 2004 Feb 27, 560(1-3), 131 - 3
Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121; Ozimek LK et al.; Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases . Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved {Meng, G . and Futterer, K., Nat . Struct . Biol . 10 (2003) 935-941} . Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified . However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme . We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins . Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5-4x10(5) times reduction of total sucrase activity.

Int J Food Microbiol, 2004 Mar 15, 91(3), 305 - 13
Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley; Helland MH et al.; A fermented probiotic maize porridge with high energy density and low viscosity was prepared, using maize flour and barley malt . The porridge was fermented with four probiotic strains (grown separately): Lactobacillus reuteri, Lb . acidophilus (LA5 and 1748) and Lb . rhamnosus GG . These strains were inoculated at two levels; to obtain approx . 7 or 6 log cfu g(-1) in the porridge at 0 h . The porridge was fermented for 24 h at 37 degrees C, and analysed for viable cell count, pH, organic acids, volatile aromatic compounds and sugar content . The inoculated cell concentration was shown to be particularly important during the first hours of the fermentation period, showing a delayed production of most metabolites in porridge inoculated with approx . 6 log cfu g(-1) . Most strains reached maximum cell count after 12-h fermentation (7.2-8.2 log cfu g(-1)), with a pH below 4.0 . Depending on the strain, lactic acid was produced in amounts ranging from 1360 to 4000 mg kg(-1) . Lb . reuteri metabolised succinate, while pyruvate and small amounts of diacetyl were detected in porridge inoculated with Lb . acidophilus LA5 and Lb . acidophilus 1748 . High amounts of diacetyl (6 mg kg(-1)) and acetoin (27 mg kg(-1)) were detected in porridge inoculated with Lb . rhamnosus GG . Porridge inoculated with Lb . acidophilus LA5 and Lb . acidophilus 1748, contained acetaldehyde, while both Lb . reuteri and Lb . rhamnosus GG reduced the acetaldehyde to ethanol . Lb . reuteri utilised both maltose and glucose as carbohydrate sources, while Lb . acidophilus LA5, Lb . acidophilus 1748 and Lb . rhamnosus GG utilised only glucose.

Int J Food Microbiol, 2004 Mar 15, 91(3), 229 - 44
Development of a novel approach for secondary modelling in predictive microbiology: incorporation of microbiological knowledge in black box polynomial modelling; Geeraerd AH et al.; This research deals with the development of a novel secondary modelling procedure within the framework of predictive microbiology . The procedure consists of three steps: (i) careful formulation of the available microbiological information, both from literature and from the experimental case study at hand, (ii) translation of these requirements in mathematical terms under the form of partial derivatives throughout the complete interpolation region of the experimental design, and (iii) determination of parameter values with suitable optimisation techniques for a flexible black box modelling approach, e.g., a polynomial model or an artificial neural network model . As a vehicle for this procedure, the description of the maximum specific growth rate of Lactobacillus sakei in modified BHI-broth as influenced by suboptimal temperature, water activity, sodium lactate and dissolved carbon dioxide concentration is under study . The procedure results in a constrained polynomial model with excellent descriptive and interpolating features in comparison with an extended Ratkowsky-type model and classical polynomial model, by combining specific properties of both model types . The developed procedure is illustrated on the description of the lag phase as well . It is stressed how the confrontation with experimental data is very important to appreciate the descriptive and interpolating capacities of new or existing models, which is nowadays not always carefully performed . Alternatively, the first two steps of the novel procedure can be used as a tool to demonstrate clearly (possible) interpolative shortcomings of an existing model with straightforward spreadsheet calculations.

Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2512 - 7
The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533; Pridmore RD et al.; Lactobacillus johnsonii NCC 533 is a member of the acidophilus group of intestinal lactobacilli that has been extensively studied for their "probiotic" activities that include, pathogen inhibition, epithelial cell attachment, and immunomodulation . To gain insight into its physiology and identify genes potentially involved in interactions with the host, we sequenced and analyzed the 1.99-Mb genome of L . johnsonii NCC 533 . Strikingly, the organism completely lacked genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors . In apparent compensation, a remarkable number of uncommon and often duplicated amino acid permeases, peptidases, and phosphotransferase-type transporters were discovered, suggesting a strong dependency of NCC 533 on the host or other intestinal microbes to provide simple monomeric nutrients . Genome analysis also predicted an abundance (>12) of large and unusual cell-surface proteins, including fimbrial subunits, which may be involved in adhesion to glycoproteins or other components of mucin, a characteristic expected to affect persistence in the gastrointestinal tract (GIT) . Three bile salt hydrolases and two bile acid transporters, proteins apparently critical for GIT survival, were also detected . In silico genome comparisons with the >95% complete genome sequence of the closely related Lactobacillus gasseri revealed extensive synteny punctuated by clear-cut insertions or deletions of single genes or operons . Many of these regions of difference appear to encode metabolic or structural components that could affect the organisms competitiveness or interactions with the GIT ecosystem.

Am J Obstet Gynecol, 2004 Feb, 190(2), 363 - 70
Time course of the regression of asymptomatic bacterial vaginosis in pregnancy with and without treatment; Klebanoff MA et al.; OBJECTIVE: The purpose of this study was to determine the effectiveness of treatment over time for bacterial vaginosis in pregnancy and the probability of spontaneous resolution with placebo . STUDY DESIGN: Women with asymptomatic bacterial vaginosis on Gram stain were assigned randomly at 16 to 23 weeks of gestation to receive two 2-g doses of metronidazole or placebo 48 hours apart and were re-evaluated for changes in Gram stain score on one occasion > or =2 weeks later . RESULTS: Of 658 women who underwent metronidazole therapy, treatment was successful (score, <7) in 78% of those women who were seen at 2 to 3.9 weeks of gestation, which was similar to women seen > or =10 weeks after treatment . In 683 women who underwent placebo therapy, spontaneous resolution increased significantly from 13% at 2 to 3.9 weeks of gestation to 36% at > or =10 weeks of gestation . Spontaneous resolution was more common with lower vaginal pH or lactobacilli on Gram stain at the time of random assignment . CONCLUSION: The effectiveness of metronidazole therapy of bacterial vaginosis persists for > or =10 weeks . Women who underwent placebo therapy had significant remission of bacterial vaginosis over > or =10 weeks . Remission was more common when the initial vaginal microbiologic disturbances were less severe.

J Contemp Dent Pract, 2004 Feb 15, 5(1), 105 - 14
The immediate antimicrobial effect of a toothbrush and miswak on cariogenic bacteria: a clinical study; Almas K et al.; The aim of this study was to assess the antimicrobial activity of the miswak chewing stick (Salvadora persica) in vivo, especially on streptococcus mutans and lactobacilli . The study was conducted clinically using patients' saliva and measuring the effect of miswak (chewing stick), miswak extract, toothbrush, and normal saline on mutans and lactobacilli . Forty male subjects aged 20-45 years were included in the study; there were four groups of ten subjects each . For the study, 50% of miswak extract (solution) was used . The levels of mutans streptococci and lactobacilli were measured using commercially available Vivacare line CRT (Caries Risk Test) bacteria 2 in 1 kit . The results showed there was a marked reduction of streptococcus mutans among all groups . When the groups were compared, the reduction of streptococcus mutans was significantly greater using miswak in comparison to toothbrushing (p = 0.013), and there was no significant difference for lactobacilli reduction (p = 0.147) . It may be concluded miswak has an immediate antimicrobial effect . Streptococcus mutans were more susceptible to miswak antimicrobial activity than lactobacilli . Dietary intake of sugar and oral health status may be considered for controlled clinical trials with special emphasis on the antibacterial activity of miswak on cariogenic bacteria for a longer period of time . A toothbrush with and without toothpaste should be compared with miswak alone . Further research is needed with a larger sample size.

J Bacteriol, 2004 Mar, 186(5), 1556 - 64
Induction of plantaricin production in Lactobacillus plantarum NC8 after coculture with specific gram-positive bacteria is mediated by an autoinduction mechanism; Maldonado A et al.; Plantaricin NC8 (PLNC8), a coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8, has recently been purified and genetically characterized . Analysis of an 8.1-kb NC8 DNA region downstream of the PLNC8 operon revealed the presence of at least four operons involved in bacteriocin production, showing high homology to the plantaricin cluster in L . plantarum C11 . However, we found a three-component regulatory operon involving a quorum-sensing mechanism . Two of these components, the induction factor (PLNC8IF) and the histidine kinase, are novel, while the response regulator is identical to PlnD from C11 . Homologous expression of plNC8IF in NC8 allowed constitutive bacteriocin production . Heterologous expression of this gene in Lactococcus lactis MG1363 produced supernatants which promoted bacteriocin production in NC8 . Reverse transcription-PCR studies indicated that cocultivation of NC8 with inducing cells promoted transcription of the bacteriocin and regulatory operons in NC8 . An identical result was obtained after addition of an external source of PLNC8IF . We propose that the presence of specific bacteria could act as an environmental signal that is able to switch on bacteriocin production in L . plantarum NC8 via a quorum-sensing mechanism mediated by PLNC8IF.

Beijing Da Xue Xue Bao, 2004 Feb, 36(1), 31 - 5
{Studies on the biotransformation of escin Ia by human intestinal bacteria and the anti-tumor activities of desacylescin I}; Yang XW et al.; OBJECTIVE: To study Biotransformation of escin Ia by the crude enzymes of human intestinal bacteria and Lactobacillus brevis, determine the structures of biotransformation products and assay the inhibitory effect of desacylescin I on the tumor cell growth . METHODS: The escin Ia was incubated with crude enzymes of human intestinal bacteria and Lactobacillus brevis in vitro, respectively . The biotransformation products were isolated and purified by the chromatographic methods and the structures were determined by the spectroscopic techniques . RESULTS: Escin Ia was converted into isoescin Ia, desacylescin I, 21beta-O-tigloylprotoaescigenin and protoaescigenin by crude enzymes of human intestinal bacteria and Lactobacillus brevis . Desacylescin I showed potentially inhibitory effects on tumor cell growth of mouse sarcoma-180, hepatic carcinoma H(22) and lung carcinoma in vivo . CONCLUSION: The results suggest that Escin Ia was a prodrug and its structure can be converted by human intestinal bacteria and Lactobacillus brevis . Desacylescin I as a biotransformation product showed potentially inhibitory effects on mouse tumor, and a potential candidate for anti tumor agents.

J Agric Food Chem, 2004 Feb 25, 52(4), 801 - 8
Formulation of low-cost fermentative media for lactic acid production with Lactobacillus rhamnosus using vinification lees as nutrients; Bustos G et al.; Lees coming from different steps in white wine and red wine vinification were characterized under physicochemical analyses to determine the content in carbon, nitrogen, ashes, solids in suspension, organic compounds, and minerals . Due to the hydrolytic activity of Lactobacillus strains, lees without autolysis treatments were used directly as the unique nutrient or in combination with corn steep liquor to carry out the glucose to lactic acid fermentation with Lactobacillus rhamnosus CECT-288 . Time courses of glucose and lactic acid were modeled according to reported models . Using 20 g/L of lees coming from the white wine technology and re-collected after the second decanting step before distillation, as the only nutrient, the values achieved (P = 105.5 g/L, Q(P) = 2.470 g/L.h) were even higher than those obtained with the costly MRS broth (P = 104.3 g/L, Q(P) = 2.251 g/L.h).

Int J Food Microbiol, 2004 Feb 15, 91(1), 19 - 29
Identification of lactic acid bacteria isolated from South African brandy base wines; du Plessis HW et al.; In brandy base wines, no sulphur dioxide is used and it therefore is ideal for the proliferation of lactic acid bacteria . As part of an extensive taxonomic survey within the ecological framework of South African vineyards and wineries, and the influence of naturally occurring lactic acid bacteria on the quality of wine and brandy, a total of 54 strains were isolated from grape juice and at different stages of brandy base wine production . The strains were identified using numerical analysis of total soluble cell protein patterns, 16S rRNA sequence analyses and polymerase chain reaction (PCR) using species-specific primers . The predominant species was Oenococcus oeni (22 strains), but Lactobacillus brevis (8 strains), Lactobacillus paracasei (8 strains) and Lactobacillus plantarum (6 strains) were also isolated frequently . Many of the O . oeni strains were isolated from brandy base wines after completion of spontaneous malolactic fermentation (MLF) . The Lactobacillus spp . were isolated from all the different stages of brandy base wine production . Lb . plantarum was the dominant species in the juice, but disappeared during the later stages of production . However, Lactobacillus hilgardii, Lb . brevis and Lb . paracasei were also isolated from base wine after spontaneous MLF . Strains identified as Lactobacillus vermiforme were isolated during the alcoholic fermentation and after MLF have been completed . Total soluble cell protein patterns grouped O . oeni strains into two phenotypic groups . Two phenotypic clusters have also been identified for the Lb . brevis isolates . The Lb . paracasei isolates all grouped in one cluster . This is the first report of the presence of Lb . paracasei and Lb . vermiforme in brandy base wines . The presence of the Lactobacillus spp . could be correlated to the decrease in quality of the base wine and distillate, while O . oeni strains were found to have a more favourable influence on the quality of base wine and distillates . These results shed some light on the ecology and oenological influence of lactic acid bacteria (LAB) on the quality of South African brandy.

Shanghai Kou Qiang Yi Xue, 2003 Oct, 12(5), 356 - 8
{Comparison of the antibacterial activity on oral pathogens among six types of nano-silver base inorganic antibacterial agents}; She WJ et al.; OBJECTIVE: To Compare the antibacterial activity on oral pathogens among six types of nano-silver base inorganic antibacterial agents.METHODS: The oral pathogens selected were Streptococcus mutans(ATCC6715),Lactobacillus (ATCC4356),Actinomyces viscosus (ATCC19246), Candida Albicans (ATCC76615), Staphylococcus aureus (ATCC25923), Escherichia coli (ATCCC25922) . Minimal bactericidal concentration (MBC) was determined by Liquid dilution method.RESULTS: Nano-silver base inorganic antibacterial agents had fine bactericidal activity against oral pathogens . The general antibacterial activity of six types were nearly equal.CONCLUSION: It is possible that nano-silver base inorganic antibacterial agents can be used in dental antibacterial materials.

J Leukoc Biol, 2004 May, 75(5), 764 - 71 Epub 2004 Feb 13.
Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells; Veckman V et al.; Dendritic cells (DCs) are the most efficient antigen-presenting cells and thus, have a major role in regulating host immune responses . In the present study, we have analyzed the ability of Gram-positive, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte-derived DCs . Stimulation of DCs with S . pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response . S . pyogenes also induced interleukin (IL)-2 and IL-12 production at mRNA and protein levels . In addition, IL-23 and IL-27 subunits p40, p19, p28, and EBI3 were induced at mRNA level . In contrast, L . rhamnosus-stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines . Furthermore, no production of IL-2 or IL-12 family cytokines was detected . Bacteria-induced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat-inactivated . Our results show that human monocyte-derived DCs respond differently to different Gram-positive bacteria . Although pathogenic S . pyogenes induced a strong Th1-type response, stimulation with nonpathogenic L . rhamnosus resulted in development of semi-mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production.

Adv Ther, 2003 Sep-Oct, 20(5), 253 - 60
Multicenter, randomized, controlled trial of heat-killed Lactobacillus acidophilus LB in patients with chronic diarrhea; Xiao SD et al.; Chronic diarrhea is a common bowel disorder; disturbance of intestinal microorganisms may play a role in its pathogenesis . This study assessed the clinical efficacy of lyophilized, heat-killed Lactobacillus acidophilus LB versus living lactobacilli in the treatment of chronic diarrhea . One hundred thirty-seven patients with chronic diarrhea were randomly allocated to receive either a 4-week course of 2 capsules of Lacteol Fort twice a day (Lacteol group, 69 patients) or a 4-week course of 5 chewable tablets of Lacidophilin three times a day (Lacidophilin group, 64 patients) . The frequency of stools was recorded quantitatively, and semiquantitative parameters such as stool consistency, abdominal pain, distention, and feeling of incomplete evacuation were evaluated . At the second and fourth week of treatment, mean bowel frequency was significantly lower in the Lacteol group than in the Lacidophilin group (1.88 +/- 1.24 vs 2.64 +/- 1.12, 1.39 +/- 0.92 vs 2.19 +/- 1.05; P<.05) . At the end of the treatment, the clinical symptoms were markedly improved in the Lacteol group, indicating that L . acidophilus LB is more effective than living lactobacilli in the treatment of chronic diarrhea.

Appl Microbiol Biotechnol, 2004 Aug, 65(3), 268 - 72 Epub 2004 Feb 13.
Evaluation of agar diffusion bioassay for nisin quantification; Pongtharangkul T et al.; The agar diffusion bioassay is the most widely used method for the quantification of nisin, due to its high sensitivity, simplicity, and cost-effectiveness . This method is based on the measurement of the inhibition zone produced in nisin-sensitive microorganisms . The size of the zone is affected by many factors, such as nisin-sensitive strain, amount of added agar and surfactant, and pre-diffusion step . This research aims to evaluate the effects of nisin-sensitive strains and pre-diffusion on the accuracy and precision of nisin quantification . Three strains of nisin-sensitive microorganisms (Micrococcus luteus, Lactobacillus sakei, Brochothrix thermosphacta) were tested along with three different incubation processes . The best combination was the method using L . sakei as an indicator strain with pre-diffusion at 4 degrees C for 24 h . Compared with M . luteus and B . thermosphacta, L . sakei gave more accurate and reproducible results . Moreover, the pre-diffusion step resulted in larger inhibition zones and more precise results . Finally, the best combination was validated and compared with the method that is usually used and the result showed that the method using L . sakei with pre-diffusion gave more accurate and precise results.

Blood, 2004 Jun 1, 103(11), 4365 - 7 Epub 2004 Feb 12.
Probiotic effects on experimental graft-versus-host disease: let them eat yogurt; Gerbitz A et al.; Acute graft-versus-host disease (aGVHD) often limits feasibility and outcome of allogeneic bone marrow transplantation . Current pathophysiologic concepts of aGVHD involve conditioning regimens, donor-derived T cells, proinflammatory cytokines, and bacterial lipopolysaccharide (LPS) as a major trigger for aGVHD . LPS derives mostly from gram-negative bacteria and can enter circulation through the impaired mucosal barrier after the conditioning regimen . Probiotic microorganisms have been shown to alter the composition of the intestinal microflora and thereby mediate anti-inflammatory effects . We hypothesized that modifying the enteric flora using the probiotic microorganism Lactobacillus rhamnosus GG, would ameliorate aGVHD . Here we show that oral administration of Lactobacillus rhamnosus GG before and after transplantation results in improved survival and reduced aGVHD . Furthermore, subculturing of mesenteric lymph node tissue revealed a reduced translocation of enteric bacteria . Our findings suggest that alteration of the intestinal microflora plays an important role in the initiation of experimental aGVHD.

J Appl Microbiol, 2004, 96(3), 593 - 602
Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp . paracasei CHCC 2115; Thage BV et al.; AIM: Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp . paracasei CHCC 2115 . METHODS AND RESULTS: The purification protocol consisted of anion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography . The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa . The AT converted isoleucine, leucine and valine at a similar rate with alpha-ketoglutarate as the amino group acceptor; minor activity was shown for methionine . The enzyme had pH and temperature optima of 7.3 and 43 degrees C, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5.2, 4% NaCl) . Hg2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5'-phosphate-dependent enzymes, when studying the effect of other metal ions, thiol- and carbonyl-binding agents . The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact . plantarum and Lactococcus lactis, respectively . CONCLUSIONS: The results suggest that Lact . paracasei subsp . paracasei CHCC 2115 may contribute to development of flavour in cheese . SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of transamination performed by cheese-related bacteria, and in the understanding and control of amino acid catabolism and the production of aroma compounds.

J Appl Microbiol, 2004, 96(3), 552 - 9
Modulation of anti-pathogenic activity in canine-derived Lactobacillus species by carbohydrate growth substrate; Tzortzis G et al.; AIMS: To investigate the effect of various carbon sources on the production of extracellular antagonistic compounds against two Escherichia coli strains and Salmonella enterica serotype Typhimurium by three canine-derived lactobacilli strains . METHODS AND MATERIALS: Cell-free preparations, pH neutralized, were used in antibiotic disc experiments as an initial screening . The bacteria/carbohydrate combinations that showed inhibition of the growth of those pathogens, were further investigated in batch co-culture experiments . The cell-free supernatants of the cultures, that decreased the population number of the pathogens in the co-culture experiments to log CFU ml-1 <or= 4, were tested for inhibition of the pathogens in pure cultures at neutral and acidic pH . CONCLUSIONS: The results showed that the substrate seems to affect the production of antimicrobial compounds and this effect could not just be ascribed to the ability of the bacteria to grow in the various carbon sources . L . mucosae, L . acidophilus and L . reuteri, when grown in sugar mixtures consisting of alpha-glucosides (Degree of Polymerization (DP) 1-4) could produce antimicrobial compounds active against all three pathogens in vitro . This effect could not be attributed to a single ingredient of those sugar mixtures and was synergistic . This inhibition had a dose-response characteristic and was more active at acidic pH . SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the effect that the carbon source has on the production of antimicrobial compounds by gut-associated lactobacilli allows the rational design of prebiotic/probiotic combinations to combat gastrointestinal pathogens.

J Appl Microbiol, 2004, 96(3), 521 - 34
Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity; Corsetti A et al.; AIMS: To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB . METHODS AND RESULTS: Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated . Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb . plantarum 4DE and 3DM and Lactobacillus spp . CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS) . BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb . sanfranciscensis . Antimicrobial production by Lb . pentosus 2MF8 and Lc . lactis M30 was also demonstrated in situ . CONCLUSIONS: BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc . lactis . Nevertheless, they are active BLIS producers under sourdough and bread-making conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of BLIS has been demonstrated in situ . It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb . sanfranciscensis, useful to confer good characteristics to the dough.

Lett Appl Microbiol, 2004, 38(3), 206 - 10
Use of folic acid casei medium reveals trimethoprim susceptibility of Lactobacillus species; Danielsen M et al.; AIM: Lactobacilli have been reported to have intrinsic resistance to trimethoprim . The susceptibility of lactobacilli to trimethoprim on different media was investigated in order to search for a phenotypic test method that could indicate the presence of acquired resistance genes . METHODS AND RESULTS: Strains of Lactobacillus acidophilus, Lact . paracasei, Lact . rhamnosus and Lact . plantarum were susceptibility tested with E-tests on folic acid casei medium (FACM), MRS and defined medium 1 . The effects of addition or removal of nucleosides and thymidine phosphorylase were investigated . E-tests on FACM yielded reproducible minimal inhibitory concentrations (MICs) for trimethoprim but addition of nucleosides was necessary for growth of Lact . acidophilus . MICs for the tested strains were 0.125-0.19, 0.25-3 and 0.064-0.19 microg ml(-1) for Lact . paracasei, Lact . rhamnosus and Lact . plantarum, respectively . With the addition of deoxyuridine and deoxyadenosine to FACM the MICs of Lact . acidophilus were 0.064-1 microg ml(-1) . CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacilli do not have intrinsic resistance to trimethoprim . The results show that trimethoprim susceptibility testing of the tested Lactobacillus species is possible and indicate that transferable resistance genes are absent in all the tested strains.

Anal Chem, 2004 Feb 15, 76(4), 1184 - 8
Polarimetric assay for the medium-throughput determination of alpha-amino acid racemase activity; Schonfeld DL et al.; A polarimetric assay has been developed for the identification of alpha-amino acid racemase activity . The setup consists of a microcuvette polarimeter (40 microL volume) connected to a pipetting robot for microtiter plates, a pump, and data processing . It could be demonstrated for a glutamate racemase from Lactobacillus fermentii, expressed in Escherichia coli, serving as model enzyme, that its activity can be determined from the time-dependent change of the optical rotation using l-glutamate as substrate . Thus, the specific activity was determined to 111.4 mdeg/min which corresponds to 45.7 micromol/min per mg purified enzyme . Moreover, a protocol was developed that allows the measurement of racemase activity from 96-well microtiter plates using purified enzymes . Thus, the method described can be used to determine racemase activity in an automatic manner . It should be also applicable for the screening of enzyme libraries created by directed evolution.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 125 - 30
Monitoring the cell number and viability of Lactobacillus helveticus GCL1001 in human feces by PCR methods; Saito Y et al.; Real-time polymerase chain reaction (PCR) and nested reverse transcription (RT) PCR were applied to demonstrate the viability of lactobacilli in the feces of volunteers fed fermented milk containing lactobacilli . Two sets of specific primers and a TaqMan probe for real-time PCR were constructed using the S-layer gene as a target . After fermented milk ingestion, Lactobacillus helveticus GCL1001 was detected in the feces of 12 volunteers over a few days, with the maximum number being between 10(4.5) and 10(7.8) cells g(-1) of feces . Moreover, mRNA from this strain was detected in the feces of all volunteers by nested RT-PCR . The results show that these methods are applicable to the demonstration of bacterial viability in feces, and that ingested L . helveticus GCL1001 can survive through the gastrointestinal tract.

Cent Eur J Public Health, 2003 Dec, 11(4), 219 - 22
Suppression of salivary Streptococcus mutans and lactobacilli by topical caries preventive agents; Juric H et al.; Reduction of cariogenic bacteria, especially salivary Streptococcus mutans and lactobacilli is a valuable clinical procedure that in many ways alleviates implementation of targeted caries preventive procedures in the entire population . The aim of this study was to investigate the caries preventive values of certain preventive procedures in in vivo conditions . Four groups of subjects, each with 18 children aged from 4-5 and 10-12 years (n = 72) were treated with different caries preventive agent (aminfluoride solution, Proxyt paste, chewing gum containing xylitol and fluoride and chlorhexidine solution) . During a period of two months five control measurements for number of salivary Streptococcus mutans (SM) and lactobacilli (LB) were performed . At the end of the study the best result in the reduction of the bacteria was achieved by the application of Proxyt paste and daily use of chewing gum (p < 0.001) . In patients treated with this preventive procedure the number of SM was reduced by 1 class and LB to < 10(4) CFU/ml saliva after two months of study . The results obtained indicate that professional teeth cleaning and use of chewing gum with xylitol and fluorides on daily basis can be very effective protocol for cariogenic bacteria reduction and in the individual caries prevention.

J Basic Microbiol, 2004, 44(1), 10 - 6
Molecular characterization of lactic acid populations associated with wine spoilage; Beneduce L et al.; We have investigated the prevalence of spoilage lactic acid bacteria (LAB) in table wines produced in the Apulia region . The occurrence of LAB was evaluated in wines produced with low sulphur dioxide doses and not supplemented with selected malolactic starters such as Oenococcus oeni . About 150 strains were isolated from wine must and a molecular characterization was performed using PCR-based techniques . Most of the strains analysed belonged to Lactobacillus plantarum species . However, some of the strains were identified as Pediococcus damnosus and Leuconostoc sp . The amplified fragments of Pediococcus damnosus were cloned and sequenced . The coding sequence was highly homologous to that of the ropy plasmid confirming that the isolated strain was a ropy(+) Pediococcus damnosus . In all the samples analysed, the final must pH value reached was relatively high (from 3.78 to 3.90) . The high pH values had probably negatively influenced (counteracted) the activity of sulphur dioxide added, allowing proliferation of spoilage wine microorganisms.

J Clin Microbiol, 2004 Feb, 42(2), 713 - 7
Identification and H(2)O(2) production of vaginal lactobacilli from pregnant women at high risk of preterm birth and relation with outcome; Wilks M et al.; Lactobacilli, principally the strains that are hydrogen peroxide (H(2)O(2)) producing, may have a protective effect against vaginal colonization by pathogenic species such as those that cause bacterial vaginosis . Previous reports have also suggested that H(2)O(2)-producing lactobacilli in the vagina may protect pregnant women against ascending infection of the chorioamniotic membranes and uterine cavity . We report the identification and H(2)O(2) production of lactobacilli isolated from vaginal swabs collected at 20 weeks' gestation from a population of pregnant women at high risk of preterm birth . We also report the correlation between identification and H(2)O(2) production in relation to the outcomes of chorioamnionitis and preterm birth . Lactobacilli were identified by partial 16S rRNA gene sequencing . H(2)O(2) production by isolates was determined by a semiquantitative method . The most commonly isolated species were L . crispatus, L . gasseri, L . vaginalis and L . jensenii . Amounts of H(2)O(2) produced by lactobacilli varied widely . The presence of lactobacilli producing high levels of H(2)O(2) in the vagina of this population of pregnant women was associated with a reduced risk of bacterial vaginosis at 20 weeks' gestation and subsequent chorioamnionitis . L . jensenii and L . vaginalis produced the highest levels of H(2)O(2) . We postulate that H(2)O(2)-producing lactobacilli are able to reduce the incidence of ascending infections of the uterus and the subsequent production of proinflammatory molecules which are important in the pathogenesis of chorioamnionitis and preterm birth.

Appl Environ Microbiol, 2004 Feb, 70(2), 1176 - 81
Colonization and immunomodulation by Lactobacillus reuteri ATCC 55730 in the human gastrointestinal tract; Valeur N et al.; Lactobacillus reuteri ATCC 55730 is a probiotic (health-promoting) bacterium widely used as a dietary supplement . This study was designed to examine local colonization of the human gastrointestinal mucosa after dietary supplementation with L . reuteri ATCC 55730 and to determine subsequent immune responses at the colonized sites . In this open clinical investigation, 10 healthy volunteers and 9 volunteers with ileostomy underwent gastroscopy or ileoscopy and biopsy samples were taken from the stomach, duodenum, or ileum before and after supplementation with 4 x 10(8) CFU of live L . reuteri ATCC 55730 lactobacilli per day for 28 days . Biopsy specimen colonization was analyzed using fluorescence in situ hybridization with a molecular beacon probe, and immune cell populations were determined by immunostaining . Endogenous L . reuteri was detected in the stomach of 1 subject and the duodenum of 3 subjects (out of 10 subjects) . After L . reuteri ATCC 55730 supplementation, the stomachs of 8 and the duodenums of all 10 subjects were colonized . Three ileostomy subjects (of six tested) had endogenous L . reuteri at baseline, while all six displayed colonization after L . reuteri supplementation . Gastric mucosal histiocyte numbers were reduced and duodenal B-lymphocyte numbers were increased by L . reuteri ATCC 55730 administration . Furthermore, L . reuteri administration induced a significantly higher amount of CD4-positive T-lymphocytes in the ileal epithelium . Dietary supplementation with the probiotic L . reuteri ATCC 55730 induces significant colonization of the stomach, duodenum, and ileum of healthy humans, and this is associated with significant alterations of the immune response in the gastrointestinal mucosa . These responses may be key components of a mechanism by which L . reuteri ATCC 55730 exerts its well-documented probiotic effects in humans.

Appl Environ Microbiol, 2004 Feb, 70(2), 670 - 4
Permanent colonization by Lactobacillus casei is hindered by the low rate of cell division in mouse gut; Lee YK et al.; Long residence times of probiotics in the intestinal tract would prolong their potential beneficial health effects and assist colonization . This study investigated the colonization potential of Lactobacillus casei Shirota in mouse intestine by using 5 (and 6)-carboxyfluorescein diacetate, succinimidyl ester (cFDA-SE) for assessment of doubling times in different parts of the intestine . The amounts of intestinal water overlying the surfaces of the duodenum, jejunum, ileum, and colon in BALB/c mice were 34.4 +/- 2.9, 58.8 +/- 6.8, 21.6 +/- 2.2, and 8.0 +/- 1.0 mg, respectively . Based on the residual concentrations of cFDA-SE-labeled lactobacilli on intestinal mucosal surfaces, the average half times for the wash-out of lactobacilli fed were estimated at 3.98, 1.55, 1.34, and 2.48 days in the duodenum, jejunum, ileum, and colon, respectively . The average doubling times of the lactobacilli, estimated from the residual fluorescent levels of surface-adhered cells, were 4.10, 4.78, 4.56, and 5.59 days in the duodenum, jejunum, ileum, and colon, respectively . It is estimated that the lactobacilli would have to achieve an average doubling time of 1.03 to 2.04 days to colonize the various sections of the mouse intestinal tract more permanently.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 248 - 54
Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp . bulgaricus; Carvalho AS et al.; The aim of this research effort was to investigate the role of various sugar substrates in the growth medium upon thermotolerance and upon survival during storage after freeze-drying of Lactobacillus bulgaricus . Addition of the sugars tested to the growth medium, and of these and sorbitol to the drying medium (skim milk) was investigated so as to determine whether a relationship exists between growth and drying media, in terms of protection of freeze-dried cells throughout storage . The lowest decrease in viability of L . bulgaricus cells after freeze-drying was obtained when that organism was grown in the presence of mannose . However, L . bulgaricus clearly survived better during storage when cells had been grown in the presence of fructose, lactose or mannose rather than glucose (the standard sugar in the growth medium) . A similar effect could not be observed in terms of thermotolerance; in this case, the growth medium supplemented with lactose was found to yield cells bearing the highest heat resistance . Supplementation of the drying medium with glucose, fructose, lactose, mannose or sorbitol led in most cases to enhancement of protection during storage, to a degree that was growth medium-dependent.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 229 - 38
Collapse temperature of freeze-dried Lactobacillus bulgaricus suspensions and protective media; Fonseca F et al.; Optimization of the freeze-drying process needs to characterize the physical state of frozen and dried products . A protocol to measure the collapse temperature of complex biological media such as concentrated lactic acid bacteria using freeze-drying microscopy was first elaborated . Afterward, aqueous solutions of one or several components as well as concentrated lactic acid bacterial suspensions were analyzed in order to study how the structure of these materials is degraded during freeze-drying . A similar behavior toward collapse was observed for all aqueous solutions, which was characterized by two temperatures: the "microcollapse" temperature (T(microc), beginning of a local loss of structure) and the "collapse" temperature (T(c), beginning of an overall loss of structure) . For aqueous solutions, these two temperatures were close, differing by less than 3 degrees C . Nevertheless, when lactic acid bacteria were added to aqueous solutions, the collapse temperatures increased . Moreover, the interval between microcollapse and collapse temperatures became larger . Lactic acid bacterial cells gave a kind of "robustness" to the freeze-dried product . Finally, comparing glass transition, measured by differential scanning calorimetry (DSC) and collapse temperature for aqueous solutions with noncrystallizable solutes, showed that these values belonged to the same temperature range (differing by less than 5 degrees C) . As suggested in the literature, the glass transition temperature can thus be used as a first approximation of the collapse temperature of these media . However, for lactic acid bacterial suspensions, because the difference between collapse and glass transition temperatures was about 10 degrees C, this approximation was not justified . An elegant physical appearance of the dried cakes and an acceptable acidification activity recovery were obtained, when applying operating conditions during freeze-drying in vials that allowed the product temperature to be maintained during primary drying at a level lower than the collapse temperature of lactic acid bacterial suspensions . Consequently, the collapse temperature T(c) was proposed as the maximal product temperature preserving the structure from macroscopic collapse and an acceptable biological activity of cells.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 179 - 92
Separation of lactic acid from acetic acid using a four-zone SMB; Lee HJ et al.; A simulated moving bed (SMB) process has been developed to separate l-(+)-lactic acid from acetic acid, a major impurity in the fermentation broth of Lactobacillus rhamnosus . Poly(4-vinylpyridine) resin (PVP) was selected as the adsorbent . Adsorption isotherms and mass transfer parameters of the organic acids were estimated from single-column frontal tests . Experimental results show that the Langmuir isotherms obtained from the frontal tests can be used in the design of an SMB process to achieve 99.9% purity and over 93% yield of lactic acid . The column profiles and effluent histories, however, deviate from rate model predictions based on the Langmuir isotherms . They agree more closely with the predictions based on a modified Langmuir isotherm for lactic acid . The standing wave design method for systems with modified Langmuir isotherms is developed in this study . Rate model simulations show that the process based on the modified design method can achieve high purity (>99.9%) and high yield (>99.9%) . For this nonlinear system, accurate isotherm model and model parameters are needed in the design, and the zone flow rates must be closely monitored and controlled in order to ensure high purity and high yield in the SMB process.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 110 - 6
Proliferation of Lactobacillus plantarum in solid-state fermentation of oats; Patel HM et al.; In an attempt to introduce probiotic functionalities to breakfast cereals and similar food products, the technique of solid-state fermentation (SSF) was applied to cultivate Lactobacillus plantarum (NCIMB 8826) on oat bran and spent oats after lipid extraction by supercritical CO(2) extraction . When compared to the frequently favored submerged processes for bacterium incubation, SSF presents not only the potential of simple downstream processing but also a more natural growth environment for the target bacterium . Preliminary studies confirmed that oat bran contained balanced nutrients to support a 25-fold bacterium propagation within a range of moisture content from 50% to 58% after a 36-h cultivation . Limited hydrolysis of the raw materials by the enzyme complex from submerged incubation of Aspergillus awamori and A . oryzae to increase nutrient accessibility extended the exponential growth phase and enhanced bacterial growth by over 183-fold . The process with the most potential, however, was to simultaneously grow both fungi aerobically on the raw materials in solid state to achieve sufficient hydrolysis, followed by controlled fungal autolysis at 65 degrees C prior to anaerobic bacterium incubation . Following this process bacterium population reached a maximum of 7.3 x 10(9) cells in each gram of the fermented solids, corresponding to a 1653-fold increase from the point of inoculation.

Poult Sci, 2004 Jan, 83(1), 45 - 8
Characterization and electrotransformation of Lactobacillus crispatus isolated from chicken crop and intestine; Beasley SS et al.; Lactic acid bacteria originating in the intestine have recently undergone intensive study for their potential probiotic properties . Here partial 16S rRNA gene sequencing of 8 Lactobacillus strains proved them to be Lactobacillus crispatus . Fatty acid analysis confirmed strains being closely related . These strains and type strain ATCC33820 were characterized for genetic engineering potential, thus determining aerobic growth, erythromycin sensitivity, and glycine tolerance . Out of 5 plasmids, a 2.9-kb plasmid (pLEB579) was successfully introduced into 4 chicken-originated wild-type L . crispatus strains . Transformation frequency was approximately 30 transformants per microgram of DNA, the first reported electrotransformation into chicken-originated L . crispatus . In spite of its low frequency, transformation enables bioengineering of these strains to improve the probiotic function in feed adsorption, chicken health, and food safety.

Curr Microbiol, 2003 Dec, 47(6), 453 - 6
Use of group-specific and RAPD-PCR analyses for rapid differentiation of Lactobacillus strains from probiotic yogurts; Schillinger U et al.; The increasing interest in probiotic lactobacilli implicates the requirement of techniques that allow a rapid and reliable identification of these organisms . In this study, group-specific PCR and RAPD-PCR analyses were used to identify strains of the Lactobacillus casei and Lactobacillus acidophilus groups most commonly used in probiotic yogurts . Group-specific PCR with primers for the L . casei and L . acidophilus groups, as well as L . gasseri/johnsonii, could differentiate between 20 Lactobacillus strains isolated from probiotic yogurts and assign these into the corresponding groups . For identification of these strains to species or strain level, RAPD profiles of the 20 Lactobacillus strains were compared with 11 reference strains of the L . acidophilus and L . casei group . All except one strain could be attributed unambigously to the species L . acidophilus, L . johnsonii, L . crispatus, L . casei, and L . paracasei . DNA reassociation analysis confirmed the classification resulting from the RAPD-PCR.

Digestion, 2004, 69(1), 53 - 6 Epub 2004 Jan 30.
Probiotic treatment increases salivary counts of lactobacilli: a double-blind, randomized, controlled study; Montalto M et al.; BACKGROUND/AIMS: Lactobacilli are used in the prevention and treatment of several diseases, but they are also known to play a role in the pathogenesis of dental caries . The aim of our study was to evaluate whether the oral administration of lactobacilli could change the salivary counts of these bacteria compared with placebo . Moreover, lactobacilli were administered in liquid and in capsule form to determine the role of direct contact with the oral cavity . METHODS: Thirty-five healthy volunteers were randomized into three groups to receive lactobacilli and/or placebo for 45 days: group A (n = 14) received probiotics in capsules and placebo in liquid form; group B (n = 16) took liquid probiotics and placebo in capsules, and group C (n = 5) used placebo in both liquid and capsule form . Streptococcus mutans populations served as control . The salivary counts of lactobacilli and S . mutans were measured semi-quantitatively using the CRT bacteria kit . RESULTS: Compared with placebo, the oral administration of probiotics, both in capsules and in liquid form, significantly increases salivary counts of lactobacilli (p = 0.005 and p = 0.02, respectively) . S . mutans populations were not significantly modified . CONCLUSIONS: The increased salivary counts of lactobacilli may indicate the need to closely monitor the dental health of patients undergoing long-term probiotics treatment, even when this treatment is administrated in a form that avoids direct contact with the oral cavity .

Food Addit Contam, 2004 Feb, 21(2), 158 - 64
Binding of aflatoxin B1 to cell wall components of Lactobacillus rhamnosus strain GG; Lahtinen SJ et al.; The surface of Lactobacillus rhamnosus strain GG (LGG) has previously been shown to bind aflatoxin B(1) (AFB(1)) effectively, it being a food-borne carcinogen produced by certain species of Aspergillus fungi . To establish which components of the cell envelope are involved in the AFB(1) binding process, exopolysaccharides and a cell wall isolate containing peptidoglycan were extracted from LGG and its AFB(1) binding properties were tested . LGG was also subjected to various enzymatic and chemical treatments and their effects on the binding of AFB(1) by LGG were examined . No evidence was found for exopolysaccharides, cell wall proteins, Ca(2+) or Mg(2+) being involved in AFB(1) binding . The AFB(1) binding activity of the cell wall isolate indicates that AFB(1) binds to the cell wall peptidoglycan of LGG or compounds tightly associated with the peptidoglycan.

Curr Pharm Des, 2004, 10(3), 315 - 36
Clinical development of microbicides for the prevention of HIV infection; D'Cruz OJ et al.; The HIV/AIDS pandemic continues its spread at a rate of over 15,000 new infections every day . Sexual transmission of HIV-1 is the dominant mode of this pandemic spread . For the first time since the disease emerged in the early 1980s, about half the 42 million people now living with HIV/AIDS worldwide are women . Worldwide, more than 90 percent of all adolescent and adult HIV infections have resulted from heterosexual intercourse . The "feminization" of the pandemic largely driven by the social, economic, and biological factors warrants urgent attention particularly for the adolescent female population . In the absence of an effective prophylactic anti-HIV therapy or vaccine, current efforts are aimed at developing intravaginal/intrarectal topical formulations of anti-HIV agents or microbicides to curb the mucosal and perinatal HIV transmission . Microbicides would provide protection by directly inactivating HIV or preventing HIV from attaching, entering or replicating in susceptible target cells as well as dissemination from target cells present in semen or the host cells that line the vaginal/rectal wall . Thus, ideally, anti-HIV microbicides should be capable of attacking HIV from different angles . In addition, a contraceptive microbicide could help prevent unintended pregnancies worldwide . To be a microbicide, these agents must be safe, effective following vaginal or rectal administration, and should cause minimal or no genital symptoms following long-term repeated usage . A safe and efficacious anti-HIV microbicide is not yet available despite the fact that more than 60 candidate agents have been identified to have in vitro activity against HIV, 18 of which have advanced to clinical testing . Targeting HIV entry has been a favored approach because it is the first step in the process of infection and several readily available anionic polymeric products seem to variably interfere with these processes are the primary candidates for potential microbicides . Formulations of some anionic polymeric antiviral agents have been tested at various doses and various durations for safety, tolerability, and acceptability in Phase I/II clinical trials (vaginal, rectal, or penile studies) in HIV-uninfected and/or HIV-infected populations . Current multicenter Phase I/II safety and Phase II/III efficacy studies that are being conducted or planned in different geographical locations by various special interest groups are designed for rapid clinical development of candidate products . The currently marketed detergent-type spermicide, nonoxynol-9 (N-9), has failed in Phase III clinical trials, due to the drug-induced formation of localized genital lesions that might in fact actually promote virus transmission . Alternative "first-generation" microbicides that have undergone Phase I/II safety and tolerability studies in HIV-uninfected and/or HIV-infected volunteers include polymeric viral fusion inhibitors (dextrin sulfate/Emmelle, carrageenans {PC-213, PC-503, PC-515/Carraguard}, cellulose sulfate/Ushercell, polystyrene sulfonate, naphthalene sulfonate {PIC 024-4/PRO 2000/5}, acidifying gel {Carbomer 974P/BufferGel}, Lactobacillus (L . crispatus) suppository/CTV-05, detergent-type dual-function barriers {ACIDFORM, GEDA Plus, SURETE, Glyminox/C31G/Savvy, Invisible Condom}, herbal extracts {Praneem}, and viral replication inhibitors {PMPA/Tenofovir} . For majority of these products, no information is available regarding their long-term mucosal safety, carcinogenicity potential, bioavailability, or efficacy following their extended vaginal or rectal exposure . The irritative genitourinary symptoms reported for a number of these first-generation products in Phase I clinical trials implies that the "soft" preclinical endpoints for mucosal safety established for the use and development of vaginal spermicides may not be rigorous enough for vaginal and rectal microbicides because of the efficient sexual tra virus diversity, and genetic environment . It is now apparent that sexually transmitted R5 HIV-1 viruses have less positive charge on their surface compared with the R4 HIV-1 viruses, which may limit the anionic polymers as topical microbicides despite extensive clinical trials . Nevertheless, their ongoing clinical trials, reviewed here, using optimized formulations, and special populations in various geographic locations are paving the way for future rigorous clinical testing of "mechanism-based" broad-spectrum anti-HIV microbicides that are currently under intense development . It is anticipated that future microbicide trials will focus on combination of products capable of attacking HIV life cycle at multiple steps intended to increase efficacy, limit cross-resistance as well as minimize microbicide-induced host toxicity.

Int J Food Microbiol, 2004 Feb 1, 90(3), 273 - 82
Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef; Jones RJ; Drip samples were collected at 4-week intervals from 10 vacuum-packaged beef striploins stored for 16 weeks at -1.5 degrees C and assayed for populations of lactic-acid bacteria (LAB), pH and spoilage-causing fermentation products . A total of 15 LAB species were identified using pulsed-field gel electrophoresis and biochemical analysis . A pattern of succession was observed during storage between strains of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus . Acetic acid production was associated with increasing LAB populations generally and butyric acid production was associated with the development of a particular strain of Leuconostoc . Changes in pH is postulated as a driver of succession.

Biosci Biotechnol Biochem, 2004 Jan, 68(1), 250 - 2
Maturation of fermented rice-koji miso can be monitored by an increase in fatty acid ethyl ester; Yamabe S et al.; A mixture of steamed soybean and boiled rice with seeded Aspergillus oryzae was naturally fermented without addition of yeasts or Lactobacilli, and kept matured for 12 months at room temperature . Chemical analysis of this rice-koji miso sample for lipid changes during maturation showed that triacylglycerol was gradually decomposed into free fatty acid, with distinct formation of fatty acid ethyl ester which, six months after the start of fermentation, came to account for 35.0% of total lipid . The ester was constituted primarily with linoleic acid (ca . 50%) and oleic acid (ca . 20%), no appreciable change in this proportion being observed during maturation . Also, the proportion was unique in that this did not reflect the fatty acid composition in a mixture of the two materials . It is possible to monitor the maturation of the rice-koji miso by following up the increase with time in fatty acid ethyl ester.

Scand J Gastroenterol Suppl, 2003, (239), 15 - 23
Probiotics in gastroenterology: indications and future perspectives; Goossens D et al.; Nowadays . there is a growing interest in probiotics as a safe way of changing the intestinal bacterial flora . Probiotics may have potential in several gastroenterological conditions, especially when the intestinal flora has been disturbed . Most scientific evidence is available for diarrhoea patients treated with Lactobacillus GG, Lactobacillus reuteri or Saccharomyces boulardii . Meta-analyses have shown an overall reduction in the risk of antibiotic-associated diarrhoea during treatment with probiotics, and benefits have also been demonstrated for patients with rota-virus-associated diarrhoea . Patients with inflammatory bowel disease, an inflammatory disorder characterized by a change in the intestinal flora, are another important target group for which probiotics may be beneficial . It has been claimed that in ulcerative colitis and Crohn disease patients, lactobacilli, S . boulardii and Escherichia coli reduce relapses . but most studies were not placebo-controlled . A reduction in relapses has also been demonstrated in pouchitis patients treated with a multispecies probiotic . Irritable bowel syndrome might be another clinical indication for probiotic therapy, but results of clinical trials performed in these patients are inconsistent . Additionally, probiotics may improve lactose absorption . Helicobacter pylori eradication and constipation . Finally, in animal models of colorectal cancer, treatment with probiotics reduces the prevalence of this disease, and in humans the amount of genotoxic substances in faeces has been reduced . In conclusion, the results of studies on the effects of probiotics in gastrointestinal conditions are encouraging . but well-designed placebo-controlled studies are warranted before recommendations for therapeutic or preventive use can be given . Many issues still have to be resolved, including optimal dose and duration of treatment, selection of and differences between the several available probiotic strains, and, importantly, their mechanisms of actions have to be elucidated.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 279 - 81
Should names reflect the evolution of bacterial species?
Dellaglio F, Felis GE, Germond JE.
Systematics is the process of characterizing and arranging bacterial diversity in an orderly manner, recognizing groups of similar organisms in a hierarchical scheme whose basic entity is the species . To allow the exchange of scientific knowledge, taxa have to be named . Taxa are not static entities since they are subject to evolution, the direction of which can be inferred by using a wide range of techniques targeting specific traits . However, it is not clear how this dynamism should be reflected in taxonomic nomenclature . In the present report, several considerations are presented that deal with the relationship between the evolution of taxa and their nomenclature; an example is given which concerns the nomenclature of the species Lactobacillus delbrueckii.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 115 - 7
Lactobacillus paracollinoides sp . nov., isolated from brewery environments; Suzuki K et al.; Three novel strains isolated from brewery environments are described . These strains were Gram-positive, facultatively anaerobic, heterofermentative rods that did not exhibit catalase activity . Phylogenetic analysis based on 16S rRNA gene sequence similarity showed that these strains belong to the genus Lactobacillus and are most closely related to Lactobacillus collinoides (approximately 99% similarity) . The novel strains could be differentiated from L . collinoides on the basis of DNA-DNA relatedness, differences in beer-spoilage ability and the inability to utilize D-fructose . These isolates represent a novel species, for which the name Lactobacillus paracollinoides sp . nov . is proposed . The type strain is LA2T (=DSM 15502T=JCM 11969T).

Appl Microbiol Biotechnol, 2004 Apr, 64(3), 326 - 32 Epub 2004 Jan 21.
Reutericyclin: biological activity, mode of action, and potential applications; Ganzle MG; Reutericyclin is an inhibitory compound produced by sourdough isolates of Lactobacillus reuteri that is structurally but not functionally related to naturally occurring tetramic acids . It is bacteriostatic or bactericidal to gram-positive bacteria based on its activity as a proton-ionophore, and a broad range of food-related spoilage organisms and pathogens is inhibited by reutericyclin . Gram-negative bacteria are resistant to reutericyclin because of the barrier properties of their outer membrane, and resistance of beer-spoiling lactobacilli towards hop bitter acids provides cross-protection to reutericyclin . Remarkably, reutericyclin-producing strains were shown to persist for a period of 10 years in an industrial sourdough fermentation, and reutericyclin was shown to be produced in concentrations active against competitors during growth of L . reuteri in sourdough . Based on the known properties of reutericyclin and L . reuteri, reutericyclin-producing strains may have applications in the biopreservation of foods . Furthermore, these strains were shown to colonize reconstituted lactobacilli-free mice at high levels . Therefore, they could serve as a suitable model system to evaluate a possible impact of antimicrobial compounds on the intestinal microflora of humans and animals.

FEMS Microbiol Lett, 2004 Jan 15, 230(1), 53 - 61
High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol; G-Alegria E et al.; A total of 76 Lactobacillus plantarum and Oenococcus oeni wild strains were recovered from traditionally elaborated Spanish red wines and were investigated with respect to their response to acid pH, lyophilisation, temperature and ethanol concentrations which are normally lethal to lactic acid bacteria . Both L . plantarum and O . oeni strains were able to grow at pH 3.2, were highly resistant to lyophilisation treatment and proliferated in the presence of up to 13% ethanol at 18 degrees C . Therefore, it is shown that both species are highly tolerant to stress conditions and that similarly to O . oeni strains, L . plantarum strains are of interest in beverage biotechnology.

J Agric Food Chem, 2004 Jan 28, 52(2), 261 - 6
Effect of fermentation and autoclaving on dietary fiber fractions and antinutritional factors of beans (Phaseolus vulgaris L.); Martin-Cabrejas MA et al.; The effect of fermentation on antinutritional factors and also on total dietary fiber (TDF), insoluble (IDF) and soluble (SDF) dietary fiber fractions was studied in beans (Phaseolus vulgaris L.) . The processes studied were two types of fermentation (lactic acid and natural), and a portion of the obtained flours were processed by autoclaving . The dietary fiber (DF) content and its components were determined using the enzymatic-gravimetric and enzymatic-chemical methods . The TDF content ranged from 24.5% dry matter (DM) in the raw to 25.2% DM in the processed beans . All the processing treatments significantly decreased the SDF content, and irrelevant changes were noticed in the IDF content of processed beans . Cellulose content of all samples was reduced by the processing treatments . Correspondingly, higher amounts of resistant starch was observed in the processed beans, except in the lactic acid fermented ones . However, the levels of pectic polysaccharides and Klason lignin were higher in the samples fermented by Lactobacillus plantarum . The action of microorganisms was determinant for the different degradation of the bean cell wall, disrupting the protein-carbohydrate integration, thus reducing the solubility of DF.

J Gastroenterol Hepatol, 2004 Feb, 19(2), 166 - 73
Lactobacillus plantarum 299: beneficial in vitro immunomodulation in cells extracted from inflamed human colon; Pathmakanthan S et al.; BACKGROUND AND AIM: The present study determined the pattern of cytokine secretion (interleukin {IL}-1beta, tumor necrosis factor {TNF}-alpha, interferon {IFN}-gamma and IL-10) and their cellular sources in mononuclear cells isolated from colonic mucosa from normal and ulcerative colitis (UC) in response to probiotic and pathogenic bacteria . METHODS: Mononuclear cells were extracted from normal and active UC colonic mucosa and incubated with pure sonicates of probiotic, commensal, and pathogenic bacteria . Cytokine secretion was measured in culture supernatant and intracellular cytokine staining measured using fluorescent-activated cytometry . RESULTS: In mononuclear cells isolated from normal mucosa, significant increases in mean IL-1beta were observed with enteropathogenic Escherichia coli (286.3 +/- 138.7 pg/mL P < 0.05) and E . coli (440.5 +/- 194.0 pg/mL P < 0.01) compared with unstimulated control cells (16.7 +/- 4.8 pg/mL) . In contrast, mononuclear cells isolated from active UC mucosa produced significant increases in mean IL-1beta in response to stimulation with Salmonella dublin (230.5 +/- 38.8 pg/mL P < 0.05), enteropathogenic E . coli (231.7 +/- 45.3 pg/mL P < 0.05) and E . coli (465.4 +/- 60.2 pg/mL P < 0.001) compared with unstimulated control cells (60.7 +/- 17.1 pg/mL) . Escherichia coli also produced significant mean increases of TNF-alpha and IFN-gamma compared with unstimulated control cells . No significant increases in IL-1beta, TNF-alpha or IFN-gamma were observed with Lactobacillus plantarum in cells derived from normal or inflamed mucosa . Strikingly, incubation of L . plantarum with mononuclear cells isolated from active UC mucosa resulted in significant increases of mean IL-10 (327 +/- 53.5 pg/mL, P < 0.05) compared with unstimulated control cells (29.7 +/- 13.2 pg/mL) . Intracellular cytokine staining confirmed T-cell and macrophage IL-10 production after L . plantarum stimulation . CONCLUSIONS: Lactobacillus plantarum demonstrates beneficial immunomodulatory activity by increasing IL-10 synthesis and secretion in macrophages and T-cells derived from the inflamed colon . This may provide a mechanism through which probiotic bacteria ameliorate inappropriate inflammation and induce tolerance.

Proteomics, 2004 Jan, 4(1), 106 - 22
Environmental stress responses in Lactobacillus: a review; De Angelis M et al.; Environmental stress responses in Lactobacillus, which have been investigated mainly by proteomics approaches, are reviewed . The physiological and molecular mechanisms of responses to heat, cold, acid, osmotic, oxygen, high pressure and starvation stresses are described . Specific examples of the repercussions of these effects in food processing are given . Molecular mechanisms of stress responses in lactobacilli and other bacteria are compared.

J Inorg Biochem, 2004 Feb, 98(2), 287 - 300
Solution structure, enzymatic, and non-enzymatic reactivity of 3-isoadenosylcobalamin, a structural isomer of coenzyme B12 with surprising coenzymic activity; Brown KL et al.; The coenzymic activity of eight analogs of coenzyme B(12) (5'-deoxyadenosyl-cobalamin, AdoCbl) with structural alterations in the Ado ligand has been investigated with the AdoCbl-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii . Six of the analogs were partially active coenzymes, and one, 3-iso-5'-deoxyadenosylcobalamin (3-IsoAdoCbl) was nearly as active as AdoCbl itself . NMR-restrained molecular modeling of 3-IsoAdoCbl revealed a highly conformationally mobile structure which required a four state model to be consistent with the NMR data . Thus, two conformations, one with the IsoAdo ligand over the eastern quadrant of the corrin, and one with the IsoAdo ligand over the northern quadrant, each undergo a facile syn/anti conformational equilibrium in the IsoAdo ligand . Spectrophotometric measurement of the kinetics of RTPR-induced cleavage of the carbon-cobalt bond of 3-IsoAdoCbl showed that it binds to the enzyme with the same affinity as AdoCbl, but its homolysis is only 20% as rapid . Investigation of the non-enzymatic thermolysis of 3-IsoAdoCbl showed that like AdoCbl, 3-IsoAdoCbl decomposes by competing homolytic and heterolytic pathways . A complete temperature-dependent kinetic and product analysis, followed by correction for the base-off species permitted deconvolution of the specific rate constant for both pathways . Eyring plots for the homolysis and heterolysis rate constant cross at 93 degrees C, so that homolysis is the predominant pathway at high temperature, but heterolysis is the predominant pathway at low temperature . At 37 degrees C, the homolysis of 3-IsoAdoCbl is 5.5-fold faster than that of AdoCbl, and the enzyme catalyzes carbon-cobalt bond homolysis in 3-IsoAdoCbl by a factor of 5.9 x 10(7), only 3.9% of the catalytic efficiency with AdoCbl itself . It seems likely that the conformational flexibility of 3-IsoAdoCbl allows it to adopt a coformation in which the hydrogen bonding patterns of the adenine moiety are similar to those of AdoCbl itself, and that this is responsible for the high enzymatic activity of this analog.

Appl Microbiol Biotechnol, 2004 Jul, 65(1), 119 - 23 Epub 2004 Jan 16.
Effect of pH on Lactobacillus fermentum growth, raffinose removal, alpha-galactosidase activity and fermentation products; LeBlanc JG et al.; In this study, the behaviour of Lactobacillus fermentum CRL 722 and CRL 251 were evaluated under different pH conditions (pH 6.0, 5.5, 5.0, 4.5) and without pH control . Growth was similar under all conditions assayed except at pH 4.5 . These microorganisms were able to eliminate raffinose, a nondigestible alpha-oligosaccharide (NDO) found in soy products, showing a consumption rate of 0.25 g l(-1) h(-1) (pH 6.0-5.0) . The removal of raffinose was due to the high alpha-galactosidase (alpha-gal) activities of these lactic acid bacteria, which was highest at pH 5.5 (5.0 U/ml) . The yield of organic acids produced during raffinose consumption was also highest at this pH . The results of this study will allow selection of the optimum growth conditions of L . fermentum with elevated levels of alpha-gal to be used in the reduction of NDO in soy products when used as starter cultures.

Arch Biochem Biophys, 2004 Feb 1, 422(1), 61 - 70
Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane; Machado MC et al.; The biochemical and biophysical properties of the membrane and some general characteristics of the response of Lactobacillus casei ATCC 393 (reclassified Lactobacillus zeae) to hyperosmotic conditions were studied . Under hypertonic conditions, the hydrophobicity and the bile salt sensitivity of the cultures were increased . The glycolipid AcylH3DG is only present in membranes of NaCl containing medium, whereas, H4DG undergoes a significant increment and H2DG a significant decrease . The fluidity of both the purified membranes and the total lipid vesicles, as determined with the fluorescent probe DPH, did not change in conditions of high salinity . This was coincident with changes in the fatty acid (FA) composition where an increase in the saturated/unsaturated FA ratio was compensated by a rise in the fluidifying 11,12-methyleneoctadecanoic FA (cyc 19:0) . Under osmotic stress conditions, Laurdan and acridine orange in total lipid vesicles showed increased lateral lipid packing and proton permeability, respectively.

J Appl Microbiol, 2004, 96(2), 384 - 97
Microbiological characterization and probiotic potential of koko and koko sour water, African spontaneously fermented millet porridge and drink; Lei V et al.; AIMS: To identify and examine the diversity of predominant lactic acid bacteria (LAB) in koko and koko sour water (KSW) from different Ghanaian production sites with regard to pattern of fermentation (API 50 CHL), genotype, antimicrobial activity, and resistance to low pH and bile salts . METHODS AND RESULTS: In total 215 LAB were isolated from koko and KSW . The isolates were identified using intergenic transcribed spacers (ITS)-PCR restriction fragment length polymorphism (RFLP), API 50 CHL, restriction enzyme analysis with pulsed-field gel electrophoresis (REA-PFGE) and sequencing of the 16S rRNA gene . The dominating micro-organisms in koko was found to be Weisella confusa and Lactobacillus fermentum, followed by Lact . salivarius and Pediococcus spp . Chemometric data analysis were used to link the LAB species to the different production stages and production sites . At intra-species level the isolates were found to have a great diversity . The isolates were investigated for antimicrobial activity using agar diffusion assays, and acid and bile tolerance . Most isolates showed low levels of antimicrobial activity towards the indicator strain Listeria innocua, but not towards the bacteriocin-sensitive Lact . sakei . Growth of all LAB isolates was unaffected by the presence of 0.3% (v/v) oxgall bile . The isolates were able to survive, but were not able to grow in growth medium adjusted to pH 2.5 . CONCLUSIONS: The dominating LAB of koko and KSW were W . confusa and Lact . fermentum showing a pronounced taxonomic biodiversity at sub-species level between stages within the production as well as between production sites . Other species observed in KSW were Lact . salivarius, Ped . pentosaceus, Ped . acidilactici and Lact . paraplantarum . They occurred in levels of 108 CFU ml-1 in fresh KSW and showed uniform antimicrobial activity, and acid and bile tolerance . SIGNIFICANCE AND IMPACT OF THE STUDY: The present study gives a detailed picture of the taxonomy and diversity of LAB in an African-fermented millet product that may have potential as a probiotic product for the local population . The chemometric tools Principal Component Analysis and anova Partial Least Squares Regression were proven to be useful in the analysis of microbial groupings and associations with specific sites and stages in the production of koko and KSW.

J Appl Microbiol, 2004, 96(2), 367 - 74
Identification and characterization of a conserved nuclease secreted by strains of the Lactobacillus casei group; Peant B et al.; AIMS: Nuclease secretion was evaluated for five species of Lactobacillus and the activity was characterized in terms of thermal resistance, molecular weight and mode of action on plasmid DNA . METHODS AND RESULTS: Assays of nuclease from L . rhamnosus ATCC 9595 on DNA of different origins indicates a broad activity spectrum . Secreted nuclease from this strain resists a thermal treatment of 20 min at 100 degrees C, is not sensitive to a treatment for disruption of disulphide bonds nor to EDTA treatment under 10 mM l(-1) . Nuclease production is not growth linked and seems to be constitutive . Extracellular nuclease of L . rhamnosus ATCC 9595 introduces a single-stranded nick in supercoiled DNA, thus potentially reducing the transformability of plasmid DNA . In seven of eight tested strains, SDS-PAGE revealed a major protein with a molecular weight of ca 35 kDa . Minor degradation products also showed nuclease activity . CONCLUSIONS: A comparative analysis of the extracellular fractions of 14 different Lactobacillus strains indicate that nuclease secretion seems to be a widely distributed function among species of milk-related lactobacilli . The production of secreted nuclease may contribute to the low ability of Lactobacillus spp . to be transformed and maintain exogenous DNA . SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the characteristics and distribution of nuclease activity contribute to developing strategies to overcome this barrier to efficient transformation of milk lactobacilli.

Appl Environ Microbiol, 2004 Jan, 70(1), 603 - 6
Cloning and heterologous expression of hematin-dependent catalase produced by Lactobacillus plantarum CNRZ 1228; Abriouel H et al.; Lactobacillus plantarum CNRZ 1228 exhibited heme-dependent catalase activity under environmental conditions similar to those encountered during sausage fermentation . The 1,455-bp catalase gene (katL) was cloned and encoded a protein of 484 amino acids . Expression of katL in a heterologous host showed that katL encodes a functional catalase . PCR screening of selected strains of lactic acid bacteria for katL indicated the presence of similar genes in other strains of lactobacilli.

Appl Environ Microbiol, 2004 Jan, 70(1), 558 - 68
Genotypic and phenotypic studies of murine intestinal lactobacilli: species differences in mice with and without colitis; Pena JA et al.; Lactobacilli represent components of the commensal mammalian gastrointestinal microbiota and are useful as probiotics, functional foods, and dairy products . This study includes systematic polyphasic analyses of murine intestinal Lactobacillus isolates and correlation of taxonomic findings with data from cytokine production assays . Lactobacilli were recovered from mice with microbiota-dependent colitis (interleukin-10 {IL-10}-deficient C57BL/6 mice) and from mice without colitis (Swiss Webster and inducible nitric oxide synthetase-deficient C57BL/6 mice) . Polyphasic analyses were performed to elucidate taxonomic relationships among 88 reference and murine gastrointestinal lactobacilli . Genotypic tests included single-locus analyses (16S ribosomal DNA sequencing and 16S-23S rRNA intergenic spacer region PCR) and genomic DNA profiling (repetitive DNA element-based PCR), and phenotypic analyses encompassed more than 50 tests for carbohydrate utilization, enzyme production, and antimicrobial resistance . From 20 mice without colitis, six Lactobacillus species were recovered; the majority of the mice were colonized with L . reuteri or L . murinus (72% of isolates) . In contrast, only, L . johnsonii was isolated from 14 IL-10-deficient mice . Using an in vitro assay, we screened murine isolates for their ability to inhibit tumor necrosis factor alpha (TNF-alpha) secretion by lipopolysaccharide-activated macrophages . Interestingly, a subpopulation of lactobacilli recovered from mice without colitis displayed TNF-alpha inhibitory properties, whereas none of the L . johnsonii isolates from IL-10-deficient mice exhibited this effect . We propose that differences among intestinal Lactobacillus populations in mammals, combined with host genetic susceptibilities, may account partly for variations in host mucosal responses.

Appl Environ Microbiol, 2004 Jan, 70(1), 518 - 26
In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota; Sgouras D et al.; We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult {Yakult Ltd., Tokyo, Japan}) on Helicobacter pylori by using (i) in vitro inhibition assays with H . pylori SS1 (Sydney strain 1) and nine H . pylori clinical isolates and (ii) the in vivo H . pylori SS1 mouse model of infection over a period of 9 months . In vitro activity against H . pylori SS1 and all of the clinical isolates was observed in the presence of viable L . casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity . In vivo experiments were performed by oral administration of L . casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H . pylori SS1 (study group; n = 25) . Appropriate control groups of H . pylori-infected but untreated animals (n = 25) and uninfected animals given L . casei strain Shirota (n = 25) also were included in the study . H . pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection . A significant reduction in the levels of H . pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H . pylori-infected control group . This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period . A trend toward a decrease in the anti-H . pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.

Appl Environ Microbiol, 2004 Jan, 70(1), 310 - 7
Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe; Bron PA et al.; This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum . Random fragments of the L . plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector . The resulting plasmid library was introduced into an L . plantarum Deltaalr strain (MD007), and 40,000 clones were selected . The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones . The library was screened for clones that were capable of complementing the D-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 micro g of the competitive Alr inhibitor D-cycloserine per ml . Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of D-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen . Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions . These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.

Appl Environ Microbiol, 2004 Jan, 70(1), 268 - 72
Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates; Dumont F et al.; This work studied the viabilities of five types of cells (two yeast cells, Saccharomyces cerevisiae CBS 1171 and Candida utilis; two bacterial strains, Escherichia coli and Lactobacillus plantarum; and one human leukemia K562 cell) as a function of cooling rate during freezing . The range of investigated cooling rates extended from 5 to 30,000 degrees C/min . Cell viability was classified into three ranges: (i) high viability for low cooling rates (5 to 180 degrees C/min), which allow cell water outflow to occur completely and do not allow any intracellular crystallization; (ii) low viability for rapid cooling rates (180 to 5,000 degrees C/min), which allow the heat flow to prevail over water outflow (in this case, cell water crystallization would occur as water was flowing out of the cell); (iii) high viability for very high cooling rates (>5,000 degrees C/min), which allow the heat flow to be very rapid and induce intracellular crystallization and/or vitrification before any water outflow from the cell . Finally, an assumption relating cell death to the cell water crystallization as water is flowing out of the cell is made . In addition, this general cell behavior is different for each type of cell and seems to be moderated by the cell size, the water permeability properties, and the presence of a cell wall.

Appl Environ Microbiol, 2004 Jan, 70(1), 129 - 36
Effects of polyunsaturated fatty acids in growth medium on lipid composition and on physicochemical surface properties of lactobacilli; Kankaanpaa P et al.; Most probiotic lactobacilli adhere to intestinal surfaces, a phenomenon influenced by free polyunsaturated fatty acids (PUFA) . The present study investigated whether free linoleic acid, gamma-linolenic acid, arachidonic acid, alpha-linolenic acid, or docosahexaenoic acid in the growth medium alters the fatty acid composition of lactobacilli and their physical characteristics . The most abundant bacterial fatty acids identified were oleic, vaccenic, and dihydrosterculic acids . PUFA, especially conjugated linoleic acid (CLA) isomers and gamma-linolenic, eicosapentaenoic, docosahexaenoic, and alpha-linolenic acids, also were identified in lactobacilli . When lactobacilli were cultured in MRS broth supplemented with various free PUFA, the incorporation of a given PUFA into bacterial fatty acids was clearly observed . Moreover, PUFA supplementation also resulted in PUFA-dependent changes in the proportions of other fatty acids; major interconversions were seen in octadecanoic acids (18:1), their methylenated derivatives (19:cyc), and CLA . Intermittent changes in eicosapentaenoic acid proportions also were noted . These results were paralleled by minor changes in the hydrophilic or hydrophobic characteristics of lactobacilli, suggesting that PUFA interfere with microbial adhesion to intestinal surfaces through other mechanisms . In conclusion, we have demonstrated that free PUFA in the growth medium induce changes in bacterial fatty acids in relation to the regulation of the degree of fatty acid unsaturation, cyclization, and proportions of CLA and PUFA containing 20 to 22 carbons . The potential role of lactobacilli as regulators of PUFA absorption may represent another means by which probiotics could redirect the delicate balance of inflammatory mediators derived from PUFA within the inflamed intestine.

J Microbiol Methods, 2004 Jan, 56(1), 27 - 35
An improved method of microencapsulation and its evaluation to protect Lactobacillus spp . in simulated gastric conditions; Chandramouli V et al.; An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions . Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions . The effects of different capsule sizes (200, 450, 1000 microm), different sodium alginate concentrations (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concentrations of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated . The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concentration . There was no significant difference (p>0.05) in the viability of encapsulated cells when the concentration of calcium chloride was increased . Increase in cell load during encapsulation increased the number of bacterial survivors at the end of 3-h incubation in simulated gastric conditions . Hardening the capsule in calcium chloride solution for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment . The release of encapsulated cells at different phosphate buffer concentrations was also studied . When encapsulated L . acidophilus CSCC 2400 and L . acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concentration (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 10(9) CFU/ml, 30 min hardening in 0.1 M CaCl(2) and capsule size 450 microm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions . Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.

J Ethnopharmacol, 2004 Jan, 90(1), 99 - 103
Evaluation of antiplaque activity of Azadirachta indica leaf extract gel--a 6-week clinical study; Pai MR et al.; Various chemical agents have been evaluated over the years with respect to their antimicrobial effects in the oral cavity; however, all are associated with side effects that prohibit regular long-term use . Therefore, the effectiveness of neem (Azadirachta indica A . Juss) leaf extract against plaque formation was assessed in males between the age group of 20-30 years over a period of 6 weeks . Present study includes formulation of mucoadhesive dental gel containing Azadirachta indica leaf extract (25 mg/g) . A 6-week clinical study was conducted to evaluate the efficacy of neem extract dental gel with commercially available chlorhexidine gluconate (0.2% w/v) mouthwash as positive control . Microbial evaluation of Streptococcus mutans and Lactobacilli species was carried out to determine the total decrease in the salivary bacterial count over a period of treatment using a semi-quantitative four quadrant streaking method . The results of the study suggested that the dental gel containing neem extract has significantly (P<0.05) reduced the plaque index and bacterial count than that of the control group.

Arch Oral Biol, 2004 Feb, 49(2), 161 - 4
Adaptation of salivary Lactobacillus strains to xylitol; Badet C et al.; OBJECTIVE: Acid production from sugars is one of the virulence factors of cariogenic bacteria . To eliminate this factor, many authors have suggested using sugar substitutes like xylitol, which is not fermented by most oral bacteria . The aim of this work was to investigate whether oral strains of lactobacilli unable to utilise xylitol could become able to utilise this polyol . DESIGN: Twenty-eight oral strains and six ATCC strains of lactobacilli were tested . None was initially able to produce acids from xylitol . Media containing only xylitol was used in order to see if an adaptation was possible . The acid production from xylitol was measured by recording the pH variations of the culture medium . The acids produced were identified by HPLC . RESULTS: After a 15-days culture, 10 strains produced acids . After a 40-days culture, 11 more strains produced acid . When acid production occurred, lactic, formic, acetic, propionic and butyric acids were the organic acids formed by all the strains . CONCLUSIONS: Adaptation of oral strains of lactobacilli to xylitol can occur in vitro.

Exp Biol Med (Maywood), 2003 Dec, 228(11), 1329 - 37
Wound healing and angiogenic properties of supernatants from Lactobacillus cultures; Halper J et al.; Extracts or supernatants from cultures of Lactobacilli are used for their medicinal effects, including wound healing and immune system stimulating activity . We have studied the in vivo and in vitro effects of supernatants from bacterial cultures of two strains of Lactobacillus (LS) on tissue repair and angiogenesis . Subcutaneous injection of LS into rodent ears led to proliferation of blood vessels that also exhibited strong immunostaining for Flk-1 receptor . Some inflammatory cells were scattered among the blood vessels . The continuous influx of polymorphonuclear leukocytes (PMNs) and macrophages into transcutaneous wounds in mice treated with LS resulted in prolonged inflammatory phase of wound healing and delayed wound closure, including reepithelialization . Subcutaneous injection of Matrigel impregnated with LS into the abdominal wall led to rapid and transient influx of PMNs in the vicinity of the gel . LS stimulated the proliferation of murine macrophage J774.A1 cell line and porcine lymphocytes but not that of murine fibroblast AKR-2B cells . LS also induced production of TNF-alpha by J774.A1 cells and by porcine kidney epithelial LLC-PK1 cells . LS did not appear to have an effect on collagen production . In conclusion, our study demonstrates the potential of LS to function as a stimulator of the inflammatory stage of tissue repair, TNF-alpha production, and of angiogenesis.

Clin Infect Dis, 2004 Jan 1, 38(1), 62 - 9 Epub 2003 Dec 04.
Lactobacillus bacteremia, clinical significance, and patient outcome, with special focus on probiotic L . rhamnosus GG; Salminen MK et al.; Lactobacillus bacteremia is a rare entity, and its clinical significance is poorly defined . We have reviewed the risk factors and outcome for 89 case patients with Lactobacillus bacteremia . Species characterization was done in 53% of the cases, revealing 25 L . rhamnosus strains and 22 other Lactobacillus species . In 11 cases, the strain was identical with the probiotic L . rhamnosus GG . In 82% of the cases, the patients had severe or fatal comorbidities . Predisposing factors to bacteremia were immunosuppression, prior prolonged hospitalization, and prior surgical interventions . No significant differences were observed in these predisposing factors or clinical features between patients with cases associated with the various Lactobacillus species, other than higher C-reactive protein values in patients with L . rhamnosus bacteremia . Mortality was 26% at 1 month and was 48% at 1 year . In multivariate analysis, severe underlying diseases were a significant predictor for mortality (odds ratio {OR}, 15.8), whereas treatment with antimicrobials effective in vitro was associated with lower mortality (OR, 0.22) . We conclude that lactobacilli in blood cultures are of clinical significance and that their susceptibility should guide decisions about antimicrobial treatment.

J Appl Microbiol, 2004, 96(1), 185 - 93
Expression analysis of putative arcA, arcB and arcC genes partially cloned from Lactobacillus plantarum isolated from wine; Spano G et al.; AIMS: The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway . METHODS AND RESULTS: Homofermentative lactic acid bacteria (LAB) isolated from a typical red wine were investigated for their ability to produce citrulline . Citrulline was formed suggesting that the arginine metabolism takes place via the arginine deiminase (ADI) pathway and not via the arginase/urease pathway . Ammonia was also detected with Nessler's reagent, and all the strains examined were able to produce ammonia . Identification of homofermentative LAB was performed using 16S ribosomal sequence analysis . The strains were further classified as belonging to L . plantarum species . Furthermore, the genes encoding for the three pathway enzymes (ADI, ornithine transcarbamylase, carbamate kinase) were partially cloned and gene expression was performed at two different pH values (3.6 and 4.5) . CONCLUSIONS: The results suggest that citrulline production in wine, could be performed by homofermentative LAB . SIGNIFICANCE AND IMPACT OF THE STUDY: Homofermentative malolactic bacteria (L . plantarum) may degrade arginine through the ADI pathway.

J Anim Sci, 2003 Dec, 81(12), 3035 - 45
Molecular ecological analysis of porcine ileal microbiota responses to antimicrobial growth promoters; Collier CT et al.; Cultivation-independent microbial molecular ecology approaches were used to examine the effects of antibiotic growth promoters on the pig ileal microbiota . Five-week-old barrows were fitted with a simple T-cannula at the distal ileum . Three diets meeting or exceeding the minimum nutrient requirements were fed for 5 wk and supplemented as follows: 1) negative control (no antibiotic; n = 5), 2) continuous tylosin administration (n = 5), and 3) an antibiotic rotation sequence (wk 1, chlorotetracycline sulfathiazole penicillin; wk 2, bacitracin and roxarsone; wk 3, lincomycin; wk 4, carbadox; wk 5, virginiamycin; n = 5) . Ileal luminal contents were collected for DNA isolation at the end of each of the 5 wk of the testing period . The V3 region of 16S rDNA was amplified by PCR and analyzed via denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR) . Resulting PCR-DGGE band numbers (bacterial species) were counted, and the banding patterns analyzed by calculating Sorenson's pairwise similarity coefficients (C(S)), an index measuring bacterial species in common among samples . Band numbers and total bacterial DNA concentrations decreased (P < 0.05) temporally in antibiotic-treated pigs compared with controls . Comparisons between treatments yielded low intertreatment C(S) indices, indicating treatment-dependent alterations in banding patterns, whereas intratreatment comparisons revealed increased homogeneity in antibiotic-treated vs . control pigs . Sequence analysis of treatment-specific bands identified three Lactobacillus, one Streptococcus, and one Bacillus species that were diminished with antibiotic rotation treatment, whereas tylosin selected for the presence of L . gasseri . Lactobacillus-specific qPCR was performed and analyzed as a percentage of total bacteria to further evaluate the effects of antibiotic administration on this genus . Total bacteria were decreased (P < 0.05) by tylosin and rotation treatments, whereas the percentage of lactobacilli increased (P < 0.05) by d 14 and through d 28 in tylosin-treated pigs . The decrease in total bacteria by antibiotics may reduce host-related intestinal or immune responses, which would divert energy that could otherwise be used for growth . Conversely, the ability of tylosin to improve animal growth may relate to its apparent selection for lactobacilli, commensals known to competitively exclude potentially pathogenic species from colonizing the intestine.

Biotechnol Lett, 2003 Nov, 25(21), 1833 - 5
Fed-batch fermentation of Lactobacillus lactis for hyper-production of L-lactic acid; Bai DM et al.; A fed-batch fermentation of Lactobacillus lactis to produce L-lactic acid was developed in which the residual glucose concentration in the culture was used to control a continuous feeding strategy . Up to 210 g L-lactic acid l(-1) (97% yield) was obtained . The maximal dry cell was 2.7 g l(-1) and the average L-lactic acid productivity was 2 g l(-1) h(-1).

Minerva Ginecol, 2003 Dec, 55(6), 483 - 92
Utilisation of hydrogen peroxide in the treatment of recurrent bacterial vaginosis; Cardone A et al.; AIM: The authors have evaluated the real efficacy of using hydrogen peroxide for previously treated recurrent bacterial vaginosis that is resistant to other forms of treatment . METHODS: The study included 58 women aged between 18 and 42 years old . Vaginal irrigations with 30 ml of hydrogen peroxide (3%) were prescribed in the evening for a week . The follow-up was carried out 3 months after the end of treatment . RESULTS: The results clearly show that the use of hydrogen peroxide in vagina can eliminate the main symptoms of bacterial vaginosis, and in particular the malodorous leucoxanthorrhea in 89% of cases at 3 months after the end of treatment, a result that is comparable to that obtained using metronidazole or clindamycin as a vaginal cream . Moreover, hydrogen peroxide facilitates the restoration of normal vaginal bacterial flora (represented by H202-producing lactobacillus) in 100% of cases and normal acid pH (pH<4.5) in 98% of cases; it also fosters the disappearance of clue cells from vaginal smears and anaerobic pathogenic flora from vaginal secretions in 100% of cases . The amine test became negative in 97.8% of cases . All results underwent statistical analysis and were found to be statistically significant . CONCLUSION: Hydrogen peroxide represents a valid alternative to conventional treatments for recurrent bacterial vaginosis, and associates the absence of collateral effects with low costs, excellent tolerability and real therapeutic efficacy.

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