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 phenomen