Lactobacillus

J Food Prot, 2000 Nov, 63(11), 1551 - 5
Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture; Fernandez-Garcia E et al.; Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispanico cheese, a semihard variety . Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp . lactis and L . lactis subsp . cremoris were added for cheese manufacture . The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments . The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture . Tyramine was detected in all batches of cheese from day 30 . Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age . After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches . Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses . The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated . Phenylethylamine and tryptamine were not found in any of the samples.

Biochim Biophys Acta, 2001 Nov 15, 1524(1), 38 - 44
Acoustic field assisted enhanced demixing of aqueous two-phase systems; Srinivas ND et al.; Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules . A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties . A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported . The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail . As phase composition increased, demixing time decreased gradually . Phase volume ratio was found to have a significant effect on demixing time at low phase compositions . However, at intermediate and high phase compositions, only a small effect on demixing time was observed . The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases . At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17-60% when compared to demixing under gravity alone . Increasing the cell concentration increased the demixing time markedly in case of yeast cells . However, it remained practically constant in the case of Lactobacillus casei cells . Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L . casei cells, respectively . Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet-droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate.

Plasmid, 2000 Nov, 44(3), 239 - 49
Nucleotide sequence and analysis of pBL1, a bacteriocin-producing plasmid from Lactococcus lactis IPLA 972; Sanchez C et al.; The complete sequence of the 10.9-kbp bacteriocinogenic plasmid pBL1 from Lactococcus lactis subsp . lactis IPLA 972 has been determined . Thirteen ORFs were encountered, of which 5 were incomplete . pBL1 proved to be a narrow-host-range plasmid which replicates neither in Bacilus subtilis nor in Lactobacillus spp . The structural organization of the pBL1 replication region was highly similar to other well-known theta-replicating plasmids of lactococci, at both the untranslated (the replication origin) and the translated (repB and orfX) sequences . As in other plasmids, the product of orfX was not necessary for plasmid replication . However, it was shown to be involved in plasmid stability . Three genes organized in an operon-like structure encompassed, most likely, the bacteriocin-encoding region . Upstream of the origin of replication a nicking site (oriT) was found . This oriT sequence proved to be functional by mobilization of plasmids wearing it . One complete and several partial IS elements were identified on pBL1 .

J Biol Chem, 2001 Mar 9, 276(10), 6998 - 7003 Epub 2000 Nov 16.
FemABX family members are novel nonribosomal peptidyltransferases and important pathogen-specific drug targets; Hegde SS et al.; Pathogen-specific antibiotics kill the offending species without inviting the patient's flora to help develop a resistance mechanism . The current scarcity of pathogen-specific antibiotics reflects the rarity of essential genes that are also not widely represented in and conserved among species . The FemX enzyme that initiates the synthesis of the interchain peptide of the peptidoglycan in a subset of bacterial species was purified from Lactobacillus viridescens . Subsequently, the encoding femX gene was cloned and sequenced using reverse genetics . The femX gene is a member of the femAB family, a large family of genes previously implicated in interchain peptide synthesis but with unknown specific functions . Mutagenesis of the femX gene identified the members of the extended FemABX family as novel nonribosomal peptidyltransferases . Determinants of FemX complex substrate recognition and a strong stimulator of FemX activity were also identified . The FemABX family members are ideal candidates for pathogen-specific antibiotic development.

Nahrung, 2000 Oct, 44(5), 364 - 7
Inhibition of Bacillus cereus ATCC 14579 by plantaricin UG1 in vitro and in food; Enan G; The inhibition of Bacillus cereus ATCC 14579 viable growth by Lactobacillus plantarum UG1 bacteriocin (plantaricin UG1) in vitro and in food (pasteurized milk and minced meat) was studied . The inhibitory effect against B . cereus food-borne pathogen noticed in this study was due to plantaricin UG1, but not due to lactic acid produced by the L . plantarum UG1 culture . Plataricin UG1 negative clone did not affect viable growth of B . cereus in both broth and meat or pasteurized milk . The inhibitory effect of L . plantarum UG1 and its bacteriocion was apparently more in liquid systems (BHI broth & pasteurized milk) than in minced meat . The inhibitory effect of plantaricin UG1 against B . cereus was dependent on its concentration . The 22880 AU/ml concentration appeared to be an ideal preservative against B . cereus ATCC 14579 in liquid systems.

J Bacteriol, 2000 Dec, 182(23), 6857 - 61
Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T); Martinez B et al.; The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L . casei ATCC 393(T) . The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium . By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L . casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.

J Bacteriol, 2000 Dec, 182(23), 6724 - 31
Inducible metabolism of phenolic acids in Pediococcus pentosaceus is encoded by an autoregulated operon which involves a new class of negative transcriptional regulator; Barthelmebs L et al.; Pediococcus pentosaceus displays a substrate-inducible phenolic acid decarboxylase (PAD) activity on p-coumaric acid . Based on DNA sequence homologies between the three PADs previously cloned, a DNA probe of the Lactobacillus plantarum pdc gene was used to screen a P . pentosaceus genomic library in order to clone the corresponding gene of this bacteria . One clone detected with this probe displayed a low PAD activity . Subcloning of this plasmid insertion allowed us to determine the part of the insert which contains a 534-bp open reading frame (ORF) coding for a 178-amino-acid protein presenting 81.5% of identity with L . plantarum PDC enzyme . This ORF was identified as the padA gene . A second ORF was located just downstream of the padA gene and displayed 37% identity with the product of the Bacillus subtilis yfiO gene . Subcloning, transcriptional analysis, and expression studies with Escherichia coli of these two genes under the padA gene promoter, demonstrated that the genes are organized in an autoregulated bicistronic operonic structure and that the gene located upstream of the padA gene encodes the transcriptional repressor of the padA gene . Transcription of this pad operon in P . pentosaceus is acid phenol dependent.

J Med Microbiol, 2000 Nov, 49(11), 1023 - 30
Inhibition of chemotaxis by organic acids from anaerobes may prevent a purulent response in bacterial vaginosis; Al-Mushrif S et al.; It has been postulated that certain organic acids produced by the anaerobes associated with bacterial vaginosis (BV) could prevent a purulent response in this infection . Varying concentrations of pure succinic, acetic and lactic acids were incubated in vitro with a monocytic cell line (MonoMac 6) . High inhibition of chemotaxis was produced by succinic acid; lower inhibition and no inhibition was shown by acetic acid and lactic acid respectively . Succinic and acetic acids were detected in high concentrations in the vaginal fluid of women with BV and in culture supernates of Prevotella and Mobiluncus spp.; these acids impaired chemotaxis of MonoMac 6 cells in vitro . The vaginal fluids of normal women and the culture supernates of Lactobacillus spp . had no effect on chemotaxis.

Eur J Pharm Biopharm, 2000 Nov, 50(3), 389 - 95
Technological and biological evaluation of tablets containing different strains of lactobacilli for vaginal administration; Maggi L et al.; Ten strains of lactobacilli were evaluated for the administration of viable microorganisms to restore the normal indigenous flora in the treatment of urogenital tract infections (UTI) in women . As the strains considered are facultative anaerobes, optimization of the production process was particularly critical to preserve bacterial viability . The microorganisms were formulated in single- and double-layer vaginal tablets . The two layers were characterized by different release properties: one is an effervescent composition that ensures a rapid and complete distribution of the active ingredient over the whole vaginal surface; while the second is a sustained release composition capable of releasing the lactobacilli over a longer period of time . Three different retarding polymers were tested, and all the formulations and tablets were evaluated in terms of technological processability, bacterial viability and stability, and cell adhesion properties of the microorganisms . From the results obtained, three out of ten strains appear particularly suitable for their application in the treatment of UTI . A larger batch of tablets made with a mixture of the three strains was then evaluated, confirming the feasibility of their industrial production and a good bacterial viability in the final dosage form.

Kokubyo Gakkai Zasshi, 2000 Sep, 67(3), 240 - 50
{The changes of oral conditions caused by artificial crown contours in severely disabled patients}; Yoshida T; The purpose of this study was to investigate the relation between the artificial crown contour and plaque accumulation in severely disabled patients who have muscle hypoactivity in the oral region . This relation was statistically analyzed by the change in CFU numbers of Mutans Streptococci, Lactobacillus, and total anaerobic bacteria . The results were as follows: 1 . In the severely disabled group, there was no significant difference in the decrease of number of microorganisms between with and without tooth brushing . When the normal contour crown was exchanged for the under contour, however, the numbers of Mutans Streptococci and total anaerobic bacteria decreased significantly (p < 0.05) . 2 . In the normal control group, both the crown contour and tooth brushing were effective for decreasing the number of Mutans Streptococci and total anaerobic bacteria (p < 0.05) . 3 . In both groups, the number of Lactobacillus was decreased by changing the crown contours and by tooth brushing, with no statistical significance . 4 . In view of the cariogenic potential of microorganisms, it is concluded that the under contour crown prevents plaque accumulation, and that this crown should be used on severely disabled patients, as it makes tooth brushing more effective.

Can J Microbiol, 2000 Oct, 46(10), 938 - 45
Molecular analysis of mutated Lactobacillus acidophilus promoter-like sequence P15; Arsenijevic S et al.; The promoter-like sequence P15 that was previously cloned from the chromosome of Lactobacillus acidophilus ATCC 4356 is active in Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, and Escherichia coli, but not in Lactococcus lactis . N-methyl-N-nitroso-N-guanidine (MNNG) mutagenesis of P15 was used to select for a promoter active in L . lactis MG1363 . Molecular analysis of the mutated promoter (designated P16) revealed a 90 bp deletion and a T-->A transversion . This deletion, in combination with the addition to the transversion, created a promoter with putative -35 and -10 hexamers identical to the consensus promoter sequence found in E . coli and Bacillus subtilis vegetative promoters . The activity of P16 was measured by its ability to promote chloramphenicol resistance in different bacteria when inserted in the promoter-probe plasmid pBV5030 (designated pLA16) . The MIC of chloramphenicol in L . lactis, L . reuteri, L . plantarum, E . coli, and L . acidophilus harbouring pLA16 were 30, 170, 180, > 500, and 3 micrograms/mL, respectively . This represents an increase in promoter activity compared to P15 in L . reuteri of 3-fold, in L . plantarum of 9-fold, and in E . coli of at least 2.5-fold, but a decrease in L . acidophilus of 7-fold.

Can J Microbiol, 2000 Oct, 46(10), 892 - 7
Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin; Hosoi T et al.; In an effort to demonstrate the potential usefulness of Bacillus subtilis (natto) as a probiotic, we examined the effect of this organism on the growth of three strains of lactobacilli co-cultured aerobically in vitro . Addition of B . subtilis (natto) to the culture medium resulted in an increase in the number of viable cells of all lactobacilli tested . Since B . subtilis (natto) can produce catalase, which has been reported to exhibit a similar growth-promoting effect on lactobacilli, we also examined the effect of bovine catalase on the growth of Lactobacillus reuteri JCM 1112 and L . acidophilus JCM 1132 . Both catalase and B . subtilis (natto) enhanced the growth of L . reuteri JCM 1112, whereas B . subtilis (natto) but not catalase enhanced the growth of L . acidophilus JCM 1132 . In a medium containing 0.1 mM hydrogen peroxide, its toxic effect on L . reuteri JCM 1112 was abolished by catalase or B . subtilis (natto) . In addition, a serine protease from B . licheniformis, subtilisin, improved the growth and viability of L . reuteri JCM 1112 and L . acidophilus JCM 1132 in the absence of hydrogen peroxide . These results indicate that B . subtilis (natto) enhances the growth and (or) viability of lactobacilli, possibly through production of catalase and subtilisin.

J Anim Sci, 2000 Nov, 78(11), 2980 - 9
Intake, digestibility, and composition of orchardgrass and alfalfa silages treated with cellulase, inoculant, and formic acid fed to lambs; Nadeau EM et al.; The objectives of this study were to determine the effect of a cellulase (from Trichoderma longibrachiatum) alone or combined with a bacterial inoculant (Lactobacillus plantarum and Pediococcus cerevisiae) or formic acid on composition, intake, and digestibility of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages . Orchardgrass and alfalfa were harvested at the early heading stage and at the early bloom stage of maturity and wilted to approximately 22 and 32% DM, respectively . Forages were then ensiled in 100-L sealed barrels for at least 60 d before they were fed to lambs . Silage treated with cellulase had lower (P < .001) pH and lower (P < .001) acetic acid and NH3 N concentrations than untreated silage of both plant species and a higher (P = .004) lactic acid concentration than the control treatment of alfalfa silage . Fermentation characteristics of cellulase-treated silages, especially of alfalfa, were further enhanced by use of inoculant . Formic acid addition increased (P < .001), reducing sugar concentration of cellulase-treated orchardgrass and alfalfa silage by 90 and 154%, respectively, and decreased (P < .001) NH3 N concentration of cellulase-treated alfalfa silage by 19% . Averaged across plant species, cellulase, combined with inoculant or formic acid, resulted in 8 and 13% greater (P = .03) DMI, respectively, than the control silage . Extensive enzymatic cell-wall degradation during ensiling decreased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 25% and decreased (P = .001) cellulose intake by 23%, when averaged across plant species . Addition of formic acid increased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 19% . Averaged across species, cellulase application decreased (P < .05) silage NDF digestibility by 18% . Greater sugar and lower acetic acid, NH3 N, and NDF concentrations resulted in greater DMI of cellulase-treated silage than of control silage, when cellulase was combined with formic acid or inoculant.

New Microbiol, 2000 Oct, 23(4), 423 - 31
Conservation in probiotic preparations of Lactobacillus with inhibitory capacity on other species; Paraje MG et al.; Strains of Lactobacillus isolated from dairy products and genital tract competed with Candida albicans through a membrane of 12000 dalton cut-off . This inhibition was due to hydrogen peroxide and was trypsin-stable, heat-sensitive and antagonized by catalase . Lactobacillus coming from "starters" showed antimicrobial activity against fungus isolated in a yogurt factory . Penicillium, Alternaria, Phialophora, Microsporum and Candida spp . were inhibited when 10(2) spores were inoculated in the assay . No inhibition was observed with 10(5) spores . Besides, one of 21 Lactobacillus strains isolated from the vaginas of healthy women inhibited pathogenic bacteria by means a bacteriocin trypsin-sensitive, heat-stable and retained by dialysis membrane . Tablets for future probiotic use were prepared and the viability of bacteria was assayed using media with different compositions . Pharmaceutical preparations with polyethyleneglycol was the best formulation for the Lactobacillus viability, the counts remained between 10(7) and 10(6) cfu/tablet for up to 1 year.

Eur J Gastroenterol Hepatol, 2000 Oct, 12(10), 1077 - 88
Micro-organisms administered in the benefit of the host: myths and facts; Marchand J et al.; OBJECTIVE: To evaluate the published literature on the potential benefit of micro-organisms on the general well being of the host . STUDY DESIGN: All published prospective, randomized, placebo-controlled trials with micro-organisms to improve the health of the host were critically reviewed . RESULTS: According to published data, there is evidence suggesting that Lactobacillus rhamnosus strain GG or Lactobacillus casei strain GG and Saccharomyces boulardii may be of possible benefit for the treatment of several medical conditions . However, published data on the therapeutic effect of other micro-organisms are almost non-existent . CONCLUSION: Better designed prospective, randomized, and placebo-controlled studies are needed . Most of the present strains have not been selected in a rational way, but apparently represent rather randomly picked isolates . Although the theoretical advantages of micro-organisms administered to the benefit of the host are extremely interesting and promising, results of clinical trials are disappointing.

Appl Environ Microbiol, 2000 Nov, 66(11), 5030 - 4
Colonization of the stratified squamous epithelium of the nonsecreting area of horse stomach by lactobacilli; Yuki N et al.; Selective adhesion to only certain epithelia is particularly common among the bacterial members of the indigenous microflora of mammals . We have found that the stratified squamous epithelium of the nonsecreting area of horse stomach is colonized by gram-positive rods . The microscopic features of a dense layer of these bacteria on the epithelium were found to be similar to those reported in mice, rats, and swine . Adhering microorganisms were isolated and identified as Lactobacillus salivarius, L . crispatus, L . reuteri, and L . agilis by DNA-DNA hybridization and 16S rRNA gene sequencing techniques . These lactobacilli associated with the horse, except for L . reuteri, were found to adhere to horse epithelial cells in vitro but not to those of rats . A symbiotic relationship of these lactobacilli with the horse is suggested.

Appl Environ Microbiol, 2000 Nov, 66(11), 4822 - 8
Integrative food-grade expression system based on the lactose regulon of Lactobacillus casei; Gosalbes MJ et al.; The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering . An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L . casei . This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3' end of lacG and the complete lacF gene . Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments . Then, integration of the cloned genes into the lactose operon of L . casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants . This procedure has been successfully used for the expression of the E . coli gusA gene and the L . lactis ilvBN genes in L . casei . Following the same expression pattern as that for the lactose genes, beta-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose . This integrative vector represents a useful tool for strain improvement in L . casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1868 - 73
Preventive effect of lactobacillus delbrueckii subsp . bulgaricus on the oxidation of LDL; Terahara M et al.; Lactobacillus delbrueckii subsp . bulgaricus 2038 was examined for its activity to prevent the oxidation of the erythrocyte membrane in vitro, and the oxidation of LDL in vivo . Strain 2038 produced radical scavengers that reacted with 1,1-diphenyl-2-picrylhydrazl (DPPH) during cultivation . Moreover, the ethereal extract from the supernatant of the culture prevented the oxidation of the erythrocyte membrane in vitro . As an in vivo study, male F344 rats were fed on diets containing 20% fresh soybean oil (or 13% oxidized oil and 7% fresh oil) with 10% freeze-dried powder of the 2038 culture (or with skim milk powder) for 4 weeks . The level of thiobarbituric acid-reactive substances was lower in the low-density lipoproteins (per milligram of cholesterol) from rats fed on the oxidized oil with freeze-dried powder of the 2038 culture than without it . The level of vitamin E in the plasma was higher in the rats fed on the oxidized oil with the freeze-dried powder than without it.

Biosci Biotechnol Biochem, 2000 Sep, 64(9), 1836 - 41
Preparative 2'-reduction of ATP catalyzed by ribonucleotide reductase purified by liquid-liquid extraction; Brunella A et al.; Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.1.17.4.2) constitutively expressed by E . coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16% (w/w))/phosphate (7% (w/w)) liquid-liquid extraction . A high yield of 75.1% RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system . The RTPR-containing top phase was used to reduce ATP in the 2'-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2'-deoxyribonucleotides . High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR . 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR.

J Appl Microbiol, 2000 Oct, 89(4), 678 - 86
Extrusion of wheat or sorghum and/or addition of exogenous enzymes to pig diets influences the large intestinal microbiota but does not prevent development of swine dysentery following experimental challenge; Durmic Z et al.; A study was made of dietary influences on the large intestinal microbiota of pigs and on the incidence of swine dysentery (SD) after experimental infection with Brachyspira hyodysenteriae, the aetiological agent of SD . Animals were fed diets based either on wheat (expts 1 and 2) or sorghum (expt 2) . Grains were ground and fed either raw or after high temperature and pressure extrusion and/or after addition of exogenous enzymes to the whole diet to reduce the starch and soluble non-starch polysaccharides available for fermentation in the large intestine . Limiting fermentation creates conditions that apparently reduce the incidence of SD after infection with B . hyodysenteriae . The diets were fed to weaned pigs for 4-6 weeks, then half the animals on each diet were killed and gut samples collected for microbiology . The treatments had little effect on bacterial numbers . In expt 1, dietary extrusion of wheat reduced lactobacilli in the large intestine . Addition of enzymes to extruded wheat-based diets in expt 2 reduced facultative anaerobes and increased non-sporing anaerobes . Addition of enzymes to a raw sorghum diet in expt 3 decreased numbers of facultative anaerobes, while extrusion of sorghum increased total anaerobes . Bacteroides spp . and Fusobacterium spp., which act in synergy with B . hyodysenteriae in SD, were isolated at a higher percentage in pigs fed the untreated wheat diet than in pigs fed the treated wheat diets . Following experimental infection the incidence of SD amongst pigs fed treated wheat diets was slightly lower than those fed the untreated diet, but with sorghum-based diets the opposite was found . Overall, the different dietary treatments used did not significantly reduce SD.

J Appl Microbiol, 2000 Oct, 89(4), 553 - 63
Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistance; Grill JP et al.; Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation . Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth . The CBSH activity increased during growth but its course depended on bile salts type and culture conditions . A Lact . amylovorus mutant was isolated from the wild-type strain of Lact . amylovorus DN-112 053 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine . An agar plate assay was used to detect mutants without CBSH activity . In resting cell experiments, the strain showed reduced activity . Differences between growth parameters determined for wild-type and mutant strains were not detected . Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant . Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant . The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain . Nevertheless, bile was more toxic for the wild-type strain . In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner . No difference was apparent between the two strains . In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant . The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (ZDeltapH) was involved in Lactobacillus bile salt resistance.

J Bacteriol, 2000 Nov, 182(22), 6525 - 8
Cholic acid is accumulated spontaneously, driven by membrane deltapH, in many lactobacilli; Kurdi P et al.; Many lactobacilli from various origins were found to apparently lack cholic acid extrusion activity . Cholic acid was accumulated spontaneously, driven by the transmembrane proton gradient . Accumulation is a newly identified kind of interaction between intestinal microbes and unconjugated bile acids and is different from extrusion and modification, which have been described previously.

J Bacteriol, 2000 Nov, 182(22), 6440 - 50
Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus; Sillanpaa J et al.; The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli . The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein . The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens . Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced . In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion . Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule . The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA . Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA . RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions . S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L . crispatus.

J Med Chem, 2000 Oct 19, 43(21), 3837 - 51
Design, synthesis, and X-ray crystal structure of a potent dual inhibitor of thymidylate synthase and dihydrofolate reductase as an antitumor agent; Gangjee A et al.; A novel N- inverted question mark2-amino-4-methyl{(pyrrolo{2, 3-d}pyrimidin-5-yl)ethyl}benzoyl inverted question mark-L-glutamic acid (3a) was designed and synthesized as a potent dual inhibitor of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as an antitumor agent . Compound 3b, the N7-benzylated analogue of 3a, was also synthesized as an antitumor agent . The synthesis of 3a was accomplished via a 12-step sequence which involved the synthesis of 2-amino-4-methylpyrrolo{2,3-d}pyrimidine (10) in 5 steps from 2-acetylbutyrolactone . Protection of the 2-amino group of 10 and regioselective iodination at the 5-position followed by palladium-catalyzed coupling afforded intermediate 14 which was converted to 3a by reduction and saponification . Similar synthetic methodology was used for 3b . X-ray crystal structure of the ternary complex of 3a, DHFR, and NADPH showed that the pyrrolo{2, 3-d}pyrimidine ring binds in a "2,4-diamino mode" in which the pyrrole nitrogen mimics the 4-amino moiety of 2,4-diaminopyrimidines . This is the first example of a classical pyrrolo{2,3-d}pyrimidine antifolate shown to have this alternate mode of binding to DHFR . Compounds 3a and 3b were more inhibitory than LY231514 against TS from Lactobacillus casei and Escherichia coli . Analogue 3a was also more inhibitory against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii . Evaluation of 3a against methotrexate (MTX)-resistant cell lines with defined mechanisms indicated that cross-resistance of 3a was much lower than that of MTX . Metabolite protection studies and folylpoly-gamma-glutamate synthetase studies suggest that the antitumor activity of 3a against the growth of tumor cells in culture is a result of dual inhibition of TS and DHFR . Compound 3a inhibited the growth of CCRF-CEM and FaDu cells in culture at ED(50) values of 12.5 and 7.0 nM, respectively, and was more active against FaDu cells than MTX . In contrast, compound 3b was inactive against both cell lines . Compound 3a was evaluated in the National Cancer Institute in vitro preclinical antitumor screening program and afforded IG(50) values in the nanomolar range against a number of tumor cell lines.

Arch Latinoam Nutr, 2000 Jun, 50(2), 157 - 63
{Obtaining a fermented chickpea extract (Cicer arietinum L.) and its use as a milk extensor}; Morales de Leon J et al.; Chickpea (Cicer arietinum L) is cultivated in the North part of Mexico and it is considered a good source of vegetal protein of low cost (20% average), nevertheless, the 80% used for the exportation and only the 20% less was used for animal feeding . The main objective in this study is to obtain a fermented chickpea extract for using in dairy extensor . Chickpea water absorbtion kinetics were carried out in e temperature conditions:while the conditions were established, chickpea was grounded and fermented in different amounts with its natural flora, L . casei, L . plantarum and a mixture culture of both microorganism in logarithmic phase . The results showed that the presence of microorganism of chickpea natural flora interferes during the fermentation, so before the inoculation it was necessary treat the chickpea extract (CE) terminally in a dilution 1:4 during 20 min at 7.7 kg/cm2 of pressure . The use of a mixture culture of 5% of L . casei and 5% L . plantarum inoculated in MRS broth was used to decrease fermentation time . Its addition in logarithmic phase to the sterile chickpea extract increased the lactic acid production and decreased the pH value in 6 h which was less time that one obtained with each of lactobacillus . The fermented extract obtained finally, presented similar sensory characteristics to the ones of dairy products . Therefore, chickpea is a good alternative as a extensor for this kind of products.

J Pediatr Gastroenterol Nutr, 2000 Oct, 31(4), 453 - 7
Is lactobacillus GG helpful in children with Crohn's disease? Results of a preliminary, open-label study; Gupta P et al.; BACKGROUND: Lactobacillus GG is a safe probiotic bacterium known to transiently colonize the human intestine . It has been found to be useful in treatment of several gastrointestinal conditions characterized by increased gut permeability . In the current study, the efficacy of Lactobacillus GG was investigated in children with Crohn's disease . METHODS: In this open-label pilot evaluation viewed as a necessary preliminary step for a possible subsequent randomized placebo-controlled trial, four children with mildly to moderately active Crohn's disease were given Lactobacillus GG (10(10) colony-forming units {CFU}) in enterocoated tablets twice a day for 6 months . Changes in intestinal permeability were measured by a double sugar permeability test . Clinical activity was determined by measuring the pediatric Crohn's disease activity index . RESULTS: There was a significant improvement in clinical activity 1 week after starting Lactobacillus GG, which was sustained throughout the study period . Median pediatric Crohn's disease activity index scores at 4 weeks were 73% lower than baseline . Intestinal permeability improved in an almost parallel fashion . CONCLUSIONS: Findings in this pilot study show that Lactobacillus GG may improve gut barrier function and clinical status in children with mildly to moderately active, stable Crohn's disease . Randomized, double-blind, placebo-controlled trials are warranted for a final assessment of the efficacy of Lactobacillus GG in Crohn's disease.

J Food Prot, 2000 Oct, 63(10), 1338 - 46
Surface application of lysozyme, nisin, and EDTA to inhibit spoilage and pathogenic bacteria on ham and bologna; Gill AO et al.; A study was conducted to determine if the effectiveness of an antimicrobial treatment for cooked ham and bologna would be increased or maintained when applied in a surface coating . Cooked 10-g disks of ham and bologna sausage received one of three treatments: no coating (control), coating with 0.2 g of 7% (wt/vol) gelatin gel (gel-control), or coating with 0.2 g of 7% gelatin gel containing 25.5 g/liter of lysozyme-nisin (1:3) plus 25.5 g/liter of EDTA (gel-treated) . The samples were then inoculated with one of six test organisms: Brochothrix thermosphacta, Escherichia coli O157:H7, Lactobacillus sakei, Leuconostoc mesenteroides, Listeria monocytogenes, or Salmonella Typhimurium . Inoculated samples were vacuum packed and stored at 8 degrees C for 4 weeks . The antimicrobial gel treatment had an immediate bactericidal effect up to 4 log CFU/cm2 on the four gram-positive organisms tested (B . thermosphacta, Lactobacillus sakei, Leuconostoc mesenteroides, and Listeria monocytogenes) and inhibited the growth of these organisms during the 4 weeks of storage . The antimicrobial gel treatment also had a bactericidal effect on the growth of Salmonella Typhimurium during storage . The numbers of E . coli O157:H7 on ham were reduced by 2 log CFU/cm2 following treatment with both antimicrobial-containing and non-antimicrobial-containing gels during the 4-week storage period . No effect was observed on the growth of E . coli O157:H7 on bologna.

Adv Biochem Eng Biotechnol, 2000, 68, 21 - 60
Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis; Sablon E et al.; A survey is given of the main classes of bacteriocins, produced by lactic acid bacteria: I . lantibiotics II . small heat-stable non-lanthionine containing membrane-active peptides and III . large heat-labile proteins . First, their mode of action is detailed, with emphasis on pore formation in the cytoplasmatic membrane . Subsequently, the molecular genetics of several classes of bacteriocins are described in detail, with special attention to nisin as the most prominent example of the lantibiotic-class . Of the small non-lanthionine bacteriocin class, the Lactococcus lactococcins, and the Lactobacillus sakacin A and plantaricin A-bacteriocins are discussed . The principles and mechanisms of immunity and resistance towards bacteriocins are also briefly reported . The biosynthesis of bacteriocins is treated in depth with emphasis on response regulation, post-translational modification, secretion and proteolytic activation of bacteriocin precursors . To conclude, the role of the leader peptides is outlined and a conceptual model for bacteriocin maturation is proposed.

Curr Microbiol, 2000 Mar, 40(3), 181 - 4
Purification and characterization of invertase from Lactobacillus reuteri CRL 1100; Cuezzo de Gines S et al.; The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa . The enzyme was stable below 45 degrees C over a wide pH range (4.5-7.0) with maximum activity at pH 6.0 and 37 degrees C . The invertase activity was significantly inhibited by bivalent metal ions (Ca(++), Cu(++), Cd(++), and Hg(++)), beta-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid . The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17% . The K(m) and V(max) values for sucrose were 6.66 mM and 0.028 micromol/min, respectively . An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a beta-fructofuranosidase.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1803 - 9
Lactobacillus arizonensis sp . nov., isolated from jojoba meal; Swezey JL et al.; Five strains of simmondsin-degrading, lactic-acid-producing bacteria were isolated from fermented jojoba meal . These isolates were facultatively anaerobic, gram-positive, non-motile, non-spore-forming, homofermentative, rod-shaped organisms . They grew singly and in short chains, produced lactic acid but no gas from glucose, and did not exhibit catalase activity . Growth occurred at 15 and 45 degrees C . All strains fermented cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, melibiose, D-ribose, salicin, D-sorbitol, sucrose and trehalose . Some strains fermented L-(-)-arabinose and L-rhamnose . D-Xylose was not fermented and starch was not hydrolysed . The mean G+C content of the DNA was 48 mol% . Phylogenetic analyses of 16S rDNA established that the isolates were members of the genus Lactobacillus . DNA reassociation of 45% or less was obtained between the new isolates and the reference strains of species with G+C contents of about 48 mol% . The isolates were differentiated from other homofermentative Lactobacillus spp . on the basis of 16S rDNA sequence divergence, DNA relatedness, stereoisomerism of the lactic acid produced, growth temperature and carbohydrate fermentation . The data support the conclusion that these organisms represent strains of a new species, for which the name Lactobacillus arizonensis is proposed . The type strain of L . arizonensis is NRRL B-14768T (= DSM 13273T).

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1789 - 95
Lactobacillus kimchii sp . nov., a new species from kimchi; Yoon JH et al.; A bacteriocin-producing lactic acid bacterium, which was isolated from the Korean fermented-vegetable food kimchi, was subjected to a polyphasic taxonomic study using phenotypic characterization and phylogenetic and genetic methods . This organism (MT-1077T) has phenotypic properties that are consistent with the description characterizing the genus Lactobacillus . Phylogenetic analysis based on 16S rDNA sequences showed clearly that strain MT-1077T is a member of the genus Lactobacillus . The closest phylogenetic relatives are Lactobacillus alimentarius KCTC 3593T and Lactobacillus farciminis LMG 9200T, with levels of 16S rDNA similarity of 98.4 and 98.2%, respectively . Levels of 16S rDNA similarity between strain MT-1077T and other Lactobacillus species were less than 93.0% . Differences in some phenotypic characteristics and DNA-DNA relatedness data indicated that strain MT-1077T should be distinguished from L . alimentarius KCTC 3593T and L . farciminis LMG 9200T . On the basis of the data presented, it is proposed that strain MT-1077T should be placed in the genus Lactobacillus as a new species, Lactobacillus kimchii sp . nov . The type strain of the new species is strain MT-1077T (= KCTC 8903PT = JCM 10707T).

Microbios, 2000, 103(404), 31 - 41
The minimum inhibitory concentration of oral antibacterial agents against cariogenic organisms; Botelho MG; The minimum inhibitory concentrations (MIC) of eight common dental antibacterial agents against three genera of bacteria which have been implicated in dentine caries, namely streptococci, lactobacilli and actinomycetes were investigated . The ultimate aim was to determine the most appropriate antibacterial agent which could be added to dental restorative materials for filling cavities where there was residual dentine caries . The antibacterial agents tested were chlorhexidine diacetate, chlorhexidine dihydrochloride, chlorhexidine gluconate, benzalkonium chloride, cetrimide, cetylpyridinium chloride, thymol and sodium hypochlorite . Thymol and sodium hypochlorite did not inhibit microbial growth at any of the concentrations tested . For the active antibacterial agents tested the MIC values against lactobacilli and streptococci were 0.25 microg/ml to 8.0 microg/ml and for actinomycetes 0.125 to 8.0 microg/ml . These results illustrate the wide spectrum of sensitivity of caries associated bacteria against dental antibacterial agents . From the MIC values alone, it is difficult to recommend which of the active antibacterial agents would be most effective in eliminating cariogenic organisms.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 335 - 9
A Lactobacillus helveticus plasmid detects restriction fragment length polymorphism in different bacterial species; Gancheva AG et al.; A small cryptic Lactobacillus helveticus plasmid, pLBL4, was able to reveal restriction fragment length polymorphism in different bacterial species including Lactobacillus species, Bacillus species, and Escherichia coli when used as a DNA probe . The observed polymorphism was a result of the combined hybridization of several microsatellite sequences . The 6-bp sequence (TTGTTT) was repeated 12 times, seven of which were concentrated within the region between 1791 and 1997 bp of the plasmid sequence . The polymorphic patterns generated with pLBL4 differed from those obtained with M13 DNA in the larger number of bands observed . The results presented here open the possibility of using pLBL4 as a new broad-spectrum polymorphic DNA probe for fingerprint analysis.

FEMS Microbiol Lett, 2000 Sep 15, 190(2), 305 - 8
Production kinetics of acidophilin 801, a bacteriocin produced by Lactobacillus acidophilus IBB 801; Zamfir M et al.; Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801 . Studying the relationship between growth and bacteriocin biosynthesis revealed primary metabolite kinetics of bacteriocin production with a peak activity at the end of the exponential growth phase followed by a decrease during the stationary phase . Both microbial growth and bacteriocin production was inhibited by lactic acid . Whereas volumetric bacteriocin production (activity units (AU) ml(-1)) was favoured under pH-controlled conditions, bacteriocin titres rapidly decreased because of strong adsorption of the bacteriocin molecules to the producing cells under less acidic conditions.

Cleft Palate Craniofac J, 2000 Sep, 37(5), 447 - 52
Dental health indices and caries associated microflora in children with unilateral cleft lip and palate; Lucas VS et al.; OBJECTIVE: To investigate the dental health and caries related microflora of children with unilateral cleft lip and palate . STUDY GROUP: Sixty children with unilateral cleft lip and palate and matched controls . OUTCOME MEASURES: The decayed, missing, and filled teeth and surfaces in both the deciduous and permanent dentitions . The presence of developmental defects and plaque and gingivitis scores were also recorded . Plaque was collected from 25 of the children and their matched controls from three different sites, which were (1) the first approximal site distal to the cleft, (2) a contralateral anterior site, and (3) a remote site . It was cultured for Streptococcus mutans and lactobacilli . Plaque was collected from two sites in the matched controls . RESULTS: There was no significant difference in the caries, plaque, and gingivitis scores between the children with cleft palate and the controls . A greater number of enamel opacities were recorded in the control group, and there was a higher prevalence of enamel discoloration in the children with cleft lip and palate . There was no significant difference in the proportion of S . mutans or lactobacilli at the cleft site, compared with the unaffected site in the study group, although there was an anterior-posterior gradient in the proportion of S . mutans . There was no significant association between the stagnation area at the cleft site and the bacteria associated with dental caries.

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 311 - 8
Intestinal receptors for adhesive fimbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine--a review; Jin LZ et al.; Determining the structure of the intestinal receptor for enterotoxigenic Escherichia coli (ETEC) K88 fimbriae will make it possible to develop new strategies to prevent K88+ ETEC-induced disease in pigs . Putative K88 adhesin receptors have been identified in both intestinal brush border and mucus preparations as either glycoproteins or glycolipids . Proteins with sizes of 25, 35, 40-42, 60, and 80 kDa in the intestinal mucus and 16, 23, 35, 40-70, 74, 210, and 240 kDa in brush border membranes were reported to bind specifically to K88ab and K88ac fimbriae . The factors accounting for these variable results may include the variants of K88, ages, breeds, and phenotypes of pigs, and even the sampling sites in the small intestine . Of the reported K88 receptors, only three brush border receptors, i.e., a pair of mucin-type sialoglycoproteins (210 kDa or 240 kDa), an intestinal neutral glycosphingolipid (IGLad), and a 74-kDa transferrin glycoprotein (GP74), have fulfilled the criteria as phenotype-specific K88 fimbrial receptors . Inhibiting the attachment of ETEC to intestine by modifying the receptor attachment sites has been the key for developing novel approaches to preventing ETEC-induced diarrhea in pigs . These include: (1) receptor analogs from a variety of biological sources, (2) an enteric protected protease, (3) chicken egg-yolk containing anti-K88 fimbrial antibodies, and (4) some Lactobacillus isolates producing proteinaceous components or carbohydrates interacting with mucus components . Future studies should be directed to further characterize the carbohydrate and protein moieties of receptors recognized by the K88 adhesin variants and to identify the genes responsible for susceptibility to K88+ infections.

Prim, Care Update Ob Gyns . 2000 Sep 1, 7(5), 181 - 185
Bacterial vaginosis; Wang J; Bacterial vaginosis is the most common cause of vaginitis, affecting over 3 million women in the United States annually . Depopulation of lactobacilli from the normal vaginal flora and overgrowth of Gardnerella vaginalis and other anaerobic species are the presumed etiology . To date, no scientific evidence shows that bacterial vaginosis is a sexually transmitted disease . Malodorous vaginal discharge is the most common symptom . Differential diagnoses include trichomoniasis, moniliasis, and allergic or chemical dermatitis . The diagnosis is confirmed when at least three of the following four findings are present (Amsel's criteria): 1) thin, homogenous discharge, 2) pH greater than 4.5, 3) positive amine test, and 4) presence of clue cells . The sensitivity and positive predictive value are both 90% . Vaginal Gram stain is also reliable and allows for permanent record . Cultures are nonspecific because G . vaginalis resides in normal vaginal flora as well . Papanicolaou smears are not particularly sensitive, but their positive predictive value is very high . The Centers for Disease Control and Prevention recommend three treatment regimens in nonpregnant patients: oral metronidazole (500 mg twice daily for 7 days), intravaginal 2% clindamycin cream (one applicatorful at bedtime for 7 days), or intravaginal metronidazole gel (one to two applicatorfuls per day for 5 days) . Alternative regimens include a single 2-g oral dose of metronidazole or a 7-day course of oral clindamycin, 300 mg twice daily . The association between bacterial vaginosis and adverse pregnancy outcomes has satisfied many criteria for a causal inference . Treatment of bacterial vaginosis in women with previous history of preterm labor results in fewer preterm deliveries than in untreated women from the same population.

J Appl Microbiol, 2000 Sep, 89(3), 547 - 52
Kinetics of the arginine metabolism of malolactic wine lactic acid bacteria Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo111; Mira de Orduna R et al.; The excretion of citrulline, a precursor of carcinogenic ethyl carbamate, formed from arginine degradation by malolactic bacteria in wine is of toxicological concern . The arginine metabolism of resting cells of Lactobacillus buchneri CUC-3 and Oenococcus oeni Lo1l1 was examined . The citrulline excretion rate was found to be linearly correlated to the arginine degradation rate . It was possible to calculate an arginine to citrulline conversion ratio which could be used to predict the amount of citrulline expected after the degradation of a known quantity of arginine . The conversion ratios determined in this study were similar to data calculated from other authors for fermentations in wine and ranged between 4.0% and 7.7% . Ribose, fructose and glucose inhibited the degradation of arginine in Lact . buchneri CUC-3, and inhibition of arginine degradation by glucose correlated with higher arginine to citrulline conversion ratios . The work presents new results of arginine metabolism in malolactic bacteria and gives starting points for investigations in wine.

J Appl Microbiol, 2000 Sep, 89(3), 511 - 6
Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex; Kullen MJ et al.; The Lactobacillus acidophilus complex includes Lact . acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii . The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact . acidophilus complex . A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction . The sequence of this region of the 16S rRNA gene from the type strains of the Lact . acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains . As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates . The approach described here represents a rapid and definitive method for the identification of Lact . acidophilus complex members.

J Appl Microbiol, 2000 Sep, 89(3), 442 - 51
Effects of lactic acid bacteria in inoculants on changes in amino acid composition during ensilage of sterile and non-sterile ryegrass; Winters AL et al.; A study was carried out on the changes occurring in the amino acid fraction of a hybrid ryegrass during ensilage in laboratory-scale silos to help to establish the relative roles of plant and microbial proteases on protein degradation in the silo . Herbage treatments included (i) normal grass without treatment (ii) lambda-irradiated grass (sterile) without treatment (iii) sterile, inoculated with a strain of Lactobacillus plantarum and (iv) sterile, inoculated with a strain of Lactobacillus paracasei subsp . paracasei . These treatments had a significant effect on silage amino acid profiles . Concentrations of free amino acids and the extent of amino acid catabolism varied with treatment . However, levels were notably higher in control silages after 90 days (free amino acid nitrogen constituting 54% of total amino acid nitrogen compared with 37, 32 and 22% for treatments i, ii and iv, respectively) . These results indicate that the extent of protein hydrolysis during ensilage is influenced by factors other than rate of pH decline and plant protease activity, and that microbial proteases play a role.

Curr Opin Pediatr, 2000 Oct, 12(5), 477 - 81
Probiotics in pediatric gastrointestinal disorders; Davidson GP et al.; Probiotics have been defined most recently as living microorganisms which, upon ingestion in certain numbers, exact health benefits beyond inherent general nutrition . They have been a part of human nutrition for centuries, but in recent years they have been more closely studied for their potential to improve health and treat disease . This review of probiotics is not extensive, highlighting the most recent reviews and well controlled clinical studies in both animals and humans . The safety issues are also discussed as well as potential mechanisms of action . The importance of studying each probiotic bacterium individually in each condition where a health benefit is claimed is highlighted by Lactobacillus GG, the most widely studied probiotic which has proven benefit in reducing the severity and duration of viral diarrhea but no benefit against bacterial diarrhea.

Int J Food Microbiol, 2000 Sep 10, 59(3), 241 - 7
Study of the cryotolerance of Lactobacillus acidophilus: effect of culture and freezing conditions on the viability and cellular protein levels; Baati L et al.; Slow cooling rate and pre-freezing stress brings about a high increase in the cell resistance and preservation of their physiological characteristics . A brutal decrease in temperature (from 37 degrees C to - 80 degrees C) causes a considerable loss of cell viability, in contrast a slow one preserves a survival rate of 75% . Pre-incubation of cells at low temperature (22 degrees C) during 6 h led to the development of cryotolerance indicated by an enhanced capacity to survive after a freezing treatment of 24 h at - 80 degrees C . Exposure of the cells to low pH (5.5) caused a large decrease in cell resistance but did not lead to any significant decrease of survival rate after freezing treatment . However, an increase of 15 +/- 3% in protein level compared to cells cultivated at regulated pH was observed.

Int J Food Microbiol, 2000 Sep 10, 59(3), 185 - 209
Structural model requirements to describe microbial inactivation during a mild heat treatment; Geeraerd AH et al.; The classical concept of D and z values, established for sterilisation processes, is unable to deal with the typical non-loglinear behaviour of survivor curves occurring during the mild heat treatment of sous vide or cook-chill food products . Structural model requirements are formulated, eliminating immediately some candidate model types . Promising modelling approaches are thoroughly analysed and, if applicable, adapted to the specific needs: two models developed by Casolari (1988), the inactivation model of Sapru et al . (1992), the model of Whiting (1993), the Baranyi and Roberts growth model (1994), the model of Chiruta et al . (1997), the model of Daughtry et al . (1997) and the model of Xiong et al . (1999) . A range of experimental data of Bacillus cereus, Yersinia enterocolitica, Escherichia coli O157:H7, Listeria monocytogenes and Lactobacillus sake are used to illustrate the different models' performances . Moreover, a novel modelling approach is developed, fulfilling all formulated structural model requirements, and based on a careful analysis of literature knowledge of the shoulder and tailing phenomenon . Although a thorough insight in the occurrence of shoulders and tails is still lacking from a biochemical point of view, this newly developed model incorporates the possibility of a straightforward interpretation within this framework.

Cryobiology, 2000 Aug, 41(1), 17 - 24
Stabilization and preservation of Lactobacillus acidophilus in saccharide matrices; Conrad PB et al.; Lyophilization and vacuum- or spray-drying are some of the most useful techniques for preserving foods, agricultural products, and pharmaceuticals . Biological materials, however, can be irreversibly damaged during these treatments . Therefore, it is essential to design protective agents to preserve protein activity and cell viability . In this paper we examine the use of alpha, alpha-trehalose-borate systems as protectants for Lactobacillus acidophilus during freeze- and vacuum-drying . Trehalose was found to be an effective protectant for freeze-dried and vacuum-dried samples, and it is equivalent to a protective formulation which is in current industrial use . It is known from our previous work on enzymes that the presence of borate can dramatically enhance the protective ability of trehalose . In this work, the addition of trehalose-borate to bacterial concentrate greatly improves the recovery of viable cells after storage . This improvement was seen in freeze-dried samples stored at 37 degrees C as well as for vacuum-dried samples held at room temperature . A tailored buffering strategy was tested to counteract the high pH resulting from the addition of borate to the mixture . Use of citric or lactic acids in combination with ammonium hydroxide gave a protectant solution with high pH (resulting in effective crosslinking between trehalose and borate) but a dry product with reduced pH upon rehydration (conducive to cell survival) . These results raise exciting possibilities for protection of more labile prokaryotic species as well as simple eukaryotes .

Cryobiology, 2000 Aug, 41(1), 10 - 6
Changes in the surface potential of Lactobacillus acidophilus under freeze-thawing stress; Fernndez Murga ML et al.; The zeta potential of Lactobacillus acidophilus CRL 640, a measure of the net distribution of electrical charges on the bacterial surface, is a function of the glucose concentration in the growing media . With 2% glucose, cells in the stationary phase showed a zeta potential of -45 +/- 2 mV . With these cells, the zeta potential after freezing and thawing decreased to -32 +/- 2 mV and there was a decrease in viability . The changes in the surface potential correlated with damage to the cell surface as shown by electron microscopy . Freeze-thawed cells incubated in a rich medium recovered a zeta potential of -38 +/- 2 mV without cell growth . L . acidophilus CRL 640 showed the same value of surface potential as control cells when they were frozen and thawed in 2 M glycerol .

Antonie Van Leeuwenhoek, 2000 Jul, 78(1), 73 - 85
Numerical phenetic study of the genus Carnobacterium; Lai S et al.; Eighty-nine strains representing the genus Carnobacterium, Enterococcus durans, Vagacoccus salmoninarum and atypical Lactobacillus strains MT12 and MT13 were examined for 92 unit characters . Computer analysis of the data resulted in the recovery of four major, five minor and thirteen single membered clusters . Three cluster-groups contained seventy-four of the Carnobacterium strains, Enterococcus durans NCFB 596T and Lactobacillus maltaromicus NCFB 2382T . Cluster-group A was equated with Carnobacterium piscicola and cluster-group B with Carnobacterium divergens . Lactobacillus maltaromicus NCFB 2382T shared many properties in common with the C . piscicola strains . The recovery of several Carnobacterium strains as single membered clusters suggests that the genus Carnobacterium is underspeciated . Further work is also required to determine the subspecific structure of Carnobacterium divergens and Carnobacterium piscicola.

Int J Food Microbiol, 2000 Sep 15, 60(1), 91 - 7
Cluster analysis, richness and biodiversity indexes derived from denaturing gradient gel electrophoresis fingerprints of bacterial communities demonstrate that traditional maize fermentations are driven by the transformation process; Amp F et al.; The bacterial communities of maize fermented foods (pozol, poto-poto and ogi) from Mexico, Congo and Benin was compared using a culture-independent approach {denaturing gradient gel electrophoresis (DGGE) analysis of total DNA} . Foods produced following the same flow chart (i) grouped in distinct clusters, (ii) shared similar richness and biodiversity indexes and (iii) exhibited a high intra-specific variability . Structural biodiversity was higher in pozol samples, probably due to oxic conditions and higher initial pH . DGGE bands found in foods of different origins suggest that Lactobacillus plantarum, Lb . delbrueckii and Lb . fermentum are particularly well adapted to the fermentation of maize.

Int J Food Microbiol, 2000 Sep 15, 60(1), 75 - 81
Chemical, physical and enzymatic pre-treatments of probiotic lactobacilli alter their adhesion to human intestinal mucus glycoproteins; Tuomola EM et al.; Intestinal mucus glycoproteins extracted from faeces of healthy adult subjects were used as a substratum for bacterial adhesion to investigate the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion . The strains studied were Lactobacillus acidophilus 1 (LCI, Nestle), L . rhamnosus strain GG (ATCC 53103), L . rhamnosus LC-705, and L . casei strain Shirota (Yakult, Yakult Ltd) . Hereafter the strains are referred to as LA1, LGG, LC-705, and Shirota, respectively . Strains LA1 and LGG adhered greatly whereas the adhesion of strains LC-705 and Shirota to intestinal mucus glycoproteins was low . Adhesion of LA1 and LGG was reduced by boiling, autoclaving and by pepsin and trypsin treatments suggesting that the bacterial protein structures are essential for their adhesion . Treatment in ethanol and in propanol prior to adhesion significantly increased the adhesion of LA1 and LC-705, respectively . Adhesion of Shirota strain was not altered by any of the treatments.

Int J Food Microbiol, 2000 Sep 15, 60(1), 25 - 32
Action of lysozyme and nisin mixtures against lactic acid bacteria; Chun W et al.; Lysozyme was formulated together with nisin for usage against food spoilage lactobacilli . The mixtures demonstrated improved minimal inhibitory concentrations (MIC), compared to the parent compounds, for many of the bacteria and media tested, including high salt media in which lysozyme lost virtually all of its activity . Synergy was also observed through measurement of the kinetics of bacterial killing of L . curvatus 845, in which strain synergy had been observed in MIC assays . The combination of lysozyme and nisin caused more severe cell damage as viewed by scanning electron microscopy, and a consequent change in optical density at 600 nm, compared to the parent compounds, effects that were presumed to reflect the action of lysozyme . In addition, the combination caused more rapid permeabilization (depolarization) of the cytoplasmic membranes of Staphylococcus aureus, an effect that reflected the mechanism of action of nisin . Thus, nisin and lysozyme appear to demonstrate synergy against gram-positive bacteria because they reinforce each others mechanisms of bacterial killing.

Appl Environ Microbiol, 2000 Oct, 66(10), 4427 - 32
Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects; Pavan S et al.; The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today . For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus . We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis . The original two-plasmid NICE system turned out to be poorly suited to L . plantarum . In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L . plantarum NCIMB8826 . Moreover, recombinant L . plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice . The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.

Appl Environ Microbiol, 2000 Oct, 66(10), 4396 - 400
Identification of collagen-binding proteins in Lactobacillus spp . with surface-enhanced laser desorption/ionization-time of flight ProteinChip technology; Howard JC et al.; Biosurfactants produced by Lactobacillus fermentum RC-14, L . rhamnosus GR-1 and 36, and L . casei Shirota were found to contain proteins that bind to both collagen types III and VI, as determined by surface-enhanced laser desorption/ionization (SELDI)-time of flight mass spectrometry . Both collagen types III and VI immobilized on SELDI preactivated ProteinChip arrays detected several different sizes (2 to 48 kDa) of collagen-binding proteins . Overall, the RC-14-produced biosurfactant contained the greatest number of collagen-binding proteins (RC-14 > GR-1 > 36 > Shirota), including the mature form of a previously cloned 29-kDa collagen-binding protein (referred to in its mature 26-kDa form) . Although biosurfactants isolated from L . casei Shirota and L . rhamnosus 36 and GR-1 also contain several collagen-binding proteins, they do not contain the 26-kDa collagen-binding protein . Together, these results demonstrate the utility of the SELDI system as a means of rapidly characterizing clinically important but complex biosurfactant solutions.

Appl Environ Microbiol, 2000 Oct, 66(10), 4272 - 8
Development of genetic tools for Lactobacillus sakei: disruption of the beta-galactosidase gene and use of lacZ as a reporter gene To study regulation of the putative copper ATPase, AtkB; Stentz R et al.; Downstream from the ptsHI operon of Lactobacillus sakei, the genes atkY and atkB, organized in an operon, were observed . The two putative proteins, AtkB and AtkY, show sequence similarity to the Enterococcus hirae copper P-type ATPase, responsible for copper efflux, and its negative regulator . Characterization of AtkB as a copper P-type ATPase could not be demonstrated since an atkB mutant did not show any phenotype . Thus, another strategy was followed in order to investigate the transcriptional regulation of the atkYB locus, leading to the development of new genetic tools for L . sakei . A plasmid was constructed, the use of which allowed gene replacement at the lacLM locus in L . sakei by two successive crossovers . A strain deleted of the lacLM operon encoding the beta-galactosidase of L . sakei was constructed by this method, and the Escherichia coli lacZ gene could then be used as a reporter gene to investigate the regulation of atkYB . Results show that the atkYB operon is induced by small concentrations of CuSO(4) (30 to 40 microM) but not when CuSO(4) is omitted or added at higher concentrations.

Appl Environ Microbiol, 2000 Oct, 66(10), 4230 - 6
Biochemical and genetic characterization of propionicin T1, a new bacteriocin from Propionibacterium thoenii; Faye T et al.; A collection of propionibacteria was screened for bacteriocin production . A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii . This bacteriocin shows no sequence similarity to other bacteriocins . Propionicin T1 was active against all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii . The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing . The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids . An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA . The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1 . Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.

Appl Environ Microbiol, 2000 Oct, 66(10), 4187 - 92
Urea hydrogen peroxide reduces the numbers of lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation; Narendranath NV et al.; Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp . (Lactobacillus plantarum, L . paracasei, Lactobacillus sp . strain 3, L . rhamnosus, and L . fermentum) from approximately 10(7) to approximately 10(2) CFU/ml in a 2-h preincubation at 30 degrees C of normal-gravity wheat mash at approximately 21 g of dissolved solids per ml containing normal levels of suspended grain particles . Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L . paracasei at approximately 10(7) CFU/ml . There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added) . However, the presence of L . paracasei at approximately 10(7) CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control . The bactericidal activity of UHP is greatly affected by the presence of particulate matter . In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present . No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30 degrees C whether the bactericidal agent was added as H(2)O(2) or as urea hydrogen peroxide . NADH peroxidase activity (involved in degrading H(2)O(2)) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H(2)O(2) . H(2)O(2)-resistant mutants were not expected or found when lethal levels of H(2)O(2) or UHP were used . Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca . 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.

Mikrobiologiia, 2000 Jul-Aug, 69(4), 471 - 7
{Manganese-dependent ribonucleotide reductase from Propionibacterium freudenreichii subsp . Shermanii: partial purification, characteristics and role in DNA biosynthesis}; Iordan EP et al.; Like Lactobacillus leichmanii, Rhizobium meliloti, and Euglena gracilis, P . freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase . However, in the absence of corrinoids, P . freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin . This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter . Experiments with crude ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein subunits, a feature that is typical of all metal-containing reductases activated by molecular oxygen . Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells . This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases . It was concluded that, in adenosylcobalamin-deficient P . freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.

FEMS Microbiol Lett, 2000 Oct 1, 191(1), 51 - 5
Acetaldehyde metabolism by wine lactic acid bacteria; Osborne JP et al.; Acetaldehyde is a volatile flavor compound present in many fermented foods and is important in the production of red and white wines . Nine strains of the genera Lactobacillus and Oenococcus were able to metabolize acetaldehyde in a resting cell system, whereas two Pediococcus strains were not . Acetic acid and ethanol were produced from its degradation . A Lactobacillus and an Oenococcus were able to degrade SO(2)-bound acetaldehyde, as well . A coincubation of resting cells of Saccharomyces bayanus Premiere Cuvee and Oenococcus oeni Lo111 showed that strain Lo111 metabolized acetaldehyde produced by the yeast . The ability of malolactic bacteria to degrade free and SO(2)-bound acetaldehyde has implications for sensory and color qualities and the use of SO(2) in wine.

Arch Virol, 2000, 145(8), 1521 - 34
Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes; Kashige N et al.; The genes encoding the host cell wall-lytic proteins were searched in the genome DNA of phage PL-1 active against Lactobacillus casei ATCC 27092 by comparing the amino acid sequences with those of others using a computer software of the DDBJ data base . The gene regions found were cloned into E . coli by inserting PCR-amplified DNA fragments into the EcoRI site of pUC 19, and the nucleotide sequences were determined . One of the ORFs (hol) consisted of 270 bp encoding 90 amino acids . The hol product (holin) possessed a putative secretion signal, two putative transmembrane helices, and a highly charged C-terminus . Another ORF (lys) consisted of 1050 bp encoding an N-acetylmuramoyl-L-alanine amidase of 350 amino acids . The gene lys was expressed in E . coli using pCALn expression vector, and the purified gene product hydrolysed the amide linkage in the peptidoglycans of L . casei . The amino acid sequence of PL-1 amidase showed a high homology to those of Lactococcus lactis phage rlt and Listeria monocytogenes phage A511 . It was suggested that the N-terminal region was involved in enzyme activity and the C-terminal region in binding the enzyme to the cell wall substrate, respectively.

Oral Dis, 2000 Sep, 6(5), 297 - 302
Coptidis rhizoma inhibits growth and proteases of oral bacteria; Hu JP et al.; OBJECTIVE: The aim of this study was to investigate the antibacterial effect of Coptidis Rhizoma (CR), a traditional medicinal plant, on oral bacteria . MATERIALS AND METHODS: CR extract was prepared by boiling CR in water for 2 h . Alkaloids contained in CR extract were assayed by high performance liquid chromatography (HPLC) . Antibacterial activity of CR extract was estimated from the lowest concentration that did not permit bacterial growth (minimum inhibitory concentration, MIC) and the concentrations that inhibited 50% of bacterial proteolytic activity (IC50) . RESULTS: CR extract inhibited the growth of Actinomyces naeslundii, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Actinobacillus actinomycetemcomitans at MIC of 0.031-0.25 mg ml(-1), whereas it had less inhibitory effect (MIC: 0.5-2 mg ml(-1)) on the growth of Streptococcus and Lactobacillus . The major active component of CR extract was berberine (Ber), an alkaloid, and its inhibiting specificity to bacterial growth was similar to that of CR extract . CR extract and Ber were bacteriostatic at the MICs against most of the bacteria, and bacteriocidal at the concentrations higher than the MICs . Ber inhibited the activities of collagenase from P . gingivalis and A . actinomycetemcomitans . CONCLUSION: CR extract and Ber had an inhibitory effect on periodontopathogenic bacteria . These results suggest the possibility of their clinical application for the treatment of periodontal diseases.

Chem Biol Interact, 2000 Aug 15, 128(1), 39 - 49
Factors affecting the sequestration of aflatoxin by Lactobacillus rhamnosus strain GG; Haskard C et al.; The interaction of a potent carcinogen, aflatoxin B(1) (AFB(1)), with a probiotic strain of lactic acid bacteria, Lactobacillus rhamnosus strain GG (GG), has been investigated . The binding of AFB(1) to GG in the late exponential-early stationary phase was studied for viable, heat-killed and acid-killed bacteria . In general, viable, heat-killed and acid-killed GG responded in a similar manner . The effects of pronase E, lipase and m-periodate on AFB(1) binding and release were consistent with AFB(1) binding predominantly to carbohydrate components of the bacteria . The effect of urea suggested hydrophobic interactions play a major role in binding . Increasing concentration (0.01-1 M) of NaCl or CaCl(2) had minor effects on AFB(1) binding suggesting some involvement of electrostatic interactions . An increase in pH from 2.5 to 8.5 had no effect on AFB(1) binding but decreased binding of AFB(2a), possibly due to hydrogen bonding interactions.

Antibiot Khimioter, 2000, 45(7), 17 - 21
{The effect of netilmicin, amikacin, ceftazidime and cefotaxime on the adhesive properties of microorganisms isolated from newborn infants}; Kushnareva MV; The effect of netilmycin, amikacin, ceftazidime and cefotaxime on adhesion of Lactobacillus spp . (14 strains), Escherichia coli (21 strains), Klebsiella pneumonia (15 strains), Enterococcus sp . (18 strains), Candida albicans (15 strains) was investigated . The strains were isolated from respiratory tract and feces of the newborns . Antibiotics were used in the following subtherapeutic and therapeutic concentrations: netilmycin--1.2 and 12.0 micrograms/ml, amikacin--1.8 and 18 micrograms/ml, ceftazidime--7.5 and 75 micrograms/ml, cefotaxime--6.5 and 75 micrograms/ml . Adhesion of C . albicans was investigated with buccal epithelium cells, adhesion of other microorganisms--on formalinized human erythrocytes (1(0)Rh(+)) . It was shown that antibiotics in subtherapeutic and therapeutic concentrations inhibited adhesion of the most strains . Cefalosporins demonstrated maximum inhibitory activity . The number of the strains inhibited by cefalosporins and by aminoglycosides enhanced along with antibiotics concentrations enhancement from subtherapeutic to therapeutic concentrations.

J Dairy Sci, 2000 Aug, 83(8), 1684 - 91
Ripening of cheddar cheese with added attenuated adjunct cultures of lactobacilli; Madkor SA et al.; We made Milled curd Cheddar cheese with Lactococcus starter and an adjunct culture of Lactobacillus helveticus I or Lactobacillus casei T subjected to different attenuation treatments: freeze shocking (FS), heat shocking (HS), or spray drying (SD) . Proteolysis during cheese ripening (0 to 6 mo), measured by urea-PAGE and water-soluble nitrogen, indicated only minor differences between control and most adjunct-treated cheeses . However, there were significant differences in the effect of Lactobacillus adjuncts on the level of free amino nitrogen in cheese . Cheeses made with FS or HS Lb . helveticus adjunct exhibited significantly greatest rates of free amino group formation . Lipolysis as measured by total free fatty acids was consistently highest in adjunct-treated cheeses, and FS Lb . casei-treated cheeses showed the highest rate of free fatty acid formation followed by FS Lb . helveticus treated cheeses . Mean flavor and aroma scores were significantly higher for cheeses made with Lb . helveticus strain . Freeze-shocked Lb . helveticus-treated cheeses obtained the highest flavor and aroma scores . Sensory evaluation indicated that most of the adjunct-treated cheeses promoted better texture and body quality.

Indian J Biochem Biophys, 2000 Apr, 37(2), 121 - 9
Purification and characterization of dihydrofolate reductase from Lactobacillus leichmannii; Rao KN; Dihydrofolate reductase (DHFR) (5,6,7,8-THF: NADDP+ oxidoreductase, EC 1.5.1.3) was purified 205-fold to apparent homogeneity from the crude extracts of Lactobacillus leichmannii . It has UV absorption maxima at 280 nm, M(r) of 20,000, Stokes radius of 0.34 nm and a S20.w value of 0.12 S . The preparation showed the presence of 168 amino acid residues with threonine and lysine as the NH2- and COOH- terminal end-groups respectively and a single reactive sulfhydryl group . pCMB inhibited the enzyme activity (IC50 = 2 microM) . The enzyme has a pH optimum of 7.4 and is thermally inactivated at > 35 degrees C . It is activated by 0.1 M KCl and KI and 2 M urea . 3-4 M urea completely inactivated the enzyme . Enzyme has Km values of 3.5 microM and 6.2 microM for NADPH and DHF respectively, and a Ki value of 7 nM for MTX, the inhibition being competitive.

Microbiol Immunol, 2000, 44(7), 551 - 6
Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker; Fu X et al.; A chromosome-plasmid balanced lethal gene delivery system for Lactobacillus acidophilus based on the thyA gene was developed . The selected L . acidophilus DOM La strain carries a mutated thyA gene and has an obligate requirement for thymidine . This strain can be used as a host for the constructed shuttle vector pFXL03, lacking antibiotic-resistant markers but having the wild-type thyA gene from L . casei which complements the thyA chromosomal mutation . The vector also contains the replicon region from plasmid pUC19 and that of the Lactococcus plasmid pWV01, which allows the transfer between Escherichia coli, L . casei and L . acidophilus . Eight unique restriction sites (i.e., PstI, HindIII, SphI, SalI, AccI, XbaI, KpnI and SacI) are available for cloning . After 40-time transfers in modified MRS medium, no plasmid loss was observed . The vector pFXL03 is potentially useful as a food-grade vaccine delivery system for L . acidophilus.

Biochemistry, 2000 Sep 5, 39(35), 10747 - 54
Mechanism of elementary catalytic steps of pyruvate oxidase from Lactobacillus plantarum; Tittmann K et al.; Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies . Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1) . Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1) . Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process . Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer . A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation . In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1) . This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.

Microbiology, 2000 Sep, 146 ( Pt 9), 2155 - 60
The synthesis of the bacteriocin sakacin A is a temperature-sensitive process regulated by a pheromone peptide through a three-component regulatory system; Diep DB et al.; Sakacin A is a bacteriocin produced by Lactobacillus sakei Lb706 . The gene cluster (sap) encompasses a regulatory unit composed of three consecutive genes, orf4 and sapKR . sapKR encode a histidine protein kinase and a response regulator, while orf4 encodes the putative precursor of a 23-amino-acid cationic peptide (termed Sap-Ph) . The authors show that Sap-Ph serves as a pheromone regulating bacteriocin production . Lb706 produced bacteriocin when the growth temperature was kept at 25 or 30 degrees C, but production was reduced or absent at higher temperatures (33.5-35 degrees C) . Production was restored by lowering the growth temperature to 30 degrees C, but at temperatures of 33-34 degrees C also by adding exogenous Sap-Ph to the growth medium . A knock-out mutation in orf4 abolished sakacin A production . Exogenously added Sap-Ph complemented this mutation, unambiguously showing the essential role of this peptide for bacteriocin production . Another sakacin A producer, Lactobacillus curvatus LTH1174, had a similar response to temperature and exogenously added Sap-Ph.

Lett Appl Microbiol, 2000 Sep, 31(3), 193 - 7
Solvent extraction of bacteriocins from liquid cultures; Burianek LL et al.; A solvent extraction method was developed to concentrate lacidin from the culture of Lactobacillus acidophilus OSU133 . The new method concentrates the bacteriocin at the interface between chloroform and the aqueous culture of the producing bacterium . Compared with other extraction procedures, the new method effectively recovers higher bacteriocin yield and results in relatively clean preparations . Recovery of lacidin by the chloroform extraction procedure, compared with ammonium sulphate precipitation and cell acidification methods, was >10-fold and about 100-fold greater, respectively . The new extraction procedure saves time and is easy to perform . This method is also effective in recovering subtilin, bacillicin, pediocin and nisin from cultures of Bacillus subtilis ATCC 6633, B . subtilis OSY1115/C, Pediococcus acidilactici PO2 and Lactococcus lactis ATCC 11454, respectively.

Lett Appl Microbiol, 2000 Aug, 31(2), 129 - 33
The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage; Yost CK et al.; A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact . sakei and Leuconostoc spp . Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact . curvatus and Lact . sakei in multiplex PCR reactions . The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C . Seventy isolates were selected for identification and 52 were determined to be Lact . sakei, while the remaining 18 isolates were identified as Leuconostoc spp.

J Mol Biol, 2000 Sep 15, 302(2), 427 - 40
Structural and functional similarities in the ADP-forming amide bond ligase superfamily: implications for a substrate-induced conformational change in folylpolyglutamate synthetase; Sheng Y et al.; Comparison of the three-dimensional structures of folylpolyglutamate synthetase (FPGS) and the bacterial cell wall ligase UDP-N-acetylmuramoyl-l-alanine:d-glutamate ligase (MurD) reveals that these two enzymes have a remarkable structural similarity despite a low level of sequence identity . Both enzymes have a modular, multi-domain structure and catalyse a similar ATP-dependent reaction involving the addition of a glutamate residue to a carboxylate-containing substrate, tetrahydrofolate in the case of FPGS, and UDP-N-acetylmuramoyl-l-alanine in the case of MurD . Site-directed mutations of selected residues in the active site of Lactobacillus casei FPGS (P74A, E143A, E143D, E143Q, K185A, D313A, H316A, G411A and S412A) showed that most of these changes resulted in an almost complete loss of activity . Several of these amino acid residues in FPGS are found in structurally equivalent positions to active-site residues in MurD . Some insights into the function of these residues in FPGS activity are proposed, based on the roles surmised from the structures of two MurD . UDP-N-acetylmuramoyl-l-alanine.ADP complexes and a MurD . UDP-N-acetylmuramoyl-l-alanine-d-glutamate complex . Furthermore, the comparison has led us to propose that conformational changes induced by substrate binding in the reaction mechanism of FPGS result in a movement of the domains towards each other to more closely resemble the orientation of the corresponding domains in MurD . This relative domain movement may be a key feature of this new family of ADP-forming amide bond ligases .

Appl Microbiol Biotechnol, 2000 Aug, 54(2), 243 - 7
Bacterial response to acetate challenge: a comparison of tolerance among species; Lasko DR et al.; Although acetate formation and tolerance are important criteria for various aspects of biotechnological process development, available studies on acetate tolerance in different species are disparate . We evaluate the response of eight bacterial strains, including two variants of Escherichia coli, two variants of Staphylococcus capitis, and one each of Acetobacter aceti, Gluconobacter suboxydans, Lactobacillus acetotolerans, and L . bulgaricus, to acetate challenges under identical conditions . Our findings were: (1) wild-type organisms of species that are considered tolerant of acetate perform only slightly better than E . coli in unadapted shaker cultures; (2) the ability to tolerate acetate is strongly dependent on the carbon source, and is, especially for E . coli, much greater on glycerol than on glucose; (3) respiration is not as important to acetate tolerance in E . coli and S . capitis as has been reported for the acetic acid bacteria; (4) S . capitis was the least affected by acetate under all conditions and grew at up to 44 g/l acetate without any preconditioning; and (5) qualitative high-throughput screening of growth characteristics can be achieved with relatively inexpensive multiwell plate readers.

J Environ Sci Health B, 2000 Sep, 35(5), 599 - 610
Salmonella enteritidis hilA gene fusion response after incubation in spent media from either S . enteritidis or a poultry Lactobacillus strain; Durant JA et al.; The purpose of this study was to determine if growth of a poultry probiotic lactobacilli strain can influence S . enteritidis virulence expression by measuring the response of a hilA-lacZY transcriptional fusion . beta-galactosidase activity was not detected when S . enteritidis was incubated in Lactobacillus-spent medium (24 h growth, pH 4.1, 50.4 mM lactate) but was detectable in spent medium from 4 h growth cultures of Lactobacillus sp . (final OD of 0.213, pH 5.7, 12 mM lactate) when pH and lactate were adjusted to that of the 24 h-pH 4 spent media levels . Adjusting the pH of the 24 h spent medium from 4 to 6, resulted in a measurable beta-galactosidase activity that was significantly higher than expression in LB broth . When S . enteritidis was grown in Salmonella-spent media (24 h growth, pH 4.2, 78 mM acetate), hilA expression was increased 4-fold over expression in the LB broth.

Infect Dis Obstet Gynecol, 2000, 8(3-4), 184 - 90
Gynecologic conditions and bacterial vaginosis: implications for the non-pregnant patient; Sweet RL; Bacterial vaginosis is characterized by a shift from the predominant lactobacillus vaginal flora to an overgrowth of anaerobic bacteria . Bacterial vaginosis is associated with an increased risk of gynecologic complications, including pelvic inflammatory disease, postoperative infection, cervicitis, human immunodeficiency virus (HIV), and possibly cervical intraepithelial neoplasia (CIN) . The obstetrical risks associated with bacterial vaginosis include premature rupture of membranes, preterm labor and delivery, chorioamnionitis and postpartum endometritis . Despite the health risks associated with bacterial vaginosis and its high prevalence in women of childbearing age, bacterial vaginosis continues to be largely ignored by clinicians, particularly in asymptomatic women.

FEMS Immunol Med Microbiol, 2000 Sep, 29(1), 47 - 52
Modulation of humoral immune response through probiotic intake; Fang H et al.; Thirty healthy volunteers were randomised into three different treatment groups and consumed Lactobacillus GG, Lactococcus lactis or placebo (ethyl cellulose) for 7 days . On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection . All subjects responded well to the vaccine, but no significant differences were observed in numbers of IgA-, IgG- and IgM-secreting cells among the different groups . There was a trend towards a greater increase in specific IgA among the subjects receiving the vaccine in combination with Lactobacillus GG . Those receiving L . lactis with their vaccine evinced significantly higher CR3 receptor expression on neutrophils than those receiving either the placebo or Lactobacillus GG . These results indicate that probiotics may influence differently the immune response to oral S . typhi vaccine and that the immunomodulatory effect of probiotics is strain-dependent.

Appl Environ Microbiol, 2000 Sep, 66(9), 4084 - 90
Purification and characterization of novel antifungal compounds from the sourdough Lactobacillus plantarum strain 21B; Lavermicocca P et al.; Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay . The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228, Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2, Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226, Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623 . The nonconcentrated culture filtrate of L . plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity . The activity was fungicidal . Calcium propionate at 3 mg ml(-1) was not effective under the same assay conditions, while sodium benzoate caused inhibition similar to L . plantarum 21B . After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L . plantarum 21B . Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity . It inhibited all the fungi tested at a concentration of 50 mg ml(-1) except for P . roqueforti IBT18687 and P . corylophilum IBT6978 (inhibitory concentration, 166 mg ml(-1)) . L . plantarum 20B, which showed high antimold activity, was also selected . Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B . Growth of A . niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S . cerevisiae and Lactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S . cerevisiae and selected L . plantarum 21B.

Appl Environ Microbiol, 2000 Sep, 66(9), 3974 - 80
Dissolution of xylose metabolism in Lactococcus lactis; Erlandson KA et al.; Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon . The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci . Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L . lactis subsp . lactis B-4449 (formerly Lactobacillus xylosus), L . lactis subsp . lactis IO-1, and L . lactis subsp . lactis 210, respectively . The two environmental isolates, L . lactis B-4449 and L . lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose . L . lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-) . Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains . Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L . lactis 210, IO-1, and B-4449 . None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity . Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L . lactis unable to metabolize xylose.

Appl Environ Microbiol, 2000 Sep, 66(9), 3966 - 73
Effects of high pressure on survival and metabolic activity of Lactobacillus plantarum TMW1.460; Ulmer HM et al.; The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death . The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa . Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds . HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells . During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture . Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability . Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation . An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells . Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.

Appl Environ Microbiol, 2000 Sep, 66(9), 3898 - 904
Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp . bulgaricus SS1 and Lactococcus lactis subsp . cremoris FT4; Gobbetti M et al.; Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp . bulgaricus SS1 and L . lactis subsp . cremoris FT4 . The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography . The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry . The most inhibitory fractions of the milk fermented by L . delbrueckii subsp . bulgaricus SS1 contained the sequences of beta-casein (beta-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82 . Those from the milk fermented by L . lactis subsp . cremoris FT4 contained the sequences of beta-CN f7-14, f47-52, and f169-175 and kappa-CN f155-160 and f152-160 . Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature . In particular, the beta-CN f47-52 sequence had high homology with that of angiotensin-II . Some of these peptides were chemically synthesized . The 50% inhibitory concentrations (IC(50)s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter) . When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC(50)s increased considerably . A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed . Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.

Appl Environ Microbiol, 2000 Sep, 66(9), 3835 - 41
Metabolic engineering of Lactobacillus helveticus CNRZ32 for production of pure L-(+)-lactic acid; Kyla-Nikkila K et al.; Expression of D-(-)-lactate dehydrogenase (D-LDH) and L-(+)-LDH genes (ldhD and ldhL, respectively) and production of D-(-)- and L-(+)-lactic acid were studied in Lactobacillus helveticus CNRZ32 . In order to develop a host for production of pure L-(+)-isomer of lactic acid, two ldhD-negative L . helveticus CNRZ32 strains were constructed using gene replacement . One of the strains was constructed by deleting the promoter region of the ldhD gene, and the other was constructed by replacing the structural gene of ldhD with an additional copy of the structural gene (ldhL) of L-LDH of the same species . The resulting strains were designated GRL86 and GRL89, respectively . In strain GRL89, the second copy of the ldhL structural gene was expressed under the ldhD promoter . The two D-LDH-negative strains produced only L-(+)-lactic acid in an amount equal to the total lactate produced by the wild type . The maximum L-LDH activity was found to be 53 and 93% higher in GRL86 and GRL89, respectively, than in the wild-type strain . Furthermore, process variables for L-(+)-lactic acid production by GRL89 were optimized using statistical experimental design and response surface methodology . The temperature and pH optima were 41 degrees C and pH 5.9 . At low pH, when the growth and lactic acid production are uncoupled, strain GRL89 produced approximately 20% more lactic acid than GRL86.

Appl Environ Microbiol, 2000 Sep, 66(9), 3764 - 72
Characterization of Leuconostoc gasicomitatum sp . nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions; Bjorkroth KJ et al.; Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs) . A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product . Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected . An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids . Protein swell has not previously been associated with any kind of meat product . A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species . In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L . gelidum was most similar to the spoilage-associated species . The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L . gelidum type strain . DNA-DNA reassociation, however, clearly distinguished the two species . The same strains showed only 22 and 34% hybridization with the L . gelidum type strain . These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp . nov . for this spoilage-associated Leuconostoc species.

Protein Eng, 2000 Aug, 13(8), 557 - 63
Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase; Kawase S et al.; Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2 . 1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP . In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS . Mutations of R23 increase K:(m) for dUMP 2-20-fold, increase K:(m) for cofactor 8-40-fold and decrease k(cat) 9-20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme . Mutations of R178 increase K:(m) for dUMP 40-2000-fold, increase K:(m) for cofactor 3-20-fold and do not significantly affect k(cat) . These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues . Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues . R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive.

Biol Pharm Bull, 2000 Aug, 23(8), 973 - 8
Effect of intranasal administration of Lactobacillus fermentum on the respiratory tract of mice; Cangemi de Gutierrez RC et al.; This paper studied the effect of intranasal inoculation of Lactobacillus fermentum, a microorganism belonging to the normal flora of the mouse pharynx, on the respiratory tract of mice . Optimal temporary colonization in different areas of the tract was obtained through administration of 4 times a dose of 5 x 10(7) CFU . L . fermentum remained in the trachea and bronchi up to the 7th day after inoculation . Re-inoculation of lactobacilli on the 10th day produced a transient colonization of the respiratory tract . Histological modifications produced in the trachea were mainly observed as an increased lymphocyte population at sub-mucosa level on the 4th day after inoculation . There was an increased number of activated macrophages in cytological slides of lung tissues on days 2 and 4 . Re-inoculation also produced stimulation of the G2 macrophages on days 12, 14 and 17 . From a histological point of view there were no other important changes in the organs studied . These results suggest stimulation of the immune system, especially that of the mucosal surfaces, after intranasal administration of L . fermentum in the experimental model employed . Stimulation was reflected in tracheal lymphocyte proliferation and increased lung macrophage population which have to be further studied in more detail.

Obstet Gynecol, 2000 Sep, 96(3), 431 - 9
Depomedroxyprogesterone-induced hypoestrogenism and changes in vaginal flora and epithelium; Miller L et al.; OBJECTIVE: To identify the effects of depomedroxyprogesterone acetate (DMPA) on vaginal microbial flora and epithelium . METHODS: Women who desired DMPA for contraception were evaluated before and at 3 and 6 months after initiation of 150-mg DMPA injections every 3 months . At each visit, we assessed genital symptoms, vaginal signs, vaginal microflora, and histopathology by vaginal biopsies . RESULTS: Among 38 women observed for 6 months, there was significant reduction in mean serum estradiol level (99.9 +/- 9.3 pg/mL to 26.6 +/- 1.6 pg/mL, P <.001) . The number of subjects with any Lactobacillus did not change, but the number with hydrogen peroxide (H(2)O(2))-positive Lactobacillus decreased from 20% before to 12% after 6 months of DMPA (P =.005) . The log concentration in colony-forming units per milliliter of vaginal fluid of H(2)O(2)-positive Lactobacillus decreased in a linear manner from 4 . 0 +/- 0.6 at baseline to 2.5 +/- 0.6 after 6 months of DMPA use (P = . 006) . The mean number of cell layers in the epithelium was reduced slightly from 28.1 +/- 0.7 to 25.9 +/- 0.9 (P =.05), epithelial thickness decreased from 1.02 +/- 0.04 mm to 0.89 +/- 0.05 mm (P = . 005), and the glycogen-positive thickness decreased from 0.81 +/- 0 . 04 mm at baseline to 0.66 +/- 0.05 after 6 months of DMPA use (P = . 005) . CONCLUSION: Depomedroxyprogesterone acetate produced a systemic hypoestrogenic state associated with decreased H(2)O(2)-positive Lactobacillus colonization and slight thinning of the glycogen vaginal epithelial layer . Such changes possibly compromise the vaginal barrier to infection.

J Bacteriol, 2000 Sep, 182(18), 5105 - 13
Changes in cspL, cspP, and cspC mRNA abundance as a function of cold shock and growth phase in Lactobacillus plantarum; Derzelle S et al.; An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in Lactobacillus plantarum . In addition to the two previously reported cspL and cspP genes, a third gene, cspC, has been cloned and characterized . All three genes encode small 66-amino-acid proteins with between 73 and 88% identity . Comparative Northern blot analyses showed that the level of cspL mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of cspC and cspP remain unchanged or increase only slightly (about two- to threefold) . Cold induction of cspL mRNA is transient and delayed in time as a function of the severity of the temperature downshift . The cold shock behavior of the three csp mRNAs contrasts with that observed for four unrelated non-csp genes, which all showed a sharp decrease in mRNA level, followed in one case (bglH) by a progressive recovery of the transcript during prolonged cold exposure . Abundance of the three csp mRNAs was also found to vary during growth at optimal temperature (28 degrees C) . cspC and cspP mRNA levels are maximal during the lag period, whereas the abundance of the cspL transcript is highest during late-exponential-phase growth . The differential expression of the three L . plantarum csp genes can be related to sequence and structural differences in their untranslated regions . It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.

J Med Chem, 2000 Aug 10, 43(16), 3125 - 33
Effect of C9-methyl substitution and C8-C9 conformational restriction on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro{2,3-d}pyrimidines; Gangjee A et al.; N-{4-{1-methyl-2-(2,4-diaminofuro{2, 3-d}pyrimidin-5-yl)ethyl}benzoyl}-L-glutamic acid (5) and its C8-C9 conformationally restricted E- and Z-isomers (6 and 7) were designed and synthesized in order to investigate the effect of incorporating a methyl group at the C9 position and of conformational restriction at the C8-C9 bridge of N-{4-{2-(2,4-diaminofuro{2, 3-d}pyrimidin-5-yl)ethyl}benzoyl}-L-glutamic acid (1) with respect to dihydrofolate reductase (DHFR) inhibitory activity as well as antitumor activity . The compounds were synthesized by a Wittig reaction of 2,4-diamino-5-(chloromethyl)furo{2,3-d}pyrimidine with ethyl 4-acetylbenzoate followed by catalytic reduction, hydrolysis, and standard peptide coupling with diethyl L-glutamate . The biological results indicated that the addition of a 9-methyl group to the C8-C9 bridge, as in 5, increased recombinant human (rh) DHFR inhibitory potency (IC(50) = 0.42 microM) as well as the potency against the growth inhibition of tumor cells in culture (CCRF-CEM EC(50) = 29 nM, A253 EC(50) = 28.5 nM, and FaDu EC(50) = 17.5 nM) compared with the 9-desmethyl analogue 1 . However, the conformationally restricted 4:1 Z/E mixture of 7 and 6 was less potent than 5 in both assays, and the pure E-isomer 6 was essentially inactive . These three classical analogues were also evaluated as inhibitors of Lactobacillus casei, Escherichia coli, and rat and rh thymidylate synthase (TS) and were found to be weak inhibitors . All three analogues 5-7 were good substrates for human folylpolyglutamate synthetase (FPGS) . These data suggested that FPGS is relatively tolerant to different conformations in the bridge region . Further evaluation of the cytotoxicity of 5 and 7 in methotrexate (MTX)-resistant CCRF-CEM cell sublines suggested that polyglutamylation was crucial for their mechanism of action . Metabolite protection studies of 5 implicated DHFR as the primary intracellular target . Compound 5 showed GI(50) values in 10(-9)-10(-7) M range against more than 30 tumor cell lines in culture.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2389 - 94
Comparative In vitro activities of ertapenem (MK-0826) against 1,001 anaerobes isolated from human intra-abdominal infections; Goldstein EJ et al.; By using an agar dilution method, the comparative in vitro activities of ertapenem (MK-0826) were studied against 1,001 anaerobes isolated from human intra-abdominal infections in 17 countries worldwide . MK-0826 was uniformly active against all isolates, including all Bacteroides fragilis group species isolates, with the exception of 12 of 61 (20%) strains of Bilophila wadsworthia, 3 strains of lactobacilli, and 1 isolate of Acidaminococcus fermentans . Geographical variation in activity was not observed.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 28 - 32
Characterisation of the shrinkage of calcium alginate gel membrane with immobilised Lactobacillus rhamnosus; Zhang W et al.; To quantify the shrinkage of calcium alginate gel membrane as a support matrix for immobilising cells during the fermentation of Lactobacillus rhamnosus, factors including time, pH, membrane thickness, and the concentrations of immobilised cells, lactic acid, glucose, and calcium chloride were examined by statistical experimental design . A Plackett-Burman design was used for the first screening experiment to identify the important factors which caused the divergent effects . Uniform Design, a powerful modelling design technique, was thus chosen to design the modelling experiments . Through a non-linear step-wise regression analysis, the predictive mathematical model of the shrinkage in membrane thickness was established and the significant main effects and two-factor interactions were identified . However, no significant model equations could be obtained for the shrinkage in area and volume of gel membranes . The methodology developed can be extrapolated to the quantitative characterisation of shrinkage in other immobilised gel matrices, which will be very useful in mathematical modelling, design, operation and scale-up of gel immobilised cell systems.

Arq Bras Cardiol, 2000 Mar, 74(3), 209 - 16
Effects of a novel fermented soy product on the serum lipids of hypercholesterolemic rabbits; Rossi EA et al.; OBJECTIVE: To assess the effect of a new feed soy product fermented by Enterococcus faecium and Lactobacillus jugurti on the serum lipid levels of rabbits with induced hypercholesterolemia . METHODS: Thirty-two rabbits were divided into 4 groups as follows: 1) control (C); 2) hypercholesterolemic (H); 3) hypercholesterolemic + fermented product (HPF); and 4) control + fermented product (CPF) . The H and HPF groups were fed with a diet with 0.15% (p/p) cholesterol in the first 15 days . C and CPF groups received regular food preparation . The HPF and CPF groups received 10 mL daily of the fermented 30 days . Blood samples were drawn at the beginning of the study and at the 15th and 30th days . Concentrations of total cholesterol, HDL-cholesterol, and triglycerides were analyzed . RESULTS: After 15 days, the HPF group showed a total cholesterol concentration lower (18.4%) than that of the H group (p = 0.05), but this difference disappeared after 30 days . No change was observed in total cholesterol levels of C and CPF groups . After 15 days, the HDL-cholesterol was higher (17.8%) in the HPF group, but the triglyceride levels remained unchanged in all groups during the same period of time . CONCLUSION: The soy fermented product caused an 18.4% reduction in total cholesterol and a 17.8% increase in the HDL-fraction . It may, therefore, be a possible coadjutor in the treatment of hypercholesterolemia.

Microbiol Res, 2000 Jul, 155(2), 101 - 6
Deoxyribonuclease activities in Lactobacillus delbrueckii; Azcarate Peril MA et al.; DNase activity was examined in the extracellular and subcellular fractions of six non-transformable strains belonging to Lactobacillus delbrueckii subsp . lactis (L . lactis) and Lactobacillus delbrueckii subsp . bulgaricus (L . bulgaricus) and compared with the activity present in Lactobacillus johnsonii NCK 65, a transformable strain of Lactobacillus . In the extracellular fraction of the L . delbrueckii strains, a common protein band of 36 kDa was detected, while a band of 29 kDa was found in the same fraction of L . johnsonii . No nuclease activity was detected in the cytoplasmic fraction of this strain, indicating that the localization of the DNase activity could be a key factor in the uptake of foreign DNA.

Electrophoresis, 2000 Jul, 21(13), 2660 - 9
"Early" protein synthesis of Lactobacillus delbrueckii ssp . bulgaricus in milk revealed by {35S} methionine labeling and two-dimensional gel electrophoresis; Rechinger KB et al.; The proteomes of exponentially growing and stationary cells of Lactobacillus delbrueckii ssp . bulgaricus grown in rich medium (MRS) were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and quantified after Coomassie staining . Stationary cells grown in MRS were inoculated in reconstituted skim milk, and "early" protein synthesis during the first 30 min of fermentation in milk was monitored by {35S}methionine labeling and 2-DE . In contrast to exponentially growing or stationary cells, the predominant "early" proteins were small (< 15 kDa) and of low pI (< 5.3) . Quantification of the proteome of the "early" lag phase based on 47 "spots" revealed that only three "early" proteins accounted for more than 80% of the total label . They were identified as pI 4.7 and 4.9 isoforms of the heat-stable phosphoryl carrier protein (HPr) with 45.2 and 9.4% of total label, respectively, and an unknown protein called EPr1 ("early" protein 1) with 26.6% of total label . Although an N-terminal sequence of 19 amino acids was obtained, no homologs to EPr1 could be found . De novo synthesis of the 10 and 60 kDa heat shock proteins (GroES and GroEL) was considerably lower (0.04 and 0.9% of total label, respectively), indicating only low levels of stress . Synthesis of triosephosphate isomerase (Tpi) as marker for glycolytic enzymes reached only 0.08% of total label . Our results demonstrate that inoculation in milk, resulting in a change from glucose to lactose as carbon source, imposes only little need for synthesis of stress or glycolytic enzymes, as sufficient proteins are present in the stationary, MRS-grown cells . The high level of expression of the pI 4.7 isoform of HPr suggests a regulatory function of the presumed Ser-46 phosphorylated form of HPr.

Oncol Rep, 2000 Sep-Oct, 7(5), 977 - 82
The effect of an oral administration of Lactobacillus casei strain shirota on azoxymethane-induced colonic aberrant crypt foci and colon cancer in the rat; Yamazaki K et al.; The preventive effect of oral administration of viable Lactobacillus casei strain Shirota (LcS) on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) and colon cancers in the rat was investigated . The study consisted of two experiments; in a short-term experiment (Exp-I), the inhibitory effect of 8- and 12-week treatments with LcS . Forty rats each received weekly a subcutaneous injection of AOM at a dose of 15 mg/kg of body weight for 5 weeks . Eight and twelve weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon and the mesenteric lymph nodes (MLN) were removed . The number of ACFs and the surface marker of lymphocytes derived from the MLN were investigated . The large ACF (those comprising four or more aberrant crypts per focus) had significantly decreased in the rats which had consumed the LcS diet . And oral administration of viable LcS significantly recovered CD8 positive lymphocytes to the levels in the control group . In a long-term experiment (Exp-II), 30 rats each received weekly a subcutaneous injection of AOM at a dose of 7 . 4 mg/kg of body weight for 10 weeks . Twenty-five weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon were removed . The number and incidence of colon cancers were investigated . The number of rats with colon cancers and the number of colon cancers per rat, were significantly decreased in the rats which had consumed the LcS diet . LcS inhibited chemically-induced colon carcinogenesis in the rat . CD8 positive T lymphocytes may play a key role in the preventive effect against colon carcinogenesis.

Int J Food Microbiol, 2000 Jul 25, 59(1-2), 37 - 46
Effects of Lactobacillus strains on the ripening and organoleptic characteristics of Arzúa-Ulloa cheese; Menendez S et al.; Seven batches of Arzua-Ulloa, a short-ripened soft cow's milk cheese produced in Galicia (NW Spain), were prepared from pasteurized milk . Two control batches of cheese (CB) were made with an acid-aromatic starter containing Lactococcus lactis subsp . lactis and Lactococcus lactis subsp . lactis var . diacetylactis, isolated from raw-milk Arzua-Ulloa cheeses . Five batches of cheese (LB) were made with the acid-aromatic starter plus one of five strains of mesophilic homofermentative Lactobacillus spp.: four of them isolated from raw-milk Arzua-Ulloa cheese (characterized in previous works) and the remaining was a commercial Lactobacillus strain . Higher counts of mesophilic viable bacteria, lactic acid bacteria and citrate-fermenting bacteria were found on days 1 or 15 of ripening, while higher counts of lactobacilli were found on day 30 of ripening . On day 1 of ripening the highest diacetyl-acetoin content was noted in the CB, but after day 15 the diacetyl-acetoin content was similar or higher in three of the five LB . The mean degradation of beta-casein in CB was higher than in LB, while the degradation of alpha(s1)-casein was higher in LB . The mean contents of nitrogen-soluble fractions were slightly higher in the LB than in the CB . Volatile free fatty acid (VFFA) contents were, in general, greater in LB than in CB and maximum amounts were determined on day 15 of maturation . Sensorial analysis indicated a more acid taste was in LB, while bitter and astringent tastes were more intense in CB . A positive correlation was found between beta-casein degradation and bitter taste . Yogurt and butter aromas were more intense in CB and in two of the five LB . Firmness was lower in LB and a negative correlation was found between this parameter and alpha(s1)-casein degradation . Crumbliness showed a positive correlation with beta-casein degradation . The use of the Lactobacillus strains assessed in this study is recommended for Arzua-Ulloa cheese manufacture, in order to enhance the desirable characteristics of this cheese variety, i.e., a soft texture due to alpha(s1)-casein proteolysis but without the bitter taste due to beta-casein degradation and a spicy and slightly rancid aroma and taste.

Int J Food Microbiol, 2000 Jul 25, 59(1-2), 19 - 27
Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation; Hebert EM et al.; The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied . Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp . lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA . All strains showed high levels of beta-galactosidase activity . However, proteolytic activity varied widely among isolates . Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C . According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases . No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L . helveticus- and L . delbrueckii subsp . lactis-specific bacteriophages.

Int J Food Microbiol, 2000 Jul 25, 59(1-2), 9 - 17
Biochemical characterization of lactic acid bacteria isolated from spontaneous fermentation of 'Almagro' eggplants; Sanchez I et al.; A total of 149 strains of lactic acid bacteria isolated from the spontaneous fermentation of 'Almagro' eggplants were characterized and identified . Of the isolates, 148 were determined as belonging to the genus Lactobacillus . A coccoid, gram-positive database-negative isolate was obtained in the early stages of fermentation . The Lactobacillus strains were divided into six groups based on sugar fermentation patterns and other physiological and morphological characteristics, and were identified as Lactobacillus plantarum biotype 1 (54.4%), Lactobacillus brevis biotype 2 (19.5%), Lactobacillus fermentum (9.4%), Lactobacillus brevis biotype 3 (5.4%), Lactobacillus pentosus (4.7%) and nine strains, which were not included in the previous species, were grouped as Lactobacillus spp . (6.0%) . Fermentation was initiated by Lactobacillus brevis biotype 2 and Lactobacillus fermentum . During the fermentation Lactobacillus plantarum became the predominant species.

J Mol Microbiol Biotechnol, 1999 Aug, 1(1), 165 - 73
Ribose utilization in Lactobacillus sakei: analysis of the regulation of the rbs operon and putative involvement of a new transporter; Stentz R et al.; A 7-kb DNA fragment of Lactobacillus sakei, containing the rbsD, rbsK and rbsR genes was sequenced . The genes responsible for ribose utilization are organized differently from what was previously described for model organisms such as Escherichia coli and Bacillus subtilis . No gene encoding RbsA, RbsB and RbsC, the subunits of the ribose ABC-transporter, were present in the rbs gene cluster . Instead, we found an open reading frame coding for RbsU, a protein similar to GltA, the glucose transporter of Staphylococcus xylosus . The disruption of rbsK, encoding the ribokinase, impaired growth on ribose . The disruption of rbsR, encoding the repressor, had no effect on the ability to grow on ribose, but led to overexpression of a large transcript corresponding to rbsU, rbsD and rbsK, suggesting that RbsU might be involved in ribose utilization . Ribose uptake and phosphorylation assays on the wild type strain and various mutants showed that, in ptsI mutants, both ribose uptake and phosphorylation are increased . These increased activities can explain the faster growth rate on ribose that was observed in ptsI mutants . The phosphotransferase system is thus involved in the negative regulation of ribose utilization . This regulation might not act at the transcriptional level since the overexpression of the rbs genes in the rbsR mutant did not lead to the same phenotype . A gene sharing high similarity scores with ackA genes, encoding the acetate kinase, was found upstream from the rbs gene cluster . The unusual location of this gene is maybe not fortuitous since acetate kinase is involved in ribose catabolism.

J Wildl Dis, 2000 Jul, 36(3), 460 - 8
Microbes in tree swallow semen; Lombardo MP et al.; A frequently hypothesized but poorly studied cost of multiple mating in birds is that exposure to pathogenic sexually transmitted microbes (STM's) can lower reproductive success . Conversely, female birds may benefit from high frequencies of copulation and multiple copulation partners if they receive cloacal inoculations of beneficial STM's that can either protect them against future encounters with pathogens and/or serve as therapy against present infection . We examined the semen of 30 male tree swallows (Tachycineta bicolor) in 1998 to determine the presence and prevalence of potential pathogenic and beneficial STM's . Semen was collected directly from males after applying gentle pressure to the cloaca and we used standard microbiological techniques to identify microbes . We found that 19 of 30 samples contained one or more types of microbes . In these 19 positive samples, we isolated both pathogenic and beneficial microbes from 11, only pathogenic microbes from seven, and only beneficial microbes from one . This variation among males suggests that females would benefit from considering a particular male's potential as a donor of either pathogenic or beneficial STM's as a criterion for mate choice . There were few significant differences between males with pathogen-infected semen and those without pathogens in their semen in measures of size, morphology, and ectoparasite score and feather damage . Likewise, there were few significant differences between males with beneficial Lactobacilli spp . in their semen and those without Lactobacilli spp . in their semen in measures of size, morphology, and ectoparasite score and feather damage . We were unable to determine if there was a relationship between microbe presence and prevalence on reproductive performance.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1479 - 85
Lactobacillus acidipiscis sp . nov . and Weissella thailandensis sp . nov., isolated from fermented fish in Thailand; Tanasupawat S et al.; Eleven strains of homofermentative, rod-shaped lactic acid bacteria and five strains of heterofermentative, sphere-shaped lactic acid bacteria were isolated from fermented fish (pla-ra and pla-chom) in Thailand . They were identified as new species and named Lactobacillus acidipiscis sp . nov . and Weissella thailandensis sp . nov., respectively, on the basis of phylogenetic analysis of the 16S rRNA gene sequences, DNA relatedness and phenotypic characteristics . The type strain of L . acidipiscis is FS60-1T (= PCU 207T = NRIC 0300T = HSCC 1411T = JCM 10692T = TISTR 1386T) and the type strain of Weissella thailandensis is FS61-1T (= PCU 210T = NRIC 0298T = HSCC 1412T = JCM 10695T = TISTR 1384T).

Electrophoresis, 2000 Jul, 21(12), 2557 - 61
Identification of stress-inducible proteins in Lactobacillus delbrueckii subsp . bulgaricus; Lim EM et al.; Lactobacillus delbrueckii subsp . bulgaricus (L . bulgaricus) is a homofermentative bacterium that produces lactic acid during growth . We adapted the two-dimensional electrophoresis (2-DE) technique to study the response of this bacterium to acidity . De novo protein synthesis was monitored by {35S}methionine labeling of exponentially growing cultures under standard (pH 6) and acidic (pH 4.75) conditions . After 2-DE separation, the protein patterns were compared . The protein spots showing increased radioactivity levels under acid conditions were considered acid-induced . We determined the N-terminal amino acid sequence of three highly induced proteins; comparing these proteins to databases we identified them to be the well-known heat shock proteins GroES, GroEL, and DnaK . Their induction levels were measured and compared . This is the first study by 2-DE of stress response in L . bulgaricus . We established the method and present a protein map which will be useful for future studies.

Enzyme Microb Technol, 2000 Sep 1, 27(6), 406 - 413
Purification and characterization of an extracellular alpha-amylase produced by Lactobacillus manihotivorans LMG 18010(T), an amylolytic lactic acid bacterium; Aguilar G et al.; This work presents the purification and characterization of an extracellular alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) produced by a new lactic acid bacterium: Lactobacillus manihotivorans able to produce L(+) lactic acid from starch . The molecular weight was found to be 135 kDa . The temperature and pH optimum were 55 degrees C and 5.5, respectively, and pI was 3.8 . The alpha-amylase had good stability at pH range from 5 to 6 and the enzyme was sensitive to temperature, losing activity within 1 h of incubation at 55 degrees C . Higher thermal stability was observed when the enzyme was incubated in presence of soluble starch . K(m) value and activation energy were 3.44 mg/ml and 32.55 kJ/mol, respectively . Amylose was found to be a better substrate than soluble starch and amylopectin . Al(3+), Fe(3+), and Hg(2+) (10 mM) almost completely inhibited the alpha-amylase.

Metab Eng, 1999 Apr, 1(2), 117 - 27
Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes; Emmerling M et al.; This study investigates the effect of overexpression of key glycolytic enzymes exhibiting either native or alternative allosteric regulation on glucose bioconversion by resting Escherichia coli cells previously engineered for ethanol production . Homologous and heterologous pyruvate kinases (Pyk) and phosphofructokinases (Pfk) were individually and simultaneously overexpressed . Overexpression of the E . coli Pfk led to a shift from ethanol to lactate formation (three-fold above the control level) while overexpression of Pyks accelerated lactate formation two-fold with less reduction in ethanol formation . Further increase in lactate formation (five-fold above the control level) resulted from overexpression of Pfk from Lactobacillus bulgaricus which, unlike the E . coli Pfk, is not allosterically regulated by either phosphoenolpyruvate or ADP . These effects on the carbon flux distribution were accompanied by significant changes in the intracellular concentrations of several glycolytic intermediates . Increased Pfk levels led primarily to reduced levels of hexose phosphates . Increased Pyk activity resulted in more complex changes which were different for overexpressed native Pyk and for overexpressed Bacillus stearothermophilus Pyk, which differs from E . coli Pyk in lacking activation by fructose 1,6-diphosphate, but is allosterically activated by AMP and ribose 5-phosphate . Simultaneous overexpression of native Pfk and Pyk caused a Pfk-overexpression-like phenotype with lower levels of hexose phosphates and further increased lactate formation (nine-fold above the control level) . The flux data demonstrate that overexpression of even single enzymes early in a central pathway can increase the fluxes to a particular metabolic product, although it may not affect the glucose uptake rate.

Biochemistry, 2000 Aug 15, 39(32), 9819 - 25
NMR studies of ligand carboxylate group interactions with arginine residues in complexes of Lactobacillus casei dihydrofolate reductase with substrates and substrate analogues; Birdsall B et al.; In a series of complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with substrates and substrate analogues, the (1)H/(15)N NMR chemical shifts for the guanidino group of the conserved Arg 57 residue were found to be sensitive to the mode of binding of their H(eta) protons to the charged oxygen atoms in ligand carboxylate groups . In all cases, Arg 57 showed four nonequivalent H(eta) signals indicating hindered rotation about the N(epsilon)-C(zeta) and C(zeta)-N(eta) bonds . The H(eta)(12) and H(eta)(22) protons have large downfield shifts as expected for a symmetrical end-on interaction with the ligand carboxylate group . The chemical shifts are essentially the same in the complexes with folate and p-aminobenzoyl-L-glutamate (PABG) and similar to those found previously for the methotrexate complex reflecting the strong and similar hydrogen bonds formed with the carboxylate oxygens . Interestingly, the rates of rotation about the N(epsilon)-C(zeta) bond for the complexes containing the weakly binding PABG fragment are almost identical to those measured in the complex with methotrexate, which binds 10(7) times more tightly . In the methotrexate complex, this rotation depends on correlated rotations about the N(epsilon)-C(zeta) bond of Arg 57 and the C(alpha)-C' bond of the ligand glutamate alpha-carboxylate group . Thus, even in a fragment such as PABG, which has a much faster off-rate, the carboxylate group binds to the enzyme in a similar way to that in a parent molecule such as folate and methotrexate with the rotation about the N(epsilon)-C(zeta) bond of Arg 57 being essentially the same in all the different complexes.

Rev Latinoam Microbiol, 1999 Jan-Mar, 41(1), 25 - 34
Bacterial vaginosis a "broad overview"; Gonzalez Pedraza Aviles A et al.; Bacterial vaginosis (BV) was first reported in 1995 by Gardner and Dukes, who described the unique clinical signs and symptoms and the distinctive nature of the vaginal discharge associated with it . They also described a "new" causative organism, which they named "Haemophilus vaginalis", subsequently renamed Gardnerella vaginalis . BV is currently the most prevalent cause of infectious vaginitis among women attending for genitourinary diseases . BV has a complex microbiology . Lactobacillus populations, which are usually dominant in healthy women, are replaced by a polymicrobial group of organisms that includes G . vaginalis, anaerobic Gram-negative rods such as Prevotella species, Peptostreptococcus species, Mycoplasma hominis, Ureaplasma urealyticum, and often Mobiluncus species . Anaerobic bacteria produce enzymes, aminopeptidases, that degrade protein and decarboxylases that convert amino acids and other compounds to amines . Those amines contribute to the signs and symptoms associated with the syndrome, raising the vaginal pH and producing a discharge odor . The excessive amounts of bacteria characteristic of the syndrome attach to epithelial cell surfaces, resulting in "clue cell" . Nearly half the patients report no noticeable symptoms, but many develop a characteristic copious, malodorous discharge if untreated . Results from epidemiologic studies have associated BV with serious upper genital tract infections and adverse pregnancy outcome . In particular, the presence of BV in pregnant women increases the risk of preterm delivery, and evidence is now compelling that BV is a cause of preterm delivery . The interest in potential invasiveness of G . vaginalis has increased . However, virulence determinants have not been studied enough . The most important therapy includes clindamycin and metronidazole.

Mikrobiol Z, 2000 May-Jun, 62(3), 49 - 55
{AF, a new bacterial preparation based on lactobacilli and its biological properties}; Kigel' NF; A highly efficient bacterial composition "AF" which includes representatives of two genera of microorganisms Enterococcus faecium and Lactobacillus acidophilus has been created as a result of analysis of biological properties of a number of strains of lactic-acid bacteria . Their biological properties--antagonistic activity in respect to pathogenic and conditionally pathogenic microorganisms, ability to adhesion and function in the intestine, sensitivity to antibiotics, etc., have been studied in the laboratory conditions . Prospects of the use of the given composition in production of milk products of functional effects have been shown.

Microbiology, 2000 Aug, 146 ( Pt 8), 1941 - 8
Expression of the phospho-beta-glycosidase ArbZ from Lactobacillus delbrueckii subsp . lactis in Lactobacillus helveticus: substrate induction and catabolite repression; Weber BA et al.; ArbZ from Lactobacillus delbrueckii subsp . lactis was previously shown to enable utilization of the beta-glucoside arbutin by Escherichia coli . The arbZ gene was cloned and expressed in the industrially used beta-glucoside-negative strain Lactobacillus helveticus 3036(62) . The transformants were able to ferment not only arbutin, but also cellobiose, salicin and methyl-beta-glucoside (MbetaGlc) . Cleavage of beta-glucosides by the transformants depended on the integrity of the cytoplasmic membrane, whereas in cell-free extracts only C(6)-phosphorylated substrates were hydrolysed . This suggested that ArbZ is a phospho-beta-glycosidase . ArbZ activity in transformants of Lb . helveticus was subject to substrate induction mediated by the beta-glucosides arbutin, salicin and MbetaGlc, whereas cellobiose or the beta-galactoside lactose had no inducing effect . Northern blot analysis proved that induction by MbetaGlc was due to enhanced transcription of arbZ . Catabolite repression of arbZ induction was observed with glucose, mannose, fructose and galactose . The induction kinetics observed in the presence of these sugars indicated that at least two different mechanisms are operative in catabolite repression of arbZ in Lb . helveticus.

Mol Microbiol, 2000 Aug, 37(3), 619 - 28
Functional analysis of promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus; Risoen PA et al.; Bacteriocin production in Lactobacillus sake LTH673 involves at least four operons: a regulatory operon (sppIPKR); two operons encoding bacteriocins and their immunity proteins (sppAiA and orfX); and an operon needed for secretion (sppTE) . We show here that the response regulator encoded by sppR in L . sake LTH673, as well as the homologous response regulators encoded by plnC and plnD in Lactobacillus plantarum C11, bind to characteristic repeats found in the -80 to -40 regions of spp operons . The promoters controlling bacteriocin operons are strictly regulated, and their activity is increased more than 1000-fold upon activation . Constitutive expression for the regulatory and transport operons is driven, at least in part, by promoters upstream of the -80 to -40 regions . Peak promoter activity of the regulatory and transporter operons precedes that of the two bacteriocin operons . The results reveal how promoters involved in quorum sensing-based regulation of bacteriocin production in Lactobacillus differ in strength, leakiness and timing of their activity.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 275 - 9
S-layer gene as a molecular marker for identification of Lactobacillus helveticus; Ventura M et al.; The paper describes two methods that allow rapid and accurate identification of Lactobacillus helveticus strains based on the nucleotide sequence of the gene coding for the surface layer (S-layer) protein . The first method is based on a polymerase chain reaction amplification using primers targeting a specific fragment of the S-layer gene . The second method involved a portion of the S-layer gene of L . helveticus as a probe in a hybridisation test . The specificity of these protocols was checked for DNA samples isolated from various Lactobacillus strains.

Syst Appl Microbiol, 2000 Jun, 23(2), 285 - 91
Molecular diversity of lactic acid bacteria from cassava sour starch (Colombia); Omar NB et al.; Lactic acid bacteria and more particularly lactobacilli and Leuconostoc, are widely found in a wide variety of traditional fermented foods of tropical countries, made with cereals, tubers, meat or fish . These products represent a source of bacterial diversity that cannot be accurately analysed using classical phenotypic and biochemical tests . In the present work, the identification and the molecular diversity of lactic acid bacteria isolated from cassava sour starch fermentation were assessed by using a combination of complementary molecular methods: Randomly Amplified Polymorphic DNA fingerprinting (RAPD), plasmid profiling, hybridization using rRNA phylogenetic probes and partial 16S rDNA sequencing . The results revealed a large diversity of bacterial species (Lb . manihotivorans, Lb . plantarum, Lb . casei, Lb . hilgardii, Lb . buchneri, Lb . fermentum, Ln . mesenteroides and Pediococcus sp.) . However, the most frequently isolated species were Lb . plantarum and Lb . manihotivorans . The RAPD analysis revealed a large molecular diversity between Lb . manihotivorans or Lb . plantarum strains . These results, observed on a rather limited number of samples, reveal that significant bacterial diversity is generated in traditional cassava sour starch fermentations . We propose that the presence of the amylolytic Lb . manihotivorans strains could have a role in sour starch processing.

Syst Appl Microbiol, 2000 Jun, 23(2), 279 - 84
Isolation and identification of tetracycline resistant lactic acid bacteria from pre-packed sliced meat products; Gevers D et al.; In recent years, the food chain has been recognised as one of the main routes for transmission of antibiotic resistant bacteria between the animal and human population . In this regard, the current study aimed to investigate if tetracycline resistant (tetR) lactic acid bacteria (LAB) are present in ready-to-eat modified atmosphere packed (MAP) sliced meat products including fermented dry sausage, cooked chicken breast meat and cooked ham . From population graphs based on doubling tetracycline concentrations between 0 and 256 microg ml(-1), only fermented dry sausage was shown to contain a high-level retR LAB population (5.10(1) - 2,23.10(4) CFU/g), and this in four out of ten examined sausages . From these four positive sausages, a total of 100 strains were isolated on de Man, Rogosa and Sharpe-sorbic acid (MRS-S) agar without tetracycline (n = 45) and on MRS-S agar supplemented with a tetracycline breakpoint concentration of 64 microg ml(-1) (n = 55) . Using resistance histograms derived from the disc diffusion method, all these strains were grouped as sensitive to rifampicin, erythromycin and ampicillin . All strains from the tetracycline-containing MRS-S plates were resistant to tetracycline . Identification with whole-cell protein profiling revealed that the total strain set represented four different species: Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sakei subsp . carnosus and Lactobacillus curvatus . All species are commonly associated with fermented dry sausage, either as starter culture or as natural contaminants . The latter three species were found to comprise all tetracycline resistant strains . To our knowledge, this is the first report providing evidence for the presence of tetR LAB in final ready-to-eat pre-packed fermented dry sausages.

Syst Appl Microbiol, 2000 Jun, 23(2), 251 - 9
Identification of lactobacillus delbrueckii and subspecies by hybridization probes and PCR; Lick S et al.; Three methods addressing two different target sites were compared for identification and differentiation of the subspecies Lactobacillus delbrueckii subsp . bulgaricus and Lactobacillus delbrueckii subsp . lactis/delbrueckii . A PCR method - three primer pairs that enable direct identification of the species and the two subspecies, respectively - was derived from a DNA fragment showing significant similarities to parts of the addAB genes of Bacillus sutbtilis . In addition, two oligonucleotide probes for the two subspecies were designed from that DNA region . Further, two oligonucleotide probes targeting the 16S rDNA were developed for subspecies differentiation by a one base-pair difference following identification of the species . Moreover, these probes were demonstrated to be applicable for in situ hybridization experiments . The results obtained by the different methods were in good agreement.

J Public Health Dent, 2000 Spring, 60(2), 82 - 4
Identification of adult populations at high risk for dental caries using a computerized database and patient records: a pilot project; Powell V et al.; OBJECTIVES: The purpose of this study is to test the usefulness of dental insurance claims history, supplemented with radiographic caries diagnoses, as a means of identifying caries-active and caries-inactive working adults, as determined by bacterial levels . Computerized identification of at-risk groups may facilitate subject selection for clinical trials designed to test caries-preventive strategies . METHODS: Two groups of subjects were initially selected from an insurance database based upon their dental service utilization during a one-year period: a "low restorative" group of individuals defined as persons who had received no restorative treatment, and a "high restorative" group comprised of individuals who had received at least three multisurfaced restorations . A chart review confirmed a diagnosis of caries in the high restorative group and an absence of caries in the low restorative group . Subjects were then approached for saliva collection . The low and high restorative groups were compared for salivary mutans streptococci and lactobacilli levels, stimulated flow rate, and buffer capacity (n = 48) . RESULTS: The high and low restorative groups differed in mutans streptococci levels, but not on other measures . CONCLUSIONS: A group of subjects who had recently received multisurfaced restorations that were placed for reasons of caries had significantly higher levels of mutans streptococci and potential for continued caries activity when compared to a group of subjects who had received no restorations and were caries free.

Immunology, 2000 Aug, 100(4), 510 - 8
Engineering the microflora to vaccinate the mucosa: serum immunoglobulin G responses and activated draining cervical lymph nodes following mucosal application of tetanus toxin fragment C-expressing lactobacilli; Shaw DM et al.; The delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options . In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein . Our data indicate that L . plantarum is more effective in this respect than L . casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression . Immunization of mice with live recombinant lactobacilli induced significant levels of circulating TTFC-specific immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains . In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage fluids, as were antigen-specific antibody-secreting cells and antigen-specific T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.

Appl Environ Microbiol, 2000 Aug, 66(8), 3427 - 31
Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp . bulgaricus grown in a chemically defined medium; Petry S et al.; We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp . bulgaricus . The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source . EPS production was greatest during the stationary phase . Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same . The EPS from strain L . delbrueckii subsp . bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L . delbrueckii subsp . bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose . However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations . Under pH-controlled fermentation conditions, L . delbrueckii subsp . bulgaricus strains produced as much EPS in the CDM as in milk . Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar . The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.

Appl Environ Microbiol, 2000 Aug, 66(8), 3368 - 75
Knockout of the p-coumarate decarboxylase gene from Lactobacillus plantarum reveals the existence of two other inducible enzymatic activities involved in phenolic acid metabolism; Barthelmebs L et al.; Lactobacillus plantarum NC8 contains a pdc gene coding for p-coumaric acid decarboxylase activity (PDC) . A food grade mutant, designated LPD1, in which the chromosomal pdc gene was replaced with the deleted pdc gene copy, was obtained by a two-step homologous recombination process using an unstable replicative vector . The LPD1 mutant strain remained able to weakly metabolize p-coumaric and ferulic acids into vinyl derivatives or into substituted phenyl propionic acids . We have shown that L . plantarum has a second acid phenol decarboxylase enzyme, better induced with ferulic acid than with p-coumaric acid, which also displays inducible acid phenol reductase activity that is mostly active when glucose is added . Those two enzymatic activities are in competition for p-coumaric and ferulic acid degradation, and the ratio of the corresponding derivatives depends on induction conditions . Moreover, PDC appeared to decarboxylate ferulic acid in vitro with a specific activity of about 10 nmol . min(-1) . mg(-1) in the presence of ammonium sulfate . Finally, PDC activity was shown to confer a selective advantage on LPNC8 grown in acidic media supplemented with p-coumaric acid, compared to the LPD1 mutant devoid of PDC activity.

Appl Environ Microbiol, 2000 Aug, 66(8), 3350 - 6
Comparative characterization of complete and truncated forms of Lactobacillus amylovorus alpha-amylase and role of the C-terminal direct repeats in raw-starch binding; Rodriguez Sanoja R et al.; Two constructs derived from the alpha-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared . These products correspond to the complete (AmyA) and truncated (AmyADelta) forms of alpha-amylase; AmyADelta lacks the 66-kDa carboxyl-terminal direct-repeating-unit region . AmyA and AmyADelta exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and alpha-cyclodextrin, and soluble starch) . The specific activities of the enzymes towards soluble starch are similar, but the K(M) and V(max) values of AmyADelta were slightly higher than those of AmyA, whereas the thermal stability of AmyADelta was lower than that of AmyA . In contrast to AmyA, AmyADelta is unable to bind to beta-cyclodextrin and is only weakly active towards glycogen . More striking is the fact that AmyADelta cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.

Appl Environ Microbiol, 2000 Aug, 66(8), 3174 - 9
Requirement of autolytic activity for bacteriocin-induced lysis; Martinez-Cuesta MC et al.; The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains . Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis . The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co(2+) and sodium dodecyl sulfate) . When L . lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmADelta1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium . Incubation of cell wall fragments of L . lactis MG1363, or of L . lactis MG1363acmADelta1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation . This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA . The autolysin was also responsible for the observed lysis of L . lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M . The results presented here show that lysis of L . lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.

Curr Microbiol, 2000 Jul, 41(1), 21 - 6
Temperature-dependent variation in API 50 CH fermentation profiles of Lactobacillus species; Nigatu A et al.; API 50 CH fermentation profiles of 45 Lactobacillus, one Atopobium, and three Weissella strains incubated at 30 degrees C and 37 degrees C were evaluated . Atopobium uli and ten species of Lactobacillus showed stable patterns despite the change in temperature . The rest of the type strains showed discrepancy between the two incubation temperatures: 18 strains lost, 12 additionally fermented another sugar, and 7 others fermented a different one in lieu . The variation was maximum in L . delbrueckii subsp . delbrueckii . L . malefermentans failed to ferment any of the substrates at 37 degrees C . Majority of the food and plant-associated strains (mainly heterofermenters) retained distinctive traits at 30 degrees C, while most of the animal-associated strains (mostly homofermenters) did so at 37 degrees C . No general trend was observed; 30 degrees C appeared to promote heterofermentation, while 37 degrees C favored homofermenters . Use of API 50 CH profiles for taxonomic purpose in most lactobacilli appears reproducible if a specific temperature for a species is strictly followed.

Appl Microbiol Biotechnol, 2000 Jun, 53(6), 709 - 14
Protective effect of the bile salt hydrolase-active Lactobacillus reuteri against bile salt cytotoxicity; De Boever P et al.; Bacterial bile salt hydrolysis is considered a risk factor for the development of colon cancer because of the risk of forming harmful secondary bile salts after an initial deconjugation step . In this study, the influence of enhanced bacterial bile salt transformation by the bile salt hydrolase-active Lactobacillus reuteri was studied in batch culture using the microbial suspension of the Simulator of the Human Intestinal Microbial Ecosystem; (SHIME), which was supplemented with oxgall at 5 g/l or 30 g/l . Changes in the fermentative capacity of the microbial ecosystem and the (geno)toxic properties of the SHIME supernatants were investigated . Increasing concentrations of oxgall inhibited the fermentation . Transient cell toxicity was observed for samples supplemented with 5 g oxgall/l, while samples with 30 g oxgall/l exhibited toxicity . The results of the haemolysis test suggest that the detrimental effects were probably due to the membrane-damaging effects of bile salts . In all cases, the adverse effects could be counteracted by the addition of 7.5 +/- 0.5 log10 CFU L . reuteri/ml . Plausible mechanisms for the protective properties of L . reuteri could involve a precipitation of the deconjugated bile salts and a physical binding of bile salts by the bacterium, thereby making the harmful bile salts less bioavailable.

Oral Dis, 2000 Jul, 6(4), 227 - 33
Surface properties of lactobacilli isolated from healthy subjects; Colloca ME et al.; OBJECTIVE: Lactobacilli are considered cariogenic micro-organisms . As oral species of lactobacilli have not been thoroughly described, the aim of this work was to isolated and identify these organisms from teeth, tongue, saliva and gum of healthy patients and to describe some of their surface properties . SUBJECTS: Forty-four subjects from Tucuman, Argentina, with D, d and M, m indices equal to 0 . MATERIALS AND METHODS: Samples were obtained from different areas of the oral cavity . Microorganisms were cultured in lactobacilli selected media (LBS) and identified morphologically and biochemically . Hydrophobicity was analysed by partition in organic solvents, acidity by affinity with chloroform and basicity with ethyl acetate (MATH method), aggregation and coaggregation in presence of (NH4)2SO4, and haemagglutination with ABO erythrocytes in microplates . RESULTS: Eighty-five lactobacilli were isolated; 29.4% were homofermenter, 44.7% facultative heterofermenter and 25.9% obligate heterofermenter . Predominant species were L . fermentum, L . plantarum, L . salivarius, and L . rhamnosus . Most of the strains showed moderate to high hydrophobicity and demonstrated high acid and basic surface charges with almost 40% showing salt aggregation . Few strains haemagglutinated . CONCLUSIONS: A variety of Lactobacillus species were isolated from healthy mouths, some of whom showed adhesion-related properties such as high hydrophobicity and charged surfaces . Probable mechanisms related to the ecological behaviour of lactobacilli in the oral cavity are discussed.

Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1217 - 22
Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790; Yamamoto N et al.; A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector . The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity . About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced . Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids . The coding region contained a Gram-positive-type signal peptide of 30 amino acids . The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38 . The putative amino acid sequence showed a significant similarity to a surface layer protein of L . helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.

J Am Dent Assoc, 2000 Jul, 131(7), 887 - 99
The science and practice of caries prevention; Featherstone JD; BACKGROUND AND OVERVIEW: Dental caries is a bacterially based disease . When it progresses, acid produced by bacterial action on dietary fermentable carbohydrates diffuses into the tooth and dissolves the carbonated hydroxyapatite mineral--a process called demineralization . Pathological factors including acidogenic bacteria (mutans streptococci and lactobacilli), salivary dysfunction, and dietary carbohydrates are related to caries progression . Protective factors--which include salivary calcium, phosphate and proteins, salivary flow, fluoride in saliva, and antibacterial components or agents--can balance, prevent or reverse dental caries . CONCLUSIONS: Caries progression or reversal is determined by the balance between protective and pathological factors . Fluoride, the key agent in battling caries, works primarily via topical mechanisms: inhibition of demineralization, enhancement of remineralization and inhibition of bacterial enzymes . CLINICAL IMPLICATIONS: Fluoride in drinking water and in fluoride-containing products reduces caries via these topical mechanisms . Antibacterial therapy must be used to combat a high bacterial challenge . For practical caries management and prevention or reversal of dental caries, the sum of the preventive factors must outweigh the pathological factors.

J Spinal Cord Med, 2000 Summer, 23(2), 136 - 41
Bacterial interference for prevention of urinary tract infection: an overview; Darouiche RO et al.; Urinary tract infection (UTI) is the most common infection in patients with spinal cord injury (SCI) and is a major cause of morbidity and mortality in this population . The bladders of patients with SCI, particularly those with indwelling bladder catheters, can become colonized by a variety of organisms, including those that may, and others that may not, cause symptoms of infection . The latter group of bacteria, so-called benign colonizers, are often left untreated because they may provide some protection against symptomatic infection with more pathogenic bacteria . In recent years, deliberate urogenital tract colonization with benign bacterial strains was studied with the objective of offering some protection against invasion by uropathogenic strains . When well-characterized strains of Lactobacillus sp . were used to colonize the vagina of women prone to frequent UTI, a moderate reduction in the rate of recurrent UTI was observed . In other studies, a non-pathogenic prototype of Escherichia coli (strain 83,972) causing asymptomatic bacteriuria was used for deliberate bladder colonization . These preliminary observations encourage the examination of the safety and preventive efficacy of this approach in human subjects.

Harefuah, 1999 Mar 1, 136(5), 347 - 9, 420
{D-lactic acidosis in short bowel syndrome}; Sela BA et al.; A boy of 20 months and a girl of 27 months who had previously undergone massive bowel resection due to congenital intestinal obstruction are described . During gradual reduction of total parenteral nutrition and initiation of oral feeding, both developed severe, acute metabolic acidosis, accompanied by encephalopathy and ataxia . After the laboratory identification of massive amounts of the d-isomer of lactic acid in urine and blood, both were successfully treated with i.v . bicarbonate, and metronidazole to suppress the overgrowth of colonic lactobacilli responsible for the metabolic crisis.

Med Wieku Rozwoj, 1999 Apr-Jun, 3(2), 293 - 301
{Microbiological evaluation of the vagina in women being screened for inflammatory and precancerous conditions of female genitalia}; Kuczynska K; The aim of this work was to study the microbiology of vaginal secretion in 3665 (19 to 72 year old) women investigated in the screening programme for inflammatory and malignant diseases . Microbiological examinations of the vaginal contents were carried out using direct preparations (Miller and Gram methods) and different media for cultures . Normal biocenosis (I and II grade of cleanliness) of the vagina was observed in 40.74% investigated patients . Abnormal biocenosis was classified as early, advanced and chronic state infections . In our study we present data on isolated strains of saprophytic organisms and pathogens including yeasts and Trichomonas vaginalis in early and chronic infections . There were homogeneous and mixed infections . The role of the physiological Lactobacillus species inhibiting development of pathogenic flora is suggestive of beneficial influence on the vaginal environment . Candidiasis of the vagina was identified in 11.35% examined women . High percentage Candida growth was found together with Lactobacillus vaginalis . It is noteworthy that trichomoniasis was less frequent compared to our previous reports . Totally we were able to isolate 2597 strains of bacteria . Bacteria type of abnormal biocenosis was found in 46.82% . The data from this study indicated that microbiological pictures (stained acc . to both Miller and Gram methods) are still of importance for the evaluation of vaginal contents . The second significant factor in microbiology of human vagina is the role of culture . This role of cultures for microbiologic studies of vaginal contents is beyond any doubt.

Acta Otolaryngol Suppl, 2000, 543, 215 - 9
Persistence of Lactobacillus plantarum DSM 9843 on human tonsillar surface after oral administration in fermented oatmeal gruel . A pilot study; Stjernquist-Desatnik A et al.; The occurrence of Lactobacillus plantarum DSM 9843 on tonsillar scrapings was studied after single-dose administration . Six healthy volunteers gargled 100 ml of fermented oatmeal gruel containing 2 x 10(11) colony forming units (cfu) of Lb . plantarum DSM 9843 for 2 min and then swallowed it . Two healthy volunteers drank 50 ml fermented oatmeal gruel (containing 1 x 10(11) cfu of Lb . plantarum DSM 9843) mixed with 50 ml fruit juice, and in another experiment, 5 ml fermented oatmeal gruel (containing 1 x 10(10) cfu of Lb . plantarum DSM 9843) mixed with 95 ml fruit juice . Lb . plantarum DSM 9843 were found in tonsillar scrapings 4-8 h after intake of 2 x 10(11) cfu, for 5-8 h after intake of 1 x 10(11) cfu, and finally up to 4 h after intake of 1 x 10(10) cfu . On electron microscopy micrographs, short rod-shaped bacteria were visible 1 h after intake of the fermented oatmeal gruel, but not 2 h after intake . The results suggest that Lb . plantarum DSM 9843 possess an ability to adhere to tonsillar cells.

J Dairy Sci, 2000 Jul, 83(7), 1487 - 502
Enzyme, bacterial inoculant, and formic acid effects on silage composition of orchardgrass and alfalfa; Nadeau EM et al.; We evaluated the effects of cellulase (from Trichoderma longibrachiatum) application rates on neutral detergent fiber (NDF) concentration and fermentation products of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages harvested with decreasing dry matter (DM) digestibility . Additionally, the impacts of inoculant (Lactobacillus plantarum and Pediococcus cerevisiae), pectinase (from Aspergillus niger), or formic acid on silage composition were studied . Forages wilted to a DM content of about 320 g/kg were ensiled in laboratory silos for 60 d . Cellulase, combined with inoculant, was applied at 2, 10, and 20 ml/kg of herbage (at least 2500 IU/ml) . Cellulase at 10 ml/kg was also applied alone or in combination with pectinase and inoculant or formic acid . The NDF concentration of orchardgrass silage decreased with increasing cellulase up to 20 ml/kg, at which NDF content was decreased by 30% . The NDF concentration of alfalfa silage decreased with increasing cellulase application up to 10 ml/kg, at which NDF content was decreased by 13% . Immature plants were more responsive to cellulase treatment than mature plants . Cellulase at 2 ml/kg combined with inoculant improved fermentation characteristics of the silages but generally, there was no effect on silage fermentation by higher cellulase applications, resulting in an accumulation of sugar . The improved fermentation of orchardgrass treated with cellulase and inoculant was mostly related to the effect of inoculant, whereas cellulase alone improved fermentation characteristics of alfalfa silage and this effect was enhanced by addition of inoculant . Decreased NDF and increased sugar concentrations did not improve the in vitro DM digestibility of cellulase-treated silages.

Can Vet J, 2000 Jul, 41(7), 547 - 54
Characterization of acute interstitial pneumonia in cattle in southern Alberta feedyards; Ayroud M et al.; Field data were collected over 2 consecutive years to characterize acute interstitial pneumonia (AIP) in feedyard cattle . Thirty-eight cattle with clinical symptoms of AIP were examined following emergency slaughter; 31 (all heifers) were confirmed to have AIP on the basis of gross and histological lung pathology . The 7 without AIP, plus 17 asymptomatic penmates, were used as contemporary controls . Plasma concentrations of 3-methylindole (3MI) metabolites were higher (P < 0.001) in heifers afflicted with AIP than in the control animals, and concentrations of 3MI mercapturates in the urine were lower (P < 0.007) in affected heifers . Concentrations of 3MI adducts in lung tissue and in microsomal protein did not differ (P > 0.05) between the 2 groups, and 3MI was not detected in ruminal fluid from either group . Total ruminal bacterial numbers and populations of lactobacilli and protozoa were similar (P > 0.05) between the AIP-positive and unafflicted groups, but fewer (P < 0.05) cellulolytic bacteria were present in the positive group . Bovine respiratory syncytial virus antigen was not found in lung tissue from any of the heifers confirmed to have AIP . To our knowledge, this study is the first to implicate 3MI metabolites as having a role in feedyard AIP . Further research is required to determine the factors responsible for the elevation in 3MI adducts in plasma and urine of feedyard cattle afflicted with AIP.

Anal Biochem, 2000 Aug 1, 283(2), 266 - 75
Determination of blood folate using acid extraction and internally standardized gas chromatography-mass spectrometry detection; Dueker SR et al.; Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level . Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status . Here, an acid extraction GCMS detection method that measures total folate whole blood is presented . Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of {(13)C(6)}pABA as internal standard (IS) . The hydrolysate is passed over a C18 resin to remove heme . The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated . Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives . The abundance of these derivatives are measured at m/z 324 for {(13)C(6)}pABA as IS and m/z 318 for pABA from whole blood folate . Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay . The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method . This feature makes it useful in monitoring folate status of patients undergoing chemotherapy . Before using our method, pABA supplements must be discontinued for a few days .

Minerva Ginecol, 2000 Apr, 52(4), 111 - 21
{Role of pH as a regulator of vaginal physiological environment}; Melis GB et al.; In the vagina there are several microorganisms . Their survival is fundamental to have a physiological environment in the vagina (ecosystem) . The same vaginal environment playg a fundamental role to guarantee tho survival of resident microorganisms . Several factors, like changes of temperature and oxygenation, can interfere on vaginal ecosystem, but the resident vaginal microorganisms are the main factors to stabilize the vaginal ecosystem . The Doderlein's lactobacillus is the predominant vaginal microorganism . It is capable of fermenting the glycogen deriving from the decline of the eutrophic vaginal mmucosa, to lactic acid with release of hydrogen ions . The final result of that metabolism is an acid pH with values between 4-4.5 . Vaginal pH undergoes physiologically changes from birth to menopause, according to changes of ovarian steroids occurring during woman's life . Adequate levels of estrogens play a fundamental role in the trophism of vaginal mucosa . In fact, estrogens increase the cellular content of glycogen . Exogenous acbvities on vaginal pH can be exerted by several factors, such as sexual activily, oral contraceptives, systemic diseases, vaginal infections (candidosis, thrichomonias, vaginosis), systemic or local therapies . They incrcase vaginal pH by acting through different mechanisms . The increase of vaginal pH is detrimental for the survival of Doderlein's lactobacillus, but not for the pathogenetic microorganisms whose replication, on the contrary, is favored by the absence of contraction exerted by Doderlein's bacillus . It has been showed that local acidifying substances (lactic acid, lactobacillus and substances recently synthesized like alpha-aminovalerianic acid, policarbophil and carbopol 934) are useful in restoring the biological and chemical characteristics of the vaginal ecosystem.

Micron, 2001 Jan, 32(1), 59 - 65
Phage-related DNA polymorphism in dairy and probiotic Lactobacillus; Brandt K et al.; Various DNA-based methods are presently being applied for identification of industrial bacterial cultures including dairy starter and probiotic strains of Lactobacillus . The success of strain-specific identification depends on the power of the DNA-based methods to reveal intraspecies DNA polymorphism . This study reveals that all eleven arbitrarily chosen Lactobacillus rhamnosus starter, laboratory and probiotic strains contain Lb . rhamnosus phage Lc-Nu related nucleotide sequences . One of these highly homologous regions in the genome of phage Lc-Nu was the 2.4kb HindIII fragment, which has been sequenced . Nucleotide sequence analysis suggested that one side of the 2.4kb HindIII fragment encodes a phage Lc-Nu helicase and accordingly represents an early gene region of phage Lc-Nu genome . Five forward and five reverse primers were derived from the nucleotide sequence of the 2.4kb HindIII fragment of phage Lc-Nu DNA for PCR-based identification of the eleven Lb . rhamnosus strains included in this study . Six different types of PCR product patterns were obtained . Among the patterns three were unique to particular Lb . rhamnosus strains . The results suggest that phage-related DNA sequences are, surprisingly, distributed widely among the Lb . rhamnosus strains, and that these sequences could also be a source of DNA polymorphism to apply for DNA-based identification of bacterial strains . Phage Lc-Nu related DNA homology was also found in the chromosome of Lb . casei, the species closely related to Lb . rhamnosus.

J Agric Food Chem, 2000 Jul, 48(7), 3044 - 51
Combined effect of sourdough lactic acid bacteria and additives on bread firmness and staling; Corsetti A et al.; The effect of various sourdoughs and additives on bread firmness and staling was studied . Compared to the bread produced with Saccharomyces cerevisiae 141, the chemical acidification of dough fermented by S . cerevisiae 141 or the use of sourdoughs increased the volume of the breads . Only sourdough fermentation was effective in delaying starch retrogradation . The effect depended on the level of acidification and on the lactic acid bacteria strain . The effect of sourdough made of S . cerevisiae 141-Lactobacillus sanfranciscensis 57-Lactobacillus plantarum 13 was improved when fungal alpha-amylase or amylolytic strains such as L . amylovorus CNBL1008 or engineered L . sanfranciscensis CB1 Amy were added . When pentosans or pentosans, endoxylanase enzyme, and L . hilgardii S32 were added to the same sourdough, a greater delay of the bread firmness and staling was found . When pentosans were in part hydrolyzed by the endoxylanase enzyme, the bread also had the highest titratable acidity, due to the fermentation of pentoses by L . hilgardii S32 . The addition of the bacterial protease to the sourdough increased the bread firmness and staling.

Int J Food Microbiol, 2000 Jun 30, 58(1-2), 93 - 106
Shelf life of modified atmosphere packed cooked meat products: addition of Na-lactate as a fourth shelf life determinative factor in a model and product validation; Devliegher F et al.; Cooked meat products are often post-contaminated because of a packaging and/or slicing step after the pasteurisation process . The shelf life is therefore limited and can be extended by adding Na-lactate . A previously developed model for the spoilage of gas packed cooked meat products, including temperature, water activity and dissolved CO2 as independent variables, was extended with a fourth factor: the Na-lactate concentration in the aqueous phase of the meat product . Models were developed for the maximum specific growth rate mu(max) and the lag phase lambda of the specific spoilage organism Lactobacillus sake subsp . carnosum . Quadratic response surface equations were compared with extended Ratkowsky models . In general, response surface equations fitted the experimental data best but in the case of mu(max) the response surface model predicted illogical growth behaviour at low water activities and high Na-lactate concentrations . A extensive product validation of the mathematical models was performed by means of inoculated as well as naturally contaminated industrially prepared cooked meat products . The deviations of the experimentally determined versus predicted growth parameters in inoculated cooked meat products were in general small . Both types of models were also able to predict the shelf life of naturally contaminated cooked meat products, except for pate where an under-estimation of the shelf life was predicted by the response surface equations . The validation studies revealed higher accuracy of the extended Ratkowsky models in comparison to the response surface equations . A significant shelf life extending effect of Na-lactate was predicted, which was more pronounced at low refrigerated temperatures . A synergistic effect has also been noticed between Na-lactate and carbon dioxide which, at least partly, could be explained by the pH-decreasing effect of CO2.

Pediatr Surg Int, 2000, 16(4), 237 - 42
Evaluation of probiotic treatment in a neonatal animal model; Lee DJ et al.; The clinical use of probiotic agents such as enteral Lactobacillus to enhance intestinal defense against potential luminal pathogens has been tested in vivo; however, an understanding of the mechanisms responsible for the observed protection is lacking . The purpose of this study was to evaluate the effects of Lactobacillus on bacterial translocation (BT) in a neonatal animal model . Newborn New Zealand white rabbit pups were enterally fed a 10% Formulac solution inoculated with or without a 10(8) suspension of ampicillin-resistant Escherichia coli K1 (E . coli K1A) and/or Lactobacillus casei GG (Lacto GG) . Pups received either no bacteria (n = 10), Lacto GG (n = 8), E . coli K1A (n = 26), or a combination of Lacto GG and E . coli K1A (n = 33) . On day 3, representative tissue specimens from the mesenteric lymph nodes (MLN), spleen (SPL), and liver (LIV) were aseptically harvested in addition to a small-bowel (SB) sample that was rinsed to remove luminal contents . The specimens were then cultured in organism-specific media . Statistical analysis was by one-way ANOVA with P values less than 0.05 considered significant . Neonatal rabbits receiving Lacto GG-supplemented formula exhibited a 25% decrease (P < 0.05) in small-bowel colonization by E . coli K1A . In addition, Lacto GG decreased the frequency of extraintestinal BT by 46% (P < 0.05), 61% (P < 0.05), and 23%, respectively, in the MLN, SPL, and LIV . We have shown that enterally-administered Lacto GG decreases the frequency of E . coli K1A translocation in a neonatal rabbit model . These results may have significant implications for the treatment of BT and sepsis in the human neonate and provide a model for further studies.

Arch Microbiol, 2000 May-Jun, 173(5-6), 383 - 9
Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species; McCracken A et al.; Promoter-active fragments were isolated from the genome of the probiotic organism Lactobacillus rhamnosus strain GG using the promoter-probe vector pNZ272 . These promoter elements, together with a promoter fragment isolated from the vaginal strain Lactobacillus fermentum BR11 and two previously defined promoters (Lactococcus lactis and Lactobacillus acidophilus ATCC 4356 slpA), were introduced into three strains of Lactobacillus . Primer-extension analysis was used to map the transcriptional start site for each promoter . All promoter fragments tested were functional in each of the three lactobacilli and a purine residue was used to initiate transcription in most cases . The promoter elements encompassed a 52- to 1,140-fold range in promoter activity depending on the host strain . Lactobacillus promoters were further examined by surveying previously mapped sequences for conserved base positions . The Lactobacillus hexamer regions (-35: TTgaca and -10: TAtAAT) closely resembled those of Escherichia coli and Bacillus subtilis, with the highest degree of agreement at the -10 hexamer . The TG dinucleotide upstream of the -10 hexamer was conserved in 26% of Lactobacillus promoters studied, but conservation rates differed between species . The region upstream of the -35 hexamer of Lactobacillus promoters showed conservation with the bacterial UP element.

Oral Microbiol Immunol, 1999 Dec, 14(6), 391 - 2
Antimicrobial effect of acidified nitrite on cariogenic bacteria; Silva Mendez LS et al.; The antimicrobial agent nitric oxide (NO) is formed in the mouth and its concentration is directly related to salivary nitrite, which in turn is related to dietary nitrate intake . The aim of this study was to determine whether nitrite under acidic conditions will have an inhibitory effect, possibly occurring through NO production, on Streptococcus mutans, Lactobacillus casei and Actinomyces naeslundii . Whereas the growth of S . mutans was inhibited by a more acid pH, the addition of nitrite caused a marked, further dose-dependent reduction in bacterial numbers after 24 h of exposure . Similar effects were observed with A . naeslundii and L . casei . The ability of these bacteria to recover from nitrite exposure was also markedly affected by nitrite concentration . At acidity levels below pH 7, low concentrations of nitrite (0.2 mM) caused effective complete killing of S . mutans, with similar effects on the other organisms tested . These results demonstrate the possibility that nitrite in saliva has an effect on the growth and survival of cariogenic bacteria.

Virology, 2000 Jul 20, 273(1), 101 - 11
Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei; Moscoso M et al.; Adjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication . Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand . Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase . Orf770 showed similarity to putative plasmid and phage DNA primases . Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection . When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation . It is deduced that this stretch contains the origin of replication of phage A2 .

Lett Appl Microbiol, 2000 Jul, 31(1), 82 - 6
Adhesion of inactivated probiotic strains to intestinal mucus; Ouwehand AC et al.; It has been suggested that probiotics should be viable in order to elicit beneficial health effects . Inactivation of probiotics has been suggested to interfere with the binding to the mucosa and thereby with the immune modulating activity of probiotics . The effect of different inactivation methods on the mucus adhesion of nine probiotic strains was studied . Inactivation by heat or gamma-irradiation generally decreased the adhesive abilities . However, heat treatment increased the adhesion of Propionibacterium freudenreichii and gamma-irradiation enhanced the adhesion of Lactobacillus casei Shirota . Inactivation by u.v . was not observed to modulate the adhesion of the tested strains and it was concluded to be the most appropriate method for studying non-viable probiotics and preparing control products.

Cancer Immunol Immunother, 2000 Jun, 49(3), 157 - 64
Antitumor effect of heat-killed Lactobacillus plantarum L-137 through restoration of impaired interleukin-12 production in tumor-bearing mice; Murosaki S et al.; We have previously reported that heat-killed Lactobacillus plantarum L-137 is a potent inducer of interleukin-12 (IL-12) in vivo as well as in vitro in mice . In order to develop effective usage of L . plantarum L-137 for tumor immunotherapy, we examined its antitumor effect against DBA/2 mice inoculated with syngenic P388D1 tumor cells in different treatment schedules . Daily injection of L . plantarum L-137 from the day of tumor inoculation induced a steep increase in plasma IL-12 only after the first injection but not after subsequent injections, and had no effect on tumor growth and survival time . In contrast, daily injection of L . plantarum L-137 from the 7th day after tumor inoculation exerted a marked antitumor effect but such an effect was not evident in mice treated with L . plantarum L-137 twice a week from the 7th day . IL-12 production was considerably impaired at the first injection but steeply increased after the third injection in the mice injected daily with L . plantarum L-137 from the 7th day . Our results suggest that daily administration of L . plantarum L-137 is required to exert an antitumor effect at the late stages of tumor development when IL-12 production is considerably impaired.

Biosci Biotechnol Biochem, 2000 May, 64(5), 1032 - 7
Application of a bioluminescence method for the beer industry: sensitivity of MicroStar-RMDS for detecting beer-spoilage bacteria . Rapid Microbe Detection System; Takahashi T et al.; We set up the original operating conditions of the MicroStar-Rapid Microbe Detection System (RMDS) to suppress false positives, which have kept this system from practical . The detection limit of our system was between 6.3 x 10(-16) mol and 3.1 x 10(-16) mol in terms of the amount of ATP, which is approximately equal to the ATP content of one yeast cell or 50 lactic acid bacteria cells . The detection time and the detection count were compared between the RMD method and the conventional plate count method (C.P.C . method) using 23 test samples of beer-spoilage Lactobacillus brevis . Judging from the detection time and detection count, 16-24 hours of cultivation for the RMD method corresponded to 40-96 hours of cultivation for the C.P.C . method . The RMD method reached a useful level for our practical use at the point of sensitivity.

Appl Environ Microbiol, 2000 Jul, 66(7), 3093 - 7
Isolation of tannin-degrading lactobacilli from humans and fermented foods; Osawa R et al.; Lactobacilli with tannase activity were isolated from human feces and fermented foods . A PCR-based taxonomic assay revealed that the isolates belong to Lactobacillus plantarum, L . paraplantarum, and L . pentosus . Additional studies on a range of Lactobacillus species from established culture collections confirmed that this enzymatic activity is a phenotypic property common to these three species.

Appl Environ Microbiol, 2000 Jul, 66(7), 2951 - 8
Gene cloning and expression and secretion of Listeria monocytogenes bacteriophage-lytic enzymes in Lactococcus lactis; Gaeng S et al.; Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions . Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan . Ply118 is a 30.8-kDa L-alanoyl-D-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-L-alanine amidase . In order to establish dairy starter cultures with biopreservation properties against L . monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone . The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study . High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels . Therefore, ply511 was genetically fused with the (SP)slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide . Expression of (SP)slpA-ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L . lactis cells . One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3'-end coding sequence of (SP)slpA-ply511, which caused a frameshift mutation and generated a stop codon . Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511 Delta(S262-K341) mutant polypeptide strongly increased its lytic activity . Proteolytic processing of the secretion competent (SP)SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity . Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L . monocytogenes cells . Moreover, transformation of pSL-PL511 delta C into L . lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.

J Dairy Sci, 2000 Jun, 83(6), 1396 - 401
Influence of salting procedure on the composition of Muenster-type cheese; Ponce De Leon-Gonzalez LP et al.; Muenster-type cheeses were salted with a traditional saturated brine solution or by direct addition of salt to the curd . Cheeses were evaluated at 0, 30, 60, 90, 120, and 180 d of age for numbers and type of microflora, casein hydrolysis, and amounts of free fatty acids . No significant differences were found in the populations of starter, lactobacilli, or yeast for the brine- and direct-salted cheeses . The amounts of free fatty acids liberated were similar for both cheeses . The hydrolysis of alpha s1-casein was complete at 90 d of age, whereas only 40% of the beta-casein was hydrolyzed at 180 d of age . The inner layer of the brine-salted cheeses had the highest number of starter microorganisms, followed by the middle and outer layers, respectively . The salt concentrations were similar in the three layers after 4 mo of age . Results of this study showed that comparable Muenster-type cheese could be produced with either of the salting procedures . With direct salt addition to curd, a 59% reduction was observed in salt emissions from the Muenster manufacturing process.

Carbohydr Res, 2000 Jun 2, 326(2), 113 - 9
Structural elucidation of the viscous exopolysaccharide produced by Lactobacillus helveticus Lb161; Staaf M et al.; A viscous extracellular polysaccharide produced by Lactobacillus helveticus Lb161 isolated from raw milk has been investigated . Sugar and methylation analysis, and 1H and 13C NMR spectroscopy revealed that the polysaccharide is composed of a heptasaccharide repeating unit . The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond connectivity experiments . The structure of the repeating unit of the exopolysaccharide from L . helveticus Lb161 is as follows: carbohydrate structure {see text} . The polysaccharide contains approximately 0.6 equivalents of O-acetyl group per repeating unit (not located).

Clin Chim Acta, 2000 Aug, 298(1-2), 99 - 109
Assay of D-lactate in urine of infants and children with reference values taking into account data below detection limit; Haschke-Becher E et al.; Accumulation of D-lactic acid produced by intestinal bacteria such as streptococci and lactobacilli has been extensively studied in ruminants {1-4} . In humans an increased production of D-lactate by intestinal bacteria under pathological conditions such as the short bowel syndrome can cause metabolic acidosis {5-8} . Since the lactate assays routinely used only measure L-lactate we developed a sensitive method of D-lactate quantification and established reference values in spot urines of infants and children (0 to 4 years of age) . The enzymatic method with fluorimetric quantification of NADH is linear up to 2 mmol/l . It has a detection limit of 3.4 micromol/l . Among structurally related organic acids an interference was found only for L-lactate and DL-2-hydroxybutyrate at concentrations which are way beyond their physiological excretion . One hundred and sixty five spot urines of healthy Swiss (S), Austrian (A), German (G) and Chilean (CHI) infants aged from 0 to 4 years were analyzed . The distribution of the data is close to a lognormal one . Values below the detection limit were simulated and age groups were formed . In all populations D-lactate excretion was found highest during the first year of life; it declines with age during infancy and remains stable from 2.5 to 4 years of age . We show that D-lactate is excreted physiologically by healthy infants and children below 4 years of age and present reference values for D-lactate excretion which show some differences between the populations tested.

Eur J Oral Sci, 2000 Jun, 108(3), 255 - 8
Decreasing prevalence of salivary lactobacilli in Swedish schoolchildren 1987-1998; Nylander A et al.; The prevalence and number of salivary lactobacilli was determined in all schoolchildren starting the 7th grade (12-13 yr; n = 1,578) in comprehensive school located in Kumla, Sweden between 1987 and 1998 . Whole saliva samples were collected and transferred to dip-slides (Dentocult-LB) and incubated in room temperature for 7 d . Caries data were collected from the dental records and from bitewing radiographs . Both the number of lactobacilli and the prevalence of manifest caries and restorations exhibited a decreasing tendency and were significantly lower in 1998 than in 1987 . The mean caries prevalence (DFS) declined from 5.2 to 1.8 . In 1987, 45% of the children harboured high or very high lactobacilli counts ( > or =10(5) CFU/ml) in their saliva compared to approximately 10-15% at the later examinations . The proportion of children with no detectable counts varied between 4-29% during the study period, while children with low counts (10(3)-10(4) CFU/ml saliva) constituted a clear majority at the recent samplings . With such low levels in the population, the use of lactobacilli counts as a didactic tool in dietary counselling must be called into question.

J Med Primatol, 2000 Feb, 29(1), 36 - 41
Vaginal myeloperoxidase and flora in the pig-tailed macaque; Lichtenwalner AB et al.; Myeloperoxidase (MPO) is an enzyme in neutrophils and monocytes which reacts with H2O2 and chloride to kill microbes after phagocytosis . Instillation of MPO into the vagina may augment vaginal defenses against sexually transmitted diseases, since the normal vaginal flora is characterized by the presence of H2O2-producing lactobacilli . We assessed the menstrual cycle stage, vaginal flora, pH, macroscopic appearance, and endogenous MPO in the adult female pig-tailed macaque (Macaca nemestrina) at baseline (n = 26; 60 observations) and at 0, 4, and 24 hours in untreated animals (n = 6) or in animals treated with intravaginal MPO gel at time 0 (n = 5) . Baseline MPO levels were highly variable, and there was no detectable effect of cycle stage . In untreated animals, there was no significant effect of vaginal swab collection on vaginal flora or MPO levels . MPO treatment did not reduce vaginal H2O2-producing organisms, and vaginal MPO levels tended to increase at 4 hours in treated animals . Vaginal/cervical colposcopic changes were not detected in either group.

Int J Food Microbiol, 2000 Jun 15, 57(3), 183 - 92
Predictive microbiology and food safety; Soboleva TK et al.; The evaluation of risk in food safety requires knowledge of the probability that microbial population sizes will not exceed defined levels . This probability is evaluated assuming that the growth of the microbial population can be described by the Gompertz equation with the variance of growth depending on the population size . It is shown that the probability density associated with this phenomenon is skewed, so that the risk of a high microbial population is greater than that which would be estimated using a symmetrical probability distribution such as the Gaussian . Maximum likelihood estimates of the parameters of the Gompertz equation based on the derived probability density are calculated using data published by Zwietering et al . {23} for the growth of Lactobacillus plantarum under different temperatures . The probability that a microbial population of a given size will exceed an unacceptable level within a given time is calculated for growth at two temperatures, 10 and 25 degrees C . The implication of these theoretical results for the management of risk in food safety and in the design of hazard analysis critical control point procedures is discussed.

Caries Res, 2000 May-Jun, 34(3), 268 - 74
Effects of dental treatment and single application of a 40% chlorhexidine varnish on mutans Streptococci in young children under intravenous anaesthesia; van Lunsen DM et al.; Clinical studies suggest that application of a highly concentrated chlorhexidine varnish results in a decrease in the number of mutans streptococci and thereby a decrease in the caries risks . The aim of this study was to determine the effect of dental treatment on the levels of mutans streptococci (MS) and lactobacilli (LB) and the additional effect of a single application of 40% chlorhexidine varnish (EC40) on the level of MS . Twenty-three children under the age of 5 years scheduled for full dental treatment were selected . Of these 23 children (mean dmf-s 27.1, SD 19.3), 11 children had nursing bottle caries . The mean sugar exposure was 6.4 . Subjects were randomly distributed into two groups of approximately equal size . One group received an EC40 application after dental treatment, while the other group received only full dental treatment, both with intravenous anaesthesia with propofol as a single drug . An unstimulated saliva sample and a plaque sample were taken prior to dental treatment . The saliva and plaque sampling of the subjects was repeated after 6 weeks . MS and LB were isolated and counted . The number of children harbouring more than 10(6) MS in a pooled plaque sample decreased significantly from 8 to 2 children 6 weeks after dental treatment . No additional effect of EC40 was found . The number of salivary MS did not change significantly between the groups or before and after treatment . The figures for LB remained at a high level of more than 10(4)/ml saliva before (21 children) and 6 weeks after treatment (17 children) . The results of this study indicate that dental treatment results in a significant suppression of plaque MS, while a single application of EC40 showed no significant additional suppression after 6 weeks.

Microbes Infect, 2000 Apr, 2(5), 543 - 6
Role played by lactobacilli in controlling the population of vaginal pathogens; Boris S et al.; The role of Lactobacillus species in the female urogenital tract as a barrier to infection is of considerable interest . These organisms are believed to contribute to the control of vaginal microbiota by competing with other microorganisms for adherence to epithelial cells and by producing antimicrobial compounds . These bactericidal compounds include organic acid, which lowers the vaginal pH, hydrogen peroxide, bacteriocin-like substances and possibly biosurfactants.

Gut, 2000 Jul, 47(1), 79 - 87
Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures; Haller D et al.; BACKGROUND AND AIM: Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis . Reactivity of IEC to bacterial signals may depend on interactions with immunocompetent cells . To address the question of whether non-pathogenic bacteria modify the immune response of the intestinal epithelium, we co-cultivated enterocyte-like CaCO-2 cells with human blood leucocytes in separate compartments of transwell cultures . METHODS: CaCO-2/PBMC co-cultures were stimulated with non-pathogenic bacteria and enteropathogenic Escherichia coli . Expression of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, IL-8, monocyte chemoattracting protein 1 (MCP-1), and IL-10 was studied by enzyme linked immunosorbent assays (cytokine secretion) and by semiquantitative reverse transcription-polymerase chain reaction . RESULTS: Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1beta, and TNF-alpha mRNA in the presence of underlying leucocytes . Leucocyte sensitised CaCO-2 cells produced TNF-alpha and IL-1beta whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells . CaCO-2 cells alone remained hyporesponsive to the bacterial challenge . Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mRNA in leucocyte sensitised CaCO-2 cells . TNF-alpha was identified as one of the early mediators involved in cellular cross talk . In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenic E coli and non-pathogenic bacteria . CONCLUSION: The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes . These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.

Cryobiology, 2000 May, 40(3), 264 - 9
Cryoprotectants lead to phenotypic adaptation to freeze-thaw stress in Lactobacillus delbrueckii ssp . bulgaricus CIP 101027T; Panoff JM et al.; This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp . bulgaricus CIP 101027T . Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose . Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol . Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties . These results suggest that bacteria such as L . delbrueckii ssp . bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress .

Int J Food Microbiol, 2000 May 25, 56(1), 71 - 80
Microgradients in bacterial colonies: use of fluorescence ratio imaging, a non-invasive technique; Malakar PK et al.; Fluorescence ratio imaging is a non-invasive technique for studying the formation of microgradients in immobilised bacterial colonies . These gradients can be quantified easily when combined with the gel cassette system designed at the Institute of Food Research, Norwich, UK . Colonies of Lactobacillus curvatus were observed using this technique and relevant pH gradients were present when the colonies reached a diameter of about 100 microm . These pH gradients were due to production of lactic acid by L . curvatus cells in the colonies . The spatial resolution of the images was about 1.5 microm (scale of bacterial cells) and therefore very suitable for observing local effects in colonies which ranged in sizes from 1 to 500 microm.

Int J Food Microbiol, 2000 Jun 1, 56(2-3), 227 - 30
UV-induced Lactobacillus gasseri mutants resisting sodium chloride and sodium nitrite for meat fermentation; Arihara K et al.; Lactobacillus gasseri, one of the predominant lactobacilli in human intestinal tracts, is utilized for probiotics and dairy starter cultures . However, since L . gasseri is relatively sensitive to sodium chloride and sodium nitrite (essential compounds for meat products), it is difficult to utilize this species for conventional fermented meat products . In this study, efforts were directed to generate mutants of L . gasseri resisting sodium chloride and sodium nitrite . UV irradiation of the strain of L . gasseri JCM1131(T) generated several mutants resisting these compounds . A mutant strain 1131-M8 demonstrated satisfactory growth in meat containing 3.3% sodium chloride and 200 ppm sodium nitrite . Although proteins extracted from the cell surface of 1131-M8 were slightly different from those of the original strain, other biochemical characteristics of both strains were indistinguishable . These results suggest that the L . gasseri mutant obtained in this study could be utilized as a starter culture to develop probiotic meat products.

Int J Food Microbiol, 2000 Jun 1, 56(2-3), 219 - 25
High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef; Vold L et al.; The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated . The background flora from eight different commercial ground beef were added to ground beef spiked with E . coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C . The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E . coli O157:H7 both aerobically and anaerobically . Inhibition was more pronounced under anaerobic conditions . The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei . These results show the importance of the natural background flora in meat for inhibition of growth of E . coli O157:H7.

Int J Food Microbiol, 2000 Jun 1, 56(2-3), 133 - 43
The spoilage microflora of cured, cooked turkey breasts prepared commercially with or without smoking; Samelis J et al.; Lactobacillus sakei subsp . carnosus was predominant in the spoilage flora of sliced, vacuum-packed, smoked, oven-cooked turkey breast fillets which developed mild, sour spoilage flavors after 4 weeks storage at 4 degrees C . In contrast, Leuconostoc mesenteroides subsp . mesenteroides predominated in the spoilage flora of sliced, vacuum-packed, unsmoked, boiled turkey breast fillets from the same plant which were also stored at 4 degrees C . The spoilage flora of the unsmoked breasts grew faster than that of the smoked breasts and was more diverse . Lactobacillus sakei, Weissella viridescens and an atypical group of leuconostoc-like bacteria were also members of the unsmoked turkey breasts flora . Consequently, the unsmoked breasts spoiled after 2 weeks at 4 degrees C: the packs swelled and the meat developed strong sour odors and flavors and abundant slime . Except for the unidentified leuconostocs, which apparently survived boiling of the unsmoked turkey, all the spoilage organisms contaminated the meats during the slicing and vacuum packaging operations . From their biochemical reactions and cellular fatty acid profiles, the atypical leuconostocs were more closely related to Leuconostoc carnosum than W . viridescens . Carnobacteria and Brochothrix thermosphacta were present in relatively large numbers on the raw turkey, but were not numerous in the spoilage flora of the cooked, vacuum-packed meat products.

FEMS Microbiol Lett, 2000 Jun 15, 187(2), 167 - 73
Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA; Song Y et al.; Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level . Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp . bulgaricus and ssp . lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp . paracasei and ssp . tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp . salicinius and ssp . salivarius) . The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay . These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

Curr Microbiol, 2000 Aug, 41(2), 136 - 41
Adherence of Lactobacillus to intestinal 407 cells in culture correlates with fibronectin binding; Kapczynski DR et al.; Lactobacilli are members of the normal mucosal microflora of most animals . Many isolates of Lactobacillus spp . are adherent to epithelial cells . In this study, using Lactobacillus acidophilus and L . agilis, we detected adherence in a pattern that suggested that the bacteria were binding to extracellular matrix proteins . Fluorescent microscopy, by using anti-fibronectin antibody, demonstrated that the isolates localize in those areas where fibronectin was detected . In addition, fibronectin pretreatment of the bacterial cells decreased adherence to Intestinal 407 epithelial cell monolayers . Cellular binding to fibronectin was detected by enzyme-linked immunosorbent assay and affinity binding to radio-labeled fibronectin . Fibronectin may be one of the eukaryotic receptors mediating attachment of Lactobacillus to mucosal surfaces.

J Biol Chem, 2000 Aug 25, 275(34), 26599 - 606
Molecular analysis of murine leukemia cell lines resistant to 5, 10-dideazatetrahydrofolate identifies several amino acids critical to the function of folylpolyglutamate synthetase; Zhao R et al.; Four L1210 murine leukemia cell lines resistant to 5, 10-dideazatetrahydrofolate (DDATHF) and other folate analogs, but sensitive to continuous exposure to methotrexate, were developed by chemical mutagenesis followed by DDATHF selective pressure . Endogenous folate pools were modestly reduced but polyglutamate derivatives of DDATHF and ALIMTA (LY231514, MTA) were markedly decreased in these mutant cell lines . Membrane transport was not a factor in drug resistance; rather, folypolyglutamate synthetase (FPGS) activity was decreased by >98% . In each cell line, FPGS mRNA expression was unchanged but both alleles of the FPGS gene bore a point mutation in highly conserved domains of the coding region . Four mutations were in the predicted ATP-, folate-, and/or glutamate-binding sites of FPGS, and two others were clustered in a peptide predicted to be beta sheet 5, based on the crystal structure of the Lactobacillus casei enzyme . Transfection of cDNAs for three mutant enzymes into FPGS-null Chinese hamster ovary cells restored a reduced level of clonal growth, whereas a T339I mutant supported growth at a level comparable to that of the wild-type enzyme . The two mutations predicted to be in beta sheet 5, and one in the loop between NH(2)- and COOH-terminal domains did not support cell growth . When sets of mutated cDNAs were co-transfected into FPGS-null cells to mimic the genotype of drug-selected resistant cells, clonal growth was restored . These results demonstrate for the first time that single amino acid substitutions in several critical regions of FPGS can cause marked resistance to tetrahydrofolate antimetabolites, while still allowing cell survival.

J Bacteriol, 2000 Jun, 182(12), 3416 - 22
In Lactobacillus plantarum, carbamoyl phosphate is synthesized by two carbamoyl-phosphate synthetases (CPS): carbon dioxide differentiates the arginine-repressed from the pyrimidine-regulated CPS; Nicoloff H et al.; Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis . Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO(2) . Lactobacillus plantarum, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagoz et al., Gene 182:37-43, 1996) and an arginine-repressed CPS (CPS-A) . Sequencing has shown that CPS-A is encoded by carA (GLN) and carB (SYN) . Transcriptional studies have demonstrated that carB is transcribed both monocistronically and in the carAB arginine-repressed operon . CP biosynthesis in L . plantarum was studied with three mutants (DeltaCPS-P, DeltaCPS-A, and double deletion) . In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed . CPS-P produced enough CP for both pathways . In CO(2)-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis . Therefore, the uracil sensitivity observed in prototrophic wild-type L . plantarum without CO(2) enrichment may be due to the low affinity of CPS-A for its substrate CO(2) or to regulation of the CP pool by the cellular CO(2)/bicarbonate level.

Clin Infect Dis, 2000 Jun, 30(6), 901 - 7 Epub 2000 Jun 13.
Influence of the normal menstrual cycle on vaginal tissue, discharge, and microflora; Eschenbach DA et al.; The objective of this study was to examine genital tissue, vaginal fluid, and vaginal microbial flora at 3 phases of the menstrual cycle in asymptomatic women . Vaginal examinations were performed 3 times in 74 women: at the menstrual phase (days 1-5), the preovulatory phase (days 7-12), and the postovulatory phase (days 19-24) . Flora of 50 women without bacterial vaginosis (BV) was analyzed separately from flora of 24 women with BV . The volume of vaginal discharge increased and the amount of cervical mucus decreased over the menstrual cycle . Among subjects without BV, the rate of recovery of any Lactobacillus changed little (range, 82% to 98%; P = .2); however, a small increase occurred in the rate of recovery of heavy (3+ to 4+ semiquantitative) growth of Lactobacillus over the menstrual cycle (P = .04) . A linear decrease occurred in the rate of recovery of heavy growth of any non-Lactobacillus species, from 72% at days 1-5 to 40% at days 19-24 (P = .002) . A linear decrease also occurred in the rate of recovery of Prevotella species, from 56% on days 1-5 to 28% on days 19-24 (P = . 007), while a small linear increase occurred in the rate of recovery of Bacteroides fragilis (P=.05) . Among subjects with BV, the only significant change was an increase in the rate of recovery of Lactobacillus, from 33% at days 1-5 to 54% at days 19-24 (P = .008) . Among all subjects, the rate of recovery of heavy growth of Lactobacillus increased over the menstrual cycle and, in contrast, the concentration of non-Lactobacillus species tended to be higher at menses, which is evidence that the vaginal flora becomes less stable at this time.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 721 - 30
Lactobacillus plantarum amylase acting on crude starch granules . Native isoforms and activity changes after limited proteolysis; Florencio JA et al.; The microheterogeneous native amylolytic complex secreted by the isolate A6 of Lactobacillus plantarum revealed a selective enzyme specificity loss when submitted to a limited proteolysis under a suboptimum pH condition . A clear electrophoretic profile change toward just one shorter, more acidic, and equally active polypeptide fragment resulted from the pronase E pretreatment . Although the whole enzyme activity remained apparently unaffected for soluble starch, the native parallel activity on intact and non-gelatinized starch granules either from cereals or tubers was dramatically reduced . This phenomenon was more clearly documented by scanning electron microscopy using the easiest accessible native substrate: wheat starch granules . The anion-exchange-purified native enzymes from L . plantarum displayed a different optimum pH curve when compared with the thermotolerant alpha-amylase from Bacillus licheniformis . The alpha-amylases from the lactic-acid-producing A6 isolate presented an electrophoretic profile easily distinguishable from those from B . liqueniformis and B . subtilis species.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 665 - 77
High-yield fermentation of pentoses into lactic acid; Iyer PV et al.; Lactobacillus species capable of fermenting glucose are generally incapable of utilizing xylose for growth or fermentation . In this study, a novel aspect of a well-known Lactobacillus strain, L . casei subsp . rhamnous (ATCC 10863), was uncovered: it can ferment xylose as efficiently as glucose . This strain is a registered organism, extremely stable on long-term operation . Fermentation by this strain is characterized by an initial lag phase lasting 24-72 h before xylose consumption takes place . The yield (grams/gram) of lactic acid from xylose is in excess of 80% with initial volumetric productivity of 0.38 g/(L.h) . Acetic acid is the primary byproduct formed at the level of about 10% of the lactic acid . In addition to xylose, it can ferment all other minor sugars in hemicellulose except arabinose . Subjected to mixed sugar fermentation, this strain consumes glucose first, then mannose, followed by almost simultaneous utilization of xylose and galactose . It shows high tolerance for lactic acid as well as extraneous toxins . It can ferment the mixed sugars present in acid-treated hydrolysate of softwood, giving yields similar to that of pure sugar but at a slower rate.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 455 - 68
Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445); Thomas S; Using the simultaneous saccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lactic acid using NRRL B445 . SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97% . The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium . On prolonged incubation, depletion of xylose with lactic acid production was observed . An experimental procedure with a nonglucose medium was developed to eliminate the lag phase . From xylose fermentation, Lactobacillus delbrueckii yielded 88-92% lactic acid and 2-12% acetic acid.

Lett Appl Microbiol, 2000 Jun, 30(6), 453 - 5
Stimulatory effect of honey on multiplication of lactic acid bacteria under in vitro and in vivo conditions; Shamala TR et al.; The effect of honey and sucrose on lactic acid bacteria in vitro and in rat gut was studied to determine whether these organisms were affected differently by honey compared with sucrose . Under in vitro conditions, the number of Lactobacillus acidophilus and Lactobacillus plantarum counts increased 10-100 fold in the presence of honey compared with sucrose . Feeding of honey to rats also resulted in significant increase in counts of lactic acid bacteria . Although there was no significant difference in the counts of lactic acid bacteria in the small and large intestines of different groups, the honey-fed group showed a significant increase (P<0.05) in counts over the control and sucrose-fed animals . The results support the fact that consumption of honey has a beneficial effect on the physiological constitution of animals fed with it.

J Appl Microbiol, 2000 Jun, 88(6), 1056 - 64
Heterogeneity of Lactobacillus plantarum isolates from feta cheese throughout ripening; Xanthopoulos V et al.; Thirty-two Lactobacillus plantarum strains isolated from Feta cheese throughout ripening were studied for their phenotypic characteristics, protein profile of cell-free extracts, enzyme profiles, plasmid profiles, proteolytic and acidifying abilities and ability to grow at low pH and in the presence of bile . Results showed that some biotechnologically important characteristics, such as acidifying and proteolytic activities, can differ between strains . In addition, different plasmid profiles suggest the presence of different Lact . plantarum strains in Feta cheese throughout ripening . The results suggest the possibility of choosing strains with specific biotechnologically interesting properties.

J Appl Microbiol, 2000 Jun, 88(6), 919 - 29
Predictive models of the combined effects of curvaticin 13, NaCl and pH on the behaviour of Listeria monocytogenes ATCC 15313 in broth; Bouttefroy A et al.; Thirty-three strains of Listeria monocytogenes belonging to different serotypes were tested for their sensitivity to curvaticin 13, an antilisterial bacteriocin produced by Lactobacillus curvatus SB13, using the well diffusion method in Institut Pasteur agar plates at 37 degrees C . No relationship between serotype and sensitivity was observed . The sensitivity of this species was strain-dependent and a large variation in tolerance to curvaticin 13 was observed . The combined effects of curvaticin 13 (0-160 AU ml-1), NaCl (0-6% w/v), pH values (5.0-8.2) and incubation time (0-24 h) were investigated on L . monocytogenes ATCC 15313 in trypcase soy-yeast extract broth at 22 degrees C . For this study, two Doehlert matrices were used in order to investigate the main effects of these factors and their different interactions . The results were analysed using the Response Surface Methodology . Curvaticin 13 had a major inhibitory effect and the response was NaCl concentration-, time- and pH-dependent . This inhibitory activity was the same at pH values between 6.6 and 8.2 . Curvaticin 13 was bactericidic at acidic pH values, but the surviving cells resumed growth . For a short incubation time (12 h), the effectiveness of curvaticin 13 was maximal in the absence of NaCl . For longer incubation times (12-48 h), with high NaCl (6%) and curvaticin 13 concentrations (160 AU ml-1), the inhibition of L . monocytogenes was greater than that observed with NaCl or curvaticin 13 alone.

J Med Microbiol, 2000 Jun, 49(6), 543 - 52
Characterisation and selection of a Lactobacillus species to re-colonise the vagina of women with recurrent bacterial vaginosis; McLean NW et al.; This paper reports the results of characterising and selecting a strain of Lactobacillus for potential use as a probiotic in regenerating the vaginal flora of women with recurrent episodes of bacterial vaginosis (BV) . BV is a condition characterised by a depletion of vaginal lactobacilli accompanied by an overgrowth of a mixed vaginal flora of aerobic, anaerobic and micro-aerophilic species in very large numbers . BV has been associated with various gynaecological and obstetric complications and has an extremely high recurrence rate, due in part to the failure to establish a normal vaginal flora after antimicrobial therapy . A total of 60 vaginal isolates of lactobacilli was assessed for characteristics considered important for vaginal re-colonisation . The characteristics studied were the in-vitro inhibitory activity of the lactobacilli against bacterial species isolated from women with recurrent BV, acid production after growth of the lactobacilli in liquid culture, production of hydrogen peroxide (H2O2) and adhesiveness of the lactobacilli to exfoliated vaginal epithelial cells (VEC) . Four strains of lactobacilli, L . acidophilus (61701 and 61880), L . crispatus (55730) and L . delbrueckii subsp . delbrueckii (65407), demonstrated the greatest inhibitory activity against the BV-associated bacterial species . Two of these isolates (55730 and 61880) produced H2O2 . All four isolates produced a highly acidic environment after growth in liquid medium (pH <4) . Only one of these (strain 61701) was strongly adherent to VEC (>100 bacteria/VEC) . A further isolate (L . acidophilus 48101) did not demonstrate maximum inhibitory activity against BV-associated bacteria, but was found to be a strong producer of H2O2 and was also highly adherent to VEC . Isolates 61701 and 48101 could be candidates for use as probiotics for vaginal re-colonisation.

Indian J Biochem Biophys, 1999 Dec, 36(6), 442 - 8
Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine; Rao KN et al.; The activity of thymidylate synthase (TS) purified in our laboratory from Lactobacillus leichmannii was inhibited by pergularinine (PGL) and tylophorinidine (TPD) and deoxytubulosine (DTB) isolated from the Indian medicinal plants Pergularia pallida and Alangium lamarckii respectively . Cytotoxicity studies showed that cell growth of L . leichmannii was inhibited (IC50 = 40-45 microM) by all the three alkaloids, the concentrations > 80-90 microM resulting in complete loss of the enzyme activity . Ki values of the enzyme calculated from Lineweaver-Burk and Dixon plots for PGL, TPD and DTB were 10 x 10(-6) M, 9 x 10(-6) M and 7 x 10(-6) M respectively . These are typed as 'non-competitive' inhibitors of TS . All the three alkaloids inhibited (IC50 = 50 microM) the elevated TS activity of leukocytes in cancer patients with clinically diagnosed chronic myelocytic leukemia (n = 10), acute lymphocytic leukemia (n = 8) and metastatic solid tumours (n = 3).

Mol Microbiol, 2000 May, 36(3), 570 - 84
Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion; Viana R et al.; We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) . Northern blot analysis revealed that these two genes are organized in a single-transcriptional unit whose expression is partially induced . The PTS plays an important role in sugar transport in L . casei, as was confirmed by constructing enzyme I-deficient L . casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose) . Phosphorylation of HPr at Ser-46 is assumed to be important for the regulation of sugar metabolism in Gram-positive bacteria . L . casei ptsH mutants were constructed in which phosphorylation of HPr at Ser-46 was either prevented or diminished (replacement of Ser-46 of HPr with Ala or Thr respectively) . In a third mutant, Ile-47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P-Ser-HPr for its target protein CcpA . The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non-PTS sugars ribose or maltose . The ptsH mutants synthesizing Ser-46-Ala or Ile-47-Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P-Ser-HPr/CcpA-mediated mechanism of CCR is common to most low G+C Gram-positive bacteria . In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P-Ser-HPr participates also in inducer exclusion.

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1253 - 8
Lactobacillus fornicalis sp . nov., isolated from the posterior fornix of the human vagina; Dicks LM et al.; Twelve strains isolated from the posterior fornix fluid of the human vagina were identified as Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus gallinarum and Lactobacillus crispatus based on numerical analyses of total soluble cell protein profiles and randomly amplified polymorphic DNA (RAPD)-PCR banding patterns . Five strains grouped with the type strains of Lactobacillus gasseri (DSM 20077T) and Lactobacillus jensenii (DSM 20557T) at r > or = 0.83 in one protein profile cluster, well separated from the other species included in this study . However, numerical analysis of the RAPD-PCR banding patterns of representative strains selected from the L . gasseri-L . jensenii protein cluster clearly indicated that they belong to two different species . Four strains (TV 1010, TG 1013, TV 1018T and TV 1045) grouped into another well separated protein profile cluster at r > or = 0.87 . Strains selected from this cluster displayed very similar RAPD-PCR banding patterns and clustered at R2 > or = 0.78, separate from the other strains examined . Sequencing of the 16S rRNA of two representative strains, TV 1018T and TG 1013, of this group indicated that it represents a new member of rRNA group I Lactobacillus, which includes Lactobacillus delbrueckii, the type of the genus, and close relatives Lactobacillus acetotolerans, Lactobacillus kefiranofaciens, Lactobacillus iners, L . jensenii, L . crispatus, L . acidophilus, Lactobacillus helveticus, Lactobacillus amylovorus, Lactobacillus hamsteri, L . johnsonii, L . gasseri and Lactobacillus amylolyticus . The name Lactobacillus fornicalis sp . nov . is proposed for strains TV 1010 (DSM 13172), TG 1013, TV 1018T and TV 1045, with strain TV 1018T (= DSM 13171T = ATCC 700934T) as the type.

Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1143 - 9
Lactobacillus algidus sp . nov., a psychrophilic lactic acid bacterium isolated from vacuum-packaged refrigerated beef; Kato Y et al.; Lactobacillus algidus sp . nov . is described on the basis of 40 strains isolated as one of the predominant bacteria from five specimens of vacuum-packaged beef collected from different meat shops and stored at 2 degrees C for 3 weeks . These strains were quite uniform in the overall characteristics examined . They are facultatively anaerobic, psychrophilic, Gram-positive, non-spore-forming, non-motile, lactic acid-homofermentative rods . The cells occurred singly and in pairs on agar media and in rather long chains in broth media . They differed in several cultural and biochemical characteristics from the authentic meso-diaminopimelic acid-positive or psychrophilic lactic acid bacteria in the genera Lactobacillus, Carnobacterium and Brochothrix . The SDS-PAGE whole-cell protein pattern was clearly distinctive . DNA-DNA hybridization and phylogenetic analysis of 16S rDNA also failed to associate these strains closely with any of the validly described organisms used . The phylogenetic analysis showed that these strains are rather remotely but most closely related to Lactobacillus mali (93% sequence similarity), which belongs to the Lactobacillus casei/Pediococcus group . Therefore, these strains should be included in the genus Lactobacillus and considered to represent a new species, Lactobacillus algidus sp . nov . The type strain is M6A9T (= JCM 10491T).

Biotechnol Prog, 2000 May-Jun, 16(3), 305 - 14
Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus; Burgos-Rubio CN et al.; Lactic acid fermentation includes several reactions in association with the microorganism growth . A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus . Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate . During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates . Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose . The inoculum preparation was performed in such a way that healthy young cells were obtained . By using this inoculum, shorter fermentation times with very little lag phase were observed . The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production . A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production . A good fit with a simple equation was obtained . It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9 . The Monod kinetic parameters were obtained . The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.

Hum Cell, 1999 Dec, 12(4), 189 - 96
{Dietary fiber inhibits the incidence of hepatic metastasis with the anti-oxidant activity and portal scavenging functions}; Tazawa K et al.; Dietary fiber is described as the proportion of plant foods not digested in the human small intestine . Among the various kinds of pectin, apple pectin exerts a bacteriostatic action and therefore may change the composition of the intestinal flora . The diet supplemented with 20% apple pectin significantly decreased the number and the incidence of AOM-induced colon tumors in rats . The prostaglandin E2 (PGE2) level in the distal colonic mucosa and blood of portal vein was lower in rats fed 20% pectin than those fed the basal diet . The ability of apple pectin to decrease PGE2 was dose-dependent and those results suggest an anti-inflammatory effect in the bowel . Rats fed apple pectin showed a significantly lower incidence of hepatic metastasis than those fed the basal diet . To determine whether the anti-inflammatory effect of Lactobacillus on hepatic metastasis model same as apple pectin, Lactobacillus casei was selected . Metastatic nodules were significantly reduced, especially in the group receiving pretreatment . Apple pectic oligosaccharides with small molecular weights showed highly inhibitory effects on hypoxanthin-xanthin oxidase and ferrous sulfate-hydrogen peroxide . The scavenging activity of apple pectic oligosaccharides was suitable and accelerated at high temperatures (120 degrees C, 30 min.) . Apple pectin and Lactobacillus have a scavenger effect in the intestinal digestion and portal circulation system as an anti-inflammatory food and have very important function for the prevention of hepatic metastasis.

Microbiology, 2000 May, 146 ( Pt 5), 1163 - 9
Transposition in Lactobacillus sakei: inactivation of a second lactocin S operon by the insertion of IS1520, a new member of the IS3 family of insertion sequences; Skaugen M et al.; The analysis of spontaneous bacteriocin-negative mutants has led to the identification and characterization of a new, transpositionally active, insertion sequence of the IS3 family in the lactocin-S-producing Lactobacillus sakei strain L45 . The element, which has been designated IS1520, is 1302 bp long with 10 bp perfect inverted repeat ends and generates direct repeats of a trinucleotide of target sequence upon transposition to the lactocin S locus . IS1520 encodes two consecutive, partially overlapping, major ORFs, which are frameshifted in a manner typical of the IS3 family . Despite a low overall DNA sequence similarity, the putative frameshifting region of IS1520 is highly similar to the corresponding region of IS1163, which is a related element previously shown to be active in L . sakei L45.

Gene, 2000 May 16, 249(1-2), 161 - 9
Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e; Kakikawa M et al.; The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro . In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es) . In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected . Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer . On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes . Thus, the present and previous {Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166} results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).

Gene, 2000 May 16, 249(1-2), 127 - 34
Insertion of bacteriophage phiFSW into the chromosome of Lactobacillus casei strain Shirota (S-1): characterization of the attachment sites and the integrase gene; Shimizu-Kadota M et al.; The integrase gene (int) on the genome of φFSW, which is a temperate bacteriophage of Lactobacillus casei strain Shirota (formerly denoted as S-1), and the four attachment sites on the genomes of the phage and its host were characterized by sequencing . The φFSW integrase was found to belong to the integrase family of site-specific tyrosine recombinase . The attachment sites shared a 40bp common core within which an integrative site-specific recombination occurs . The common core was flanked on one side by an additional segment of high sequence similarity . An integration plasmid, consisting of int, the phage attachment site (attP), and a selectable marker, inserted stably into the bacterial attachment site (attB) within the common core, as did the complete prophage genome at a frequency of more than 10(3)/microg of plasmid DNA . This plasmid was used as a test system for a preliminary mutational analysis of int and attP . The attB common core was located within and near the end of an open reading frame that appears to encode a homolog to glucose 6-phosphate isomerase, an enzyme of the glycolytic pathway . It is unlikely that the prophage integration inactivates this protein, since a change of only the C-terminal amino acid is predicted because of the sequence similarity between attP and attB.

Appl Environ Microbiol, 2000 Jun, 66(6), 2605 - 12
Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L . salivarius strains during heat treatment and spray drying; Gardiner GE et al.; Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential . Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59 degrees C . An air outlet temperature of 80 to 85 degrees C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 x 10(9) CFU/g for NFBC 338 and 5.2 x 10(7) CFU/g for UCC 118 . Thus, L . paracasei NFBC 338 survived better than L . salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining . In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles . Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide . The level of survival of NFBC 338 remained constant at approximately 1 x 10(9) CFU/g during 2 months of powder storage at 4 degrees C, while a decline in the level of survival of approximately 1 log (from 7.2 x 10(7) to 9.5 x 10(6) CFU/g) was observed for UCC 118 stored under the same conditions . However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature . Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.

Appl Environ Microbiol, 2000 Jun, 66(6), 2599 - 604
Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination; Martin MC et al.; The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (beta-recombinase) and their respective target sites (attP-attB and six, respectively) is reported . The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2 . The clearing system provides a plasmid-borne gene encoding beta-recombinase . The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain . The transfer of the clearing system into this strain, with the subsequent expression of the beta-recombinase, led to site-specific DNA resolution of the non-food-grade DNA . These methods were validated by the construction of a stable food-grade L . casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.

Appl Environ Microbiol, 2000 Jun, 66(6), 2548 - 54
Surface of lactic acid bacteria: relationships between chemical composition and physicochemical properties; Boonaert CJ et al.; The surface chemical composition and physicochemical properties (hydrophobicity and zeta potential) of two lactic acid bacteria, Lactococcus lactis subsp . lactis bv . diacetilactis and Lactobacillus helveticus, have been investigated using cells harvested in exponential or stationary growth phase . The surface composition determined by X-ray photoelectron spectroscopy (XPS) was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbonlike compounds . The concentration of the last was always below 15% (wt/wt), which is related to the hydrophilic character revealed by water contact angles of less than 30 degrees . The surfaces of L . lactis cells had a polysaccharide concentration about twice that of proteins . The S-layer of L . helveticus was either interrupted or crossed by polysaccharide-rich compounds; the concentration of the latter was higher in the stationary growth phase than in the exponential growth phase . Further progress was made in the interpretation of XPS data in terms of chemical functions by showing that the oxygen component at 531.2 eV contains a contribution of phosphate in addition to the main contribution of the peptide link . The isoelectric points were around 2 and 3, and the electrophoretic mobilities above pH 5 (ionic strength, 1 mM) were about -3.0 x 10(-8) and -0.6 x 10(-8) m(2) s(-1) V(-1) for L . lactis and L . helveticus, respectively . The electrokinetic properties of the latter reveal the influence of carboxyl groups, while the difference between the two strains is related to a difference between N/P surface concentration ratios, reflecting the relative exposure of proteins and phosphate groups at the surface.

Appl Environ Microbiol, 2000 Jun, 66(6), 2302 - 10
Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation; Pham PL et al.; The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated . EPS production by L . rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars . The product yield coefficient (Y(EPS/S)) was 3.15 (0.0315 g of EPS {g of lactose}(-1)) and 2.88 (0.0288 g of EPS {g of glucose}(-1)) . It was clearly shown that the amount of EPS produced declined upon prolonged fermentation . Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities . As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown . The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium . Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucuronidase, and some traces of alpha-L-rhamnosidase) . These enzymes were localized, two of them (alpha-D-glucosidase and beta-D-glucuronidase) were partially purified and characterized . When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars . Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.

Acta Obstet Gynecol Scand, 2000 May, 79(5), 390 - 6
Treatment with 2% clindamycin vaginal cream prior to first trimester surgical abortion to reduce signs of postoperative infection: a prospective, double-blinded, placebo-controlled, multicenter study; Larsson PG et al.; BACKGROUND: Bacterial vaginosis (BV) and intermediate flora is known risk-factor for postoperative infection after surgical termination of pregnancy . Vaginal application of 2% clindamycin cream is an efficacious treatment for BV, but it is not known whether preoperative administration of clindamycin cream might reduce the signs of post-abortion infection after surgical termination of pregnancy . AIM: To evaluate whether preoperative treatment with clindamycin cream might reduce the signs of post-abortion infection after legal abortion . DESIGN: Prospective, double-blinded, placebo-controlled, multicenter study . MATERIAL AND METHODS: Consecutive women attending for surgical termination prior to 11+4 gestational weeks were approached . We randomized participants to preoperative vaginal treatment with 2% clindamycin cream or placebo cream in a double-blinded fashion . At all visits vaginal smears were air dried on microscopy slides to be stored . The rate of postoperative pelvic infection according to our definition was the main outcome variable, the cure rates of BV and of intermediate flora were secondary outcome variables . RESULTS: Of 1655 enrolled women, 1102 were evaluable for analyses . Fifty-eight women developed signs of post-abortion infection . Preoperative treatment with clindamycin cream significantly (RR: 4.2, 95% C.I . 1.2-15.9) reduced the risk of post-abortion infection among women with abnormal vaginal flora (BV and intermediate flora) . Treatment with clindamycin cream in women with normal lactobacilli flora did not demonstrate any difference compared to the non-treatment group . CONCLUSION: Preoperative treatment for at least three days with clindamycin cream significantly reduced the risk for developing signs of post-abortion infection only among women with preoperative abnormal vaginal flora (BV and intermediate flora).

Eur Urol, 2000, 37 Suppl 3, 10 - 5
Biological response modifiers for the treatment of superficial bladder tumors; Zlotta AR et al.; BACKGROUND: For more than 20 years, superficial bladder tumors have been demonstrated to be sensitive to several biological response modifiers and especially to immunomodulators . The best-known and studied immunomodulator is the bacillus Calmette-Guerin (BCG) . However, despite its well-recognized efficacy, BCG is not a universal panacea and is associated with potentially significant side effects . METHODS: New perspectives in BCG therapy aiming to increase BCG efficacy or to decrease side effects include the use of genetically engineered BCG strains producing cytokines as well as the use of purified BCG subcomponents . Because a cascade of immunological reactions including the secretion of several cytokines has been demonstrated in the BCG mode of action, many other biological response modifiers and especially immunomodulators have been studied for superficial transitional cell carcinoma therapy . Some were investigated in human trials, others are still in laboratory studies; some are administered intravesically whereas others are given orally . Interferon-alpha (IFN-alpha) intravesical instillations have been evaluated in several controlled studies . RESULTS: Although toxicity of intravesical IFN is minimal, its optimal dose, schedule and efficacy remain to be defined . Recent prospective studies comparing IFN to BCG intravesical therapy have been somewhat disappointing although this cytokine may be effective in some patients with T(a)-T(1) disease who have failed BCG therapy . Other immunomodulators administered intravesically investigated in clinical studies include interleukin 2 (recently used in a clinical study with a marker tumor response), levamisole, Rubratin, a Nocardia rubra cell wall skeleton, and keyhole limpet hemocyanin . Several biological response modifiers administered orally such as vitamin A (and its derivatives), Lactobacillus casei or bropirimine have been tested in clinical trials as well . In contrast, Allium sativum (garlic) or OK-432 (a streptococcal preparation) or BCG subfractions have been tested in laboratory studies only . CONCLUSIONS: Published reports on several of these biological response modifiers suggest that these compounds may be an alternative in patients with superficial bladder cancer who have failed or have not tolerated BCG, but further evaluation to improve efficacy, durability and understand their mechanism of action is warranted .

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 251 - 8
Lactobacillus mucosae sp . nov., a new species with in vitro mucus-binding activity isolated from pig intestine; Roos S et al.; A new Lactobacillus species from pig small intestine has been identified . In an attempt to isolate Lactobacillus reuteri strains carrying the putative colonization-factor gene (mub, for mucus binding) a mub-derived gene probe was used to screen pig intestinal material . A number of isolates were obtained and primary characterization showed that they were Gram-positive, catalase-negative, non-spore-forming, non-motile rods . Growth occurred at 45 degrees C but not at 15 degrees C and the DNA G+C content was 46 mol% . Cell wall analysis together with DNA-DNA hybridization and analysis of the 16S rRNA sequence revealed that the new isolates represent a previously undescribed Lactobacillus species closely related to L . reuteri, Lactobacillus fermentum and Lactobacillus pontis . The name Lactobacillus mucosae is proposed for this species and the type strain is S32T.

Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 19 - 24
Description of Paralactobacillus selangorensis gen . nov., sp . nov., a new lactic acid bacterium isolated from chili bo, a Malaysian food ingredient; Leisner JJ et al.; Paralactobacillus selangorensis gen . nov., sp . nov . is described . This organism, isolated from a Malaysian food ingredient called chili bo, is an obligatory homofermentative, rod-shaped lactic acid bacterium . The G+C content is 46.1-46.2+/-0.3 mol% . Earlier 16S rRNA studies showed that this organism constitutes a new taxon distantly related to the Lactobacillus casei-Pediococcus group . A phenotypic description that distinguishes Paralactobacillus selangorensis from other genera of lactic acid bacteria is presented . The type strain of Paralactobacillus selangorensis is LMG 17710T.

J Food Prot, 2000 May, 63(5), 638 - 44
Probiotic effects of feeding heat-killed Lactobacillus acidophilus and Lactobacillus casei to Candida albicans-colonized immunodeficient mice; Wagner RD et al.; Probiotic bacteria can protect immunodeficient mice from orogastric candidiasis but cause some pathology of their own . Severely immunodeficient patients may be at risk if fed viable probiotics, so this study evaluated the probiotic potential of nonviable probiotic bacteria to protect immunodeficient mice from Candida albicans infections . Heat-killed probiotic bacteria were fed to gnotobiotic bg/bg-nu/nu and bg/bg-nu/+ mice to ascertain if they could protect the mice from mucosal and systemic candidiasis . Both heat-killed Lactobacillus acidophilus (HKLA) and heat-killed Lactobacillus casei (HKLC), in comparison to control mice not fed the probiotic bacteria but challenged (oral) with C . albicans, suppressed the severity of orogastric candidiasis in bg/bg-nu/nu mice at 2 weeks after colonization with C . albicans, inhibited disseminated candidiasis in C . albicans-colonized bg/bg-nu/+ mice at 4 weeks after colonization, and suppressed the number of viable C . albicans in the alimentary tract . HKLA, but not HKLC, treatment inhibited disseminated candidiasis in bg/bg-nu/nu mice at 2 weeks after oral challenge and enhanced the proliferative responses of splenocytes from C . albicans-colonized bg/bg-nu/+ mice to C . albicans antigens . Neither HKLA nor HKLC were able to prolong the survival of gnotobiotic bg/bg-nu/nu mice after oral challenge with C . albicans . These results demonstrate that heat-killed lactobacilli can induce some (limited) protection (probiotic effect) against candidiasis in mice.

J Infect Dis, 2000 May, 181(5), 1574 - 80 Epub 2000 May 15.
Induction of human immunodeficiency virus type 1 expression by anaerobes associated with bacterial vaginosis; Hashemi FB et al.; Bacterial vaginosis (BV) is a common disorder characterized by increased levels of anaerobic bacteria in the genital tract . BV has been associated with an increased rate of sexual transmission of human immunodeficiency virus (HIV) . The effects of BV-associated anaerobic bacteria on HIV expression in monocytoid cells and T cells were examined . Peptostreptococcus asaccharolyticus and Prevotella bivia stimulated HIV expression in monocytoid cells, whereas Bacteroides ureolyticus, Peptostreptococcus anaerobius, and Lactobacillus acidophilus did not enhance HIV expression . P . asaccharolyticus also enhanced HIV expression in T cells and activated HIV long-terminal-repeat transcription in U38 cells . This report suggests a mechanism by which disturbances in vaginal flora could lead to a higher rate of sexual transmission of HIV . Furthermore, this study supports the idea that treatment of BV might serve as a preventive measure to reduce the risk of HIV transmission.

J Clin Pathol, 2000 Apr, 53(4), 308 - 13
Wet mount microscopy reflects functional vaginal lactobacillary flora better than Gram stain; Donders GG et al.; AIM: The status of vaginal lacto-bacillary flora, an indicator of possible genital infection and pregnancy complications, can be assessed on wet mount or Gram stained specimens . The former is quick, the latter more routine . The accuracy of the two preparative techniques to detect normal vaginal lacto-bacillary microflora was compared for 646 patients . The effect of delay in transport medium before Gram staining was also investigated . METHODS: Patients presented with infectious vaginitis or for a routine prenatal visit . After placement of a speculum, duplicate smears were taken from the upper vaginal vault and examined fresh or after Gram staining . Lacto-bacillary grades from both methods were compared with lactate concentration in vaginal rinses . In a subgroup of 238 patients, Gram staining was performed both on fresh smears and those that had been transported in Stuart's growth medium . RESULTS: Higher lacto-bacillary grades (more disrupted flora) were diagnosed 2.9 times more frequently on Gram stained specimens than on wet mounts (p < 0.0001), a difference even more pronounced after transport in Stuart's medium (relative risk, 4.2; p < 0.0001) . Lacto-bacillary grades assessed on wet mounts correlated better with vaginal lactate concentration than those assessed on Gram stains . CONCLUSIONS: Easier recognition of lacto-bacillary morphotypes on wet mounts than on Gram stains might result from the loss of lactobacilli by the process of fixation or Gram staining . Wet mount microscopy of vaginal smears for assessment of lacto-bacillary grades, rather than Gram staining, is strongly recommended.

Oper Dent, 1999 Sep-Oct, 24(5), 297 - 305
Fluoride release and antibacterial properties of new-generation tooth-colored restoratives; Yap AU et al.; The aim of this study was to compare the amounts and pattern of fluoride release and antibacterial properties of new-generation restoratives over a 35-day period . Materials evaluated included fluoride-releasing composites (Tetric, Experimental X), compomers (Dyract, Compoglass), and a resin-modified glass-ionomer cement (Fuji II LC) . A conventional glass ionomer (Fuji II Cap) was used as a control for fluoride-release testing . Five samples of each restorative material were evaluated for daily fluoride release over a 35-day period by means of ion chromatography . Ranking of materials from least to greatest total fluoride release over 35 days was as follows: Tetric < Experimental X < Dyract < Fuji II LC < Compoglass < Fuji II Cap . Fuji II Cap had significantly greater fluoride release than all other materials evaluated . Fuji II Cap, Fuji II LC, and Compoglass had similar patterns of fluoride release characterized by a high initial release that was many times that released later . The fluoride-releasing composites evaluated stopped releasing fluoride by day 14 . Antibacterial testing was conducted using the agar diffusion inhibitory test . Five samples of each restorative were assessed at baseline and weekly intervals up to 35 days . The microorganisms used were Lactobacillus casei, Streptococcus mutans, and Streptococcus sobrinus . IRM, a zinc oxide/eugenol cement, was used as the baseline control . None of the restorative materials evaluated affected the growth of L casei, S sobrinus, or S mutans at all time periods including baseline, where fluoride was detected in the agar beneath the specimen disks . There was no correlation noted between fluoride-release potential and antibacterial properties.

Biol Pharm Bull, 2000 May, 23(5), 527 - 31
Cloning of the gene of beta-N-acetylglucosaminidase from Lactobacillus casei ATCC 27092 and characterization of the enzyme expressed in Escherichia coli; Senba M et al.; The gene encoding beta-N-acetylglucosaminidase (GlcNAcase) of Lactobacillus casei ATCC 27092 was cloned and expressed in Escherichia coli . The gene consisted of 1581 nucleotides, and had a promoter, Shine-Dalgarno, and rho-independent type transcription termination sequences typical in bacteria . The protein deduced from the sequence consisted of 526 amino acids, and had a putative signal peptide of 14 amino acids and 5 possible asparagine-linked glycosylation sites . A conserved sequence was homologous to the 12 other hexosaminidases from different origins . The recombinant GlcNAcase (r-GlcNAcase) purified from the transformed E . coli had a MW of 39 kDa and lacked oligosaccharide chains . The isoelectric point and the optimum pH for the activity of r-GlcNAcase were similar to those of original GlcNAcases (o-GlcNAcase) . However, the thermal stability was lower, and sensitivity to Cd2+, Fe2+, Cu2+ and sodium dodecyl sulfate (SDS) was higher than those of o-GlcNAcases, suggesting that the oligosaccharide moieties of the enzyme contribute to their stability . The Km value for p-nitrophenyl-N-acetyl-beta-1,4-D-glucosamine (PNP-GlcNAc) of r-GlcNAcase (6.4 microM) implied that the affinity of r-GlcNAcase for the substrate was 200-fold higher than that of the original ones.

J Dairy Sci, 2000 May, 83(5), 915 - 22
Transit of radical scavenging activity of milk products prepared by Maillard reaction and Lactobacillus casei strain Shirota fermentation through the hamster intestine; Nishino T et al.; Oxidative stress in the colon is associated with the incidence of colon cancer . In situ, the suppression of oxidative stress in the colon would be an effective form of prevention of the cancer . In this study we investigated the transit of the radical scavenging activity of milk products through the hamster intestinal tract . Two types of skim milk products were prepared by Maillard reaction and then lactic acid fermentation . Heat treatment enhanced the radical scavenging activity for 2,2-diphenyl-1-picrylhydrazyl radical of skim milk . The activity was further increased by fermentation with Lactobacillus casei strain Shirota . Normal hamsters were fed these milk products for 14 d . For potential radical scavenging activity per unit dry weight of feces and cecal content, the groups ranked in the order of fermented product-fed hamsters > heated product-fed hamsters > control hamsters, reflecting the order of the potential of the corresponding diets . Approximately 12% of the activity of the heated and the fermented product diets passed through the gastrointestinal tract . These results suggest that some of the radical scavenging activity generated by food processing reached the colon in nonabsorbable products.

J Agric Food Chem, 2000 May, 48(5), 1630 - 6
Flavor enhancement of reduced fat cheddar cheese using an integrated culturing system; Midje DL et al.; Mild cheese flavor in reduced fat Cheddar cheese was enhanced by using an integrated starter culture system . Three cultures, Lactococcus lactis subsp . cremoris SK11, L . lactis subsp . lactis biovar . diacetylactis JVI, and Lactobacillus casei 7A, were carefully selected to obtain a nonbitter, mildly acid, buttery flavored cheese . Cheeses were produced from all possible combinations of these cultures with the constraint that L . lactis subsp . cremoris SK11 was used as the primary acid-producing culture . Cheeses made with SK11 were compared to cheeses produced using an L . lactis subsp . cremoris commercial starter culture . Cheeses were ripened for 150 days and periodically sampled for chemical, microbiological, and sensory analysis . Cheeses produced with L . lactis subsp . cremorisSK11 had substantially lower bitterness intensity than the cheeses produced with commercial starter culture . L . lactis subsp . lactis biovar . diacetylactis JVI significantly increased diacetylacetoin and acetate concentrations . Sensory results indicate that these cheeses had increased buttery (diacetyl) flavor.

Infect Immun, 2000 Jun, 68(6), 3535 - 40
Bacterial cell wall-induced arthritis: chemical composition and tissue distribution of four Lactobacillus strains; Simelyte E et al.; To study what determines the arthritogenicity of bacterial cell walls, cell wall-induced arthritis in the rat was applied, using four strains of Lactobacillus . Three of the strains used proved to induce chronic arthritis in the rat; all were Lactobacillus casei . The cell wall of Lactobacillus fermentum did not induce chronic arthritis . All arthritogenic bacterial cell walls had the same peptidoglycan structure, whereas that of L . fermentum was different . Likewise, all arthritogenic cell walls were resistant to lysozyme degradation, whereas the L . fermentum cell wall was lysozyme sensitive . Muramic acid was observed in the liver, spleen, and lymph nodes in considerably larger amounts after injection of an arthritogenic L . casei cell wall than following injection of a nonarthritogenic L . fermentum cell wall . The L . casei cell wall also persisted in the tissues longer than the L . fermentum cell wall . The present results, taken together with those published previously, underline the possibility that the chemical structure of peptidoglycan is important in determining the arthritogenicity of the bacterial cell wall.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Mar-Apr, (2), 67 - 73
{Cloning and gene expression in lactic acid bacteria}; Bondarenko VM et al.; The possibility of using the genera Lactobacillus and Lactococcus as vector representatives is widely discussed at present . The prospects of the construction of recombinant bacteria are closely connected with the solution of a number of problems: the level of the transcription of cloned genes, the effectiveness of the translation of heterologous mRNA, the stability of protein with respect to bacterial intracellular proteases, the method by protein molecules leave the cell (by secretion or as the result of lysis) . To prevent segregation instability, the construction of vector molecules on the basis of stable cryptic plasmids found in wild strains of lactic acid bacteria was proposed . High copying plasmids with low molecular weight were detected in L . plantarum and L . pentosus strains . Several plasmids with molecular weights of 1.7, 1.8 and 2.3 kb were isolated from bacterial cells to be used as the basis for the construction of vector molecules . Genes of chloramphenicol- and erythromycin-resistance from Staphylococcus aureus plasmids were used as marker genes ensuring cell transformation . The vector plasmids thus constructed exhibited high transformation activity in the electroporation of different strains, including L . casei, L . plantarum, L . acidophilus, L . fermentum and L . brevis which could be classified with the replicons of a wide circle of hosts . But the use of these plasmids was limited due to the risk of the uncontrolled dissemination of recombinant plasmids . L . acidophilus were also found to have strictly specific plasmids with good prospects of being used as the basis for the creation of vectors, incapable of dissemination . In addition to the search of strain-specific plasmids, incapable of uncontrolled gene transmission, the use of chromosome-integrated heterologous genes is recommended in cloning to ensure the maximum safety.

Mikrobiologiia, 2000 Jan-Feb, 69(1), 98 - 104
{Comparison of methods of storing lactic acid bacteria}; Stoianova LG et al.; The study was undertaken to elucidate how different methods of storage (immersing in mineral oil, lyophilization, and subculturing) of lactic acid bacteria belonging to the genera Lactobacillus and Lactococcus affect their viability, antibiotic activity, and ability to accumulate organic acids . Storage of the lactic acid bacterium L . lactis subsp . lactis by immersion in mineral oil proved to be ineffective . Lyophilization allowed the survival of a sufficiently large number of cells, although their antibiotic activity somewhat decreased . The resuscitation of lyophilized bacteria by subculturing them in rich nutrient media, such as skim milk, led to the restoration of their physiological activity, including the effective antimicrobial spectrum.

Mikrobiologiia, 2000 Jan-Feb, 69(1), 75 - 80
{A family of shuttle vectors for lactobacilli and other gram-positive bacteria based on the plasmid pLF1311 replicon}; Aleshin VV et al.; A set of broad-host-range single-replicon shuttle vectors for cloning nucleotide sequences in gram-positive bacteria (lactobacilli, enterococci, lactococci, bacilli, etc.) was created . The vectors are based on the cryptic plasmid pLF1311 from Lactobacillus fermentum VKM 1311 belonging to a family of the sigma-type pE194-like plasmids . The vectors can replicate in gram-positive bacteria and Escherichia coli . They are stable in many gram-positive bacteria, have small sizes, and allow the selection of recombinants on media with X-Gal . The vectors that contain the region of initiation of the conjugal transfer of plasmid RP4 belonging to the incompatibility group IncP alpha can be mobilized in a great number of bacteria using a helper plasmid from E . coli but not from gram-positive bacteria.

AIDS Patient Care STDS, 2000 Apr, 14(4), 215 - 9
Microbicide for prevention of sexually transmitted diseases using a pharmaceutical excipient; Neurath AR; Preferred microbicides are expected to inactivate most sexually transmitted viral and nonviral pathogens, including HIV-1, without affecting lactobacilli, components of the natural defense system against sexually transmitted diseases (STDs), be widely available, be inexpensive, and have an established safety record for human use . We show here that cellulose acetate phthalate {C-A-P enteric coating polymer (Eastman)}, a compound used for coating of enteric tablets, meets all these criteria.

Infect Dis Obstet Gynecol, 2000, 8(2), 99 - 104
In vitro inhibition of commercial douche products against vaginal microflora; Pavlova SI et al.; Recently, vaginal douching has been associated with many health risks in women . The aim of this study was to analyze the effect of commercial douche products against various vaginal microorganisms, including lactobacilli . Seven commercial douches were tested against eight Lactobacillus clinical isolates and three type strains from the American Type Culture Collection . BV-associated bacteria included six strains of five genera: Gardnerella, Mobiluncus, Mycoplasma, Peptostreptococcus, and Ureaplasma . Two isolates of group B Streptococcus, and three species of Candida were also tested . The minimal inhibition concentrations and minimal contact times for these products against vaginal microorganisms were determined in broth cultures . Four antiseptic-containing douche products showed a strong inhibitory effect against all vaginal microorganisms tested with a short contact time (less than 1 min) . Three vinegar-containing douche products selectively inhibited vaginal pathogens associated with bacterial vaginosis, group B streptococcal vaginitis, and candidiasis, but not lactobacilli . The antimicrobial effects of the commercial douche products varied among different brands and microbial species tested.

Obstet Gynecol Surv, 2000 May, 55(5 Suppl 1), S1 - 19
Bacterial vaginosis in pregnancy; McGregor JA et al.; Bacterial vaginosis is a clinical condition caused by replacement of the normal hydrogen peroxide producing Lactobacillus sp . in the vagina with high concentrations of characteristic sets of aerobic and anaerobic bacteria . Bacterial vaginosis is the most prevalent cause of vaginal discharge or malodor, although 50 percent of women who meet the criteria for this condition are asymptomatic . Bacterial vaginosis is reported in 10 to 41 percent of women, and new evidence has shown association with maternal and fetal morbidity . Studies have shown that spontaneous abortion, preterm labor, premature birth, preterm premature rupture of the membranes, amniotic fluid infection, postpartum endometritis, and postcesarean wound infections are increased because of infection with bacterial vaginosis during pregnancy . Clinical trials demonstrated important reductions in many of these adverse events with appropriate screening and antimicrobial treatment protocols . New low-cost, diagnostic, point-of-care screening tools are available for rapid screening of patients, affording the physician the opportunity to potentially make a dramatic clinical and cost impact in preventing preterm birth and the costly sequelae of prematurity . Practicing physicians need to be aware of current guidelines for screening and treating pregnant patients for bacterial vaginosis . The authors recommend that all pregnant women be screened and treated with the Centers for Disease Control and Prevention (CDC-P) recommended oral regimens early in pregnancy . Each treated women should be evaluated for "test of cure" 1 month after treatment . Mothers likely to benefit from "screen and treat" approaches include 1) those with the highest concentrations of genital anaerobes and mycoplasmas, 2) women with prior preterm birth or who have low body mass (BMI < 19.8 kg/m2), 3) those with evidence of endometritis before pregnancy, and 4) those who are treated with oral agents effective for both presumed intrauterine mycoplasmas and other bacterial vaginosis flora (i.e., oral clindamycin or erythromycin and metronidazole).

Biosci Biotechnol Biochem, 2000 Mar, 64(3), 466 - 75
Lower plasma triglyceride level in Syrian hamsters fed on skim milk fermented with Lactobacillus casei strain Shirota; Kikuchi-Hayakawa H et al.; The effect of fermented skim milk (FSM) by Lactobacillus casei strain Shirota on plasma lipids in hamsters was examined . Hamsters fed on cholesterol-free and -enriched diets containing 30% FSM had lower levels of plasma triglyceride than those fed on the control diet . In the experiment with the cholesterol-enriched diet-fed hamsters, the plasma triglyceride level was suppressed by FSM at concentrations of 10% to 30% . Unfermented milk tended to lower the level of triglyceride, but not significantly . The plasma cholesterol concentration was not affected by an FSM and unfermented skim milk supplement to the diet . L . casei strain Shirota grew well in the presence of mixed lipid micelles containing bile acid, but did not have the ability to remove cholesterol from the culture broth . These results indicate that FSM lowered the plasma triglyceride level in hamsters.

FEMS Microbiol Lett, 2000 May 15, 186(2), 269 - 73
A physical and functional analysis of the newly-identified bglGPT operon of Lactobacillus plantarum; Marasco R et al.; A newly-identified bglGPT operon of Lactobacillus plantarum was isolated and expressed in Escherichia coli . The sequence analysis of the cloned DNA fragment showed three open reading frames encoding (i) a 237-amino acid protein (BglG), (ii) a 577-amino acid protein (BglP) and (iii) a 486-amino acid protein (BglT) . BglG, BglP and BglT were shown to be homologous to the BglG family of transcriptional antiterminators, to permeases of the phosphoenolpyruvate-dependent phosphotransferase system and to beta-glucosidases, respectively . Complementation of E . coli mutant strains showed that BglP and BglT are a permease and a beta-glucosidase active on the beta-glucosides, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-glucoside, respectively . BglG was also shown to promote expression of a bglG-lacZ gene fusion in an E . coli bglG(-) background . A ribonucleic antiterminator sequence, the antiterminator-responsive cis-element and a 'catabolite responsive element', were found downstream of the transcriptional start point . Transcription of the operon was repressed 10-fold in L . plantarum cells grown on glucose as compared to ribose.

J Natl Med Assoc, 2000 Feb, 92(2), 83 - 6
Lactobacillemia: an emerging cause of infection in both the immunocompromised and the immunocompetent host; Antony SJ; The bacterium, lactobacillus, is found in the mucosal surfaces of the mouth and the gastrointestinal and genitourinary tracts . There have been increasing reports of the micro-organism being a cause of serious infection in immunocompromised individuals . This article reviews the clinical presentation, laboratory characteristics and treatment of patients with lactobacillemia.

Can J Gastroenterol, 2000 Apr, 14(4), 327 - 32
Mechanisms of alcoholic liver injury; French SW; There have been numerous recent advances in the understanding of the mechanisms of alcoholic liver disease pathogenesis . Endotoxin-induced Kupffer cell activation plays a role in cytokine-mediated inflammatory changes in the liver, and this can be blocked by a diet high in saturated fat, by a diet containing lactobacillus, which does not produce endotoxin, by neomycin antibiotic sterilization of the gut, by eliminating Kupffer cells, or by removing tumour necrosis factor-alpha with antibody or by using tumour necrosis factor-alpha knockout mice . The fatty liver component is mainly the result of the nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide redox shift to the reduced state by ethanol oxidation generation of reduced nicotinamide adenine dinucleotide, although this too can be blocked by a diet high in saturated fat . Hepatocytic enlargement occurs due to ethanol-induced inhibition of the ubiquitin-proteasome pathway of cytoplasmic protein degradation and the retention of oxidized proteins in hepatocytes . The liver is scarred by stellate cells that have been activated by inflammatory cytokines and growth factors produced by activated Kupffer cells, and by bile ductule metaplasia . Mallory bodies and balloon cell degeneration develop through the ethanol-induced oxidative stress-protein kinase activation pathway, inhibition of phosphatase activity and inhibition of the ubiquitin-proteasome pathway.

Indian J Pediatr, 1999 Sep-Oct, 66(5), 663 - 8
Effect of Lactobacillus supplementation on immune status of malnourished pre-school children; Devi S et al.; The present study was undertaken to assess the effect of Lactobacillus supplementation on immune response of pre-school children . Thirty malnourished (grade I and grade II) pre-school (3-5 years old) children were selected for the study . These were further categorized into three groups 'A', 'B' and 'C', each group comprising of 10 individuals . Groups 'A' and 'B' were given lactobacillus supplementation 1.0 x 10(8) million viable spores/day/child and 2.0 x 10(8) million of viable spores/day/child for 90 days respectively . Group 'C' served as control . Serum adenosine deaminase (ADA) which is an important immuno enzyme marker for assessing lymphocyte function and proliferation was determined at different intervals . The results revealed that lactobacillus supplementation at level of 1.0 x 10(8) million viable spores/day/child for 15 days along with regular diet significantly improved the ADA levels of pre-school children indicating therapeutic value of lactobacillus as supplementation to malnourished pre-school children.

Diagn Microbiol Infect Dis, 2000 May, 37(1), 75 - 6
Chondritis attributable to Lactobacillus after ear piercing; Razavi B et al.; A diabetic patient was admitted with ear pain and purulent discharge after "high ear piercing" by a friend . After investigation she was found to have chondritis, complicated by an abscess . Wound cultures grew Lactobacillus species . Patient responded to parenteral antibiotics without suffering cosmetic deformity . Sterile technique, adherence to post-piercing ear hygiene, and avoiding piercing the auricular cartilage may prevent complications associated with ear piercing . Lactobacillus should be included in the differential of pathogens involved in post-piercing chondritis.

J Agric Food Chem, 1999 Jun, 47(6), 2341 - 3
Dried green and purple lavers (Nori) contain substantial amounts of biologically active vitamin B(12) but less of dietary iodine relative to other edible seaweeds; Watanabe F et al.; Vitamin B(12) concentrations of dried green (Enteromorpha sp.) and purple (Porphyra sp.) lavers (nori) were determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor chemiluminescence methods . The values determined by using the microbiological method (63.58 +/- 2.90 and 32.26 +/- 1.61 microg/100 g of dry weight) were identical to those found by using the chemiluminescence method (69.20 +/- 2.21 and 25.07 +/- 0.54 microg/100 g of dry weight) in both dried green and purple lavers, respectively . A silica gel 60 thin-layer chromatography of both laver extracts shows that non-coenzyme forms (hydroxo and cyano forms) of vitamin B(12) predominate in both dried lavers . The dried lavers contained lesser amounts of dietary iodine ( approximately 4-6 mg/100 g of dry weight) relative to other seaweeds, suggesting that excessive intake of the dried lavers is unlikely to result in harmful intake of dietary iodine . These results indicate that the dried lavers (nori) are the most excellent source of vitamin B(12) among edible seaweeds, especially for strict vegetarians.

Syst Appl Microbiol, 1999 Dec, 22(4), 573 - 85
A polyphasic approach towards the identification of strains belonging to Lactobacillus acidophilus and related species; Gancheva A et al.; A set of 98 strains belonging to nine species of the Lactobacillus acidophilus rRNA-group have been analysed by SDS-PAGE of cellular proteins, RAPD-PCR and AFLP with fluorescently labeled primers in order to find improved methods for their identification . Strains of the following phenotypically highly similar species were examined: L . acidophilus, L . amylovorus, L . crispatus, L . johnsonii, L . gasseri, L . gallinarum, L . helveticus, L . iners and L . amylolyticus . Although the majority of the species can be differentiated by SDS-PAGE of whole-cell proteins, the latter technique showed poor discrimination between L . gasseri and L . johnsonii strains and between some strains of L . amylovorus and L . gallinarum . However, this study shows that the RAPD-PCR (using at least 3 different primers followed by numerical analysis of the combined patterns) and AFLP are most suitable genomic fingerprinting techniques for the differentiation of all the species listed above, and that databases for identification can be constructed, particularly when commercially available molecular tool-kits are used . The separate species status of the recently described L . amylolyticus and L . iners was fully confirmed.

Chir Ital, 1999 May-Jun, 51(3), 221 - 6
{A probiotic as an antagonist of bacterial translocation in experimental pancreatitis}; Mangiante G et al.; Infection is the most common cause of death in acute pancreatitis . Earlier studies have demonstrated that early enteral nutrition decreases microbial translocation, upregulates the immune function and reduces septic complications and mortality . Lactobacillus plantarum (Lp) has been shown to be effective in reducing egress of endotoxin and microbial strain that showed very high adherence power to gut mucosa . We adopted a model of acute pancreatitis induced by isolation and ligation of biliopancreatic duct in adult Lewis rats . Three groups were studied: A . control group (sham operation); B . induced pancreatitis, no further treatment; C . Induced pancreatitis + gavage with 5 ml/day of a suspension of Lp 299 v in a dose of 0.5-1.0 x 10(9)/ml during 4 days before and 4 days after induction of pancreatitis . All animals were sacrificed after 96 hours . Histological studies and microbiological analyses were performed . Forty out of 55 animals showed signs of severe pancreatitis on sacrifice after 96 hours . Only these animals were further studied . In group A, we found only 1/20 bacteria in mesenteric nodes (MN) . Pathogenic microrganisms were found in the non-treated group in MN in 14/20 and in the pancreatic tissue in 10/20 . In contrast, when kept on an umbrella of Lp 299 v, only 4/20 animals demonstrated growth of enteric bacteria in MN and 3/20 in pancreatic tissue . All of these results showed a significant reduction of infection in the treated groups . In our model, Lp 299 v is effective in preventing microbial translocation in experimental pancreatitis . Treatment with probiotic bacteria, such as Lactobacillus spp, seems to be a promising alternative as problems with antibiotic-resistant bacteria seem to accumulate.

Lett Appl Microbiol, 2000 May, 30(5), 370 - 4
Amino acid fermentation in non-starter Lactobacillus spp . isolated from cheddar cheese; Tammam JD et al.; Amino acid fermentation profiles of nine strains of Lactobacillus spp., initially isolated from a 3-year-old Cheddar cheese, were determined using the Biolog MT microplatetrade mark method . Eight of the isolates were able to ferment amino acids, but only when incubated in the presence of exogenously supplied alpha-ketoglutaric acid that served as an acceptor in the initial transamination step in the fermentative degradation . The range of amino acids catabolized was strain dependent . Amino acid catabolites were detected by gas chromatography mass spectrometry (GCMS) in culture supernatant fluids of a representative non-starter lactic acid bacteria isolate Lactobacillus paracasei CI6.

Lett Appl Microbiol, 2000 Apr, 30(4), 325 - 9
Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755; Kerovuo J et al.; Phytase enzymes can increase the nutritional value of food and feed by liberating inorganic phosphate from phytate, the major storage form of phosphorus in plants . The phytase (phyC) from Bacillus subtilis VTT E-68013 was expressed in Lactobacillus plantarum strain 755 using Lact . amylovorus alpha-amylase secretion signals . In an overnight cultivation in MRS medium containing cellobiose for induction of the alpha-amylase promoter, catalytically active phytase was secreted as a predominant extracellular protein . However, Western blot analysis revealed unprocessed and processed phytase in the cell fraction . Pulse chase experiments showed that the recombinant phytase was secreted at a slower rate in comparison to the native proteins of Lact . plantarum 755.

J Appl Microbiol, 2000 Apr, 88(4), 695 - 703
Iron requirement of Lactobacillus spp . in completely chemically defined growth media; Elli M et al.; A completely chemically-defined growth medium, containing guanine, thymine, cytidine, 2'-deoxyadenosine and 2'-deoxyuridine as DNA precursors, was developed for Lactobacillus johnsonii, on the basis of statistically designed techniques suitable for other lactobacilli . Particular focus was given to the nucleotide composition of different defined media, and to the specific nucleotide requirements of Lact . johnsonii . Most of the lactobacilli tested grew in a medium containing five free bases, four ribonucleosides or five deoxyribonucleosides . Adenine and guanine were replaceable by inosine . The requirement for thymine and cytosine was satisfied with uracil . The presence of inosine and uracil was identified as being essential for the growth of different Lactobacillus species, displaying their inability to synthesize purines and pyrimidines de novo . Defined recipes with different nucleotide composition were used to investigate iron requirements of lactobacilli . Only marginal differences in growth were observed in iron-depleted media supplemented with five free bases, four ribonucleosides or five deoxyribonucleosides; iron depletion had a greater effect on growth when inosine and uracil were supplied as the only nucleotide sources . The results suggest that iron plays a role in the pyrimidine and purine metabolism of lactobacilli . Lactobacillus spp., particularly Lact . johnsonii, require iron under particular environmental conditions with limited or specific nucleotide sources.

J Appl Microbiol, 2000 Apr, 88(4), 584 - 93
Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine; Moreno-Arribas V et al.; Wines containing high levels of biogenic amines were investigated for the presence of tyramine-producing strains . Two different Lactobacillus brevis (IOEB 9809 and IOEB 9901) able to produce the amine were isolated . None of the isolated strains identified as Oenococcus oeni formed tyramine . In addition, other Lact . brevis and Lact . hilgardii strains from our collection (IOEB) and the American Type Culture Collection (ATCC) were strong tyramine producers . Lactobacillus brevis IOEB 9809 and Lact . hilgardii IOEB 9649 were found to produce tyramine and phenylethylamine simultaneously . The conditions that can influence tyramine formation in wine were evaluated for three strains of Lact . brevis (IOEB 9809 and IOEB 9901) and Lact . hilgardii (IOEB 9649) . Tyrosine was the major factor affecting tyramine formation and was enhanced by the presence of sugars, mainly glucose . Tyrosine decarboxylase (TDC) activity greatly depended on the presence of the precursor, which suggested that tyrosine induced the TDC system . These results indicate that Lactobacillus could be the lactic acid bacteria responsible for tyramine production in wine.

J Appl Microbiol, 2000 Apr, 88(4), 572 - 83
Characterization of an arylesterase from Lactobacillus helveticus CNRZ32; Fenster KM et al.; An esterase gene (estA) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32 . The estA gene consisted of a 558 bp open reading frame encoding a putative peptide of 21.3 kDa . Protein sequence homology searches using BLAST revealed that EstA had low amino acid sequence identity with the serine-dependent arylesterases TesI (24%) and EtpA (26%) from Escherichia coli and Vibrio mimicus, respectively . A recombinant EstA fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity . Characterization of EstA revealed that it was a serine-dependent enzyme having a monomeric Mr of 22.6-25.1 kDa . Optimum temperature, NaCl concentration and pH for EstA activity were determined to be 35-40 degrees C, 3.5% NaCl and 7.5-8.0, respectively . EstA had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1) . EstA hydrolysed a variety of ester compounds and preferred those with substituted phenyl alcohol and short-chain fatty acid groups . Site-directed mutagenesis suggested that the S10 and H164 residues were essential for EstA activity.

J Dairy Sci, 2000 Apr, 83(4), 674 - 82
High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents; Gervilla R et al.; High hydrostatic pressure inactivation of Escherichia coli, Pseudomonas fluorescens, Listeria innocua, Staphylococcus aureus, and Lactobacillus helveticus were studied . These microorganisms were inoculated at a concentration between 10(7) and 10(8) cfu/ml in Ringer solution and in ovine milk adjusted to 0, 6, and 50% fat content to evaluate the baroprotective effect of fat content on inactivation of microorganisms . Treatments of pressurization consisted of combinations of pressure (100 to 500 MPa) and temperature (4, 25, and 50 degrees C) for 15 min . Gram-negative microorganisms were more sensitive than were Gram-positive ones (more destruction P . fluorescens > E . coli > or = List . innocua > Lb . helveticus > S . aureus) . Pressurizations at low temperature (4 degrees C) produced greater inactivation on P . fluorescens, List . innocua, and Lb . helveticus than at room temperature (25 degrees C), whereas for E . coli and S . aureus the results were opposite . Ovine milk per se (0% fat) showed a baroprotective effect on all microorganisms, but percentage of fat (6 and 50%) did not show a progressive baroprotective effect in all pressurization conditions or for all microorganisms.

J Dairy Sci, 2000 Apr, 83(4), 628 - 32
Comparison of newly isolated strains of Lactobacillus delbrueckii subsp . lactis for hydrogen peroxide production at 5 degrees C; Yap PS et al.; Isolates of Lactobacillus delbrueckii subsp . lactis obtained from raw milk samples were compared for the ability to produce hydrogen peroxide (H2O2) at 5 degrees C . Nineteen out of 101 lactobacilli isolated were identified as L . delbrueckii subsp . lactis . The isolates of L . delbrueckii subsp . lactis from most raw milk samples produced more H2O2 than did isolates of other species of lactobacilli from the same samples . Seven isolates of L . delbrueckii subsp . lactis, which produced the highest levels of H2O2 at 5 degrees C were selected for comparison with a laboratory strain, L . delbrueckii subsp . lactis I . In 24 h, isolate RM2-5 produced 7.0 microg/10(9) cfu in buffer containing 5 mM sodium lactate and 4.4 microg/10(9) cfu in buffer containing 5 mM glucose . Three other isolates also produced more H2O2 on sodium lactate than on glucose . However, three remaining new isolates produced more H2O2 on glucose than on sodium lactate . All seven of the most active new isolates of L . delbrueckii subsp . lactis produced significantly higher concentrations of H2O2 than did L . delbrueckii subsp . lactis I in both solutions . Strain RM2-5 produced more H2O2 than did the other six most active newly isolated strains of L . delbrueckii subsp . lactis in this comparison.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 249 - 53
Adaptation of Lactobacillus alimentarius to environmental stresses; Lemay MJ et al.; Lactobacillus alimentarius BJ33 has been tested for its biopreservative capacities to improve quality and safety in many meat products . The combination of different preservatives such as NaCl, glucono-delta-lactone and citric acid with this protective culture during the manufacture of sausages represent an interesting alternative to control microbial spoilage and to extend product shelf life . The use of these preservatives may also limit the growth of L . alimentarius . In this study, the sublethal doses of these preservatives were determined and tested in combination to verify if the organism was able to adapt to these stresses . The sublethal doses of gluconic acid, citric acid, and NaCl were 100-110 mM, 50-55 mM and 8%, respectively . When the culture was first grown in MRS broth containing citric acid (50 or 55 mM) or gluconic acid (100 or 110 mM) and then transferred in MRS broth containing NaCl (8%), only limited growth was observed (O.D.(600 nm) = 0.2-0.3) after 6 days at 30 degrees C . However, when the culture was first grown in NaCl and then transferred in MRS broth containing gluconic or citric acid, growth was observed after 1 day (O.D.(600 nm) = 0.4-0.5) and after 5 days an O.D.(600 nm) of 0.8 was reached . Cell filamentation was also observed under electron microscopy when cells were grown for 2 days in presence of gluconic and citric acid at their sublethal doses and with a combination of 18 mM gluconic acid and 37 mM citric acid, but cellular elongation was not observed with cultures exposed to 8% NaCl . These results suggest that two different adaptation mechanisms are induced in L . alimenatrius when treated with organic acids and NaCl.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 235 - 8
Influence of culture pH on survival of Lactobacillus reuteri subjected to freeze-drying; Palmfeldt J et al.; L . reuteri was cultivated at pH 5 and 6 . The cells were harvested at 0.5, 2.5 and 4.5 h after entering the stationary phase and their viability after freeze-drying was compared with their viability prior to freeze-drying . The highest viability--approximately 80% of the viability prior to freeze-drying--was obtained for cells from the pH 5 cultures harvested 2.5 h after entering the stationary phase . The time after entering the stationary phase did not influence the viability of the cells from the pH 6 cultures where the viability was approximately 50% irrespective of harvest time . Product formation was the same for pH 5 and pH 6 grown cells, whereas the pH 6 grown cells exhibited a more elongated morphology.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 167 - 70
Glycerol metabolism in Lactobacillus collinoides: production of 3-hydroxypropionaldehyde, a precursor of acrolein; Sauvageot N et al.; Lactobacillus collinoides is a lactic acid bacterium commonly found in fermenting apple juice . Although this bacterium is not particularly involved in malolactic conversion, the presence of L . collinoides in cider may have serious consequences on the product . L . collinoides is indeed considered to be responsible for the transformation of glycerol to 3-hydroxypropionaldehyde (3-HPA), a precursor of acrolein that spoils the product quality by generating bitter tastes . The purpose of our work was to evaluate the influence of environmental and culture conditions on the conversion of glycerol to 3-HPA in L . collinoides, and to obtain a DNA probe of the gene coding for glycerol dehydratase, the enzyme responsible for this conversion.

Appl Environ Microbiol, 2000 May, 66(5), 2224 - 6
Design and evaluation of a Lactobacillus manihotivorans species-specific rRNA-targeted hybridization probe and its application to the study of sour cassava fermentation; Ampe F; Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation . The probe recognized the rRNA obtained from all the L . manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria . This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L . manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.

Appl Environ Microbiol, 2000 May, 66(5), 2113 - 6
Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation; Muller MR et al.; A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed . Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis . They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough . Other strains isolated together with L . pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer . For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed . To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

J Dent, 2000 Jul, 28(5), 307 - 12
Caries-associated micro-organisms in infants from different socio-economic backgrounds in Scotland; Radford JR et al.; OBJECTIVES: The aims of this study were: (1) to compare the frequency of isolation of mutans streptococci, (Streptococcus mutans, Steptococcus sobrinus), lactobacilli and yeasts (caries-associated micro-organisms) in the saliva of 1-year-old infants with and without dental caries; and (2) to determine if socio-economic background influenced the frequency isolation of bacteria and caries status . METHODS: 1393 1-year-old consented infants, who comprised 70 . 3% of children born in Dundee during a 1year period, had saliva samples taken (tongue-loop method) for microbiological culture and were examined for dental caries (d(1)-threshold: enamel and dentine diagnostic threshold) . Thirty-nine infants were diagnosed with caries and the frequencies of isolation of caries-associated micro-organisms (and absolute microbial counts) were compared with infants who were caries-free . In addition, associations were sought between the infants' socio-economic background, the frequency of isolation of caries-associated micro-organisms and caries status . RESULTS: Streptococcus mutans, lactobacilli and yeasts were isolated more frequently from those infants with caries compared to those who were caries-free (S . mutans: 29.7 vs 9.8%, P=0.0008; lactobacilli: 15.4 vs 4.3%, P=0.0073; yeasts: 23.7 vs 10.4%, P=0.0016-Fisher's exact test) . There were no significant differences between the isolation frequencies of S . sobrinus (2.7 vs 1.3%, P=0.39) from those with and without caries . Significantly, more infants living in areas of high deprivation had caries compared to those from more affluent areas (DEPCAT 6 and 7 vs 1-5: 3.6 vs 1.9%, P=0.049), but, apart from yeasts, socio-economic background was not significantly associated with the isolation frequencies of any of the caries-associated micro-organisms . CONCLUSIONS: In infants as young as 1year of age, salivary S . mutans, lactobacilli and yeasts but not S . sobrinus were isolated significantly more frequently from those with caries compared to those who were caries-free . Apart from yeasts, socio-economic background did not influence the frequency of isolation of caries-associated micro-organisms . However, infants living in areas of highest deprivation had significantly higher frequencies of caries compared to those from more affluent areas.

Plasmid, 2000 May, 43(3), 190 - 9
Expression of bacillar glutamyl endopeptidase genes in Bacillus subtilis by a new mobilizable single-replicon vector pLF; Shevelev AB et al.; The pLF1311 natural plasmid from Lactobacillus fermentum 1311 was used to construct a single-replicon vector suitable for rapid cloning in a wide range of gram-positive hosts and Escherichia coli . The new vector is capable of conjugative mobilization from E . coli to various hosts by conjugal transfer . The final vector (3.4 kb) showed a high segregational and structural stability and a high copy number . Glutamyl endopeptidase genes from Bacillus licheniformis (gseBL) and B . intermedius (gseBI) were cloned in both pLF9 and pLF14 vectors and introduced to B . subtilis . The yield of enzymes in the pLF-derived producers was 6- to 30-fold more than in the natural producers and reached 100-150 mg/L of mature protease .

J Bacteriol, 2000 May, 182(10), 2855 - 64
Biosynthesis of lipoteichoic acid in Lactobacillus rhamnosus: role of DltD in D-alanylation; Debabov DV et al.; The dlt operon (dltA to dltD) of Lactobacillus rhamnosus 7469 encodes four proteins responsible for the esterification of lipoteichoic acid (LTA) by D-alanine . These esters play an important role in controlling the net anionic charge of the poly (GroP) moiety of LTA . dltA and dltC encode the D-alanine-D-alanyl carrier protein ligase (Dcl) and D-alanyl carrier protein (Dcp), respectively . Whereas the functions of DltA and DltC are defined, the functions of DltB and DltD are unknown . To define the role of DltD, the gene was cloned and sequenced and a mutant was constructed by insertional mutagenesis of dltD from Lactobacillus casei 102S . Permeabilized cells of a dltD::erm mutant lacked the ability to incorporate D-alanine into LTA . This defect was complemented by the expression of DltD from pNZ123/dlt . In in vitro assays, DltD bound Dcp for ligation with D-alanine by Dcl in the presence of ATP . In contrast, the homologue of Dcp, the Escherichia coli acyl carrier protein (ACP), involved in fatty acid biosynthesis, was not bound to DltD and thus was not ligated with D-alanine . DltD also catalyzed the hydrolysis of the mischarged D-alanyl-ACP . The hydrophobic N-terminal sequence of DltD was required for anchoring the protein in the membrane . It is hypothesized that this membrane-associated DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with D-alanine and that the resulting D-alanyl-Dcp is translocated to the primary site of D-alanylation.

Med Microbiol Immunol (Berl), 1999 Dec, 188(3), 111 - 6
Inhibitory effect of oral administration of Lactobacillus casei on 3-methylcholanthrene-induced carcinogenesis in mice; Takagi A et al.; The present study was designed to determine whether tumor induction by 3-methylcholanthrene (MC), a carcinogenic hydrocarbon, can be inhibited by oral administration of Lactobacillus casei strain Shirota (LC) . C3H/HeN mice were divided into four groups and assigned to the following treatments: treated with MC and given control or LC-containing diet; treated with vehicle only and given control or LC-containing diet . MC (1 mg) was injected intradermally at 7 weeks of age and the tumor incidence was monitored; LC was mixed into a diet at a concentration of 0.05% (w/w) and the diet was fed from the day of MC injection throughout the study . Spleen cells were analyzed for the immune parameters at 12 and 16 weeks after the MC injection . Oral feeding of mice with LC reduced tumor incidence (P < 0.05) . MC treatment lowered the in vitro response to concanavalin A (Con A) of spleen cells, the secretion of interleukin-2 in spleen cell culture after stimulation of the cells with Con A and the proportions of CD3+ CD4+ and CD8 + splenic cells . However, the analysis of the spleen cells obtained from the mice treated with MC and given the LC-containing diet revealed that these disrupted host immune parameters were maintained at the level of normal controls . These results suggest that oral feeding of mice with LC inhibits MC-induced tumorigenesis by modulating the disrupted host immune responses during MC carcinogenesis.

Vaccine, 2000 May 22, 18(23), 2613 - 23
Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains; Maassen CB et al.; Different Lactobacillus strains are frequently used in consumer food products . In addition, recombinant lactobacilli which contain novel expression vectors can now be used in immunotherapeutic applications such as oral vaccination strategies and in T cell tolerance induction approaches for autoimmune disease . Both for food and clinical applications of lactobacilli, proper selection of wild type strains is crucial.For that purpose, eight different common Lactobacillus strains were analysed with respect to mucosal induction of pro- and anti-inflammatory cytokines, IgA-producing plasma cells in the gut, as well as systemic antibody responses against a parenterally administered antigen . Immunohistochemical analysis of cytokine-producing cells in the gut villi showed no significant induction of the cytokines IL-1alpha, IFN-gamma, IL-4 or IL-10 after oral administration of wild type Lactobacillus strains . In contrast, oral administration of L . reuteri and L . brevis induced expression of the proinflammatory/Th1 cytokines TNF-alpha, IL-2 and/or IL-1beta . Oral administration of these two strains and L . fermentum also significantly enhanced the IgG response against parenterally administered haptenated chicken gamma globulin (TNP-CGG) . The five other strains did not show this adjuvanticity . L . reuteri induced relatively high levels of IgG2a compared to L . murines, a nonadjuving Lactobacillus strain.These findings imply that different Lactobacillus strains induce distinct mucosal cytokine profiles and possess differential intrinsic adjuvanticity . This suggests that rational Lactobacillus strain selection provides a strategy to influence cytokine expression and thereby influence immune responses.

Am J Physiol Heart Circ Physiol, 2000 May, 278(5), H1717 - 24
A new method of long-term preventive cardioprotection using Lactobacillus; Oxman T et al.; Potential long-term cardioprotection was investigated in an extensive experimental study . Lactobacillus cultivation components (LCC) were administered intravenously in anesthetized rats 1, 7, and 21 days before global ischemia (GI) . GI was produced by full stop flow in isolated Langendorff-perfused hearts for 20 min and was followed by reperfusion . Control animals were injected with saline . LCC reduced reperfusion tachyarrhythmia significantly and improved functional recovery of the ischemized rat heart . These beneficial effects were associated with reduction of release of norepinephrine (NE) and prostacyclin at the first minute of reperfusion, activation of myocardial catalase, and overexpression of 70-kDa heat stress protein (HSP-70) at ischemia and reperfusion (P < 0.05) . This cardioprotection was documented up to 21 days after a single injection of LCC . Thus Lactobacillus cultivation components are new nontoxic materials that produce marked long-term cardioprotection against ischemia-reperfusion damage . This effect is attributed to an activation of the cellular defense system, manifested by activation of the antioxidant pathway and by expression of protective proteins . NE is involved in this process, and the data also suggest a role for prostacyclin in this model of cardioprotection . The potential of LCC and related compounds working through similar mechanisms in the prevention and therapy of various ischemic heart syndromes should be explored.

Annu Rev Med, 2000, 51, 349 - 56
Bacterial vaginosis; Sobel JD; Bacterial vaginosis represents a unique upheaval of the complex vaginal bacterial flora with disappearance of lactobacilli and overgrowth of Gardnerella vaginalis and resident anaerobic vaginal bacteria . Little progress has occurred in identifying causal factors, although the pathophysiology of this syndrome is better understood . Although symptoms are easily recognizable, obstetric and gynecologic complications continue to increase in number . Bacterial vaginosis is far more than a nuisance infection . Problems with diagnosis continue to dominate clinical practice, although new tests have been introduced . Therapeutic options have increased, although recurrent disease remains common, and management of this common complication constitutes a major challenge.

Acta Odontol Scand, 1999 Dec, 57(6), 301 - 5
Dental caries and caries-associated microorganisms in Uruguayan preschool children; Angulo M et al.; The prevalence of dental caries was studied in 3-5-year-old Uruguayan children (n = 76) living in 2 areas with different socioeconomic and cultural conditions . More children from the low socioeconomic area of Las Acacias had caries (68%) than children from the middle- to high-class neighborhood of Pocitos (19%) . They also had poorer oral hygiene and a significantly higher caries prevalence (P < 0.05) than those from Pocitos . The occurrence of mutans streptococci and lactobacilli was determined in whole unstimulated saliva and compared with that in debris collected with a loop from the dorsum of the tongue . Mutans streptococci were detected in 42% of the children with significant correlations between the salivary levels of the microorganism and caries experience . Lactobacilli were recovered less frequently (18%) . The detection of mutans streptococci in the tongue-loop samples was significantly correlated with that in whole saliva.

Arch Biochem Biophys, 2000 Apr 15, 376(2), 299 - 312
Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae; DeSouza L et al.; Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments . The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme . In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide . The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP . A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol . A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype . A met7 shm2 strain is nonviable . In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam) . All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain . The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine . Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP . Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene .

J Food Prot, 2000 Apr, 63(4), 549 - 52
Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B, from the chicken duodenum; El-Nezami H et al.; The ability of Lactobacillus rhamnosus strains GG and LC-705 to remove AFB1 from the intestinal luminal liquid medium has been tested in vivo using a chicken intestinal loop technique . In this study, the GG strain of L . rhamnosus decreased AFB1 concentration by 54% in the soluble fraction of the luminal fluid within 1 min . This strain was more efficient in binding AFB1 compared with L . rhamnosus strain LC-705 (P < 0.05) that removed 44% of AFBl under similar conditions . Accumulation of AFB1 into the intestinal tissue was also determined . There was a 74% reduction in the uptake of AFB1 by the intestinal tissue, in the presence of L . rhamnosus strain GG compared with 63% and 37% in the case of Propionibacterium freudenreichii ssp . shermanii JS and L . rhamnosus strain LC-705, respectively . The complexes formed in vitro between either L . rhamnosus strain GG or L . rhamnosus strain LC-705 and AFB1 were stable under the luminal conditions for a period of 1 h.

J Food Prot, 2000 Apr, 63(4), 502 - 8
Effect of salt and smoke on the microbiological quality of cold-smoked salmon during storage at 5 degrees C as estimated by the factorial design method; Leroi F et al.; The simultaneous effect of salt and smoke on the natural flora of cold-smoked salmon was studied during 5 weeks of vacuum storage at 5 degrees C . The quadratic polynomial, as a function of factors, was used to express total viable count (TVC), total lactic acid bacteria, lactobacilli numerated on Rogosa agar, H2S-producing bacteria, and yeasts at different sampling times . TVC and total lactic acid bacteria were mainly inhibited by the salt concentration (5% wt/wt) in the meat and to a lesser extent by the phenol content . Inhibition was linearly proportional to salt and smoke content (the higher the concentration, the greater the inhibition) . No synergistic effect on inhibition was observed between the two factors . In our working conditions, the TVC French standard (<10(6) CFU g(-1)) was maintained during 4 weeks of storage at 5 degrees C, with a minimum concentration of 2.4% (wt/wt) of salt in meat and smoking treatment corresponding to 0.6 mg 100 g(-1) of phenol . When the salt level was higher than 3%, the TVC standard was maintained, regardless of phenol level . A negative interaction between the two factors was found for H2S-producing bacteria and a positive one for yeasts.

J Food Prot, 2000 Apr, 63(4), 462 - 72
Development of a simple recovery-enrichment system for enhanced detection of heat-injured Listeria monocytogenes in pasteurized milk; Teo AY et al.; A simple anaerobic recovery-enrichment system, semisolid Penn State University (ssPSU) broth, that enhances recovery of heat-injured Listeria monocytogenes, was rapidly achieved in 10-ml screw-capped tubes by adding Bacto-agar (2.5 g/liter) and L-cysteine (0.5 g/liter) to Penn State University broth . Glucose was removed from the formulation for ssPSU broth to prevent the growth of thermoduric lactobacilli . Ferric ammonium citrate was added to ssPSU broth to detect esculin hydrolysis and to indicate the presumptive presence of L . monocytogenes . Replacement of phosphate buffer with 3-{N-morpholino}propanesulfonic acid (MOPS) buffer and addition of magnesium sulfate (15 mM) enhanced recovery and detection of L . monocytogenes heat treated at 62.8 degrees C for 20 min . D-Serine, at a concentration of 150 mM, was found to inhibit germination of Bacillus spp . spores but did not inhibit severely heat-injured L . monocytogenes . Finally, ssPSU broth was modified (to mPSU broth) to contain the following: (i) Bacto-agar, 2.5 g/liter; (ii) ferric ammonium citrate, 0.5 g/liter; (iii) MOPS buffer, pH 7.0; (iv) D-serine, 13.7 g/liter; (v) D-alanine, 11.6 g/liter; and (iv) magnesium sulfate, 1.81 g/liter . Incubation temperature significantly affected the recovery and detection of severely heat-injured L . monocytogenes . L . monocytogenes that were heat challenged in filter-sterilized whole milk at 62.8 degrees C for 20, 25, and 30 min could not be detected at incubation temperatures > or = 30 degrees C but were consistently detected after incubation at 25 degrees C for 174, 199, and 330 h, respectively . Heat-injured cells of L . monocytogenes that were added to various commercial brands of pasteurized whole milk were also detected using mPSU broth . When clostridial spores (10(4) spores per ml) were added to filter-sterilized milk containing either heat-injured or non-heat-injured L . monocytogenes, only the latter could be detected in mPSU broth . The mPSU broth system requires no purging with nitrogen gas to create anaerobic conditions and permits recovery, growth, and detection of L . monocytogenes in one vessel in the presence of thermoduric background microflora commonly found in pasteurized milk.

Microbiol Res, 2000 Mar, 154(4), 313 - 20
Citrate utilization by homo- and heterofermentative lactobacilli; Medina de Figueroa R et al.; Citrate utilization by several homo- and heterofermentative lactobacilli was determined in Kempler and McKay and in calcium citrate media . The last medium with glucose permitted best to distinguish citrate-fermenting lactobacilli . Lactobacillus rhamnosus ATCC 11443, Lactobacillus zeae ATCC 15820 and Lactobacillus plantarum ATCC 8014 used citrate as sole energy source, whereas in the other strains, glucose and citrate were cometabolized . Some lactobacilli strains produced aroma compounds from citrate . Citrate transport experiments suggested that all strains studied presented a citrate transport system inducible by citrate . The levels of induction were variable between several strains . Dot blot experiment showed that lactobacilli do not present an equivalent plasmid coding for citrate permease.

Eur J Oral Sci, 2000 Apr, 108(2), 154 - 61
Effects of fluoride- and chlorhexidine-containing varnishes on plaque composition and on demineralization of dentinal grooves in situ; Zaura-Arite E et al.; The aim of the study was to compare the effects of Cervitec, containing 1% chlorhexidine (CHX) and 1% thymol, Fluor Protector, containing 0.1% fluoride, their 1:1 mixture, and a placebo varnish on the percentage of mutans streptococci and lactobacilli in plaque and on the underlying dentin demineralization, as assessed by microradiography . Bovine dentine discs, fitted with three parallel grooves, received one of the varnish treatments into the first groove and on the adjacent part of the dentin surface . Volunteers (n = 23) wore the discs fixed to their partial dentures for four consecutive 3 wk periods . Microbiological analysis of plaque accumulated in the grooves showed no difference between groups . Fluoride varnishes (Fluor Protector and mixed varnish) had a significantly larger inhibitory effect on mineral loss in the treated groove than Cervitec or placebo . All treatment varnishes had more pronounced effect in panelists (n = 14) with higher degree of demineralization (mineral loss in placebo group > or = 1,200 vol% x microm) . In these panelists, CHX-containing varnishes showed an inhibitory effect on demineralization in all grooves, also in the two non-varnished grooves . As fluoride varnishes had the largest localized effect on demineralization, and CHX varnishes were showing a peripheral effect, a combined treatment could be the preferred method to obtain an optimal caries preventive effect in caries-prone individuals.

J Inorg Biochem, 2000 Jan 30, 78(2), 149 - 60
Hydrogen abstraction from thiols by adenosyl radicals: chemical precedent for thiyl radical formation, the first catalytic step in ribonucleoside triphosphate reductase from Lactobacillus leichmannii; Sirovatka JM et al.; Aqueous solutions of adenosylcobalamin (AdoCbl) were thermolyzed with excess beta-mercaptoethanol under anaerobic conditions . The product studies reveal that approximately 90% Co-C bond homolysis occurs, to yield Co(II)cobalamin, 5'-deoxyadenosine, and the disulfide product from the combination of two HOCH2CH2S* radicals, 2,2'-dithiodiethanol; there is also approximately 10% Co-C bond heterolysis, yielding Co(III)cobalamin, adenine, and 2,3-dihydroxy-4-pentenal . The kinetic studies show there is a first-order dependence on AdoCbl and zero-order dependence on thiol under the higher {RSH} experimental conditions used, consistent with the rate-determining step at high {RSH} being the generation of Ado* . The kinetic results require that, in enzyme-free AdoCbl solution, adenosyl radical (Ado*) is formed as a discrete intermediate which then abstracts H* from the added thiol . The activation parameters for Co-C bond homolysis in the presence of thiol trap are the same within experimental error as the activation parameters for Co-C bond homolysis without trap, standard delta H(obs) = 29(2) kcal mol(-1) and standard delta S(obs) = -1(5) e.u . The results, in comparison to the rate of Co-C bond homolysis in ribonucleoside triphosphate reductase (RTPR), reveal that RTPR accelerates Co-C bond cleavage in AdoCbl by approximately 10(10+/-1) . The recent literature evidence bearing on the exact mechanism of RTPR enzymic cleavage of the Co-C bond of AdoCbl is briefly discussed, notably the fact that this mechanism is presently controversial, but does involve at least coupled (and possibly concerted) Co-C cleavage, -S-H cleavage, and C-H (Ado-H) formation steps.

Am J Obstet Gynecol, 2000 Apr, 182(4), 872 - 8
Pathogenesis of abnormal vaginal bacterial flora; Donders GG et al.; OBJECTIVE: This study was undertaken to determine the relationships between microscopy findings on wet mounts, such as lactobacillary grade or vaginal leukocytosis, and results of vaginal culture, lactate and succinate content of the vagina, and levels of selected cytokines . STUDY DESIGN: In a population of 631 unselected women seeking treatment at an obstetrics and gynecology outpatient clinic, vaginal fluid was obtained by wooden Ayre spatula for wet mounting and pH measurement, by high vaginal swab for culture, and by standardized vaginal rinsing with 2 mL 0.9% sodium chloride solution for measurements of lactate, succinate, interleukin 1beta, interleukin 8, leukemia inhibitory factor, and interleukin 1 receptor antagonist concentrations . Lactate and succinate levels were measured by gas-liquid chromatography and the cytokine concentrations were measured by specific immunoassays . Both univariate analysis (Student t test, Welch test, chi(2) test, and Fisher exact test) and multivariate regression analysis (Cox analysis) were used . RESULTS: Increasing disturbance of the lactobacillary flora (lactobacillary grades I, IIa, IIb, and III) was highly correlated with the presence of Gardnerella vaginalis, Trichomonas vaginalis, enterococci, group B streptococci, and Escherichia coli . Vaginal pH and interleukin 8 and interleukin 1beta concentrations increased linearly with increasing lactobacillary grade, whereas lactate concentrations and the presence of epithelial cell lysis decreased . A similar pattern of associations with increasing leukocyte count was clear, but in addition there was an increase in leukemia inhibitory factor concentration . Multivariate analysis of vaginal leukocytosis, lactobacillary grades, and the presence of positive vaginal culture results showed that interleukin 1beta concentration was most closely related to the lactobacillary grade, leukemia inhibitory factor concentration was most closely related to the lactobacillary grade and positive culture results, interleukin 8 concentration was most closely related to positive culture results, and interleukin 1 receptor antagonist concentration was most closely related to vaginal leukocytosis and positive culture results . The concentration ratio of interleukin 1beta to interleukin 1 receptor antagonist remained stable, except when vaginal leukocytosis increased . In its most severe form, with >10 leukocytes per epithelial cell present, a decompensation of the vaginal flora with a collapse in interleukin 1beta and interleukin 1 receptor antagonist concentrations was seen, but there was a concurrent sharp increase in leukemia inhibitory factor concentration . This pattern was completely different from the course of the cytokine concentrations associated with a lactobacillary grade increase . CONCLUSION: Both disturbed lactobacillary grade and the presence of increasing vaginal leukocytosis were correlated with lactobacillary substrate (lactate) concentration, pH, and the concentrations of a variety of cytokines . There was a remarkably linear increase in these cytokines as either leukocytosis or lactobacillary grade became more severe . In circumstances in which leukocytosis was extreme, however, interleukin 1beta was no longer produced but leukemia inhibitory factor concentrations increased . We speculate that in extreme inflammation the body tries to limit the damage that can be done by exaggerated cytokine production.

J Bacteriol, 2000 May, 182(9), 2582 - 90
Phosphorylation of HPr by the bifunctional HPr Kinase/P-ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion; Dossonnet V et al.; We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P) . Purified recombinant L . casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) . The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity . A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities . When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished . In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant . However, inducer expulsion of methyl beta-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.

Mol Microbiol, 2000 Mar, 35(6), 1383 - 93
Characterization of the Lactococcus lactis transcription factor FlpA and demonstration of an in vitro switch; Scott C et al.; The commercially important bacterium Lactococcus lactis contains two FNR-like proteins (FlpA and FlpB) which have a high degree of identity to each other and to the FLP of Lactobacillus casei . FlpA was isolated from a GST-FlpA fusion protein produced in Escherichia coli . Like FLP, isolated FlpA is a homodimeric protein containing both Zn and Cu . However, the properties of FlpA were more like those of the E . coli oxygen-responsive transcription factor FNR than the FLP of L . casei . As prepared FlpA recognized an FNR site (TTGAT-N4-ATCAA) but not an FLP site (CCTGA-N4-TCAGG) in band-shift assays . In contrast to FLP, DNA binding by FlpA did not require the formation of an intramolecular disulphide bond . However, despite containing only two cysteine residues per monomer, FlpA was able to acquire an FNR-like, oxygen-labile {4Fe 4S} cluster . But, whereas the incorporation of a {4Fe 4S} cluster into FNR enhances interaction with target DNA, it abolished DNA binding by FlpA . An FlpA variant (FlpA') with an N-terminal region designed to be more FLP-like failed to incorporate an iron-sulphur cluster but could now form an intramolecular disulphide . This simple example of protein engineering, converting an oxygen-labile {4Fe 4S} containing FNR-like protein into a dithiol-disulphide FLP-like redox sensor demonstrates the versatility of the basic CRP structure . Attempts to demonstrate an FlpA-based aerobic-anaerobic switch in the heterologous host E . coli were unsuccessful . However, studies with a series of FNR-dependent lac reporter fusions in strains of E . coli expressing flpA or flpB revealed that both homologues were able to activate expression of FNR-dependent promoters in vivo but only when positioned 61 base pairs upstream of the transcription start.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 699 - 702
Lactobacillus nagelii sp . nov., an organism isolated from a partially fermented wine; Edwards CG et al.; A Gram-positive rod was isolated from a commercial grape wine undergoing a sluggish/stuck alcoholic fermentation . The organism produced DL-lactic acid from glucose without gas formation, produced dextran from sucrose, hydrolysed aesculin and fermented galactose, D-glucose, D-fructose, D-mannose, L-sorbose, rhamnose, mannitol, sorbitol, methyl alpha-D-glucoside, N-acetylglucosamine, amygdalin, salicin, cellobiose, maltose, sucrose, trehalose and beta-gentiobiose . 16S rRNA gene sequence analysis revealed that the isolate was phylogenetically a member of the genus Lactobacillus and formed a distinct subline within the Lactobacillus casei cluster of species . On the basis of phenotypic and phylogenetic evidence, Lactobacillus nagelii sp . nov . ATCC 700692T is proposed as a new species.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 471 - 8
Comparative analysis of the genes encoding 23S-5S rRNA intergenic spacer regions of Lactobacillus casei-related strains; Chen H et al.; In this study, investigations into the 23S-5S rRNA intergenic spacer regions (ISRs) of the Lactobacillus casei group were performed . A 1.6 kb fragment, from Lactobacillus paracasei strain ATCC 27092, containing part of the 5S rRNA gene (60 bp), the 5S-23S spacer region (198 bp) and part of the 23S rRNA gene (1295 bp) was cloned and sequenced (GenBank no . AF098107) . This fragment was used as a probe to determine the rRNA restriction fragment length polymorphism (RFLP) patterns of nine strains belonging to the Lactobacillus casei group, along with four other non-Lactobacillus casei lactobacilli species . A pair of PCR primers, 23-Fl and 5-Ru, was designed and used for PCR amplification of the 23S-5S rRNA ISRs of these strains . The ISR length and sequence polymorphisms provided additional information for the taxonomic study of the Lactobacillus casei group . The spacer-length polymorphism of Lactobacillus rhamnosus was distinct from those of the other strains and this observation is consistent with the classification of Lactobacillus rhamnosus proposed by Mori et al . For all Lactobacillus casei and Lactobacillus paracasei strains, two major bands (approx . 250 and 170 bp in size) were obtained except in the case of Lactobacillus paracasei subsp . tolerans strain NCIMB 9709T, which yielded only one amplified product (250 bp) . The sequencing data of the PCR products of seven well-characterized Lactobacillus casei and Lactobacillus paracasei strains revealed the presence of a 76/80 bp insertion/deletion with some random, single-base substitutions between the longer and shorter spacers for each respective strain . A few base variations were also detected within different strains in this group although the overall sequence similarity was very high (95.9-99.5%) . The rRNA RFLP and the spacer sequence of Lactobacillus casei type strain ATCC 393T exhibited unique identities in this cluster . On the other hand, Lactobacillus casei strain ATCC 334 showed a high level of similarity with the other Lactobacillus casei strains tested . These observations lend some support to the request for the reassignment of strain ATCC 334 as the neotype of Lactobacillus casei subsp . casei in place of strain ATCC 393T.

FEMS Microbiol Lett, 2000 Apr 15, 185(2), 157 - 61
Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705; Cuozzo SA et al.; The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced . Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each . These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis . However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.

J Dairy Sci, 2000 Mar, 83(3), 526 - 35
The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage; Ranjit NK et al.; Several microorganisms and one chemical preservative were tested for their effects on the fermentation and aerobic stability of corn silage . Whole-plant corn (one-half milk line, 31.3% dry matter) was ensiled in quadruplicate 20-L laboratory silos untreated or after the following treatments: Lactobacillus buchneri at 1 x 10(5) and 1 x 10(6) cfu/g of fresh forage; two different strains of L . plantarum, each at 1 x 10(6) cfu/g; and a buffered propionic acid-based product at 0.1% of fresh forage weight . After 100 d of ensiling, silage treated with L . buchneri (1 x 10(6) cfu/g) had a lower concentration of lactic acid compared with the untreated silage, but was similar to other treated silages . The silage treated with the high (1 x 10(6) cfu/g), but not the moderate rate (1 x 10(5) cfu/g) of L . buchneri also had a greater concentration of acetic acid (3.60%) and less yeasts (2.01 log cfu/g) when compared with other treatments (average of 1.88% acetic acid and 5.85 log cfu of yeasts/g) . Silages treated with L . plantarums, the moderate rate of L . buchneri, and the chemical preservative took longer to heat than untreated silage when exposed to air, but improvements were numerically small (6.3 to 10.5 h) . In contrast, silage treated with the high rate of L . buchneri never heated throughout a 900-h period of monitoring . Inoculating corn silage with 1 x 10(6) cfu/g of L . buchneri resulted in a more heterolactic fermentation and dramatically improved the aerobic stability of corn silage.

J Dairy Sci, 2000 Mar, 83(3), 401 - 3
Effect of Lactobacillus reuteri on the prevention of hypercholesterolemia in mice; Taranto MP et al.; Administration of Lactobacillus reuteri CRL 1098 (10(4) cells/d) to mice for 7 d before inducing hypercholesterolemia (by feeding mice with a fat-enriched diet for the subsequent 7 d) was evaluated . At this low dose, L . reuteri was effective in preventing hypercholesterolemia in mice, producing a 17% increase in the ratio of high-density lipoprotein to low-density lipoprotein . Total cholesterol and triglycerides decreased by 22 and 33%, respectively, in the group that was not fed the lactobacilli . The hypocholesterolemic effect produced by L . reuteri CRL 1098 might be considered as indirect evidence of the permanency of the lactobacilli in the gut.

J Antimicrob Chemother, 2000 Apr, 45(4), 447 - 52
Ecological effects of triclosan and triclosan monophosphate on defined mixed cultures of oral species grown in continuous culture; Saunders KA et al.; The effects of triclosan and its phosphorylated derivative, triclosan monophosphate were studied using a continuous culture microcosm model . Two conditions were simulated, a caries-like state (pH 5.5 with artificial saliva plus glucose as growth medium) and a periodontal disease-like state (pH 7.5 with BHI plus yeast extract, haemin and cysteine as growth medium) . Both cultures were maintained anaerobically at 37 degrees C at a growth rate of 0.1/h . Steady-state chemostats were pulsed with triclosan or triclosan monophosphate (initial concentrations between 20 and 40 mg/L) and changes in the ecological composition noted after 6 h . The caries-like microcosm steady state was dominated by streptococci, Lactobacillus and Veillonella sp . with low but detectable levels of Neisseria, Actinomyces and Fusobacterium sp . No significant ecological shifts occurred following pulses of either antimicrobial agent; all species were affected to approximately the same degree . The periodontal disease-like microcosm steady state was dominated by streptococci, Fusobacterium, Veillonella, Actinomyces, Prevotella and Porphyromonas sp . with low numbers of Neisseria and Lactobacillus sp . Significant ecological shifts were apparent following pulses of triclosan . The streptococci became the dominant group followed by Fusobacterium sp . For triclosan monophosphate, the streptococci again became dominant although Lactobacillus and Actinomyces were now the main sub-dominant species and Gram-negative anaerobes including Fusobacterium sp . were markedly inhibited . It is concluded that in the periodontal disease state, both triclosan and triclosan monophosphate affected the Gram-negative anaerobes to a greater extent than the Gram-positive groups and that this effect was more marked for triclosan monophosphate.

Lett Appl Microbiol, 2000 Mar, 30(3), 197 - 202
Lactobacillus farciminis MD, a newer strain with potential for bacteriocin and antibiotic assay; Halami PM et al.; A native isolate Lactobacillus farciminis MD isolated from fermenting mushroom exhibited a high degree of sensitivity to the majority of the bacteriocins produced by strains of lactobacilli, leuconostoc and pediococci . Also, the efficacy of Lact . farciminis MD as a sensitive strain for antibiotic assay was established against different antibiotics including ampicillin, cefazoline, chloramphenicol and nitrofurantoin at concentrations of 30 microg each, showing an inhibition zone of 30 mm diameter . The high degree of sensitivity towards bacteriocins and antibiotics provide potential for the exploitation of Lact . farciminis MD in establishing very well-defined bacteriocin producers.

Lett Appl Microbiol, 2000 Mar, 30(3), 173 - 7
Selective control of lactobacilli in kimchi with nisin; Choi MH et al.; The use of nisin to control the lactobacilli responsible for the over-ripening of kimchi, traditional Korean fermented vegetables, was studied . Of the 40 strains of lactic acid bacteria studied, most were sensitive to nisin at a concentration of 100 IU ml-1, while two strains appeared to be resistant . In MRS broth containing nisin at concentrations of 100-300 IU ml-1, the growth of sensitive strains of Lactobacillus plantarum was delayed for 2-3 d at 20 degrees C . When nisin was added to kimchi at a concentration of 100 IU ml-1, the growth of Lactobacillus spp . was inhibited more than the growth of Leuconostoc spp . Scanning electron micrograph observations confirmed the results, demonstrating the predominance of cocci in kimchi containing nisin . These results suggest that at recommended levels, nisin can be used to preserve kimchi by inhibiting lactobacilli more effectively than other lactic acid bacteria involved in kimchi fermentation.

J Appl Microbiol, 2000 Mar, 88(3), 536 - 45
Production of sakacin P by Lactobacillus sakei in a completely defined medium; Moretro T et al.; In order to investigate factors influencing the production of the bacteriocin, sakacin P, Lactobacillus sakei CCUG 42687 was grown in a completely defined medium (DML-B) with 33 components . Although the maximum sakacin P concentration obtained was higher on a complex medium due to higher cell mass, the production per cell mass was higher in DML-B . Sakacin P was produced at 4-30 degrees C, with the highest specific production at low temperatures . More sakacin P was produced at uncontrolled pH compared with cultivation at pH 6.3 . Tween-80 had a positive effect on sakacin P production, while addition of sodium chloride and trace metals had negative effects . The decrease in sakacin P concentration during the late growth and stationary phases was shown to be cell-independent and promoted at high temperature and pH . Some differences in production levels of sakacin P were found among six strains of Lactobacillus sakei tested.

J Appl Microbiol, 2000 Mar, 88(3), 365 - 70
Antagonistic effect of Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli combinations against experimental infections with Shigella flexneri and Salmonella enteritidis subsp . typhimurium in gnotobiotic mice; Filho-Lima JV et al.; Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli are probiotic strains used individually to protect against enteropathogenic agents . In order to determine if a synergistic effect of the individual protective mechanisms ordinarily attributed to each of these biotherapeutic agents is possible, we orally administered Lact . acidophilus H2B20, S . boulardii and E . coli EMO (LSE) to germfree mice . Ten days after colonization of the digestive tract, groups of animals associated (experimental) or not (control) with LSE were challenged orally with streptomycin resistant (Sfr) or streptomycin sensitive (Sfs) Shigella flexneri strains or Salmonella enteritidis subsp . typhimurium . Bacterial counts in faeces from experimental mice showed that the Sfr strain was eliminated 11 d after challenge while Sfs and S . enteritidis subsp . typhimurium colonized the digestive tract and continued to be present at high population levels (108 CFU g-1 of faeces), which is similar to that observed in control animals . All possible di- and monoassociations of the three probiotics with gnotobiotic mice were also performed before experimental oral infection with Sfr . The data showed that antagonism was obtained only when E . coli EMO was present . Different sensitivity of Sh . flexneri Sfr and Sfs to E . coli EMO antagonism could be explained by the different generation times between Sfr and Sfs, as shown by colonization kinetic experiments in the digestive tract of gnotobiotic mice.

AIDS Patient Care STDS, 1999 Dec, 13(12), 747 - 51
Potential role of lactobacilli as prophylactic agents against genital pathogens; Barbes C et al.; The characteristics required of lactobacilli as probioties are the following: beneficial function, easy cultivation, nonpathogenicity, adhesion, and population stability . Several studies have assessed the potential of lactobacilli in the prevention or treatment of certain genitourinary tract infections such as bacterial vaginosis, vaginitis, or urinary tract infections . The main goal of therapy with biotherapeutic agents should be to prevent overgrowth of a pathogen until such a time that the normal microbiota can be reestablished . The possibility of using lactobacilli is promising, especially in pregnant women and in the case of patients with recurrent genitourinary tract infections produced by strains with resistance to several antibiotics . In addition, probiotic therapy is considered as "natural" and without side effects in contrast with conventional pharmaceutical treatments, but there is a limited array of tested biotherapeutic agents and a lack of pharmacokinetic data.

ASDC J Dent Child, 2000 Jan-Feb, 67(1), 42 - 6, 9
Cariogenic oral flora and its relation to dental caries; Llena-Puy MC et al.; Many attempts have been made to establish the risk profile of dental caries; however, no diagnostic procedures are yet available to reliably predict such risk particularly among the pediatric and adolescent population . Age of subject, a history of caries affecting the primary dentition, the prevalence of Streptococcus mutans, pH values, salivary flow, and the frequency and amount of sugar consumption have been the factors most studied . A cross-sectional study is made of schoolchildren in the 12-13-year age-range to evaluate the relationship between dental caries and CFU/ml of S . mutans and Lactobacillus, salivary buffer capacity, and salivary flow . Likewise, an evaluation is made of the predictive value of the variables, bacterial count and salivary pH with respect to caries . A total of 167 children were subjected to oral examination to establish the DMFT and DMFS indices, followed by the collection of saliva for quantitating S . mutans, Lactobacillus, pH and salivary flow, by the Dentocult (SM)(LB) and Dentobuff systems (Vivadent) . The data obtained were subjected to descriptive analysis, comparisons among variables were made by nonparametric testing with a confidence level of 95 percent . Test predictive value was studied in terms of sensitivity, specificity, positive and negative predictive values, and probability ratio . Statistically significant (p < 0.001) correlations were observed between the caries indices and bacterial counts . No significant association was recorded with the rest of the variables studied . Bacterial counts as well as salivary buffer capacities exhibited greater negative than positive predictive values, i.e., they were more effective in identifying healthy individuals than patients who required treatment.

Lett Appl Microbiol, 2000 Feb, 30(2), 126 - 9
Development of a rapid method for the identification of Lactobacillus spp . isolated from naturally fermented italian sausages using a polymerase chain reaction-temperature gradient gel electrophoresis; Cocolin L et al.; A rapid method for the identification of Lactobacillus spp . isolated from naturally fermented Italian sausages was developed . It is based on the amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis (TGGE) . Lactobacillus sakei, L . curvatus, L . alimentarius, L . casei, L . plantarum and L . brevis, obtained from International Collections, were used to optimize the method . Thiry-nine strains of Lactobacillus spp . were isolated from naturally fermented sausages and, after traditional identification, were tested by the PCR-TGGE protocol developed . No differences were observed comparing the results obtained, apart from five strains identified as L . curvatus that showed a PCR-TGGE profile identical to L . sakei.

J Appl Microbiol, 2000 Feb, 88(2), 342 - 8
Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus; Murga ML et al.; In order to correlate the lipid composition of the membrane of Lactobacillus acidophilus CRL 640 with the freeze-thaw behaviour of the cultures grown at different temperatures, fatty acid methyl esters (FAMEs) from extracts grown at 25, 30, 37 and 40 degrees C were obtained and compared . Cultures grown at 25 degrees C (M25) exhibited more resistance to the freeze-thaw process probably because of an increase in C18:2 and C16:0 fatty acids . This culture also exhibited a lesser amount of phospholipids as shown by the sugar: phosphorus ratio . In all cases, the presence of the uncommon 10-hydroxyoctadecanoic acid was determined . From the extracts of the M25 and M37 cultures, diacylphosphatidylglycerol, cardiolipin, diglycosyldiglycerides, triglycosyldiglycerides and neutral lipids were isolated and identified . The structural elucidation was carried out by FAMEs and sugar analysis and by mass spectrometry using fast atom bombardment ionization . The changes in lipid composition due to different growth temperatures could be indicative of the resistance of the bacteria to freeze-thaw processes.

J Appl Microbiol, 2000 Feb, 88(2), 317 - 24
Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid; Gobbetti M et al.; Sixty-five strains of obligately and facultatively heterofermentative sourdough lactic acid bacteria were screened for their capacity to grow optimally in the presence of arabinose, ribose and xylose as carbon sources . Lactobacillus alimentarius 15F, Lact . brevis 10A, Lact . fermentum 1F and Lact . plantarum 20B showed higher growth rate, cell yield, acidification rate and production of acetic acid when some pentoses instead of maltose were added to the SDB medium . Lactobacillus plantarum 20B used arabinose also in a synthetic medium where complex growth factors such as yeast extract were omitted . Other Lact . plantarum strains did not show the same property . Pentosan extract was treated with alpha-L-arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates containing mainly arabinose and xylose, respectively . In particular, the hydrolysate containing arabinose substantiated the growth and the production of lactic acid and, especially, of acetic acid by Lact . plantarum 20B . Sourdough fermentation by Lact . plantarum 20B with addition of pentosan extract and alpha-L-arabinofuranosidase increased the acidification rate, titratable acidity and acetic acid content compared with traditional sourdough . A facultatively heterofermentative strain, Lact . plantarum 20B, also produced a sourdough with an optimal fermentation quotient.

J Appl Microbiol, 2000 Feb, 88(2), 286 - 92
Identification and enumeration of oleic acid and linoleic acid hydrating bacteria in the rumen of sheep and cows; Hudson JA et al.; The diversity and population densities of facultative anaerobic bacteria with the capacity to hydrate oleic acid and linoleic acid in the rumen of sheep and dairy cows were determined . The screening of representative colonies, from rumen fluid plated aerobically on a range of agar media, revealed that sheep rumen fluid contained hydration-positive strains of Streptococcus, Staphylococcus, Enterococcus, Lactobacillus and Pediococcus, whereas cow rumen fluid contained hydration-positive strains of Streptococcus, Lactobacillus and Staphylococcus . Mean counts of facultative anaerobic bacteria in sheep and cattle rumen were log10 7.29 and log10 6.40, respectively, and were independent of diet . Approximately 56% of facultative anaerobic bacteria were able to hydrate oleic and/or linoleic acid in anaerobic broth culture . For both sheep and cows, the most numerous hydration-positive isolates were strains of Strep . bovis . The results, which are the first to show that pediococci have the capacity to hydrate unsaturated fatty acids, suggest that lactic acid bacteria are the major unsaturated fatty acid hydrating bacteria in the rumen.

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