The Value of Certain Tests in the Differentiation of Lactobacillus bulgaricus from Lactobacillus acidophilus.
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Journal Article↗
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Comparative Study of Lactobacillus acidophilus and Lactobacillus bulgaricus.
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Journal Article↗
[Biological characteristics of Lactobacillus. II. Tolerance of a multiple antibiotic resistant strain, Lactobacillus casei PSR 3002, to artificial digestive fluids].
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Bile Acids and Salts↗
Nucleic acids of species of Lactobacillus.
Nucleic acid composition of 17 cultures representing the type or neotype strains of 15 named Lactobacillus species was studied. Nucleic acid characterization of these isolates was accompanied by a comparative study of conventional phenotypic reactions. The overall guanine plus cytosine mean deoxyribonucleic acid base composition ranged from 33 to 50% and genome sizes varied between 700 and 1500 X 10(6) daltons. Sporolactobacillus inulinus contained 2500 X 10(6) daltons of chromosomal deoxyribonucleic acid and was therefore similar in size to members of the genus Bacillus. Deoxyribonucleic acid hybridization at a temperature 15 degrees C below the thermal melting temperature confirmed the extreme molecular heterogeneity of many species. The genus can be divided into three major groups, each containing four or more species based on a combination of nucleic acid characteristics and conventional phenotypic reactions. Group I (33 to 39% guanine plus cytosine) contains Lactobacillus jensenii, Lactobacillus helveticus, Lactobacillus helveticus nov. ssp. jugurt, Lactobacillus acidophilus, Lactobacillus salivarius, and Lactobacillus sanfrancisco. Group II (42 to 48% guanine plus cytosine contains Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus brevis, and Group III (48 to 50% guanine plus cytosine) contains Lactobacillus delbrueckii, Lactobacillus delbrueckii nov. ssp. bulgaricus, Lactobacillus delbrueckii nov. ssp. lactis, Lactobacillus delbrueckii nov. ssp. leichmanii, Lactobacillus fermentum, and Lactobacillus fermentum nov. ssp. cellobiosis.
Base Composition↗
Comparative analysis of the genes encoding 23S-5S rRNA intergenic spacer regions of Lactobacillus casei-related strains.
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.
Base Sequence↗
Lactobacillus fornicalis sp. nov., isolated from the posterior fornix of the human vagina.
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.
Bacterial Proteins↗
Use of polymerase chain reaction techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis for differentiation of oral Lactobacillus species.
BACKGROUND/AIMS: The genus Lactobacillus has been associated with dental caries in humans, although it is seldom speciated due to lack of simple and nonlaborious identification methods. A considerable heterogeneity among Lactobacillus species has been demonstrated. The purpose of this study was to develop simple methods combining restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rRNA (16S rRNA gene PCR-RFLP) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the identification of 13 reference strains of Lactobacillus. METHODS: The 16S rRNA gene sequences were amplified by PCR using universal primers and digestion of PCR products with the restriction endonucleases, HpaII and HaeIII. The 16S rRNA gene PCR-RFLP is reproducible and has been proved to be useful for differentiating Lactobacillus strains to species level. Seventy-seven Lactobacillus isolates from a Thai population were used to show the applicability of the identification test. RESULTS: PCR-RFLP alone had limitations, because the RFLP patterns of Lactobacillus casei and Lactobacillus rhamnosus and of Lactobacillus acidophilus and Lactobacillus crispatus showed similar patterns; however, these could be differentiated by SDS-PAGE. Of the 77 isolates, 38 were identified as Lactobacillus fermentum, 25 as L. rhamnosus, 5 as Lactobacillus salivarius, 5 as L. casei, 3 as L. acidophilus and 1 as Lactobacillus plantarum. CONCLUSION: 16S rRNA gene PCR-RFLP, using HpaII and HaeIII, together with SDS-PAGE protein profiles could be an alternative method for the identification of oral Lactobacillus strains to species level, and may be applicable for large-scale studies on the association of Lactobacillus to dental caries.
Adult↗
Lactobacillus paracasei normalizes muscle hypercontractility in a murine model of postinfective gut dysfunction.
BACKGROUND & AIMS: The effects of probiotics on gut dysfunction in postinfective irritable bowel syndrome are unknown. We tested whether probiotics influence persistent muscle hypercontractility in mice after recovery from infection with Trichinella spiralis and analyzed the underlying mechanisms. METHODS: Mice were gavaged with Lactobacillus paracasei, Lactobacillus johnsonii, Bifidobacterium longum, or Bifidobacterium lactis in spent culture medium from days 10 to 21 after infection. Additional mice received heat-inactivated Lactobacillus paracasei, Lactobacillus paracasei -free spent culture medium, or heat-inactivated Lactobacillus paracasei -free spent culture medium. Lactobacilli enumeration, immunohistochemistry, and cytokine detection (enzyme-linked immunosorbent assay) were performed. Mice were also treated with Lactobacillus paracasei or Lactobacillus paracasei -free spent culture medium from days 18 to 28 after infection. Contractility was measured on days 21 and 28 after infection. RESULTS: Lactobacillus paracasei, but not Lactobacillus johnsonii, Bifidobacterium lactis, or Bifidobacterium longum, attenuated muscle hypercontractility. This was associated with a reduction in the Trichinella spiralis -associated T-helper 2 response and a reduction in transforming growth factor-beta1, cyclooxygenase-2, and prostaglandin E 2 levels in muscle. Attenuation of muscle hypercontractility by Lactobacillus paracasei -free spent culture medium was abolished after heat treatment. Improvement of muscle hypercontractility at day 28 after infection was also observed after the administration of Lactobacillus paracasei or Lactobacillus paracasei -free spent culture medium from day 18 after infection. CONCLUSIONS: Probiotics show strain-dependent attenuation of muscle hypercontractility in an animal model of postinfective irritable bowel syndrome. This likely occurs via both a modulation of the immunologic response to infection and a direct effect of Lactobacillus paracasei or a heat-labile metabolite on postinfective muscle hypercontractility. Lactobacillus paracasei may be useful in the treatment of postinfective irritable bowel syndrome.
Animals↗
Impact of dried skim milk in production diets on Lactobacillus and pathogenic bacterial shedding in growing-finishing swine.
AIMS: To determine the possible effects of inclusion of dried skim milk (DSM) in swine diets on indigenous Lactobacillus spp. and Escherichia coli, and its potential for controlling pathogen shedding and affect animal growth in growing-finishing swine. METHODS AND RESULTS: Animals were fed over three dietary phases to match production needs from age 10-14 weeks, 14-18 weeks and 18-22 weeks. For each feeding phase, diets were formulated to contain 0 or 10% DSM (balanced for metabolizable energy and true ileal digestible amino acids). Animals were weighed every 2 weeks and faecal samples were collected from 40 animals (20 with DSM and 20 without DSM) at week 10 (d 0 on diets), 14, 18 and 22 of age, and were analysed for Lactobacillus spp., Enterobacteriaceae, coliforms, E. coli, Salmonella, Campylobacter and E. coli O157:H7. At the start of the study (week 10), faecal bacterial counts (log10 CFU g(-1) faeces) were 9.55, 7.26, 7.01 and 6.93 for Lactobacillus, Enterobacteriaceae, coliforms and E. coli populations respectively. The Enterobacteriaceae, coliform and E. coli populations decreased through week 14 and 18, but were higher in animals fed with the DSM diet compared with the basal diet without DSM. The Lactobacillus populations at weeks 14 and 18 were lower in the animals fed the diet without DSM, whereas feeding DSM maintained the Lactobacillus counts from week 10. At week 22, populations of Enterobacteriaceae, coliforms and E. coli were >week 18 for the animals fed the diet without DSM, less change was observed with the feeding of DSM, and no differences between the diets were observed at week 22. However, in week 22 the animal gain was positively correlated with Lactobacillus numbers and negatively correlated with E. coli numbers. Subtraction of the E. coli population (log10) from the Lactobacillus population (log10) yielded a positive value termed 'effective'Lactobacillus that correlated well with animal gain and may better define a beneficial function in the intestine. Salmonella were detected in over 60% of the animals at week 10 and 14, and <20% at week 18 and 22. Campylobacter were detected rarely at weeks 10, 14 and 18, but were found in 25% of the animals at week 22. The DSM did not affect Salmonella or Campylobacter shedding, but examination of individual animals over the entire experiment indicated that fewer recurring incidences of Salmonella shedding occurred in animals that maintained higher Lactobacillus. In addition, at week 22, Salmonella and Campylobacter shedding was associated with lower levels of effective Lactobacillus and lower animal weight gains. CONCLUSIONS: The DSM did not directly affect the animal performance or pathogen shedding via the Lactobacillus spp. population at any phase of production. However, analysis of data from all animals revealed that faecal Lactobacillus affected Salmonella shedding and in the finishing phase, animal growth and pathogen shedding also were affected, as reflected by the 'effective'Lactobacillus-associated observations. SIGNIFICANCE AND IMPACT OF THE STUDY: In the swine intestine, any benefits from gastrointestinal Lactobacillus may be compromized by the E. coli population, and this antagonism may explain responses observed with prebiotics or probiotics in some swine.
Animal Feed↗
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.
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.
Bacterial Typing Techniques↗
Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula.
The developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally, Bifidobacterium and Lactobacillus predominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, the Lactobacillus species distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5' nuclease assays, targeted on rRNA intergenic spacer regions, were developed for Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of different Lactobacillus species in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% +/- 0.3% versus 4.1% +/- 1.5%) and the OSF group (0.8% +/- 0.3% versus 4.4% +/- 1.4%). The Lactobacillus species distribution in the OSF group was comparable to breast-fed infants, with relatively high levels of L. acidophilus, L. paracasei, and L. casei. The SF-fed infants, on the other hand, contained more L. delbrueckii and less L. paracasei compared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.
Dietary Supplements↗
Effects of anti-caries antibodies on Lactobacillus GG in its fermentation and storage periods.
OBJECTIVE: To investigate how antibodies influence the fermentation of Lactobacillus GG and how Lactobacillus GG influences the biological properties of antibodies during the fermentation and storage periods. METHODS: Anti-caries immune colostrum powder (IP) and control colostrum powder (CP), skimmed milk powder (SP) at concentrations of 1%, 2.5%, 5% and 10% (w/v) were added to MRS and 50 mM Hepes buffer system was used in the milk, the growth curves of Lactobacillus GG including viable cells, lactic acid concentratrion and pH, and also the titer of specific antibodies were determined during the fermentation and storage periods. RESULTS: In MRS, SP could improve the growth of Lactobacillus GG in all periods of fermentation, especially at the concentrations of 5% and 10%. CP at the concentrations of 1% and 2.5% had a good initial velocity at the beginning and CP (1%, 2.5%, 5%, 10%) in all the groups could reach a high viable cell concentration at the end of fermentation, which suggested that there were some growth factors for Lactobacillus GG in CP overcoming the inhibition of unspecific antibodies. IP at 1%, 2.5%, 5% and 10% could inhibit the growth of Lactobacillus GG in all the fermentation periods, the critical concentration point was between 2.5%-5%, and there was a competition between growth factors and the inhibition of specific antibodies. In pasteurized milk, the influence of Hepes could help the fermentation start quickly and reach the log phase earlier than in the control group; however, when combined with 5% IP, the growth of Lactobacillus GG was strongly inhibited during all the fermentation periods. The fermentation of Lactobacillus GG had no significant effect on the titer change of anti-caries antibodies during the fermentation and storage periods. CONCLUSION: SP and CP is beneficial to the growth of Lactobacillus GG in MRS, whereas there is a competition between growth factors and the inhibition of specific antibodies to the growth of Lactobacillus GG, the critical concentration of IP is 2.5%-5%. Hepes could influence the titer of antibodies through buffering Ph, thus influence the fermentation of Lactobacillus GG. The fermentation of Lactobacillus GG with IP or CP could be used for the development of functionally fermented immune milk in the future.
Animals↗
Characterisation of the microbiota of rice sourdoughs and description of Lactobacillus spicheri sp. nov.
The microbiota of two industrially processed rice sourdoughs was characterised by bacteriological culture in combination with PCR-denaturing gradient gel electrophoresis (DGGE) and 16S/28S rDNA sequence analysis. Rice sourdough I was continuously propagated for several years by back-slopping every week, whereas sourdough II was processed by using a commercial starter culture and back-slopping daily for three days. In rice sourdough II Candida krusei and Saccharomyces cerevisiae as well as Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus kimchii, Lactobacillus plantarum, and Lactobacillus pontis dominated at the first day of fermentation. RAPD analysis of lactobacilli revealed identical profiles for each of the species except for L. fermentum and L. pontis indicating the presence of different strains. Fluctuations within the LAB community during fermentation were monitored by PCR-DGGE. L. pontis decreased in numbers over time and L. curvatus became dominant after 3 days of fermentation. Rice sourdough I contained S. cerevisiae, Lactobacillus paracasei (present with three different RAPD types), Lactobacillus paralimentarius, and a Lactobacillus strain which could not be allotted to any valid species. Phylogenetic analysis based on 16S rDNA sequences revealed Lactobacillus brevis as the closest relative (97.3% sequence similarity). Differences in some phenotypic characteristics and DNA-DNA relatedness indicated that the strain represents a new Lactobacillus species, for which the name Lactobacillus spicheri is proposed.
Base Composition↗
Lactobacillus kitasatonis sp. nov., from chicken intestine.
Four strains isolated from chicken small intestine and strains JCM 1038 and JCM 1039 (designated as Lactobacillus acidophilus) were characterized by phenotypic and molecular taxonomic methods. They were Gram-positive, catalase-negative, facultatively anaerobic rods that did not produce gas from glucose. These strains had similar phenotypic characteristics and exhibited intergroup DNA relatedness values of >77 %, indicating that they comprised a single species. The 16S rRNA gene sequence of a representative strain, JCM 1039(T) (designated as type strain in this study), was determined and aligned with those of other Lactobacillus species. JCM 1039(T) was placed in the Lactobacillus delbrueckii cluster of the genus Lactobacillus on the basis of phylogenetic analysis and formed an independent cluster that was distinct from its closest neighbours, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, L. acidophilus and Lactobacillus helveticus. Results of DNA-DNA hybridization experiments and whole-cell protein profiles clearly indicated that these strains represent a novel Lactobacillus species, for which the name Lactobacillus kitasatonis sp. nov. is proposed; the type strain of this species is JCM 1039(T).
Animals↗
Lactobacillus bacteremia, species identification, and antimicrobial susceptibility of 85 blood isolates.
BACKGROUND: Data regarding antimicrobial susceptibility of clinical Lactobacillus isolates are scarce, and appropriate interpretation criteria for susceptibility tests are not available. METHODS: We examined 85 cases of Lactobacillus bacteremia, of which 47 cases have been included in our previous studies. Overall, 14 antimicrobial agents were evaluated by the E-test method, and these results were compared with disk diffusion test findings. The clinical outcomes of the patients and their antimicrobial treatments were registered. RESULTS: The antimicrobial susceptibility of Lactobacillus strains was species dependent. The considerable number of Lactobacillus rhamnosus (n=46), Lactobacillus fermentum (n=12), and Lactobacillus casei (n=12) strains available for testing made it possible to compare the susceptibilities within 1 species, as well. Of the 46 L. rhamnosus isolates, 22 were identified as L. rhamnosus GG type by pulsed-field gel electrophoresis. All Lactobacillus isolates demonstrated low minimum inhibitory concentrations (MICs) of imipenem, piperacillin-tazobactam, erythromycin, and clindamycin. MICs of vancomycin were high (>256 microg/mL) for all other species except Lactobacillus gasseri and Lactobacillus jensenii. Disk diffusion and E-test results were concordant. The MICs of cephalosporins varied; cefuroxime demonstrated a higher level of activity than did ceftriaxone. Benzylpenicillin and ampicillin MICs had variable ranges between different species. Combination therapy was given to 83% of the patients, but, in 54% of them, therapy included only 1 microbiologically active agent, according to results of the susceptibility tests. Mortality at 1 week was 12% among patients who presumably were receiving adequate treatment and 27% among patients who were receiving inadequate treatment (P=.131, by E-test). CONCLUSION: Most clinical Lactobacillus blood isolates demonstrated low MICs of imipenem, piperacillin-tazobactam, erythromycin, and clindamycin, but they had variable susceptibility to penicillin and cephalosporins.
Anti-Bacterial Agents↗
Effects of two probiotic Lactobacillus strains on jejunal and cecal microbiota of broiler chicken under acute heat stress condition as revealed by molecular analysis of 16S rRNA genes.
We examined the effects of probiotic Lactobacillus strains of Lactobacillus agilis JCM 1048 and Lactobacillus salivarius subsp. salicinius JCM 1230 on jejunal and cecal microbiota of broiler chicken under heat stress condition using terminal restriction fragment length polymorphism (T-RFLP) analysis. The jejunal bacterial community was limited to a few bacterial groups, mostly Lactobacillus spp. A relatively abundant and higher prevalence of Lactobacillus spp. were observed in the jejunal and cecal microbiota of the probiotic chickens compared with those of the control chickens under heat stress condition. In general, the probiotic strains did not significantly affect the abundance of L. agilis and L. salivarius in chicken intestine but clearly contributed to increasing their prevalence in the probiotic chickens. The probiotic Lactobacillus strains enriched the diversity of Lactobacillus flora in chicken jejunum and cecum by increasing the abundance and prevalence of Lactobacillus spp. inhabiting the intestine. The richness of Lactobacillus species tended to be similar among the jejunal and cecal microbiota. The bacterial community of cecum was complex and age-dependent. The major components of the cecal microbiota were clostridia and lactobacilli. The Clostridium subcluster XIVa was the most predominant group in chicken cecum. Probiotic Lactobacillus strains restored the microbial balance and maintained the natural stability of indigenous bacterial microbiota following heat stress-induced changes.
Age Factors↗
Genetic characterization and specific detection of beer-spoilage Lactobacillus sp. LA2 and related strains.
AIMS: Lactobacillus sp. LA2 (DSM15502) and related strains (LA2 group) possess strong beer-spoilage ability. The 16S rDNA sequence of LA2 strain is virtually indistinguishable from that of L. collinoides, generally considered to be nonbeer-spoilage bacteria. The aim of this study was to identify the genetic marker to distinguish between Lactobacillus sp. LA2 group and L. collinoides and to provide a rapid means of identifying beer-spoilage strains belonging to Lactobacillus sp. LA2 group. METHODS AND RESULTS: The 16-23S rDNA intergenic spacer (ITS) regions of Lactobacillus sp. LA2 and L. collinoides JCM1123T were sequenced to identify a genetic marker to distinguish between the two groups. As a result, 300 and 500 bp ITS regions of Lactobacillus sp. LA2 were found to be almost identical with those of L. collinoides JCM1123T. Sequence comparison analysis between Lactobacillus sp. LA2 and L. collinoides JCM1123T revealed that the two contiguously located nucleotides are absent in both ITS regions of Lactobacillus sp. LA2. Based on the sequence difference, we have designed specific PCR primers with a minor modification to the primer sequence that can differentiate between beer-spoilage Lactobacillus sp. LA2 group and nonbeer-spoilage L. collinoides. CONCLUSIONS: The PCR-based method has been developed to identify Lactobacillus sp. LA2 group, providing a rapid and sensitive means of determining the beer-spoilage ability of detected bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The substitution of one nucleotide, located at the third position to the 3'-end in the primer sequence, enhanced the specificity of the PCR method while retaining sufficient sensitivity. The nucleotide gap identified in this study appeared to serve as a useful genetic marker that can differentiate 12 beer-spoilage Lactobacillus sp. LA2 group strains from its close relatives that exhibit no beer-spoilage ability.
Bacterial Typing Techniques↗
Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces.
AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.
Animals↗