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Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii.

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.

Base Sequence↗

Lactobacillus curvatus subsp. curvatus subsp. nov. and Lactobacillus curvatus subsp. melibiosus subsp. nov. and Lactobacillus sake subsp. sake subsp. nov. and Lactobacillus sake subsp. carnosus subsp. nov., new subspecies of Lactobacillus curvatus Abo-Elnaga and Kandler 1965 and Lactobacillus sake Katagiri, Kitahara, and Fukami 1934 (Klein et al. 1996, emended descriptions), respectively.

Lactobacillus curvatus and Lactobacillus sake are each genetically homogeneous species, as indicated by the high levels of DNA homology (> or = 76%) exhibited by strains of these taxa. However, the results of a numerical analysis of total soluble cell protein patterns and biochemical test data revealed that there are two phenotypic subgroups within L, curvatus and two phenotypic subgroups within L. sake. The overall randomly amplified polymorphic DNA (RAPD)-PCR band patterns obtained for the majority of L. curvatus strains corresponded well to the pattern obtained for the type strain of L. curvatus (strain DSM 20019). However, six strains of L. curvatus had different, but similar, RAPD-PCR profiles and grouped in a separate genetic cluster, which was linked to one of the clusters of L. sake strains. On the basis of these results, differences in biochemical and physiological characteristics, and total soluble cell protein profiles, we describe the subspecies L. curvatus subsp. curvatus subsp. nov. and L. curvatus subsp. melibiosus subsp. nov. for L. curvatus Abo-Elnaga and Kandler 1965 (Klein et al. 1996, emended description). Strains of L. sake grouped in two RAPD-PCR clusters, which was consistent with previous reports of phenotypic heterogeneity. Strains of Lactobacillus bavaricus, including type strain LMG 9844, clustered with the type strain of L. sake (strain NCFB 2714), indicating that these organisms belong to the same genetic group. We propose that strains of L. sake Katagiri, Kitahara, and Fukami 1934 (Klein et al. 1996, emended description) should be reclassified as members of L. sake subsp. sake subsp. nov. and L. sake subsp. carnosus subsp. nov. Strains of L. bavaricus are reclassified as members of L. sake subsp. sake, and the name L. bavaricus Stetter and Stetter 1980 is rejected.

DNA, Bacterial↗

Taxonomic study of the Lactobacillus acidophilus group, with recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981).

Biochemical properties and DNA-DNA reassociation studies of Lactobacillus acidophilus strains isolated from humans and animals indicate that these include six genomospecies. Two new species can be differentiated from the established species of the genus Lactobacillus: L. gallinarum sp. nov. (type strain, ATCC 33199) and L. johnsonii sp. nov. (type strain, ATCC 33200). Furthermore, it was clarified that L. acidophilus group A3 (Johnson et al. 1980) is synonymous with L. amylovorus.

DNA, Bacterial↗

Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus.

AIMS: To develop a tool for rapid and inexpensive identification of the Lactobacillus casei complex. METHODS AND RESULTS: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus were identified by PCR-amplification of the segment between the U1 and U2 regions of 16S rDNA (position 8-357, Escherichia coli numbering) and temporal temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were divided into three TTGE-subgroups. CONCLUSION: TTGE successfully distinguished between the closely-related target species. TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to rapid and easy performance, TTGE of PCR-amplified 16S rDNA fragments will be useful for the identification of extended numbers of isolates.

Bacterial Typing Techniques↗

Reclassification of Lactobacillus casei subsp. casei ATCC 393 and Lactobacillus rhamnosus ATCC 15820 as Lactobacillus zeae nom. rev., designation of ATCC 334 as the neotype of L. casei subsp. casei, and rejection of the name Lactobacillus paracasei.

The type strain of Lactobacillus casei subsp. casei (ATCC 393) exhibits low levels of DNA homology with other strains of L. casei subsp. casei (8 to 46%) and strains of Lactobacillus paracasei (30 to 50%), but exhibits a level of DNA similarity of 80% with Lactobacillus rhamnosus ATCC 15820, the original type strain of "Lactobacterium zeae" Kuznetsov 1959. Strains ATCC 393T (T = type strain) and ATCC 15820T are members of one protein profile cluster that is separate from the other Lactobacillus spp. The randomly amplified polymorphic DNA PCR profile of strain ATCC 393T is also different from the profiles obtained for the other species. L. casei ATCC 334T is genetically closely related to L. casei subsp. casei strains (71 to 97%) and L. paracasei strains (71 to 91%), is a member of the same protein profile cluster as these organisms, and shares several DNA amplicons with L. paracasei strains. On the basis of these results, we propose that L. casei subsp. casei ATCC 393T and L. rhamnosus ATCC 15820 should be reclassified as members of Lactobacillus zeae nom. rev. (type strain, ATCC 15820), that strain ATCC 334 should be designated the neotype strain of L. casei subsp. casei, and that the name L. paracasei should be rejected.

Base Sequence↗

Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and propionibacteria.

Nineteen bacteriological media were evaluated to assess their suitability to selectively enumerate Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, bifidobacteria, and propionibacteria. Bacteriological media evaluated included Streptococcus thermophilus agar, pH modified MRS agar, MRS-vancomycine agar, MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS-NNLP (nalidixic acid, neomycin sulfate, lithium chloride and paramomycine sulfate) agar, reinforced clostridial agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) media, sodium lactate agar, arabinose agar, raffinose agar, xylose agar, and L. casei agar. Incubations were carried out under aerobic and anaerobic conditions at 27, 30, 37, 43, and 45 degrees C for 24, 72 h, and 7 to 9 d. S. thermophilus agar and aerobic incubation at 37 degrees C for 24 h were suitable for S. thermophilus. L. delbrueckii ssp. bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.20) and under anaerobic incubation at 45 degrees C for 72 h. MRS-vancomycine agar and anaerobic incubation at 43 degrees C for 72 h were suitable to enumerate L. rhamnosus. MRS-vancomycine agar and anaerobic incubation at 37 degrees C for 72 h were selective for L. casei. To estimate the counts of L. casei by subtraction method, counts of L. rhamnosus on MRS-vancomycine agar at 43 degrees C for 72 h under anaerobic incubation could be subtracted from total counts of L. casei and L. rhamnosus enumerated on MRS-vancomycine agar at 37 degrees C for 72 h under anaerobic incubation. L. acidophilus could be enumerated using MRS-agar at 43 degrees C for 72 h or Basal agar-maltose agar at 43 degrees C for 72 h or BA-sorbitol agar at 37 degrees C for 72 h, under anaerobic incubation. Bifidobacteria could be enumerated on MRS-NNLP agar under anaerobic incubation at 37 degrees C for 72 h. Propionibacteria could be enumerated on sodium lactate agar under anaerobic incubation at 30 degrees C for 7 to 9 d. A subtraction method was most suitable for counting propionibacteria in the presence of other lactic acid bacteria from a product. For this method, counts of lactic bacteria at d 3 on sodium lactate agar under anaerobic incubation at 30 degrees C were subtracted from counts at d 7 of lactic bacteria and propionibacteria.

Agar↗

Comparative studies of lactate dehydrogenases in lactic acid bacteria. Amino-acid composition of an active-site region and chemical properties of the L-lactate dehydrogenase of Lactobacillus casei, Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus acidophilus.

The molecular weight, the amino acid composition and the N-terminal and C-terminal amino acids of two allosteric (Lactobacillus casei, L. curvatus) and two non-allosteric (L. plantarum, L. acidophilus) L-lactate dehydrogenases, purified to homogeneity by affinity chromatography, were determined. The amino acid composition of the only tryptic peptide unequivocally common to the fingerprints of the 4 enzymes is virtually identical with that of the arginine peptide, called Arg6 of the the substratebinding site of the L-lactate dehydrogenase dehydrogenase of several animals. However, the 'essential' cysteine residue 165 is replaced by threonine, as it is in the L-lactate dehydrogenase of lobster. In addition, the 4 bacterial peptides differ by one or two changes in single amino acid residues from each other as well as from those of animals. The data indicate that not only the animal L-lactate dehydrogenases, but also the allosteric and lactate dehydrogenases from bacterial sources may have evolved from a common gene.

Amino Acids↗

Lactobacillus uli sp. nov. and Lactobacillus rimae sp. nov. from the human gingival crevice and emended descriptions of lactobacillus minutus and Streptococcus parvulus.

Lactobacillus uli sp. nov. and Lactobacillus rimae sp. nov. are described. These organisms are short, gram-positive, strictly anaerobic, rod-shaped bacteria that have DNA G+C contents of 53 and 45 mol%, respectively, produce major amounts of lactic acid, and have been isolated from human gingival crevices and periodontal pockets. The major cellular fatty acid derivatives for both species are C18:1 cis-9 fatty acid methyl ester and C18:1 cis-9 dimethylacetyl. The type strain of L. uli is strain VPI D76D-27C (= ATCC 49627), and the type strain of L. rimae is strain D140H-11A (= ATCC 49626). Emended descriptions of Lactobacillus minutus (based on selected strains) and Streptococcus parvulus (based on many additional strains) also are given.

Base Composition↗

Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction.

Lactobacillus casei, Lact. paracasei and Lact. rhamnosus form a closely related taxonomic group within the heterofermentative lactobacilli. These three species are difficult to differentiate using traditional fermentation profiles. We have developed polymerase chain reaction primers which are specific for each of these species based on differences in the V1 region of the 16S rRNA gene. Sixty-three Lactobacillus isolates from cheese were identified using these primers. The 12 Lact. rhamnosus and 51 Lact. paracasei identified in this way were also differentiated using a randomly amplified polymorphic DNA (RAPD) primer.

Bacterial Typing Techniques↗

Lactobacillus helveticus Lh59 secretes an exopolysaccharide that is identical to the one produced by Lactobacillus helveticus TN-4, a presumed spontaneous mutant of Lactobacillus helveticus TY1--2.

Lactobacillus helveticus Lh59 produces a high-molecular-mass exopolysaccharide (> or = 2 x 10(6) Da) when cultured in skimmed milk. Compositional analysis, methylation analysis and NMR experiments (1H and 13C) recorded from the native polysaccharide as well as from oligosaccharides released by partial acid hydrolysis, allowed the complete structural determination of this polysaccharide, which consists of the following hexasaccharide repeating unit: [symbol: see text] This structure is identical to the one of an EPS produced by L. helveticus TN-4, which was claimed to be a spontaneous mutant of strain TY1-2.

Carbohydrate Sequence↗

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri.

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.

Amino Acids↗

Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains.

Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition.

Acetates↗

Transport of D-xylose in Lactobacillus pentosus, Lactobacillus casei, and Lactobacillus plantarum: evidence for a mechanism of facilitated diffusion via the phosphoenolpyruvate:mannose phosphotransferase system.

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low-affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min(-1) mg of dry weight(-1). In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2-deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity.

Adenosine Triphosphate↗

Effects of Lactobacillus rhamnosus GG and Lactobacillus reuteri R2LC on acetic acid-induced colitis in rats.

BACKGROUND: Certain lactobacilli reduce the severity of experimental colitis. The aim of this study was to compare the effects of a human strain Lactobacillus rhamnosus GG and a rat strain Lactobacillus reuteri R2LC on acetic acid-induced colitis in rats. METHODS: Lactobacillus rhamnosus GG, Lactobacillus reuteri R2LC or sulphasalazine were given orally to the rats. Colitis was assessed 72 h after induction with acetic acid. RESULTS: Lactobacillus reuteri R2LC significantly antagonized body weight loss caused by inflammation compared with Lactobacillus rhamnosus GG and sulphasalazine, and oedema formation in the colon compared with sulphasalazine. Lactobacillus reuteri R2LC reduced the median value of macroscopic ulceration and the protein content of inducible nitric oxide synthase by 50% and the median of the protein content of inducible cyclooxygenase by 30% compared with that of the colitis control group, and Lactobacillus rhamnosus GG reduced the median of inducible nitric oxide protein content by 40% and increased the median of inducible cyclooxygenase protein content by 30% compared with the median value of the colitis control group, but these differences were not statistically significant. CONCLUSIONS: The rat strain Lactobacillus reuteri R2LC, but not the human strain Lactobacillus rhamnosus GG, is of benefit in reducing the severity of acetic acid-induced colitis in rats. These results suggest that it is not the total amount of Lactobacillus but the particular species or strain of Lactobacillus that is important in attenuating experimental colitis.

Acetic Acid↗

[Prevention of caries with lactobacillus (final results of a clinical trial on dental caries with killed lactobacillus [streptococcus and lactobacillus] given orally)].

Caries prevention with lactic bacteria. (Final results of a dental caries clinical trial using heat killed lactic bacteria [streptococci and lactobacilli] orally.) The results of a dental caries clinical trial in 245 seven-year-old children are reported. Chewable tablets of two different types were prepared: A) Containing pyridoxine (Vit. B6) and heat killed lactic bacteria. B) Placebo tablets with pyridoxine only. They were randomly given once a week for 16 weeks to experimental and control groups respectively. Four evaluation surveys were conducted during 24 months of follow up, using the "Decay, Missing, Filled, Surfaces" index (DMFS) for the clinical evaluation of the permanent teeth. A consistent reduction in the incidence of dental caries in the experimental group was observed in all 4 surveys. After 2 years of follow up a 42% reduction in the incidence rate of dental caries was observed in the experimental group compared to the control group. Summary tables and discussion of the clinical evaluation surveys are given. The potential use of these clinical findings as support for a future dental caries vaccine evaluation project is proposed.

Bacterial Vaccines↗

[Bacteriocin properties of Lactobacillus fermenti, Lactobacillus brevis and Lactobacillus buchneri].

Four bacteriocins of L. fermenti, 3 bacteriocins of L. brevis and 1 bacteriocin of L. buchneri were studied with respect to morphology of the inhibition growth zones of the indicator strains, capacity for diffusion through cellophane, sensitivity to high temperature, bacterial proteases, trypsin, chymotrypsin, pepsin, papain, nucleases and lysozyme. According to the differences in their properties the bacteriocins were classified as belonging to 8 types, including 4 types of L. fermenti bacteriocins and 3 types of L. brevis bacteriocins.

Antibiosis↗

Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species.

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.

Animals↗