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PubMed1993

Numerical taxonomy of Lactobacillus spp. associated with healthy and diseased mucosa of the human intestines.

Two-hundred and fifty Lactobacillus strains isolated from healthy and diseased mucosa of human intestines of 75 individuals and 49 reference strains were phenotypically classified using 49 unit characters. Data were processed by the Jaccard (SJ) and Simple Matching (SSM) coefficients, and unweighted pair group algorithm with arithmetic averages. Seventeen major clusters were defined at the 76% SJ-similarity level which approximately correspond to the SSM-level of 91%. Seven clusters could be identified: Lactobacillus plantarum (isolates recovered from 5% of the patients), Lact. casei subsp. rhamnosus (17% of the patients), Lact. casei subsp. pseudoplantarum (5% of the patients), Leuconostoc mesenteroides subsp. mesenteroides (3% of the patients), Lact. buchneri (4% of patients), Lact. reuteri (4% of the patients) and Lact. salivarius subsp. salivarius (9% of the patients). Unassigned clusters 1 and 3 both contained homofermentative Lactobacillus strains. Cluster 1 included the type strains of Lact. crispatus, Lact. acidophilus, Lact. jensenii and Lact. gasseri, and cluster 3, the type strains of Lact. delbrueckii subsp. lactis, Lact. agilis and Lact. casei subsp. tolerans. Clusters 1 and 3 were found in 15% and 25% of the patients, respectively. Unassigned clusters 2, 6, 7, 8 and 10 contained homofermentative Lactobacillus strains but no reference strains. Clusters 11, 12, 15 and 17 were made up of heterofermentative Lactobacillus strains but no reference strains. Phenotypical characteristics of the clusters are given. No obvious trends in species (cluster) composition between different intestinal locations could be noted. Most clusters contained isolates from both diseased and healthy mucosa. Exceptions were cluster 15 and cluster 17 which only included isolates from healthy mucosa, and cluster 11 which only included isolates from diseased mucosa. Cluster 15 was isolated in 12% of the patients, and cluster 11 in 8%.

Classification↗
PubMed1998

Diversity of lactic acid bacteria associated with ducks.

The diversity of lactic acid bacteria occurring in the crop and intestinal content of ducks of different ages (5 to 34 days) was evaluated using the RAPD-PCR technique and comparative sequence analysis of 16S rRNA. Cluster analysis derived from isolates grown on Rogosa agar used for the enrichment of lactic acid bacteria, revealed at least twenty six different groups of organisms representing species, subspecies and strains belonging to Streptococcus, Enterococcus, Pediococcus, Weissella and Lactobacillus. Homofermentative and heterofermentative lactobacilli were identified, belonging to the phylogenetically defined Lb. acidophilus, Lb. salivarius and Lb. reuteri groups.

Age Factors↗
PubMed1995

In vitro colonization ability of human colon mucosa by exogenous Lactobacillus strains.

We have tested five Lactobacillus strains for their in vitro colonization ability in a human colon epithelium culture model. Positive colonization occurred in different degrees in the presence of four Lactobacillus strains. We have found that L. casei GG and L. acidophilus NCFB 1748 induce an intermediate colonization. These two strains, largely investigated by others, have been reported to be beneficial to humans. The use of human intestinal tissue in our study partially reconstitutes the complex architectural specificity of human epithelium with the mucus layer and more closely simulates the in vivo situation.

Colon↗
PubMed1987

Identification of vaginal lactobacilli from asymptomatic women.

In the present work, carried out by means of phenotypic and genotypic tests, it has been demonstrated that none of the vaginal lactobacilli isolated from asymptomatic women were genetically close to L. acidophilus ATCC 4356. Strains of homofermentative lactobacilli were identified as Lactobacillus gasseri, L. jensenii and L. crispatus, while in the heterofermentative only one strain was classified as L. fermentum. Moreover a genetically homologous group of heterofermentative lactobacilli, that could represent a new species, were evidenced.

DNA, Bacterial↗
PubMed1998

Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products.

Twenty-five strains of thermophilic lactobacilli isolated from yoghurt and from semi-hard and hard cheeses (in parallel with nine type or reference strains) were identified and grouped according to their genetic relatedness. Strains were identified by sugar fermentation patterns using the "API 50 CHL" galleries, by species-specific DNA probes in dot-blot hybridization experiments, by amplification and restriction analysis of the 16S rRNA gene (ARDRA) and by polymerase chain reaction (PCR) using species-specific oligonucleotide primers. Strains were classified as Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus, L. helveticus, and L. acidophilus. Strains which were atypical by sugar fermentation patterns were also identified. Most of the strains could not be grouped using carbohydrate fermentation profiles. PCR fingerprinting was used to identify DNA profiles for the 25 lactobacilli. Experimentally obtained PCR profiles enabled discrimination of all strains, which were grouped according to the similarities in their combined patterns. In general, the clustering of the strains corresponded well with species delineation obtained by molecular identification. The dendrogram of genetic relatedness enabled the unambiguous identification of most of the strains which were shown to be atypical by the sugar fermentation profile, except for a discrepancy in one L. delbrueckii subsp. lactis strain and one atypical Lactobacillus sp. strain.

Bacterial Typing Techniques↗
PubMed1995

Development of intestinal lactobacilli in normal piglets.

The development of intestinal Lactobacillus spp. in 18 normal piglets from six litters was investigated from 1 to 45 d after birth. Faecal Lactobacillus strains isolated from piglets and their dams were identified at species and biovar levels on the basis of carbohydrate fermentation. A definite pattern was observed in the changes of four species and 20 biovars of lactobacilli appearing in faeces, with growth of the piglets. Lactobacillus reuteri colonized the animals on the first day of birth. The Lact. acidophilus group appeared 1 week after birth and maintained high levels in the intestine. Biovars B, C and E of Lact. reuteri were recovered as major biovars on the first to fourteenth days after birth. In the third and fourth weeks after birth, however, biovars F and G of Lact. reuteri were mainly recovered from piglet faeces. By the seventh week, biovars B, C and E predominated again in the intestine. No change in the composition of species and biovars of the genus Lactobacillus in the dams was detected.

Animals↗
PubMed1998

Antibacterial activity of Lactobacillus species against Vibrio species.

Forty-one Lactobacillus strains were tested for antagonistic activity against nine strains of Vibrio. L. plantarum and L. casei were the most effective, and L. brevis was the least effective in inhibiting the growth of Vibrio species. L. gasseri and L. helveticus strains showed higher activity, while L. reuteri and L. fermentum showed lower inhibitory activity against Vibrio species. L. acidophilus strains exhibited various degrees of antagonistic activities against Vibrio species. However, none of the Lactobacillus species were able to inhibit the growth of Salmonella enteritidis, S. typhimurium, Escherichia coli, and Staphylococcus aureus. Inhibition of the Vibrio species was probably due to the production of organic acids by the Lactobacillus species.

Bacteriocins↗
PubMed2001

Lactobacilli in human dental caries and saliva.

Samples (98 plaque and 72 saliva) from 93 patients with dental caries were investigated for Lactobacillus species which comprised 65 (62.5%) of 104 isolates. Yeasts (20.1%), Streptococcus spp. (8.7%), Staphylococcus spp. (2.9%) and a few unidentified species (5.8%), were also found. The Lactobacillus isolates were L. brevis (24.6%) L. fermentum (18.5%) L. casei (16.9%), L. delbrueckii (15.4%), L. plantarum (9.23%), L. acidophilus (7.69%), L. jensenii (4.62%), L. salivarius (1.54%) and L. gasseri (1.54%). The most common species was L. brevis (24.6%). The strains tested for beta-lactamase production showed 75.4% positive. All the Lactobacillus strains were tested for bacteriocin production against Escherichia coli, Salmonella spp., Shigella dysenteriae, S. sonnei, Klebsiella spp. and Campylobacter sp. All the lactobacilli except L. jensenii produced bacteriocin against at least one of the indicator organisms. The involvement of Lactobacillus in dental caries was established, although its role and mechanism is not well understood. The ability of Lactobacillus spp. to protect their host against certain diseases by inhibiting the growth of potential pathogens was evident.

Adult↗
PubMed2006

Characterisation of prototype Nurmi cultures using culture-based microbiological techniques and PCR-DGGE.

Undefined Nurmi-type cultures (NTCs) have been used successfully to prevent salmonella colonisation in poultry for decades. Such cultures are derived from the caecal contents of specific-pathogen-free birds and are administered via drinking water or spray application onto eggs in the hatchery. These cultures consist of many non-culturable and obligately anaerobic bacteria. Due to their undefined nature it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials for poultry. In this study, 10 batches of prototype NTCs were produced using an identical protocol over a period of 2 years. Traditional microbiological techniques and a molecular culture-independent methodology, polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), were applied to characterise these cultures and also to examine if the constituents of the NTCs were identical. Culture-dependent analysis of these cultures included plating on a variety of selective and semi-selective agars, examination of colony morphology, Gram-staining and a series of biochemical tests (API, BioMerieux, France). Two sets of PCR-DGGE studies were performed. These involved the amplification of universal and subsequently lactic acid bacteria (LAB)-specific hypervariable regions of a 16S rRNA gene by PCR. Resultant amplicons were subjected to DGGE. Sequence analysis was performed on subsequent bands present in resultant DGGE profiles using the Basic Local Alignment Search Tool (BLAST). Microbiological culturing techniques tended to isolate common probiotic bacterial species from the genera Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Clostridium, Escherichia, Pediococcus and Enterobacterium as well as members of the genera, Actinomyces, Bacteroides, Propionibacterium, Capnocytophaga, Proteus, and Klebsiella. Bacteroides, Enterococcus, Escherichia, Brevibacterium, Klebsiella, Lactobacillus, Clostridium, Bacillus, Eubacterium, Serratia, Citrobacter, Enterobacter, Pectobacterium and Pantoea spp. in addition to unculturable bacteria were identified as constituents of the NTCs using universal PCR-DGGE analysis. A number of the sequences detected by LAB-specific PCR-DGGE were homologous to those of a number of Lactobacillus spp., including L. fermentum, L. pontis, L. crispatus, L. salivarius, L. casei, L. suntoryeus, L. vaginalis, L. gasseri, L. aviaries, L. johnsonii, L. acidophilus, and L. mucosae in addition to a range of unculturable lactobacilli. While NTCs are successful due to their complexity, the presence of members of Lactobacillus spp. amongst other probiotic genera, in these samples possibly lends to the success of the NTC cultures as probiotics or competitive exclusion products in poultry over the decades. PCR-DGGE proved to be an effective tool in detecting non-culturable organisms present in these complex undefined cultures. In conclusion, while the culture-dependent identification methods or PCR-DGGE alone cannot comprehensively elucidate the bacterial species present in such complex cultures, their complementarity provides useful information on the identity of the constituents of NTCs and will aid in future development of defined probiotics. Moreover, for the purpose of analysing prototype NTCs during their development, PCR-DGGE overcomes the limitations associated with conventional culturing methods including their low sensitivities, inability to detect unculturable bacteria and unknown species, very slow turnabout time and poor reproducibility. This study demonstrated that PCR-DGGE is indeed more valuable in detecting predominant microbial populations between various NTCs than as an identification methodology, being more applicable as a quality control method used to analyse for batch-to-batch variation during NTC production.

Animals↗
PubMed1994

The effect of Lactobacillus spp. on the attachment of enterotoxigenic Escherichia coli to isolated porcine enterocytes.

A total of 43 strains of lactobacilli were isolated from the gastrointestinal tract of piglets at the time of weaning. Isolates, grown on solid media, were allocated to strongly adherent or non/weakly adherent groups on the basis of numbers attaching to isolated porcine enterocytes. Strains of Lactobacillus fermentum were disproportionally represented amongst the strongly-adherent strains and Lact. acidophilus and Lact. salivarius amongst the non/weakly-adherent group. Lactobacilli showed significantly better attachment ability when grown on agar than when grown in broth culture. Strongly adherent strains were not found to effect the attachment of enterotoxigenic Escherichia coli to porcine enterocytes, tested under the conditions of exclusion (lactobacilli added to the enterocytes before E. coli), competition (lactobacilli and E. coli added simultaneously) and displacement (E. coli added before lactobacilli). Tests were made with [14C]-labelled E. coli. Suspensions of bacteria and enterocytes were passed through a filter selected to retain enterocytes but pass free bacterial cells. Counts (dpm) obtained from filters after solubilization were taken as a measure of E. coli attachment. Some strains of lactobacilli coaggregated with enterotoxigenic E. coli with K88 fimbriae, but not with a K88-negative mutants strain. These were excluded from the competitive exclusion experiments. In the apparent absence of a direct effect on the association of E. coli with host tissue, removal of potential gut pathogens by aggregation could contribute to the probiotic properties ascribed to lactic acid bacteria.

Animals↗
PubMed2003

Susceptibility of Lactobacillus spp. to antimicrobial agents.

Bacteria used as probiotics or in starter cultures may serve as hosts of antibiotic resistance genes, which can be transferred to pathogenic bacteria. Before launching a starter culture or a probiotic product into the market, it is therefore important to verify that the single bacterial isolates (strains) do not contain transferable resistance genes. A study has been undertaken to establish the levels of susceptibility of Lactobacillus spp. to various antimicrobial agents. This is a prerequisite for differentiating putative transferable resistance from natural resistance. A selection of 62 strains has been screened with the use of the Etest (ABBiodisk, Stockholm, Sweden) for their susceptibility to 25 antimicrobial agents. The strains belonged to the following species: Lactobacillus plantarum/pentosus, L. rhamnosus, L. paracasei, L. sakei, L. curvatus and species of the L. acidophilus group: L. johnsonii, L. crispatus, L. gasseri, and L. acidophilus. The results from the Etests have shown that the level of susceptibility to the antimicrobial agents is species-dependent. For the following antimicrobial agents, susceptibility varied several folds between species: vancomycin, teicoplanin, tetracycline, norfloxacin, ciprofloxacin, fusidic acid, and clindamycin. The differences between the species were more subtle for the rest of the tested antimicrobial agents. On the basis of the result, it was possible to suggest minimal inhibition concentrations (MICs) for the individual Lactobacillus species to be used as a microbiological breakpoint when screening strains for transferable resistance genes.

Anti-Bacterial Agents↗
PubMed1998

Peritonitis associated with vancomycin-resistant Lactobacillus rhamnosus in a continuous ambulatory peritoneal dialysis patient: organism identification, antibiotic therapy, and case report.

A case of Lactobacillus rhamnosus-associated peritonitis in a patient undergoing continuous ambulatory peritoneal dialysis is reported. The patient was treated with vancomycin after isolation of glycopeptide-susceptible coagulase-negative staphylococci. After a skin rash developed, vancomycin was discontinued and replaced with teicoplanin. Seven weeks after the glycopeptide therapy was discontinued, a Lactobacillus strain was isolated in pure cultures. The isolate was identified first incorrectly as L. acidophilus but later correctly as L. rhamnosus. Antibiotic susceptibility testing showed that the isolate was resistant to glycopeptides but susceptible to several other antibiotics. The antibiotic treatment was then switched to imipenem and was successful.

Anti-Bacterial Agents↗
PubMed2001

Molecular discrimination of lactobacilli used as starter and probiotic cultures by amplified ribosomal DNA restriction analysis.

Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus.

Base Sequence↗
PubMed1999

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes.

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.

Bacterial Proteins↗
PubMed1989

The effect of pH on the sensitivity of species of Lactobacillus to chlorhexidine and the antibiotics minocycline and spiramycin.

The purpose of this study was to determine the sensitivity of a range of Lactobacillus species to chlorhexidine, Minocycline, and Spiramycin, at a range of pH from 5.0 to 7.4. Strains of Streptococcus were also tested for their sensitivity to chlorhexidine, as a comparison between the genera. There were both inter- and intra-species variations in the sensitivity of these strains to chlorhexidine. The strains tested were sensitive at pH 6.7 to the following levels of chlorhexidine (micrograms/mL): L. casei (6 strains), 10-60; L. plantarum (4 strains), 40; L. fermentum (13 strains), 2-20; L. brevis (1 strain), 10; and L. acidophilus (3 strains), 10-60. The Streptococcus species were sensitive to 1-4 micrograms/mL (13 strains); 4-10 micrograms/mL (3 strains); and 10-20 micrograms/mL (2 strains). One strain was able to survive 20 micrograms/mL. Chlorhexidine was found to be less effective at lower pH levels. The following examples show sensitivity (micrograms/mL) at pH 6.5 followed by sensitivity at pH 5.0: L. casei (ATCC 15008), 40,60; L. plantarum (CH 374), 40,100; L. fermentum (CH 324) 10,40; L. acidophilus (ATCC 4356), 10,40; and S. mutans (BM 52), 2,2. All of the strains of Lactobacillus tested with Spiramycin were resistant at pH 5.0. Minocycline was less affected by changes in pH, but at pH 7.4 Lactobacillus strains were more resistant to Minocycline as compared with Spiramycin. Both of these antibiotics are bacteriostatic, and therefore may have a more limited effect than a bactericidal agent such as chlorhexidine. The Lactobacillus strains tested required higher concentrations of chlorhexidine than did the strains of Streptococcus for a killing effect in vitro.

Chlorhexidine↗
PubMed1995

Influence of lactobacilli on the adhesion of Staphylococcus aureus and Candida albicans to fibers and epithelial cells.

The ability of organisms to adhere to and form biofilms on fibrous materials is believed to be an important initiating step in the induction of several diseases, such as toxic shock syndrome. Using an in vitro assay, a moderately hydrophobic strain of Staphylococcus aureus (water contact angle 35 degrees) and a hydrophilic Candida albicans (shown by a hexadecane test) were highly adherent to commercial diaper fibers. The lumen side of the diaper was porous and the fibers were very hydrophobic (> 140 degrees), but the internal section was very hydrophilic (0 degrees), presumably for lus strains was present. Surfaces precoated with lactobacilli inhibited staphylococcal adhesion by 26-97%, and candida by 0-67%. When the lactobacilli were used to challenge adherent pathogens, there was 99% displacement of the S. aureus and up to 91% displacement of C. albicans. Hydrophobic L. acidophilus 76 (54 degrees) and T-13 (80 degrees) were the most effective of five Lactobacillus isolates tested at interference by precoating. The moderately hydrophilic L. casei var rhamnosus GR-1 (33 degrees) was the most effective at displacing the yeast. Experiments with uroepithelial cells also showed that the lactobacilli could significantly interfere with the adhesion of both pathogens to the cells. The results demonstrate the rapidity with which two pathogens adhered to fibers and epithelial cells, and raised the possibility that members of the normal female urogenital flora might interfere with infections caused by these organisms.

Adult↗