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Isolation, identification and characterisation of the dominant microorganisms of kule naoto: the Maasai traditional fermented milk in Kenya.

From 22 samples of kule naoto, the traditional fermented milk products of the Maasai in Kenya, 300 lactic acid bacterial strains were isolated and phenotypically characterised by their ability to ferment different carbohydrates and by additional biochemical tests. Lactic acid bacteria (LAB), especially the genus Lactobacillus, followed by Enterococcus, Lactococcus and Leuconostoc, dominated the microflora of these samples. The major Lactobacillus species was Lactobacillus plantarum (60%), with a lower frequency of isolation for Lactobacillus fermentum, Lactobacillus paracasei and Lactobacillus acidophilus. Most strains produced enzymes such as beta-galactosidase and peptidases, which are of relevance to cultured dairy product processing, and exhibited similar patterns of enzymatic activity between species. Enterobacteriaceae could not be detected in 15 out of 22 samples (detection level 10(2)/ml). Conversely, yeasts (detection level 10(1)/ml) were detected in those samples in which Enterobacteriaceae were not found. The pH values of all these samples were < 4.5.

Colony Count, Microbial↗

Vaginal lactobacilli: self- and co-aggregating ability.

Lactic acid bacteria are the dominant bacteria of the vaginal tract in healthy women. Lactobacillus species form a barrier population that protects from pathogen colonisation by mechanisms that include adhesion to epithelial surfaces, self-aggregation and co-aggregation. In this study, factors involved in the self-aggregating ability of vaginal lactobacilli and in the co-aggregation of these microorganisms with Candida spp. are studied. Both self-aggregation and co-aggregation are monitored quantitatively by the decrease in the absorbance of suspensions of the microorganisms and qualitatively by light microscopy. The self-aggregating ability of four vaginal lactobacilli was shown to be caused by a peptide or protein sensitive to trypsin. However, in self-aggregating Lactobacillus acidophilus CRL 1294 the factor was resistant to trypsin and sensitive to pepsin. Among self-aggregating lactobacilli, L. acidophilus CRL 1294 and L. salivarius CRL 1328 were able to co-aggregate with Candida spp. The co-aggregating factor for both strains proved to be peptide of the surface and a peptide on the bacterial surface, while the receptor on the yeast was a carbohydrate. Co-aggregation of both lactobacilli and Candida spp. was inhibited by the addition of mannose but was not affected by other carbohydrates. Self and co-aggregation factors were not able to induce aggregation in non-aggregating lactobacilli.

Bacterial Adhesion↗

Production of phenyllactic acid by lactic acid bacteria: an approach to the selection of strains contributing to food quality and preservation.

The ability of lactic acid bacteria (LAB) to produce phenyllactic (PLA) and 4-hydroxy-phenyllactic (OH-PLA) acids, metabolites involved in food quality and preservation, has been evaluated by HPLC analysis in 29 LAB strains belonging to 12 species widely used in the production of fermented foods. Metabolite production was demonstrated for all strains of the species Lactobacillus plantarum, Lactobacillus alimentarius, Lactobacillus rhamnosus, Lactobacillus sanfranciscensis, Lactobacillus hilgardii, Leuconostoc citreum, and for some strains of Lactobacillus brevis, Lactobacillus acidophilus and Leuconostoc mesenteroides subsp. mesenteroides. Strains were distinguished by analysis of variance in three groups including 15 strains that produced both metabolites (0.16-0.46 mM PLA and 0.07-0.29 mM OH-PLA), five strains accumulating in culture only PLA (0.17-0.57 mM) and nine non-producer strains (< or = 0.10 mM PLA and < or = 0.02 mM OH-PLA). Improvement of phenyllactic acid production was obtained in a selected L. plantarum strain by increasing the concentration of phenylalanine in culture and using low amounts of tyrosine.

Arsenicals↗

Antibacterial effect of a hydraulic calcium phosphate cement for dental applications.

Calcium hydroxide is currently used in dentistry for endodontic treatment where its main advantage consists of its antibacterial and anti-inflammatory potency. However, it also has some drawbacks such as pulp necrosis, slight solubility, slow and low hardening, and retraction on drying. Since the studies conducted by Brown and Chow (IADR 1983, abst. 207), calcium phosphate-based cements (CPC) have attracted considerable interest in bone reconstruction because of their good osteoconductivity. By mixing calcium bis-dihydrogenphosphate monohydrate (MCPM) and calcium oxide with sodium phosphate buffer in the form of liquid phase, we obtained a CPC with better mechanical properties than calcium hydroxide. The setting reaction produced a mixture of calcium-deficient hydroxyapatite and calcium hydroxide, making this cement more suitable for dental applications than orthopedic ones. The presence of calcium hydroxide a priori confers antibacterial properties to this cement, which were investigated in agar plates (diffusion method) against Streptococcus mutans, Lactobacillus acidophilus, Candida albicans (clinical isolates) and a preparation of polymicrobial flora isolated from dental plaque. The cement samples tested were prepared at calcium-to-phosphate molar ratios (Ca/P) ranging from 1.67 to 2.75. A pure calcium hydroxide paste was used as reference material. Clear and reproducible bacterial growth inhibition was observed for cement samples with Ca/P > or = 2 against all the microorganisms tested. MCPM-CaO-based cement is therefore a potential candidate for pulp capping and cavity lining.

Anti-Infective Agents, Local↗

Study of adhesion of Lactobacillus casei CRL 431 to ileal intestinal cells of mice.

It is well known that the cell wall of Lactobacillus casei CRL 431, a strain present in probiotics, presents lectinlike surface molecules. Presence of these molecules stimulates the immune system. Given the role that lectins and lectinlike substances play in the adhesion phenomenon, it is probable that this is an initial stage in the immunostimulation produced by this bacterium. To confirm this, adhesion of this microorganism to exfoliated mouse ileal epithelial cells was studied in vitro. Other L. casei strains isolated from adult human intestines and one of dairy origin were also examined for their ability to adhere to ileal epithelial cells. Another strain, which was included in the present study, was Lactobacillus acidophilus CRL 730. L. casei strains isolated from humans showed good ability to adhere to ileal epithelial cells, whereas L. casei isolated from dairy origin did not. Adhesion was only observed at 37 degrees C and at a pH between 6 and 7.5. The exposure time needed for highest adhesion was 30 min. Presence of lectinlike substances on the surface of L. casei CRL 431 is important to this adhesion phenomenon, since adherence capacity was lost after removal of these substances.

Animals↗

Bacteriocin susceptibility of Gardnerella vaginalis and its relationship to biotype, genotype, and metronidazole susceptibility.

OBJECTIVE: Our purpose was to investigate the bacteriocin susceptibility of Gardnerella vaginalis and its relationship to biotype, genotype, and resistance to metronidazole. STUDY DESIGN: Bacteriocin susceptibility of 36 G vaginalis clinical isolates was tested against a vaginal strain of Lactobacillus acidophilus by a growth-inhibition method. The relationship to biotype, genotype, and resistance to metronidazole were analyzed by the chi2 test and Fisher exact test. RESULTS: Eight G vaginalis strains (22%) were bacteriocin-resistant. Biotypes 5 and 7 were found to be the most frequent among these resistant strains. Eight (42%) of the 19 isolates classified as biotype 5, 6, or 7 were bacteriocin-resistant compared with none of the isolates that were classified as other biotypes (P <.01). Biotype 5 was found in higher prevalence among the isolates resistant to bacteriocin (62%) than among the susceptible isolates (14%) (P =.01). Genotype B was found more frequently among the bacteriocin-resistant strains, but this finding was not statistically significant (P =.71). Seven (88%) bacteriocin-resistant strains were also resistant to metronidazole. CONCLUSION: An association between biotype and an increased resistance to bacteriocin was found. The ability of G vaginalis to resist the antibacterial activity of Lactobacillus bacteriocin may be a pivotal factor in understanding bacterial vaginosis.

Anti-Bacterial Agents↗

Antibodies in normal and diseased pulps reactive with microorganisms isolated from deep caries.

Immunoglobulin molecules in the supernatant fluids (SF) from pulpal explant cultures have been observed to react with microorganisms implicated in infections of root canals. In this study, the reactivity of immunoglobulin molecules in the SF from normal and irreversible pulpitis pulps to six strains of predominant microorganisms isolated from the immediate layer of carious lesions above the pulps used for explant cultures was investigated using an enzyme-linked immunosorbent assay. Two ATCC strains of Eubacterium were also included in this assay. Specific antibodies to Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus, Lactobacillus acidophilus (I), (II), Streptococcus mutans, Bacteroides intermedius, Eubacterium brachy, and Eubacterium alactolyticum in the SF from the normal and irreversible pulpitis tissues were observed with a large variation of antibody levels in both groups. Immunodiffusion assays of the SF revealed that IgG was the major class of immunoglobulin in the normal as well as the irreversible groups. The presence of natural antibodies in the normal pulps suggested a possible protective role of antibodies during the invasive process of caries.

Antibodies, Bacterial↗

Influence of controlled lactic fermentation on growth and sporulation of Bacillus cereus in milk.

The growth and sporulation of Bacillus cereus NVH 45 in a fermentor with controlled pH or simulated pH conditions were investigated. The study was carried out in a fermentor to measure the influence of a rapid and a slow lactic acid production on the inhibition of B. cereus in a controlled environment during the initial part of fermentation and to observe if other factors than lactic acid influenced the inhibition. In the controlled pH experiments the pH was allowed to decrease to an end pH 5.0, 5.5 or 6.0 either by Lactobacillus casei 2756 (a fast acid producer) or Lactobacillus acidophilus NCFB 1748 (a slow acid producer). In co-cultures of Lb. casei 2756 and B. cereus NVH 45, low numbers (10-70 cfu/ml) of B. cereus NVH 45 were observed at end pH 5.5 (72 h) while at pH 5.0 no viable cells (<10 cfu/ml) were detected (48-72 h). B. cereus NVH 45 did not sporulate in co-culture with Lb. casei 2756. In co-culture with Lb. acidophilus NCFB 1748, B. cereus NVH 45 sporulated and survived as spores. In these co-cultures B. cereus NVH 45 grew to higher maximum counts (>10(7) cfu/ml) than with Lb. casei 2756 (<10(7) cfu/ml). Significantly different amounts of lactic acid were observed between the two co-cultures after 7 and 12 h. A rapid decrease of pH appears to prevent B. cereus from sporulating and it seems that it is enough to just reach pH 5.0 rapidly and keep that pH to achieve the desirable inhibition of B. cereus. In the simulated pH experiments B. cereus NVH 45 was inoculated in the fermentor and the different pH developments from different LAB strains were monitored by addition of lactic acid. These experiments showed the same tendency: a fast pH reduction during the initial hours of fermentation, simulating lactococci, resulted in complete inhibition of B. cereus NVH 45 (<10 cfu/ml). However, when simulating the pH development of the two different Lactobacillus strains, complete inhibition of B. cereus NVH 45 was not seen. In co-cultures competition for nutrients with consequences for cell density appears to be important. Based on these results it seems that B. cereus must reach a certain density to induce sporulation.

Animals↗

Ex vivo effects of lactobacilli, streptococci, and bifidobacteria ingestion on cytokine and nitric oxide production in a murine model.

Increasing numbers of functional foods and pharmaceutical preparations are being promoted with health claims based on the potential probiotic characteristics of lactic acid bacteria and on their capacity for stimulating the host immune system. However, the specific immune effects of oral administration of these microbes still remains undefined. In this study, we tested the hypothesis that production of immunologic mediators by leukocytes in mice is affected by orally administered lactic acid bacteria. The specific objectives of this study were to evaluate the effects of exposure to eight different lactic acid bacteria in mice on ex vivo cytokine and nitric oxide production in leukocyte cultures. Mice were gavaged with 1 X 10(9) viable bacteria and peritoneal, Peyer's patch and splenic leukocytes were isolated 8 h later. These were cultured for 2 or 5 days in the presence or absence of mitogens and then interleukin (IL)-6, IL-12, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and nitric oxide production was measured. The results revealed that Lactobacillus acidophilus and L. casei potentiated IL-6 and IL-12 production by peritoneal cells whereas L. acidophilus upregulated IFN-gamma and nitric oxide. In contrast, L. helveticus, L. gasseri, L. reuteri, and Bifidobacterium attenuated the production of IL-6, IFN-gamma, and nitric oxide by peritoneal cells. TNF-alpha was not detectable in peritoneal cultures. None of the bacteria altered ex vivo production of cytokines or nitric oxide by Peyer's patch or spleen cell cultures. Taken together, the results suggest that prior oral exposure to lactic acid bacteria could differentially potentiate or attenuate subsequent cytokine and nitric oxide production by peritoneal cells.

Animals↗

Acid production from lactulose by dental plaque bacteria.

Representative strains of oral streptococci, lactobacilli and bifidobacteria were incubated overnight with lactulose or other carbohydrates and the final pH recorded. Most bacteria tested were able to metabolize lactulose with the exception of strains of Streptococcus salivarius, Lactobacillus acidophilus and Lact. fermentum. Streptococcus mutans produced most acid overnight but the initial rate of acid production from lactulose by uninduced cultures was very low. Plaque pH was monitored in 12 volunteers following rinsing the mouth with lactulose, sucrose or sorbitol or Lactulose BP. These studies in vivo showed both lactulose and Lactulose BP to exhibit low acidogenic potential. Thus, although plaque bacteria are capable of fermenting lactulose, the results suggest that lactulose is likely to pose a small acidogenic challenge to teeth under normal conditions of use.

Bifidobacterium↗

Reclassification of Lactobacillus kefirgranum Takizawa et al. 1994 as Lactobacillus kefiranofaciens subsp. kefirgranum subsp. nov. and emended description of L. kefiranofaciens Fujisawa et al. 1988.

Fourteen homofermentative lactic acid bacteria that were isolated from kefir grains and kefir fermented milks were assigned to either Lactobacillus kefiranofaciens or Lactobacillus kefirgranum, based on their characteristic morphotypes, phenotypic features and SDS-PAGE profiles of whole-cell proteins. Further genotypic analyses on representative strains from both taxa demonstrated that L. kefiranofaciens and L. kefirgranum share 100 % 16S rDNA sequence similarity and belong phylogenetically to the Lactobacillus acidophilus species group. DNA-DNA binding values of >79 % and analogous DNA G+C contents of 37-38 mol% showed that the strains studied belonged to one species: L. kefirgranum is a later synonym of L. kefiranofaciens. An emended description is proposed for L. kefiranofaciens. Due to the specific morphological and biochemical characteristics of these taxa in kefir grain formation, it is proposed that L. kefirgranum should be reclassified as L. kefiranofaciens subsp. kefirgranum subsp. nov.

Animals↗

[Growth and morphological characteristics of industrial strains of Bifidobacterium and Lactobacillus cultivated in hydrolysate-milk and hydrolysate-soybean media].

Growth features of industrial strains of Bifidobacterium adolescentis MC-42, B. bifidum 1, B. longum B-379, Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus 8-79 and L. plantarum 8PA3 cultivated in hydrolysate-milk or hydrolysate-soybean media (HMM and HSM respectively) were analysed comparatively. The bacterial cells were investigated morphometrically with atom strength microscopy. It was shown that HSM vs HMM provided a higher growth rate of the strains (shortened growth phases and higher growth rates) that was more evident for the bifidobacteria as compared to the lactobacilli. At the same time, the morphological features of the bacterial cells slightly depended on the medium composition and were mainly defined by the genus.

Bifidobacterium↗

Selecting lactic acid bacteria for their safety and functionality by use of a mouse colitis model.

Studies showed that specific probiotics might provide therapeutic benefits in inflammatory bowel disease. However, a rigorous screening of new probiotics is needed to study possible adverse interactions with the host, particularly when intended for administration to individuals with certain health risks. In this context, the objective of this study was to investigate the role of three lactobacilli (LAB) on intestinal inflammation and bacterial translocation using variations of the mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute colitis. We first compared the in vitro ability of LAB to survive gastrointestinal tract (GIT) conditions and their ability to persist in the GIT of mice following daily oral administration. As a control, we included a nonprobiotic Lactobacillus paracasei strain, previously isolated from an endocarditis patient. Feeding high doses of LAB strains to healthy and to TNBS-treated mice did not induce any detrimental effect or abnormal translocation of the bacteria. Oral administration of Lactobacillus salivarius Ls-33 had a significant preventive effect on colitis in mice, while Lactobacillus plantarum Lp-115 and Lactobacillus acidophilus NCFM did not. None of the three selected LAB strains translocated to extraintestinal organs of TNBS-treated mice. In contrast, L. paracasei exacerbated colitis under severe inflammatory conditions and translocated to extraintestinal organs. This study showed that evaluations of the safety and functionality of new probiotics are recommended. We conclude that not all lactobacilli have similar effects on intestinal inflammation and that selected probiotics such as L. salivarius Ls-33 may be considered in the prevention or treatment of intestinal inflammation.

Acute Disease↗

Establishment of Lactobacillus and Bifidobacterium species in germfree mice and their influence on some microflora-associated characteristics.

Germfree mice were inoculated with both Lactobacillus acidophilus A10 and Bifidobacterium bifidum B11. Both strains were established and present in more than 10(8) cells per g of cecum and colon contents. Furthermore, L. acidophilus A10 was established in high numbers in stomach and small intestine. Contents from different parts of the intestine were investigated with regard to the following microflora-associated characteristics: degradation of mucin, beta-aspartylglycine and tryptic activity, conversion of cholesterol to coprostanol and bilirubin to urobilinogen, deconjugation of bilirubin glucuronides, and reduction of the cecum size. In spite of being established, the microbes were not able to mediate any alterations of the parameters investigated. All animals retained values as found in their germfree counterparts.

Actinomycetales Infections↗

[Lactic bacteria in the digestive tract of poultry].

Lactic bacteria predominate in the microflora of the digestive tract of chicken and turkey. They are represented mainly by Lactobacillus acidophilus, L. salivarius, L. fermentum and L. buchneri. Streptococcus faecium is always isolated. L. ruminis, L. vitulinus, L. delbrueckii, L. coryniformis and L. viridescens were found in this ecological niche for the first time. S. faecium and S. faecalis prevail in the digestive tract of geese and ducks, while lactobacilli are detected in a lesser amount and are represented mainly by L. plantarum. L. salivarius cells isolated from the digestive tract of poultry are highly polymorphous. Most of the lactic acid bacteria found in the digestive tract of poultry can grow at 45-50 degrees C whatever is the species they belong to.

Animals↗

Vaginal lactobacilli in adolescents: presence and relationship to local and systemic immunity, and to bacterial vaginosis.

OBJECTIVE: The objective of this study was to survey middle adolescents for the presence of vaginal lactobacilli, lactobacilli-specific immune sensitization, and correlates of vaginal immunity with lactobacilli and bacterial vaginosis (BV). METHODS: A cohort of 89 female adolescents were evaluated for the presence of vaginal lactobacilli species, H2O2-producing species, and the prevalence of BV. Cytokines and antibodies in cervicovaginal lavages were detected and peripheral blood lymphocyte (PBL) responses to Lactobacillus crispatus were evaluated. RESULTS: The majority of lactobacillus species were H2O2-producing and predominated by Lactobacillus acidophilus. PBL responses to lactobacilli were detectable in 50% of the cohort. BV was present in 36% of adolescents and negatively correlated with the presence of vaginal lactobacilli. The majority of locally associated cytokines and antibodies were similar in those with or without BV or lactobacilli. CONCLUSIONS: Adolescents harbor vaginal lactobacilli with relationships to BV along with lactobacilli-specific immune sensitization, but with few correlates of local immunity to lactobacilli or BV.

Adolescent↗

Evolution of carbohydrate fraction in carbonated fermented milks as affected by beta-galactosidase activity of starter strains.

The influence of carbonation on the evolution of lactose, galactose and glucose in fermented milks with added probiotic bacteria (Lactobacillus casei, Lactobacillus acidophilus and/or Bifidobacterium bifidum) was evaluated and related to beta-galactosidase activity of starter strains. During incubation and first days of refrigeration, lactose hydrolysis resulting in the liberation of galactose and glucose occurred in CT (Streptococcus thermophilus/Lb. casei), AT (Str. thermophilus/Lb. acidophilus) and ABT fermented milks (Str. thermophilus/Lb. acidophilus/Bifid. bifidum). Levels of galactose were higher than those of glucose and could be related to the preferential consumption of glucose by actively growing bacteria. Through the incubation, lactose and monosaccharide levels were not affected by milk carbonation. However, during refrigerated storage the presence of this gas was associated with slightly lower content of lactose and higher levels of galactose and glucose in AT and ABT products but not in CT fermented milks. Through the refrigeration galactose was moderately utilised by Lb. acidophilus in AT products whereas the presence of Bifid. bifidum seems to prevent the consumption of this sugar in ABT fermented milks. Glucose remained constant, with minor variations in CT products but a continuous increase of this sugar occurred in carbonated AT and ABT fermented milks during storage. Beta-galactosidase activity displayed by Str. thermophilus strains was similar at pH 6.5 (initial pH of non-carbonated samples) and pH 6.3 (initial pH of carbonated samples) whereas Lb. acidophilus LaA3 showed greater beta-galactosidase activity at pH 6.3 than at higher pH values. Thus, the enhanced metabolic activity of Lb. acidophilus caused by the low initial pH of carbonated milk also promoted higher cellular beta-galactosidase activity that could have released greater amounts of galactose and glucose from lactose in AT and ABT fermented milks through the refrigerated period. In CT fermented milks, similar beta-galactosidase activity levels of Str. thermophilus at pH 6.5 and 6.3 together with the absence of beta-galactosidase activity in Lb. casei could explain the lack of differences on glucose and galactose content between carbonated and non-carbonated samples.

Animals↗

Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model.

Viable bacteria were not cultured from the mesenteric lymph nodes, spleens, or livers of specific-pathogen-free (SPF) mice. Viable enteric bacteria, primarily indigenous Escherichia coli and lactobacilli, were present in the mesenteric lymph nodes of gnotobiotic mice inoculated intragastrically with the whole cecal microflora from SPF mice but not in the nodes of control SPF mice similarly inoculated. These indigenous E. coli also were cultured from the mesenteric lymph nodes of 96% of gnotobiotic mice monoassociated with E. coli but from none of the mesenteric lymph nodes of SPF mice inoculated with the E. coli. Furthermore, viable E. coli were detected in the mesenteric lymph nodes of these monoassociated gnotobiotes for as long as 112 days after inoculation. Indigenous Lactobacillus acidophilus also translocated to the mesenteric lymph nodes of gnotobiotic mice monoassociated with L. acidophilus. Apparently, there are mechanisms active in SPF mice inhibiting translocation of indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes, spleens, and livers, whereas these mechanisms are either absent or reduced in gnotobiotic mice. Indigenous E. coli maintained higher population levels in the gastrointestinal tracts of the gnotobiotes compared with their population levels in SPF mice, suggesting that high bacterial population levels might promote translocation of certain bacteria from the gastrointestinal lumen to the mesenteric lymph nodes. Gnotobiotic and SPF mice, therefore, provide experimental models for determining the nature of the mechanisms operating to confine indigenous bacteria to the gastrointestinal tract in normal, healthy animals.

Animals↗