Selection and identification of anaerobic lactobacilli producing inhibitory compounds against vaginal pathogens

Two strains of Lactobacillus crispatus (15L08 and 21L07) and one strain of Lactobacillus jensenii (5L08) were selected from amongst 100 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization and the production of H2O2 and/or bacteriocin-like compound. All three strains self-aggregated and adhered to vaginal epithelial cells, displacing well-known vaginal pathogens, such as Gardnerella vaginalis and Candida albicans. Lactobacillus crispatus 15L08 was characterized as a potential H2O2 producer. A high level of bacteriocin-like compound was synthesized by L. jensenii 5L08, with a bactericidal mode of action for G. vaginalis, C. albicans and Escherichia coli. However, H2O2-dependent activity alone was not sufficient to inhibit the growth of C. albicans. Simultaneous actions of H2O2 and bacteriocin-like compound produced by lactobacilli may be important for antagonizing pathogenic bacteria. These strains of lactobacilli may be excellent candidates for eventual use as probiotics to restore the normal microbial communities in the vaginal ecosystem.     

The adhesion of putative probiotic lactobacilli to cultured epithelial cells and porcine intestinal mucus

Aims: To investigate the adhesion of lactobacilli and their subsequent competitive exclusion ability against pathogens. Methods and Results: Four species of putative probiotic lactobacilli were studied for their adhesion abilities. First, the adhesion to Caco‐2 cells was examined by light and electron microscopy. The four species were then labelled by [methyl‐³H] thymidine and their adhesion to porcine intestinal mucus was determined by radioactivity. The tested lactobacilli showed best adhesion on ileal mucus compared with duodenal and jujenal mucus. Oxidative compound pre‐treatment (NaIO₃ and NaIO₄) dramatically decreased the adhesion of the lactobacilli to mucus. Pre‐treating mucus with proteolytic enzymes (proteinase K and trypsin) resulted in the increase of adhesion in Lactobacillus serotype Reuteri I2021, but the results in the other species were variable. Lactobacillus serotype Fermentum I5007 showed greatest adhesion potential and exerted the best competitive exclusion against Salmonella and Escherichia. Conclusions: Adhesion ability in lactobacilli is species‐specific. Lactobacilli with higher adhesion index have better competitive exclusion ability. Significance and Impact of the Study: This study suggests that there is a positive correlation between adhesion and competitive exclusion ability of lactobacilli. Additionally, the in vitro adhesion assay is a feasible way to screen unknown lactobacilli, potentially for future industrial applications.     

Influence of lactobacilli on the adhesion ofStaphylococcus aureus andCandida 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 anin vitro assay, a moderately hydrophobic strain ofStaphylococcus aureus (water contact angle 35°) and a hydrophilicCandida 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°), but the internal section was very hydrophilic (0°), presumably for adsorption purposes. There was evidence that adhesion of the pathogens was inhibited when one of fiveLactobacillus 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 theS. aureus and up to 91% displacement ofC. albicans. HydrophobicL. acidophilus 76 (54°) and T-13 (80°) were the most effective of fiveLactobacillus isolates tested at interference by precoating. The moderately hydrophilicL. casei varrhamnosus GR-1 (33°) 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.     

Maple sap as a rich medium to grow probiotic lactobacilli and to produce lactic acid

Aims: To demonstrate the feasibility of growing lactobacilli and producing lactic acid using maple sap as a sugar source and to show the importance of oligosaccharides in the processes. Methods and Results: Two maple sap samples (Cetta and Pinnacle) and purified sucrose were used as carbon sources in the preparation of three culture media. Compared with the sucrose‐based medium, both maple sap‐based media produced increased viable counts in two strains out of five by a factor of four to seven. Maple sap‐based media also enhanced lactic acid production in three strains. Cetta sap was found to be more efficient than Pinnacle sap in stimulating lactic acid production and, was also found to be richer in various oligosaccharides. The amendment of the Pinnacle‐based medium with trisaccharides significantly stimulated Lactobacillus acidophilus AC‐10 to grow and produce lactic acid. Conclusions: Maple sap, particularly if rich in oligosaccharides, represents a good carbon source for the growth of lactobacilli and the production of lactic acid. Significance and Impact of the Study: This study provides a proof‐of‐concept, using maple sap as a substrate for lactic acid production and for the development of a nondairy probiotic drink.     

Isolation of lactobacilli with probiotic properties from the human stomach

Aims: Recent evidence suggests that the human gastric microbiota is much more diverse than previously thought. The aim of this study was to assess the potential for isolating lactobacilli from the human stomach. Methods and Results: Lactobacilli were selectively cultured from gastric biopsies from 12 patients undergoing routine endoscopy. Lactobacilli were present in four of 12 biopsies. We isolated, in total 10 different strains representing five species (Lactobacillus gasseri, L. fermentum, L. vaginalis, L. reuteri and L. salivarius). The 10 isolates varied greatly in their ability to inhibit the growth of two Gram‐positive bacteria and two Gram‐negative bacteria. Furthermore, the acid and bile resistance profiles of the 10 isolates spanned a wide range. Conclusions: Five different Lactobacillus species were cultured from human gastric biopsies for the first time. Significance and Impact of the Study: Diverse Lactobacillus species are more prevalent in the human stomach than previously recognized, representing an untapped source of bacteria with beneficial probiotic and/or biotechnological properties.     

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains—Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38—presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.     

Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus

AIMS: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. METHODS AND RESULTS: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P<0.05) the adhesion of Bacteroides, Clostridium, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. CONCLUSIONS: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection.     

Proliferation and differentiation of prepubertal mouse vaginal epithelial cells in vitro and the specificity of estrogen-induced growth retardation

Summary Mouse vaginal epithelial cells were isolated from intact 21-day-old BALB/cCrgl mice and cultured in a serum-free medium (SF₂₀: basal medium supplemented with insulin, epidermal growth factor, transferrin, and bovine serum albumin—fraction V) to examine the proliferation, differentiation, and specificity of estrogen-induced growth retardation in vitro. Histologic and ultrastructural studies showed that vaginal epithelial cells undergo differentiative changes in vitro in the absence of estrogen, and that these changes are similar to those induced in vivo by estrogen. Addition of 17β-estradiol inhibited cellular proliferation in a dose-dependent manner. Whereas other estrane derivatives (17α-estradiol and estriol) also significantly retarded cellular proliferation, cholesterol, testosterone, and progesterone had no effect. Keoxifene, an antiestrogen, significantly reversed estrogen-induced growth inhibition, resulting in proliferation of estrogen-treated cells equivalent to that of the untreated control. The results suggest that both proliferation and differentiation of prepubertal mouse vaginal epithelial cells in vitro are estrogen-independent, and that the growth inhibition is a specific estrogen-induced response. This work was supported by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD.     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

Genome shuffling of Lactobacillus plantarum C88 improves adhesion

Genome shuffling is an important method for rapid improvement in microbial strains for desired phenotypes. In this study, ultraviolet irradiation and nitrosoguanidine were used as mutagens to enhance the adhesion of the wild-type Lactobacillus plantarum C88. Four strains with better property were screened after mutagenesis to develop a library of parent strains for three rounds of genome shuffling. Fusants F3-1, F3-2, F3-3, and F3-4 were screened as the improved strains. The in vivo and in vitro tests results indicated that the population after three rounds of genome shuffling exhibited improved adhesive property. Random Amplified Polymorphic DNA results showed significant differences between the parent strain and recombinant strains at DNA level. These results suggest that the adhesive property of L. plantarum C88 can be significantly improved by genome shuffling. Improvement in the adhesive property of bacterial cells by genome shuffling enhances the colonization of probiotic strains which further benefits to exist probiotic function.     

Diversity of probiotic adhesion genes in the gastrointestinal tract of goats

Gastrointestinal (GI) microflora is an important system in the host, as it has both pathogenic and probiotic bacteria. Most of the studies were focused on the human gut microflora and the available information on the intestinal microflora of goats was limited. This urged the need to inspect the impacts of the goat's gut microflora. Metagenomic investigation of probiotic bacteria in the GI tract of goat is one of the challenging streams because of the less available data of the uncultivable bacteria. In our report, comparative analysis of metagenomic and enrichment samples of goat intestinal content was done and this approach will be helpful in analyzing the identification of uncultivable and cultivable probiotic bacteria. This study mainly focused on three key probiotic adhesion genes, such as EF-Tu, mapA, and mub. The GI of four different goats were investigated for these genes. The data from this study showed that there is a wide diversity of these genes among goat intestinal samples.     

Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances

AIMS: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products. METHODS AND RESULTS: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C. Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C. Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx. 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact. salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact. rhamnosus GG. CONCLUSIONS: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage. SIGNIFICANCE AND IMPACT OF THE STUDY: High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.     

Cytofluorescent assay to quantify adhesion of equine spermatozoa to oviduct epithelial cells in vitro

To facilitate the study of interactions between equine spermatozoa and homologous oviduct epithelial cells, we developed an assay to count labelled spermatozoa bound to oviduct epithelial cell (OEC) monolayers and used the assay to compare the binding ability of spermatozoa from different stallions. Washed spermatozoa from three stallions were incubated with the fluorochrome Hoechst 33342 (5 μg/ml) for 1 min. Spermatozoa were then layered over confluent monolayers of oviduct epithelial cells in 2 cm² culture wells. Coculture treatments comprised five concentrations of spermatozoa (10⁵, 5 × 10⁵, 10⁶, 2.5 × 10⁶, and 5 × 10⁶ per well). Cocultures were incubated for 30 min before unattached spermatozoa were aspirated in coculture supernatant. Fluorescent videoimages of attached spermatozoa were digitized, and attached spermatozoa were counted by image processing and analysis. Four wells (replicates) of each concentration were allocated within each ejaculate, and ejaculates were blocked by stallion for ANOVA. The total number of spermatozoa bound was not different between replicate wells (P > 0.1). Stallion, ejaculate, concentration, and all higher level interactions influenced total spermatozoa bound (P < 0.00001). Coefficients of variation between replicates were lowest for inseminate concentrations between 10⁶ and 5 × 10⁶ spermatozoa per well. Within the ejaculate, a log linear relationship exists between the number of bound spermatozoa and a spermatozoal concentration of the inseminate between 5 × 10⁵ and 5 × 10⁶ spermatozoa per well. This assay provides a reliable method of determining numbers of spermatozoa bound to somatic cells in vitro. Furthermore, differences exist in the ability of spermatozoa from different stallions to bind OEC monolayers. © 1996 Wiley-Liss, Inc.     

Assessment of cell surface properties and adhesion potential of selected probiotic strains

Aim:  To evaluate the physicochemical cell surface and adhesive properties of selected probiotic strains for human use.
Methods and Results:  Probiotic strains, Bifidobacterium longum B6, Lactobacillus acidophilus ADH, Lactobacillus paracasei , Lactobacillus rhamnosus GG, Lactobacillus brevis , Lactobacillus casei , Leuconostoc mesenteroides and Pediococcus acidilactici were tested for the physicochemical properties of cell surfaces and the adhesion abilities against foodborne pathogens. Bif .  longum B6 (53·6%) and Lact .  rhamnosus GG (46·5%) showed the highest hydrophobicity, while the least affinity to xylene was observed in Ped .  acidilactici (10·4%). Bifidobacterium longum B6 showed the strongest coaggregation phenotype with Listeria monocytogenes (53·0%), Shigella boydii (42·0%) and Staphylococcus aureus (45·9%). Lactobacillus rhamnosus GG had the strong binding ability to Caco-2 cells and effectively inhibited the adhesion of L .  monocytogenes , Salmonella Typhimurium, Sh .  boydii and Staph .  aureus to Caco-2 cells. The hydrophobicity was highly correlated with coaggregative abilities and competitive inhibition, suggesting a good relationship between in vitro adhesion and in vivo colonization.
Conclusion:  The results suggest that Bif .  longum B6 and Lact .  rhamnosus GG can be candidate probiotics available for human consumption.
Significance and Impact of the Study:  Because the use of probiotic strains has been more concerned with their beneficial effects in the GI tract, it is essential to examine the potential of probiotic strains based on the physicochemical properties in terms of bacterial-binding and adhesion capabilities.