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.     

Toward an Alternative Therapeutic Approach for Skin Infections: Antagonistic Activity of Lactobacilli Against Antibiotic-Resistant Staphylococcus aureus and Pseudomonas aeruginosa

The wide spread of antimicrobial resistance has urged the need of alternative therapeutic approach. In this context, probiotic lactobacilli have been reported for the prevention and treatment of many gastrointestinal and urogenital infections. However, very little is known about their antagonistic activity against skin pathogens. Accordingly, the present study aimed to investigate the potential of lactobacilli to interfere with pathogenesis features of two antibiotic-resistant skin pathogens, namely methicillin-resistant Staphylococcus aureus and multiple-resistant Pseudomonas aeruginosa. A total of 49 lactobacilli were recovered, identified and tested for their antagonistic activities against the aforementioned pathogens. Of these, eight isolates were capable of blocking the adherence of pathogens to mammalian cells independent of the skin pathogen tested or model adopted. Moreover, three Lactobacillus isolates (LRA4, LC2 and LR5) effectively prevented the pathogen internalization into epithelial cells in addition to potentiating phagocyte-mediated pathogen killing. Interestingly, the lactobacilli LC2, LF9 and LRA4 markedly inhibited the growth of P. aeruginosa and S. aureus isolates in coculture experiments. Besides, the lactobacilli LRA4, LC2, LR5 and LF9 have counteracted pathogen cytotoxicity. Taken together, the present study revealed some inhibitory activities of lactobacilli against two antibiotic-resistant skin pathogens. Moreover, it revealed two lactobacilli, namely LC2 and LRA4, with antagonistic capacity against different virulence determinants of skin pathogens. These lactobacilli are considered promising probiotic candidates that may represent an alternative therapeutic approach for skin infections.     

Ability of <Emphasis Type="Italic">Lactobacillus</Emphasis> to inhibit enteric pathogenic bacteria adhesion on Caco-2 cells

The antagonistic effects of Lactobacillus against pathogenic bacteria were evaluated in vitro on cultured Caco-2 cells. Lactobacilli were added simultaneously with enteric pathogenic strains (enterotoxigenic Escherichia coli K88 or Salmonella typhimurium SARB21 and SL1344), before pathogenic strains and after pathogenic strains for competition, exclusion and displacement assays. The six lactobacilli significantly limited the adhesion and invasion of the pathogenic bacteria. In the simulating competition and exclusion assays, the adhesion of pathogenic strains was reduced by Lactobacillus strains significantly, whereas the inhibiting effect on pathogenic strains adhesion was a little weaker in the displacement assay. Furthermore, we found that the antagonistic effects of lactobacilli against K88, SARB21, and SL1344 were various. Strain R4 showed a strong inhibitory effect on the adhesion of K88 to Caco-2 cells. In the competition assay of R4, the number of viable cell-associated K88 (3.84 ± 0.10 log CFU/well) was much lower than the control group without Lactobacillus (5.98 ± 0.02 log CFU/well). Compared to the control group (6.07 ± 0.02 log CFU/well), the six Lactobacillus strains all performed strong antagonistic effects against SL1344, particularly D17 showed a higher inhibitory effect in the displacement assays (4.15 ± 0.04 log CFU/well). These results implied that several Lactobacillus strains might be useful for protecting against enteric pathogenic infection.     

Inhibitory potential of lactobacilli against <Emphasis Type="Italic">Escherichia coli</Emphasis> internalization by HT 29 cells

A quantitative fluorescent in situ hybridization method was employed to evaluate the competitive inhibitory effect of three Lactobacillus strains (Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus plantarum) against Escherichia coli internalization in a model system of HT 29 cells. Furthermore, aggregation and adhesion abilities of the Lactobacillus strains were examined. All lactobacilli were able to attach to the HT 29 cells and aggregate with pathogens; however, the adhesion and aggregation degree was strain-dependent. L. reuteri possessed a high capacity of adhesion (6.80 ± 0.63; log CFU ± SEM per well), whereas lower capacities were expressed by L. gasseri (4.52 ± 0.55) and L. plantarum (4.90 ± 0.98). Additionally, L. reuteri showed the rapid or normal ability to aggregate with selected E. coli in comparison with remaining two lactobacilli, which showed only slow or negative aggregative reaction. Internalization of E. coli into the cell lines was markedly suppressed by L. reuteri, while L. gasseri and L. plantarum caused only a minimum anti-invasion effect. The fact that L. reuteri in our experiments showed an outstanding potential for adhering to the colon epithelial cell line, compared with the rest strains, suggested that one of the possible mechanisms of preventing pathogen adhesion and invasion is simple competitions at certain receptors and capability to block receptor binding sites, or that an avid interaction between L. reuteri and the host cell might be modulating intracellular events responsible for the E. coli internalization. Moreover, L. reuteri exhibited a strong ability to aggregate with E. coli, which could be another limiting factor of pathogen invasion.     

FISH analysis of Lactobacillus biofilms in the gastrointestinal tract of different hosts

Aims: To investigate the spatial organization of endogenous and exogenously applied Lactobacillus communities at specific locations in the adult gastrointestinal tract of different hosts. Methods and Results: Samples of the human, murine and avian gastrointestinal tract of subjects that received or not received a Lactobacillus probiotic were analysed by fluorescence in situ hybridization (FISH) with rRNA‐targeted probes. High levels of endogenous lactobacilli were observed on the nonsecretory, stratified squamous epithelia present in the forestomach of mice and crop of chickens, respectively. These epithelial associations showed characteristics of bacterial biofilms, i.e. bacteria attached to a surface and embedded in a matrix of extracellular polymeric substances. In other regions of the analysed intestines, lactobacilli seemed to occur mainly as dispersed bacterial cells or as microcolonies. Exogenous administration of a Lactobacillus probiotic did increase the levels of loosely adherent Lactobacillus cells detected. However, the probiotic strains were unable to establish themselves inside the gastrointestinal biofilms. Conclusions: Gastrointestinal biofilms of lactobacilli occur only in specific niches in certain hosts, such as the murine forestomach and avian crop. Significance and Impact of the Study: Biofilm formation by lactobacilli in specific parts of animal gastrointestinal tracts was documented for the first time by FISH.     

Relationship Between Expression of Cell Surface Hydrophobicity Protein 1 (CSH1p) and Surface Hydrophobicity Properties of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>

Candida dubliniensis and Candida albicans cause most of the oral candidiasis infections in AIDS patients. Unlike C. albicans, which variably expresses cell surface hydrophobicity (CSH) depending on environmental conditions, C. dubliniensis is hydrophobic under all environmental conditions. C. dubliniensis produces CdCSH1p, a protein related to CaCSH1p that contributes to CSH expression of C. albicans. We investigated whether environmental conditions affect CdCSH1p expression, CSH avidity, and adhesion to fibronectin (Fn). C. dubliniensis CD36 was grown at 23°C and 37°C in four different media. CdCSH1p expression was affected by growth temperature, with cells grown at 37°C expressing the protein, but cells grown at 23°C did not. Hydrophobic avidity for two media was higher in cells grown at 37°C than at 23°C. Cells grown at 23°C were generally less adherent than 37°C-grown cells to Fn. The results suggest CdCSH1p but not hydrophobic avidity may have a role in adhesion of C. dubliniensis to Fn.     

Binding of extracellular matrix proteins by lactobacilli

Ten gut and ten vaginalLactobacillus strains were investigated for their ability to bind type I collagen (Cn-I) and four selected gut lactobacilli were investigated for their binding to other extracellular matrix (ECM) molecules. Immobilized Cn-I (100 mg/L) in wells of microtitre plates was bound by all 10 autoaggregating vaginal strains and by 3 strains of gut lactobacilli from piglets in the range ofA ₅₇₀ readings 0.114–1.806.L. acidophilus strain SV31 was much more adherent than the rest of strains. All four gut lactobacilli tested for binding to other ECM molecules displayed good binding to porcine fibronectin and heparin and some of them bound weakly to fetuin and porcine mucin. No binding of these strains was observed to bovine mucin, bovine fibrinogen and bovine lactoferrin.     

The Role of 17β-Estrogen in Candida albicans Adhesion on Human Vaginal Epithelial Cells via FAK Phosphorylation

Purposes

To investigate the role of 17β-estrogen in Candida albicans (C. albicans) adhesion on human vaginal epithelial cells in vulvovaginal candidiasis (VVC).

Methods

The vaginal epithelial cell line, VK2/E6E7, was used to study the estrogen-induced molecular events between C. albicans and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation in cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα–FAK signaling cascade.

Results

A transient activation of ERα and FAK was observed following the stimulation with 1000 nM estrogen for 48 h, as well as the increased average number of C. albicans adhering to each vaginal epithelial cell. Estrogen-induced activation of ERa and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 μM concentration and allowed to act for 12 h. Simultaneously, a decrease in the number of adherent C. albicans was observed. However, this inhibitory effect diminished as the concentration of estrogen increased.

Conclusion

FAK and ERα signaling cascades were involved in the early interaction between the vaginal epithelial cells and C. albicans, which appeared to be linked with the enhanced cell adhesion leading to VVC and promoted by a certain concentration of estrogen.

     

Inhibition of Staphylococcus aureus adherence to Caco-2 cells by lactobacilli and cell surface properties that influence attachment

Staphylococcus aureus is an opportunistic pathogen that can colonize human and animal intestinal tracts, causing certain gastrointestinal diseases. The adherence of enteric pathogens to host intestinal epithelial cells is important for their pathogenesis. In the present study, Lactobacillus salivarius and Lactobacillus plantarum were investigated in vitro to examine their ability to competitively exclude S. aureus. Various factors involved in attachment, including bacterial status and cell concentration, growth phase, competition patterns, and surface-layer protein extracts, were also investigated. Live lactobacilli in the mid-log growth phase exhibited maximum inhibitory activity when lactobacilli were pre- or co-incubated with S. aureus. However, the inhibitory activity was significantly reduced when the lactobacilli were inactivated by heating or treated with LiCl. Furthermore, both lactobacilli possessed certain cell surface properties, such as hydrophobicity, autoaggregation, and coaggregation ability. L. salivarius and L. plantarum strongly inhibited S. aureus adherence to Caco-2 cells and their inhibition activity was significantly influenced by several factors that affect adhesion inhibition.     

Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin

Three human Lactobacillus strains, coded B21060, B21070 and B21190, have recently been isolated. The strains show a series of features (acid and bile resistance, adhesion to various types of mucosal cell) which make them particularly promising for the preparation of probiotic products. In the present study, the ability of the strains to inhibit the growth of pathogens in coculture was investigated. Lactobacilli were incubated simultaneously or after one overnight growth with enterotoxigenic Escherichia coli, Salmonella enteritidis or Vibrio cholerae. After 24 and 48 h, bacterial counts of the pathogens and of the lactobacilli were performed. The results showed that these Lactobacillus strains inhibited the in vitro growth of E. coli and S. enteritidis under both conditions. Moreover, a cumulative effect was observed for mixtures of lactobacilli. In contrast, no significant inhibition of Vibrio cholerae growth was observed, provided that the pH of the medium was kept constant. The presence of the pathogens did not affect the growth of the Lactobacillus strains. Moreover, each of the Lactobacillus strains showed coaggregation ability with two pathogenic E. coli strains, namely ATCC 25922 and ATCC 35401.     

Live and heat-inactivated lactobacilli from feces inhibit <Emphasis Type="Italic">Salmonella typhi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> adherence to caco-2 cells

A quantitative approach has been proposed to evaluate the competitive inhibition of Escherichia coli and Salmonella typhi by live and heat-inactivated laboratory isolated Lactobacillus sp. on adhesion to monolayer of Caco-2 cells. Three species of Lactobacillus (L. casei, L. acidophilus, L. agilis) isolated from human neonate feces and two commercial probiotic strains (L. casei, L. acidophilus) have been compared for probiotic activity. All lactobacilli were able to attach to the Caco-2 cells, however, the degree of adhesion was bacterial strain-dependent. The adhesion indices of the two commercial probiotic strains were not significantly different from the values obtained for the other two similar fecal strains (p > 0.01). The inhibition of attachment of the pathogenic bacteria by inactivated cells of fecal L. acidophilus was examined and compared to the results of live bacteria. The inhibition pattern was similar for live and heat-inactivated L. acidophilus (p > 0.01). The number of attached pathogenic bacteria to the Caco-2 cells decreased when the number of L. acidophilus increased from 10⁶ to 10⁹ CFU/mL. The heat-inactivated L. acidophilus displayed similar probiotic activity compared to the live bacteria.     

In vitro assay of the antimicrobial activity of kephir against bacterial and fungal strains

Kephir is a fermented carbonated refreshing milk, with a slightly acidic aromatic taste and creamy foam composition which contains lactobacilli, leuconostocci, acetic acid bacteria, lactostreptococci and yeasts. Recent studies have demonstrated its antibacterial, immunostimulating, antitumoral and cholesterol-lowering activities.

Purpose

The purpose of this study was to investigate the antimicrobial activity of kephir against Bacillus subtilis spp. spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 8739, Salmonella enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 9027 and Candida albicans ATCC 10231. The kephir fermented for 24 h and 48 h, as well and after 7 days preservation at 4–8 °C was tested by in vitro disk diffusion method. The intensity of the antimicrobial activity was interpreted by comparison with two antibiotics, i.e. ampicillin and neomycin.

Results

The antimicrobial activity of 24 h as well as 48 fermented kephir, fresh or after 7 days preservation at 4–8 °C was similar and observed against B. subtilis, S. aureus, E. coli, E. faecalis and S. enteritidis. For E. coli, E. faecalis and S. enteritidis the antimicrobial activity was superior to both tested antibiotics and for B. subtilis and S. aureus to one antibiotic. The tested products exhibited no activity against P. aeruginosa and C. albicans.

Conclusion

Kephir is exhibiting large spectrum and strong antibacterial activity probably due to the complex viable probiotic strains association producing antimicrobial substances.     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Interference of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains in the In Vitro Conjugative Transfer of R-Plasmids

Probiotic compounds, which are often constituted of lactobacilli, exert a number of health benefits through maintenance of the intestinal ecosystem balance. Among the important interactions that occur in the gut microbiota, plasmid transfer by mating is an increasing cause of concern, particularly when antibiotic-resistant genes are involved. Because lactobacilli seem to be able to influence this mechanism, the aim of the present work was to investigate the in vitro capability of two Lactobacillus plantarum strains (one bacteriocin producer and one nonproducer) to interfere with the conjugation processes. For this purpose different matings were performed adding to the donor and recipient cells L. plantarum 35d bac+ and L. plantarum 396/1 bac– as agents of interference. Conjugations added with a Staphylococcus aureus strain or without any agent of interference were used as controls. The results of our experiments demonstrated that both lactobacillus strains were able to decrease mating frequency. Statistically significant differences in the viable transconjugants were obtained in the presence and in the absence of the lactobacilli. The effect was almost the same with the two L. plantarum independent of bacteriocin production. In the trial performed with S. aureus, no decrease in mating frequency was observed, confirming that the capability to interfere with R-plasmid transfer ability could be a property of the tested L. plantarum strains.     

Effect of culture media onLactobacillus hydrophobicity and electrophoretic mobility

The hydrophobicity of six strains representing three species ofLactobacillus was measured using dextran-polyethylene glycol contact angle measurements. These ranged from 123.6° forLactobacillus casei douche to 26.2° forL. casei RC-17 under identical growth conditions. The results indicated that the nutritional environment affected bacterial hydrophobicity. Electrophoretic mobilities of the lactobacilli were also determined and found to be negative for all specimens, and to vary with growth media, especially when sugars were added to urine. The electrophoretic mobility histograms showed one main peak for all strains, exceptLactobacillus acidophilus T-13 which had two peaks, suggesting two morphological sizes or types within its population. In addition, strain T-13 was more positively charged than the other five strains after growth in agar, urine, and supplemented urine. The use of contact angle and electrophoretic mobility techniques allows examination of cell surface properties of lactobacilli that may have importance in the colonization of mucosal epithelia.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein

Background  

Lactic acid bacteria of the genus Lactobacillus and Bifidobacterium are one of the most important health promoting groups of the human intestinal microbiota. Their protective role within the gut consists in out competing invading pathogens for ecological niches and metabolic substrates. Among the features necessary to provide health benefits, commensal microorganisms must have the ability to adhere to human intestinal cells and consequently to colonize the gut. Studies on mechanisms mediating adhesion of lactobacilli to human intestinal cells showed that factors involved in the interaction vary mostly among different species and strains, mainly regarding interaction between bacterial adhesins and extracellular matrix or mucus proteins. We have investigated the adhesive properties of Lactobacillus plantarum, a member of the human microbiota of healthy individuals.     

Characterization of the beneficial properties of lactobacilli isolated from bullfrog (<Emphasis Type="Italic">Rana catesbeiana</Emphasis>) hatchery

The present work addresses the isolation and partial identification of the microbial population of a R. catesbeiana hatchery in spring and summer as well as some beneficial properties of Lactobacillus strains isolated in different seasons and hatchery areas. The bacterial population was grouped into the following taxa: Lactobacillus spp., Pediococcus spp., Enterococcus faecalis and Ent. faecium, and Enterobacteriaceae (Enterobacter spp., Escherichia coli) while Pseudomonas aeruginosa and Staphylococcus epidermidis were isolated from frogs displaying red-leg syndrome. The Lactobacillus plantarum and L. curvatus strains isolated showed to inhibit the growth of red-leg syndrome associated pathogens and food-borne bacteria by organic acids. While L. plantarum CRL 1606 also inhibited red-leg syndrome related pathogens by hydrogen peroxide, meat spoilage bacteria were only inhibited by acidity. However, by using a MRS medium added with tetramethyl-benzidine and peroxidase, a high percentage of H₂O₂-producing lactobacilli were detected. The surface properties of Lactobacillus strains showed that a few strains were able to agglutinate ABO human erythrocytes, while the highest number of strains had a low to medium degree of hydrophobicity. This paper constitute the first study related to the beneficial properties of Lactobacillus isolated from a bullfrog hatchery, as well as the selection criteria applied to a group of strains, which could help to control or prevent bacterial infectious diseases in raniculture.     

Screening for lactobacilli with probiotic properties in the infant gut microbiota

Lactobacilli are believed to be beneficial for the human hosts and are currently being evaluated as potentially probiotic bacteria. In this study, Lactobacillus strains were isolated from infant faeces and were examined in vitro for potential probiotic properties. Faecal specimens from 63 healthy, full-term infants were collected at 4, 30 and 90 days after delivery. Seventy-four Lactobacillus strains were isolated and one or more different phenotypes from each infant (n = 44) were selected for further testing. The bacterial isolates were identified mainly as L. gasseri, L. crispatus, Lactobacillus paracasei, L. salivarius, L. fermentum after amplification and sequencing of 16s rRNA gene. The strains were examined for acid and bile tolerance, adhesion to Caco-2 cells, antibiotic susceptibility and antimicrobial activity against selected enteric pathogens. The great majority of the isolated lactobacilli were susceptible to ampicillin, amoxicillin/clavulanic acid, tetracycline, erythromycin, cephalothin, chloramphenicol and rifampicin. Resistance to vancomycin or bacitracin was detected to 34% of the strains. Twenty strains out of forty-four exhibited significant tolerance to bile salts. Those strains were subsequently tested for resistance to low pH conditions (pH 2 and 3). Interestingly, 85% (17 strains) of the tested lactobacilli remained unaffected at pH 3 after 3 h of incubation, 6 strains were found resistant at pH 2 after 1.5 h and only 2 strains found resistant after 3 h of incubation. Two of the strains were able to adhere to Caco-2 cells. In conclusion, two isolates fulfilled the in vitro probiotic criteria and are good candidates for further in vivo evaluation.