Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.     

Optimization of culture conditions of probiotic bifidobacteria for maximal adhesion to hexadecane

Culture growth conditions were optimized for adhesion to hexadecane of the probiotic Bifidobacterium bifidum HI 39 and HI 48. Among three growth media used, MILS lactose broth was the best medium to obtain maximum cell adhesion, followed by MRS and TPY lactose broth for B. bifidum HI 39 and HI 48. Increasing the incubation time from 6 to 18 h resulted in a gradual increase in percentage adhesion at 37 °C of both organisms in MILS, MRS and TPY media. Thereafter, incubation up to 48 h showed a marked reduction in adhesion of B. bifidum HI 39 and B. bifidum HI 48. When the test cultures were grown at pH values from 5.0 to 8.0 in MILS lactose broth at 37 °C for 18 h, there was a gradual enhancement in cell adhesion up to pH 7.0; but higher pH values retarded the bacterial adhesion. The study showed that the optimum conditions for adhesion to hexadecane of the selected bifidobacterial strains were pH 7.0 and incubation at 37 °C for 18 h in MILS broth.     

Influence of probiotic vaginal lactobacilli on in vitro adhesion of urogenital pathogens to vaginal epithelial cells

AIMS: Lactobacilli, the predominant micro-organisms of the vaginal microbiota, play a major role in the maintenance of a healthy urogenital tract by preventing the colonization of pathogenic bacteria. The aim of the present study was to assess the ability of four vaginal Lactobacillus strains, previously selected for their probiotic features, to block in vitro the adherence of three human urogenital pathogens to vaginal epithelial cells (VEC). METHODS AND RESULTS: Three types of assays were performed in order to determine the inhibitory effect of lactobacilli on adhesion of urogenital pathogens to VEC: blockage by exclusion (lactobacilli and VEC followed by pathogens), competition (lactobacilli, VEC and pathogens together) and displacement (pathogens and VEC followed by the addition of lactobacilli). Bacterial adhesion to VEC was quantified by microscopy (x1000) after Gram's stain. All the strains were able to inhibit by exclusion and competition the adhesion of Staphylococcus aureus to VEC but none was able to decrease the attachment of Escherichia coli by neither of the mechanisms assayed. Only Lactobacillus acidophillus CRL 1259 and Lactobacillus paracasei CRL 1289 inhibited the attachment of Group B streptococci (GBS) to VEC by exclusion and competition respectively. CONCLUSIONS: Lactobacillus of vaginal origin were able to inhibit the attachment of genitouropathogenic Staph. aureus and GBS to the vaginal epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support the probiotic potential of these Lactobacillus strains as anti-infective agents in the vagina and encourage further studies about their capacity to prevent and manage urogenital tract infections in females.     

Effect of lactobacilli onE. coli adhesion to caco-2 cellsin Vitro

Inhibitory effect of various lactobacilli against pathogenic strains of E. coli in model system Caco2 cells was determined by enumerating the number of adhering E. coli after pre-incubation (exclusion), post-incubation (displacement) or co-incubation (competition) with lactobacilli. Porcine E. coli strain F107 (F18ab, Stx2v) in the competition assay with porcine lactobacillus strain P10 gave bacterial counts 7.25 (log CFU per well); in the exclusion test it was only 7.05 while in displacement test it reached 7.29. The lowest E. coli counts adhering to Caco-2 cells were in exclusion assay (pre-incubation, Lactobacillus inoculated as the first). Pre-treatment of E. coli with our lactobacilli strains reduced the cultivable E. coli numbers.     

Correlation between hydrophobicity and resistance to nonoxynol-9 and vancomycin for urogenital isolates of lactobacilli

Seven clinical isolates of lactobacilli were found to be relatively hydrophobic with a mean water-contact angle of 66 +/- 15 degrees, and to be susceptible to 1% nonoxynol-9 and vancomycin. However, seven other strains were relatively hydrophilic with a mean water-contact angle of 32 +/- 13 degrees, and found to be resistant to 25% nonoxynol-9 and vancomycin. Thus, the surface properties of lactobacilli that influence susceptibility to antimicrobial agents may involve surface hydrophobicity. Possibly the penetration barrier posed by the cell surface towards these two nonionic antimicrobials is lower for hydrophobic cells than for hydrophilic cells.     

The effect of orally administered viable probiotic and dairy lactobacilli on mouse lymphocyte proliferation

Four common Lactobacillus strains were screened for their effects on proliferation of mouse splenic lymphocytes. Mice received perorally 10(9) viable bacteria kg(-1) body weight for 7 days. Lactobacillus acidophilus treatment enhanced ex vivo basal proliferation (by 43%) and B-cell response at suboptimal and optimal concentrations of lipopolysaccharide (LPS) (by 27-28%). Conversely, Lactobacillus casei, Lactobacillus gasseri and Lactobacillus rhamnosus inhibited both basal proliferation (by 14-51%) and mitogen-stimulated lymphoproliferation, particularly at supra-optimal concentrations of concanavalin A (by 43-68%) and LPS (by 23-62%). Therefore, these Lactobacillus strains demonstrate strain-specific effects on B- and T-cells and may also alter the splenocyte sensitivity to the cytotoxic effects of mitogens.     

Antitumor effect of lactobacilli substances: "L. bulgaricus effect"

Scattered reports suggest a moderate effect, difficult to reproduce, of products from strains of specific lactobacilli (L. bulgaricus) used for yogurt on cancer and leukemia in mice. Skeptics have disregarded that even lyophilized preparations of demonstrated activity will lose effect when stored above -80 degrees C. This explains some inconsistencies of results and difficulties in repetition. Flow DNA fluorometry of ascitic fluid from leukemic mice has shown some accumulation of cells in the G2 + N phase after 8 hours, clearly different from the effect of 5-fluorouracil, although occasionally similar in strength. The effect depends on the strain of lactobacilli, and seems to deserve rational analysis by modern methods.     

Cell surface energy,contact angles and phase partition III. Adhesion of bacterial cells to hydrophobic surfaces

The densities of adhesion of Staphylococcus epidermis, Staphylococcus aureus and Serratia marcescens to five types of plastics were studied in relation to interfacial free energies at the aqueous interfaces of both the bacteria and the plastics. The free energy of adhesion of bacteria to plastic in an aqueous medium is a linear function of partition of the bacteria between the solid surface and the liquid phase. These results show that the thermodynamics of the partitioning of a suspended particle between two immiscible liquid phases also apply to partitioning between a liquid and a solid phase.     

Nature of the determinant responsible for the adhesion of lactobacilli to chicken crop epithelial cells.

Using an in vitro method, some factors affecting the attachment of a strain of lactobacillus to chicken crop epithelial cells have been studied. Time of contact beyond 10 min, pH value, age or growth temperature of the bacterial culture, or nature of the energy source in the growth medium had little or no effect on attachment. Heating to 100 degrees C for 10 min, or treatment with EDTA or surface active compounds was also without effect. Treatment with sodium periodate markedly decreased adhesion, proteolytic enzymes had a smaller effect but wheat germ lipase was completely inactive. The pronounced inhibition of adhesion by periodate suggested the invovement of carbohydrate. However, enzymes known to attack carbohydrate substrates were inactive in reducing adhesion. Concanavalin A, which binds specifically to certain sugar residues, reduced attachment. It is suggested that these concanavalin A receptors on the lactobacillus are responsible for its attachment to crop epithelial cells.     

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.     

Comparison of culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry

Background

Performances of different salmonella detection methods were evaluated by applying them to of artificially contaminated faecal specimens from cattle, pigs and poultry. The NMKL71 method, being the standard reference method for detection of salmonella in the official Swedish control program, was compared with the proposed ISO method using MSRV-selective enrichment for culturing, and also with three commercial ELISA- based systems, Bioline Selecta, Bioline Optima and Vidas, a commercial PCR-based method, BAX^® system, and three different strategies using PCR detection using a non-commercial PCR system.

Results

Altogether, 391 samples were tested, and the overall results clearly indicate that, when faeces from all animal species and all serotypes were included, the MSRV performed best, with a calculated accuracy of 99% and a calculated sensitivity of 98%. The second most sensitive and specific method was the BAX^® system, using the modified enrichment protocol as recommended by the manufacturer for faecal samples. However, this protocol includes one additional day of work, as compared with the standard procedure for food sample analysis by the same method. The different strategies for salmonella detection using non-commercial PCR showed a sensitivity and specificity in the same range as the BAX^® method; furthermore, results were obtained more quickly. The various commercial ELISA methods and the NMKL method showed the poorest performance of the methods included in the study, and were closely dependent on the origin of the faeces used and on which salmonella strain was to be detected.

Conclusion

The study showed that the sensitivity of the different methods depended to a great extent on the origin of the faecal matrices and the salmonella strains used to "spike" the samples.     

Applications from bacterial adhesion and biofilm studies in relation to urogenital tissues and biomaterials: A review

Summary The urogenital tract, particularly of the adult female, is the habitat for many species of microorganisms. These populations are in a state of flux, are susceptible to disruption by antibiotics and spermicides, and are exposed to many different biomaterial substrata. Infections of the genital area and bladder are common, and are invariably initiated by microbial adhesion to surfaces. This review examines the actual and potential applications to industry and to patients emerging from the study of bacterial adhesion to surfaces.     

Cell surface energy,contact angles and phase partition. II. Bacterial cells in biphasic aqueous mixtures

Partition coefficients in biphasic mixtures of poly(ethylene glycol) and Dextran are compared to cell surface energies obtained from contact angles of each liquid phase on cell layers. Linear relationships are observed between these two independent measurements for a variety of bacterial cells. The results demonstrate the importance of interfacial phenomena and contact angles in the phase-partition process.     

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.     

Growth phase, cellular hydrophobicity, and adhesion in vitro of lactobacilli colonizing the keratinizing gastric epithelium in the mouse.

Lactobacillus strains of numerous species isolated from several animal sources exhibited cellular hydrophobicities that differed from those expected on the basis of their abilities to colonize the keratinizing stratified squamous epithelium in the mouse stomach. Cells of Lactobacillus fermentum 100-33, grown to either exponential or stationary phase, were strongly hydrophilic. By contrast, cells of L. fermentum RI and six transformant derivatives of strain RI and 100-33, strains DM101 through DM106, were hydrophobic to various degrees in either growth phase. Most of them were less hydrophobic, however, when in the stationary phase than in the exponential phase. Cells of strains RI and 100-33 in the exponential phase adhered in the same number in vitro to disks of keratinized mouse gastric mucosa. By contrast, when in stationary phase, strain RI and two transformants, DM103 and DM104, adhered to the surface in higher numbers than 100-33. In contrast to their cellular progenitor, 100-33, the transformant strains share with their DNA donor, RI, the capacity to colonize the keratinizing gastric epithelium in mice. These findings indicate that lactobacilli able to colonize the surface of the keratinocytes in the murine stomach can adhere to that surface by either hydrophilic or hydrophobic molecules.