María Elena Nader-Macías

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).

Selection of Vaginal H2O2-Generating Lactobacillus Species for Probiotic Use

Abstract. Lactobacilli are believed to contribute to the control of the vaginal microflora by different mechanisms such as production of antagonistic substances like lactic acid, bacteriocins, and H2O2. This paper describes the selection of H2O2-generating lactobacilli among 35 hydrophobic isolates from the human vagina. Lactobacillus crispatus F117, which generated the highest H2O2 level, was chosen to study: (a) the kinetics of H2O2 production considering different culture conditions, and (b) the effect of this metabolite on the growth of urogenital tract pathogens. The levels of H2O2 in L. crispatus supernatant increased during its growth and were maximum at the early stationary phase (3.29 mmol H2O2L−1) under aerated conditions (agitated cultures). In nonagitated cultures there were no detectable levels of H2O2. L. crispatus F117 spent supernatant inhibited Staphylococcus aureus growth in plaque assay. Inhibition was due to H2O2 since catalase treatment of the supernatant suppressed inhibition. In mixed cultures performed with L. crispatus and S. aureus a significant decrease in pathogen growth was observed. The inhibitory effect depended on the initial inoculum of S. aureus. Further evaluation of the properties of L. crispatus F117 will be performed to consider its inclusion in a probiotic for local use in the vaginal tract.

Vaginal lactobacilli: self- and co-aggregating ability.

Abstract 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 anda 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.

Probiotic properties of vaginal lactic acid bacteria to prevent metritis in cattle

Aims:  The isolation of bovine vaginal lactic acid bacteria (LAB) and the screening of their beneficial properties to select those that could be used as probiotics in the prevention of bovine metritis were performed.

Screening of surface properties and antagonistic substances production by lactic acid bacteria isolated from the mammary gland of healthy and mastitic cows.

Bovine mastitis (BM) is a costly disease in dairy cattle production. The prevention and treatment of mastitis is performed by applying antimicrobial products that negatively affect milk quality. In the last years, the use of probiotic microorganisms to prevent infections in humans and animals has being aggressively studied. Samples from teat canal and milk (foremilk and stripping) were taken from healthy and mastitic mammary quarters. A screening of the surface properties and antagonistic substances production of lactic acid bacteria (LAB) isolated from the mammary gland was performed to select potential probiotic strains to prevent mastitis. Somatic cell count, physico-chemical and microbiological studies were carried out. Pre-selected microorganisms were genetically identified. Compared with stripping milk, foremilk showed lower levels of fat and higher levels of pH, density, microorganism numbers, lower percentage of strains with mean and high hydrophobicity and mean autoaggregation and higher number of strains able to produce hydrogen peroxide and bacteriocins. The other parameters analyzed were not statistically significant. One hundred and two LAB strains were isolated. Most of them had low degrees of hydrophobicity and autoaggregation. No correlation between these properties was found. Antagonistic metabolites were mainly produced by strains isolated from healthy quarters. Most of the pre-selected strains were identified as Streptococcus bovis and Weissella paramesenteroides. Three bacteriocin-producers were found and their products partially characterized. The results of this work are the basis for the further design of a specie-specific probiotic product able to prevent BM.

Lactic acid bacteria isolated from young calves – Characterization and potential as probiotics

Lactic acid bacteria (LAB) are widely used as probiotics in humans and animals to restore the ecological balance of different mucosa. They help in the physiological functions of newborn calves that are susceptible to a variety of syndromes. The criteria for the selection of strains for the design of probiotic products are not available. Based in the host-specificity of the indigenous microbiota, 96 LAB isolates from faeces and oral cavity of calves were obtained. The surface properties were screened showing a small number of highly hydrophobic or autoagglutinating isolates. Also, a group produced H(2)O(2) and were able to inhibit pathogens, and two strains were bacteriocin-producers. Some grew at very low pH and high bile concentrations. The strains sharing some of the specific properties evaluated were identified genetically, assayed their compatibility and exopolysaccharide production. The results allow going further in the establishment of criteria to select strains to be included in a multi-strain-probiotic-product to be further assayed in animals.

Production of Antimicrobial Substances by Lactic Acid Bacteria I

Restoration of the balance of different ecological niches has been proposed as a way to control the income of pathogenic microorganisms. The genus Lactobacillus has been used in different human and animal tracts as probiotic microorganisms with this objective in mind. The characteristics of the strains proposed as probiotics have been published or patented under the process of elaboration of different types of products. One of the mechanisms suggested to control the vaginal ecosystem is the production of antagonistic substances (lactic acid, bacteriocins, or H2O2). The H2O2-producing microorganisms present in the vagina of healthy women have been suggested as some of the bacteria responsible for maintenance of ecological balance, mainly in pregnant women. The absence of these microorganisms is related to a higher risk of: bacterial vaginosis, recurrent urinary tract infections by Escherichia coli, and acquisition of human immunodeficiency virus type 1 (HIV-1). Bauer has proposed that H2O2-producing lactobacilli also might exert control over vaginal cancer through specific interactions of reactive oxygen species, such as superoxide anion, hydroxyl radicals, and hypochlorous acid. The conversion of H2O2 into more toxic compounds during the oxidative process is potentiated by peroxidase and halures. This enzyme and some halures, such as chloride and bromide, are present in vaginal washes in sufficient amounts to allow an optimal environment for successful inhibition of pathogens. In vitro tests provide an approach for determining the ability of lactobacilli to produce H2O2. The H2O2 amounts produced in such systems are probably not a direct reflection of what happens in the vaginal tract of women or animals, which is not yet know. However, there is a registered patent with an H2O2-generating L. crispatus strain, also supporting the use of H2O2-producing lactobacilli to restore the vaginal ecosystem.Bacteriocins have been defined as proteinaceous, bactericidal substances synthesized by bacteria, which usually have a narrow spectrum of activity, only inhibiting strains of the same or closely related species. The term bacteriocin-like substance is applied to antagonistic substances that are not completely defined or do not fit the typical criteria of bacteriocins. They have been reported to inhibit a wide range of both Gram-positive and Gram-negative bacteria as well as fungi. Lactobacillus species are the dominant microorganisms isolated from the vagina of healthy premenopausal women. In this environment, they exert a protective effect against pathogenic microorganisms by different mechanisms such as production of antimicrobial agents, which include organic acids, hydrogen peroxide, and probably bacteriocins. The production of bacteriocins by vaginal lactobacilli has been demonstrated in vitro; however, it is not yet well established whether they are produced in vivo as another antagonistic mechanism exerted by the normal microflora.Bacteriocin-producing bacteria as well as bacteriocins per se are of growing interest as biological controls in the manufacture of beverages and fermented products, mainly in the area of dairy products. These bacteria have also been proposed as probiotic candidates for human or animal use. The objectives of the present chapter are to describe the methods employed for: 1. Detection of production of bacteriocins among vaginal Lactobacillus strains. 2. Characterization of the bacteriocin or bacteriocin-like substances. 3. Study of the kinetics of production and mode of action of bacteriocins. 4. Determination of the inhibition of pathogenic microorganisms by bacteriocin-producing strains in mixed cultures.

Vaginal bacterial microflora modifications during the growth of healthy cows.

The aim of this work was first, to determine the predominant groups capable of colonizing the vagina and maintaining high numbers with time. The normal microbial flora of the cows vagina and its evolution from weaning to service was then studied using standard microbiological methods. The results show that the most dominant bacteria belong to the streptococci, followed by the staphylococci, with similar levels during the whole study period. Enterobacteriaceae and lactobacilli were present at very low levels, the latter increasing during the cows growth, suggesting some kind of hormonal influence. The results will allow the selection of micro‐organisms with probiotic characteristics, classified as GRAS (Generally Regarded as Safe), to be used in the prevention of infections in the vaginal tract of cows, such as metritis, which produces delayed periods between partum and conception, and consequent economic losses.

Antibiotic Susceptibility of Potentially Probiotic Vaginal Lactobacilli

Objective. To study the antimicrobial susceptibility of six vaginal probiotic lactobacilli. Methods. The disc diffusion method in Müeller Hinton, LAPTg and MRS agars by the NCCLS (National Committee for Clinical Laboratory Standards) procedure was performed. Due to the absence of a Lactobacillus reference strains, the results were compared to those of Staphylococcus aureus ATCC29213. Minimal Inhibitory Concentration (MIC) with 21 different antibiotics in LAPTg agar and broth was also determined. Results. LAPTg and MRS agars are suitable media to study antimicrobial susceptibility of lactobacilli. However, the NCCLS procedure needs to be standardized for this genus. The MICs have shown that all Lactobacillus strains grew at concentrations above 10 μg/mL of chloramphenicol, aztreonam, norfloxacin, ciprofloxacin, ceftazidime, ceftriaxone, streptomycin and kanamycin. Four lactobacilli were sensitive to 1 μg/mL vancomycin and all of them were resistant to 1000 μg/mL of metronidazole. Sensitivity to other antibiotics depended on each particular strain. Conclusions. The NCCLS method needs to be standardized in an appropriate medium to determine the antimicrobial susceptibility of Lactobacillus. Vaginal probiotic lactobacilli do not display uniform susceptibility to antibiotics. Resistance to high concentrations of metronidazole suggests that lactobacilli could be simultaneously used with a bacterial vaginosis treatment to restore the vaginal normal flora.

Characterization of salivaricin CRL 1328, a two-peptide bacteriocin produced by Lactobacillus salivarius CRL 1328 isolated from the human vagina

Salivaricin CRL 1328 is a heat-stable bacteriocin produced by Lactobacillus salivarius CRL 1328, a strain isolated from healthy human vagina, with potential applications for preventing urogenital infections. The objective of this study was to characterize the locus responsible for salivaricin CRL 1328 production and its mechanism of action against Enterococcus faecalis MP97 as the sensitive strain. Oligonucleotides were designed based on sequences of antimicrobial peptides previously described in the literature. The salivaricin CRL 1328 cluster was identified, sequenced and analyzed. This cluster was similar to the previously described ABP118 which codified for a two-peptide bacteriocin. The putative mature peptides of salivaricin CRL 1328, Salalpha and Salbeta were chemically synthesized. These peptides did not show bacteriocin activity when assayed individually. Both peptides exhibited optimal antimicrobial activity at an equimolar ratio. Spectroscopic fluorescence assays were carried out using the synthetic peptides to study the effect of salivaricin on proton motive force. This bacteriocin was shown to dissipate membrane potential and the transmembrane proton gradient, both components of proton motive force. E. faecalis MP97 cells treated with salivaricin CRL 1328 peptides were observed in transmission electron microscopy which revealed ultrastructural modifications of the cell wall.