Virginia S. Ocaña

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.

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.

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.

Bacterial Surface Characteristics Applied to Selection of Probiotic Microorganisms

For the study of probiotic microorganisms, the in vitro selection tests need to be based on a solid scientific foundation. Surface characteristics, one of the in vitro properties are used to evaluate the potentially probiotic strains of lactobacilli. Bacterial surface properties have been associated with attachment to a variety of substrata. Bacterial adhesion to tissues is considered the first step, and such adhesion can also determine the colonization capability of a microorganism. Through adhesion ability and colonization of tissues, probiotic microorganisms can prevent pathogen access by steric interactions or specific blockage on cell receptors. One of the main characteristics studied is the hydrophobic nature of the bacterial cell surface. To test this property, Rosenberg and Doyle divided microbial cell hydrophobicity assays into two categories. The first includes contact angle measurements (CAMs), partitioning of cells into one or another liquid phase (TTP), and adsorption of individual hydrophobic molecular probes at the cell surface. The second category includes microbial adhesion to hydrocarbons (MATH), hydrophobic interaction chromatography (HIC), and adhesion to polystyrene and other hydrophobic solid surfaces. The tests included in the first category measure hydrophobic properties of the outer cell surface as a whole; those in the second measure hydrophobicity in terms of adhesion. Finally, those bacterium classified as hydrophobic can be considered as able to mediate adhesion. The objective of this chapter is to describe three different methods applied in our laboratory for the study of bacterial surface properties. They can be used to screen characteristics of lactobacillus strains for probiotic purposes. They are: Microbial adhesion to hydrocarbons (MATH); Salt aggregation test (SAT); Hemagglutination (HA) reaction.

Advances in the Knowledge and Clinical Applications of Lactic Acid Bacteria as Probiotics in the Urogenital Tract

Probiotics, defined as live microorganisms administered in high numbers to produce beneficial and physiological effects on the host, are increasingly applied to the prevention and treatment of many clinical situations in both human beings and animals. The female urogenital tract is one of the targets of probiotics, mainly by the frequency of infectious diseases that affects patients of all ages, including pregnant women and, potentially, newborns. Urogenital infections are associated with high morbidity and mortality rates as well as with high health care costs. The development of a probiotic formula to control these situations is of great interest. In this review, the mechanisms that support the probiotic action of lactic acid bacteria in the urogenital tract are summarized. Animal models used to test their effect on certain pathologies as well as clinical trials performed in humans are described. Some products currently available on the market are also mentioned.

Effect of Lactobacilli Administration in the Vaginal Tract of Mice

Lactobacilli are the predominant microorganisms in the vaginal tract of human and some homeothermic animals. They can maintain the ecological equilibrium of the tract by protecting against pathogenic microorganisms. In the last few years, there has been an increased tendency to use probiotic microorganisms to restore the ecological equilibrium and to protect against infections. This principle has been widely applied to the gastrointestinal tract. More recently, some other studies have reported the application of probiotics in different tracts, for example, the urogenital or respiratory tract. One of the objectives of our group is to design probiotic products for the urogenital tract. With this purpose, lactobacilli were isolated from the human vagina, and later some of them were selected for their probiotic characteristics (production of antagonistic substances or adhesion capability). The application of probiotic products in the vaginal tract has been approached empirically; some pharmaceuticals containing these microorganisms are available in the United States or Europe or are protected under the patent process or intellectual property rights. There are not enough studies in humans or animals to determine whether their administration can produce some type of collateral or adverse effect. Using Balb/c mice as the experimental model, the object of the present work was to study (1) whether intravaginal administration of human lactobacilli can produce colonization of the tract; (2) whether such administration produces some type of adverse or collateral effect; and (3) whether probiotics are able to stimulate the local immune system. Keeping in mind that hormones can affect the colonization or persistence ability of microorganisms, and with the purpose of having all animals at the same point in the sexual cycle, animals were cycled with estradiol 48 h before inoculation with lactobacilli. They were then inoculated im with hormones 48 h before beginning microorganism inoculations. Later they were intravaginally inoculated with the appropriate dose of each Lactobacillus strains. The animals were sacrificed on different days after inoculation to perform the following studies: 1. Microbiological assays: To determine the number of lactobacilli in the tract (in vaginal washes or in organ homogenates), by plating the samples in selective media containing antibiotic (to differentiate the resident flora from those administered experimentally).

Adhesion Ability of Lactobacillus to Vaginal Epithelial Cells

Adhesion of lactobacilli to the epithelium has been described as the first step in the formation of a barrier to prevent undesirable microbial colonization; consequently, it has been defined as a characteristic of interest for selecting probiotic strains. Several methods have been described to predict the adhesion ability of Lactobacillus. Early studies were phenomenological: it was useful to determine whether a particular bacterium could hemagglutinate, or bind to coated particles. Later, studies based on the adhesion of bacteria to epithelial cells in vitro were developed. These last assays were based on the numbers of bacteria attached to epithelial cells, which were determined by counting stained microorganisms under light microscopy or by measuring the radioactivity of previously radiolabeled bacteria. Because the microscope technique, had some disadvantages, a modification was developed in our laboratory. The technique described in this chapter is a modification of the Mardh and Weströn method. The method allows the study of the adhesion ability of bacteria, even if they are aggregating or if they are high-adherent bacteria covering a large area of the epithelial cell surface. Determination of the number of adherent bacteria by counting colonies grown in a selective media avoids the time-consuming, tedious, and hazardous counting under the light microscope and the use of radioactive methods.