Liliana Pascual

Vaginal colonization and activity of the probiotic bacterium Lactobacillus fermentum L23 in a murine model of vaginal tract infection.

A strain of Lactobacillus, identified as Lactobacillus fermentum L23, was selected from among 100 strains isolated from vaginal swabs of healthy, non-pregnant, pre-menopausal women. L. fermentum L23 was chosen on the basis of its bacteriocinogenic ability and its properties relevant to colonization, i.e. self-aggregation, adherence to vaginal epithelial cells and co-aggregation with bacterial pathogens. The antimicrobial preventative and curative effects produced by the probiotic L. fermentum L23 administered locally against Escherichia coli in a murine vaginal tract infection model were studied. One dose of the human strain L23 containing 10(8) c.f.u. ml(-1) colonized and persisted in the vaginal tract of the female BALB/c mice for 5 days. Infection with the pathogen at 10(6) c.f.u. ml(-1) in the vaginal tract was maintained for more than 7 days. A single dose of L23 administered 24 h pre-infection inhibited E. coli growth on day 3 post-infection, showing the preventative effect displayed by this Lactobacillus strain. Treatment with L. fermentum L23 during the post-infection period showed complete inhibition of pathogen growth from day 5. Thus, this in vivo study indicated that the probiotic bacterium L. fermentum L23 produced both preventative and curative effects on E. coli growth. The beneficial properties and the production of antimicrobial metabolites may act in situ to inhibit a pathogenic micro-organism within the vaginal environment. Strain L23 could be a good natural alternative to other therapies used for genital infections.

Antifungal activity of two Lactobacillus strains with potential probiotic properties

Aflatoxin (highly toxic and carcinogenic secondary metabolites produced by fungi) contamination is a serious problem worldwide. Modern agriculture and animal production systems need to use high-quality and mycotoxin-free feedstuffs. The use of microorganisms to preserve food has gained importance in recent years due to the demand for reduced use of chemical preservatives by consumers. Lactic acid bacteria are known to produce various antimicrobial compounds that are considered to be important in the biopreservation of food and feed. Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23 are producers of secondary metabolites, such as organic acids, bacteriocins and, in the case of L60, hydrogen peroxide. The antifungal activity of lactobacilli strains was determined by coculture with Aspergillus section Flavi strains by two qualitative and one quantitative methods. Both L23 and L60 completely inhibited the fungal growth of all aflatoxicogenic strains assayed. Aflatoxin B (1) production was reduced 95.7-99.8% with L60 and 27.5-100% with L23. Statistical analysis of the data revealed the influence of L60 and L23 on growth parameters and aflatoxin B (1) production. These results are important given that these aflatoxicogenic fungi are natural contaminants of feed used for animal production, and could be effectively controlled by Lactobacillus L60 and L23 strains with probiotic properties.

Antimicrobial Activity, Inhibition of Urogenital Pathogens, and Synergistic Interactions Between Lactobacillus Strains

Lactobacillus fermentum strain L23 and L. rhamnosus strain L60 were selected as an alternative treatment to prevent or treat urogenital infections based on their probiotic properties and production of bacteriocins. The objectives of the present work were to study the inhibitory activities of these two bacteriocin-producing strains, and to analyze the interactions between pairs of bacteriocins that inhibit urogenital pathogens. Antimicrobial activity tests of L23 and L60 were performed by a diffusion method with 207 bacterial strains, isolated from female patients presenting a urogenital infection. Inhibitory substances interaction tests were carried out by using a streak-diffusion method on agar plates. One hundred percent of the clinical isolates showed sensitivity to the antimicrobial substances produced by L23 and L60. The selected lactobacilli produced larger inhibition halos when compared to several antibiotics commonly used for treating these infections. Synergistic interactions and indifferent interactions were recorded in 68.6% and 31.4% of the cases, respectively. No antagonistic interactions were observed. In conclusion, the bacteriocin-producing strains L23 and L60 are potential candidates for probiotic prophylaxis and treatment of urogenital disorders in women.

Bacteriocins and other bioactive substances of probiotic lactobacilli as biological weapons against Neisseria gonorrhoeae

In the search of new antimicrobial agents against Neisseria gonorrhoeae, the bacteriocins-producing probiotic lactobacilli deserve special attention. The inhibitory effects of biosubstances such as organic acids, hydrogen peroxide and each bacteriocin-like inhibitory substance (BLIS) L23 and L60 on the growth of different gonococcal strains were investigated. Different non-treated and treated cell-free supernatants of two probiotic lactobacilli containing these metabolites were used. The aims of this work were (i) to evaluate the antimicrobial activity of the biosubstances produced by two probiotic lactobacilli, estimating the proportion in which each of them is responsible for the inhibitory effect, (ii) to define their minimum inhibitory concentrations (MICs) and, (iii) to determine the potential interactions between these biosubstances against N. gonorrhoeae. The main antimicrobial metabolites were the BLIS-es L23 and L60 in comparison with other biosubstances. Proportionally, their contributions to the inhibition on the gonococcal growth were 87.28% and 80.66%, respectively. The MIC values of bacteriocins were promising since these substances, when diluted, showed considerable inhibitory activity for all gonococci. In the interaction between bacteriocins, 100% of synergism was found on the gonococcal growth. In summary, this study indicates that both L23 and L60 could potentially serve to design new bioproducts against N. gonorrhoeae.

Surveillance of Aflatoxin and Microbiota Related to Brewer's Grain Destined for Swine Feed in Argentina

Córdoba province in the center of Argentina is an important area of swine production. The use of industry by-product (brewers grain) as feedstuff for swine is a regular practice and increases animal performance on these animals production. The occurrence of aflatoxin contamination is global, causing severe problems especially in developing countries. No reports on aflatoxin B1 production, micoflora, and potential aflatoxin B1 producing microorganism from brewers grain are available. The aims of this study were (1) to isolate the microbiota species from brewers grain, (2) to determine aflatoxin B1 natural contamination levels, and (3) to determine the ability of Aspergillus section Flavi isolates to produce aflatoxins in vitro. Physical properties, total fungal counts, lactic acid bacteria, and fungal genera distribution were determined on this substrate. In 65% of the samples, fungal counts were higher than recommended by GMP, and lactic bacterium counts ranged from 1.9 × 105 to 4.4 × 109 CFU g−1. Aspergillus spp. prevailed over other fungal genera. Aspergillus flavus was the prevalent species followed by A. fumigatus. Aflatoxin B1 levels in the samples were higher than the recommended limits (20 ng g−1) for complementary feedstuffs. Several Aspergillus section Flavi strains were able to produce aflatoxin B1  in vitro. Inadequate storage conditions promote the proliferation of mycotoxin-producing fungal species. Regular monitoring of feeds is required in order to prevent chronic and acute toxic syndromes related to this kind of contamination.

Biological control of AFB1-producing Aspergillus section Flavi strains isolated from brewer's grains, alternative feed intended for swine production in Argentina

ABSTRACT The aim of the present study was to investigate the inhibitory activity of lactic acid bacteria (LAB) isolated from brewers grains on Aspergillus section Flavi growth and aflatoxin B1 production. The Aspergillus strains tested were inhibited by all the LAB strains assayed. The isolates Lactobacillus brevis B20, P. pentosaceus B86, Lactococcus lactis subsp. lactis B87, L. brevis B131, and Lactobacillus sp. B144 completely suppressed the fungal growth and reduced aflatoxin B1 production. In conclusion, LAB isolated from brewers grains show a high inhibitory activity on fungal growth and aflatoxin biosynthesis by Aspergillus flavus and Aspergillus parasiticus. Further studies must be conducted to evaluate the success of in vitro assays under food environment conditions and to elucidate the antifungal mechanism of these strains.

Prevention Strategy of Urogenital Infections by Using Lactobacilli with Probiotic Properties

Lactic acid bacteria (LAB) constitute a group of Gram-positive nonsporing nonrespiring bacteria, cocci or rods, that produce lactic acid as the major end product during the fermentation of carbohydrates. The term LAB is associated with bacteria involved in food fermentation and bacteria normally associated with the mucosal surfaces of human and animals. The classification of lactic acid bacteria into different genera is based on morphology, mode of glucose fermentation, growth at different temperatures, configuration of the lactic acid produced, ability to grow at high salt concentrations, and acid or alkaline tolerance (Pascual, 2004). Bacteria belonging to the genus Lactobacillus are considered to be the main LAB and the predominant microorganisms in the gastrointestinal and urogenital tracts of humans as well as homeotermic animals. They are also used for elaborating different fermented foods categorized as GRAS (generally considered as safe). Although there are data on simultaneous colonization of the human vagina by two different species of Lactobacillus, which can be homofermentative, heterofermentative or a combination of both (Kaewsrichan et al., 2006; Pascual et al, 2006), only one species has been isolated from the vaginal tract. Also, there are evidences of their effectivity in the prevention of urogenital infections (Pascual, 2004; Axelsson, 2004). The urogenital microbiota of a healthy woman comprises approximately 50 species of organisms, witch differ in composition according to reproductive stages and exposure to several factors, including antibiotics and spermicides (Pascual, 2004). In the complex vaginal environment, bacteria of the lactobacilli group (107–108 CFU g-1 of vaginal fluid) are the dominant microorganisms in healthy pre-menopausal women, and play an important protective role by limiting growth of pathogenic microorganisms. When lactobacilli are reduced, eliminated, or replaced by pathogenic species, the host has an increased susceptibility to urinary tract infections (UTIs), genital tract infections (GTIs), bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and infection by Neisseria gonorrhoeae or Trichomonas vaginalis. Worldwide studies of UTIs or GTIs have revealed increasing antibiotic resistance among pathogenic microorganisms. Our research group has isolated human vaginal lactobacilli, selecting those with beneficial or probiotic properties (Czaja et al., 2007).