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Lactic acid bacteria (LAB) were isolated, identified, and characterized from pig feces at various growth stages and feed rations in order to be used as probiotic feed additives. Lactic acid bacteria numbers ranged from 7.10 ± 1.50 to 9.40 log CFUs/g for growing and lactating pigs, respectively. Isolates ( n = 230) were identified by (GTG)5-polymerase chain reaction and partial sequence analysis of 16S rRNA. Major LAB populations were Limosilactobacillus reuteri (49.2%), Pediococcus pentosaceus (20%), Lactobacillus amylovorus (11.4%), and L. johnsonii (8.7%). In-vitro assays were performed, including surface characterization and tolerance to acid and bile salts. Several lactobacilli exhibited hydrophobic and aggregative characteristics and were able to withstand gastrointestinal tract conditions. In addition, lactobacilli showed starch- and phytate-degrading ability, as well as antagonistic activity against Gram-negative pathogens and the production of bacteriocin-like inhibitory substances. When resistance or susceptibility to antibiotics was evaluated, high phenotypic resistance to ampicillin, gentamicin, kanamycin, streptomycin, and tetracycline and susceptibility towards clindamycin and chloramphenicol was observed in the assayed LAB. Genotypic characterization showed that 5 out of 15 resistance genes were identified in lactobacilli; their presence did not correlate with phenotypic traits. Genes erm(B), strA, strB, and aadE conferring resistance to erythromycin and streptomycin were reported among all lactobacilli, whereas tet(M) gene was harbored by L. reuteri and L. amylovorus strains. Based on these results, 6 probiotic LAB strains ( L. reuteri F207R/G9R/B66R, L. amylovorus G636T/S244T, and L. johnsonii S92R) can be selected to explore their potential as direct feed additives to promote swine health and replace antibiotics., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

The fungal pathogen Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis and has been a key driver in the catastrophic decline of amphibians globally. While many strategies have been proposed to mitigate Bd outbreaks, few have been successful. In recent years, the use of probiotic formulations that protect an amphibian host by killing or inhibiting Bd have shown promise as an effective chytridiomycosis control strategy. The North American bullfrog (Lithobates catesbeianus) is a common carrier of Bd and harbours a diverse skin microbiota that includes lactic acid bacteria (LAB), a microbial group containing species classified as safe and conferring host benefits. We investigated beneficial/probiotic properties: anti-Bd activity, and adhesion and colonisation characteristics (hydrophobicity, biofilm formation and exopolysaccharide-EPS production) in two confirmed LAB (cLAB-Enterococcus gallinarum CRL 1826, Lactococcus garvieae CRL 1828) and 60 presumptive LAB (pLAB) [together named as LABs] isolated from bullfrog skin.We challenged LABs against eight genetically diverse Bd isolates and found that 32% of the LABs inhibited at least one Bd isolate with varying rates of inhibition. Thus, we established a score of sensitivity from highest (BdGPL AVS7) to lowest (BdGPL C2A) for the studied Bd isolates. We further reveal key factors underlying host adhesion and colonisation of LABs. Specifically, 90.3% of LABs exhibited hydrophilic properties that may promote adhesion to the cutaneous mucus, with the remaining isolates (9.7%) being hydrophobic in nature with a surface polarity compatible with colonisation of acidic, basic or both substrate types. We also found that 59.7% of LABs showed EPS synthesis and 66.1% produced biofilm at different levels: 21% weak, 29% moderate, and 16.1% strong. Together all these properties enhance colonisation of the host surface (mucus or epithelial cells) and may confer protective benefits against Bd through competitive exclusion. Correspondence analysis indicated that biofilm synthesis was LABs specific with high aggregating bacteria correlating with strong biofilm producers, and EPS producers being correlated to negative biofilm producing LABs. We performed Random Amplified Polymorphic DNA (RAPD)-PCR analysis and demonstrated a higher degree of genetic diversity among rod-shaped pLAB than cocci. Based on the LAB genetic analysis and specific probiotic selection criteria that involve beneficial properties, we sequenced 16 pLAB which were identified as Pediococcus pentosaceus, Enterococcus thailandicus, Lactobacillus pentosus/L. plantarum, L. brevis, and L. curvatus. Compatibility assays performed with cLAB and the 16 species described above indicate that all tested LAB can be included in a mixed probiotic formula. Based on our analyses, we suggest that E. gallinarum CRL 1826, L. garvieae CRL 1828, and P. pentosaceus 15 and 18B represent optimal probiotic candidates for Bd control and mitigation., Competing Interests: The authors have declared that no competing interests exist.

Diarrhea is one of the most frequent diseases affecting newborn calves in intensive systems. Several strategies were proposed to protect and improve health, such as probiotics. This work was directed to design a product containing freeze-dried bacteria, vitamins, and minerals, as well as to optimize conditions with lyoprotectors, combine strains and add vitamins, minerals, and inulin to the product. The lyoprotectors were milk, milk-whey, and actose, and products were stored for 6 months at 4°C. Combined bacteria were freeze-dried in milk and the final products were added with minerals, vitamins, and insulin. The viable cells were determined by the plate count assay and antibiotic profiles to differentiate strains. Lactobacillus johnsonii CRL1693, L. murinus CRL1695, L. mucosae CRL1696, L. salivarius CRL1702, L. amylovorus CRL1697, and Enterococcus faecium CRL1703 were evaluated. The optimal conditions were different for each strain. Milk and milk whey maintained the viability during the process and storage after 6 months for most of the strains, except for L. johnsonii. Lactose did not improve cell's recovery. L. murinus was viable for 6 months in all the conditions, with similar results in enterococci. In strains combined before freeze-dried, the viability decreased deeply, showing that one-step process with bacteria mixtures, vitamins, and minerals were not adequate. Freeze-dried resistance depends on each strain and must be lyophilized individually.

Adhesion and biofilm formation are strain properties that reportedly contribute to the permanence of lactobacilli in the human vagina. The kinetics of biofilm formation and the chemical nature of the biofilm matrix formed by Lactobacillus reuteri CRL (Centro de Referencia para Lactobacilos Culture Collection) 1324 and Lactobacillus rhamnosus CRL 1332, vaginal beneficial strains, were evaluated in this work. Crystal violet-stained microplate assay and techniques of epifluorescence, electron and confocal microscopy were applied. The highest density and complexity of biofilms of both vaginal lactobacilli were observed at 72 h of incubation. Protease, proteinase K, α-chymotrypsin and trypsin treatments efficiently detached L. reuteri CRL 1324 biofilm that was also partially affected by α-amylase. However, L. rhamnosus CRL 1332 biofilm was slightly affected by protease, proteinase K and α-amylase. Confocal microscopy revealed greater amount of polysaccharides in L. rhamnosus CRL 1332 biofilm matrix than in L. reuteri CRL 1324 biofilm matrix. The results indicate that proteins are one of the main components of the L. reuteri CRL 1324 biofilm, while the biofilm matrix of L. rhamnosus CRL 1332 is composed of carbohydrates and proteins. The results obtained support the knowledge, understanding and characterization of two biofilm-forming vaginal Lactobacillus strains.

Problem: Maternal Group B Streptococcus (GBS) colonization is a risk factor for infectious disease in newborns. One promising strategy is the modulation of vaginal defense to increase the host's ability to combat infection., Method of Study: The effect of intravaginal (i.va.) Lactobacillus reuteri CRL1324 inoculation on different immune cell populations, cytokines, and immunoglobulin isotypes in a murine model of GBS vaginal colonization was evaluated., Results: Seven i.va. inoculations of L. reuteri CRL1324 previous to GBS challenge showed an immunomodulatory effect on the cells and mediators of innate immunity, decreasing the number of neutrophils induced by the pathogen and increasing the activated macrophage population. Moreover, increases in B lymphocytes and IgA and IgG subclasses were observed in mice inoculated with L. reuteri CRL1324 and then challenged with GBS., Conclusion: Lactobacillus reuteri CRL1324 shows a protective effect against GBS colonization that could be mediated by the modulation of the immune response., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Probiotic formulations must include a high number of viable and active microorganisms. In this work, the survival of human vaginal Lactobacillus reuteri CRL 1324 during encapsulation, lyophilization and storage, and the activity of encapsulated and/or freeze-dried bacterial cells were evaluated. Extrusion-ionic gelation technique was applied to encapsulate L. reuteri CRL 1324, using xanthan and gellan. Encapsulated and free bacterial cells were freeze-dried with or without lactose and skim milk as lyoprotectors. The different systems obtained were stored at room temperature and at 4°C for 150days. The following determinations were performed: L. reuteri CRL 1324 viability, microorganism released from capsules, survival in a medium simulating the vaginal fluid and maintenance of beneficial properties (growth inhibition of opportunistic pathogenic Streptococcus agalactiae NH 17 and biofilm formation). L. reuteri CRL 1324 encapsulation was efficient, allowing the recovery of a high number of entrapped lactobacilli. The survival of encapsulated L. reuteri during lyophilization and storage was significantly higher in the presence of lyoprotectors. At the end of storage, the highest numbers of viable cells were obtained in free or encapsulated cells freeze-dried with lyoprotectors, stored at 4°C. Encapsulated and/or lyophilized L. reuteri cells maintained their viability in simulated vaginal fluid as well as the ability to inhibit S. agalactiae NH 17 growth and to form biofilm. Encapsulated and freeze-dried L. reuteri CRL 1324 can be included in a suitable pharmaceutical form for vaginal application to prevent or treat urogenital infections in women., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Probiotics, defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, are considered a valid and novel alternative for the prevention and treatment of female urogenital tract infections. Lactobacilli, the predominant microorganisms of the healthy human vaginal microbiome, can be included as active pharmaceutical ingredients in probiotics products. Several requirements must be considered or criteria fulfilled during the development of a probiotic product or formula for the female urogenital tract. This review deals with the main selection criteria for urogenital probiotic microorganisms: host specificity, potential beneficial properties, functional specifications, technological characteristics and clinical trials used to test their effect on certain physiological and pathological conditions. Further studies are required to complement the current knowledge and support the clinical applications of probiotics in the urogenital tract. This therapy will allow the restoration of the ecological equilibrium of the urogenital tract microbiome as well as the recovery of the sexual and reproductive health of women., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Context: The administration of pharmabiotics is a promising alternative to antimicrobial drugs for the treatment and/or prevention of female urogenital infections., Objective: To design pharmabiotic formulations including bioactive ingredients of microbial origin combined with non-microbial substances and then to evaluate the stability of the combinations during freeze-drying and storage., Materials and Methods: Different formulations including Lactobacillus gasseri CRL 1263, Lactobacillus salivarius CRL 1328, salivaricin CRL 1328 (a bacteriocin) and non-microbial compounds (lactose, inulin and ascorbic acid) were assayed, and the ingredients were freeze-dried together or separately. The formulations were stored in gelatin capsules at 4 °C for 360 d., Results: The viability of lactobacilli was affected to different extents depending on the strains and on the formulations assayed. L. salivarius and ascorbic acid were successfully combined only after the freeze-drying process. Salivaricin activity was not detected in formulations containing L. gasseri. However, when combined with ascorbic acid, lactose, inulin or L. salivarius, the bacteriocin maintained its activity for 360 d. The selected microorganisms proved to be compatible for their inclusion in multi-strain formulations together with lactose, inulin and ascorbic acid. Salivaricin could be included only in a L. salivarius CRL 1328 single-strain formulation together with non-microbial substances., Conclusions: This study provides new insights into the design of urogenital pharmabiotics combining beneficial lactobacilli, salivaricin CRL 1328 and compounds with different functionalities.

Aims: To assess the preventive effect of different intravaginal (i.va.) doses of Lactobacillus reuteri CRL1324 against vaginal colonization by Group B Streptococcus (GBS) in a murine experimental model., Methods and Results: The major virulence factors of four vaginal GBS clinical isolates were determined to select the most virulent strain and set up a murine model of streptococcal vaginal colonization. Later, the effect of four and seven doses of 10(8) viable cells of Lact. reuteri CRL1324 i.va. administered, prior to the GBS challenge was studied. Seven doses of lactobacilli were able to significantly reduce the number of viable GBS cells, while four doses showed no preventive effect. Both doses reduced the leucocyte influx induced by GBS. Seven doses caused a slight increase in the Lact. reuteri CRL1324 vaginal colonization compared with four doses and reduced murine vaginal pH compared to control mice., Conclusions: Lactobacillus reuteri CRL1324 evidenced a preventive effect on GBS vaginal colonization in an experimental mouse model., Significance and Impacts of the Study: Maternal GBS colonization is one of the most important risk factors for developing disease in newborns. Lactobacillus reuteri CRL1324 could be considered as a new biological agent to reduce infections caused by this micro-organism., (© 2014 The Society for Applied Microbiology.)

Aims: To evaluate the phenotypic expression of auto-aggregation, adhesion to mucin and biofilm formation of lactobacilli isolated from human vagina and the presence of related genes., Methods and Results: Seven different strains of three Lactobacillus species (Lactobacillus gasseri, Lactobacillus rhamnosus and Lactobacillus reuteri) were evaluated. The auto-aggregation property was determined by spectrophotometric assay and flow cytometry. Adhesion and biofilm formation were assayed by crystal violet staining. The presence of the genes encoding sortases, pilin subunits and surface proteins was evaluated by polymerase chain reactions. The two Lact. reuteri strains assayed showed high auto-aggregation, adhesion to mucin and biofilm formation ability. In these strains, the genes encoding three adhesion proteins were identified. In Lact. rhamnosus CRL (Centro de Referencia para Lactobacilos Culture Collection) 1332, pilus-encoding genes were detected. In all Lact. rhamnosus strains assayed, two genes encoding for other surface proteins related to adhesion and biofilm formation were detected., Conclusions: The vaginal lactobacilli assayed exhibited phenotypic and genetic characteristics that were specific for each strain., Significance and Impact of the Study: This is the first study on auto-aggregation, adhesion and biofilm formation of vaginal Lactobacillus strains by phenotypic and genetic assays., (© 2014 The Society for Applied Microbiology.)

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery inhibits the growth of Citrobacter freundii (a bullfrog pathogen) and Listeria monocytogenes by a synergistic effect between lactic acid, hydrogen peroxide and a bacteriocin-like molecule. The chemical characterization of the bacteriocin in cell-free supernatants indicates that it has a proteinaceous nature. Hexadecane and ethyl acetate did not modify the bacteriocin activity, while 10 and 20 % (v/v) chloroform decreased the activity by 29 and 43 %, respectively. The antimicrobial peptide was heat stable since 85 % of residual activity was detected when neutralized supernatants were heated at 80 °C for 30 min. Moreover, no bacteriocin inactivation was observed when supernatants were kept at -20 °C for 3 months. The synthesis of the bacteriocin was associated with bacterial growth, highest production (2,100 AU/ml) being detected at the end of the exponential growth phase. At pH ranges of 5-6.5 and 5.0-5.5 the inhibitory molecule was stable when stored for 2 days at 4 and 25 °C, respectively. Moreover, it had a bactericidal effect on L. monocytogenes and the ultrastructural studies of pathogenic cells revealed clumping of the cytoplasmic material, increased periplasmic space and cell wall modifications. The deduced amino acid sequence of the bacteriocin was identical to nisin Z and the genetic determinants for its production are harbored in the chromosome. These results, described for the first time in L. lactis from a bullfrog hatchery, will increase knowledge of the bacteriocin under study with a view to its potential inclusion in probiotics for raniculture or biopreservatives.

Bovine Enterococcus mundtii CRL1656 (Centro de Referencia para Lactobacilos Culture Collection) produces an anti-Listeria and anti-Streptococcus dysgalactiae bacteriocin identified as mundticin CRL1656. The strain and its bacteriocin are candidates to be included in a beneficial product to prevent bovine mastitis as an alternative to antimicrobial agents. To optimize the production of biomass and mundticin CRL1656 by E. mundtii CRL1656, a complete 3 × 2(4) factorial design was applied. The effect of culture medium, initial pH, inoculum size, incubation temperature, and agitation conditions on biomass and bacteriocin production was evaluated simultaneously. Growth parameters were determined using the modified Gompertz model. A nonlinear model was used to estimate the effects of the variables on growth parameters. Bacteriocin production was analyzed using a linear mixed model. Optimal biomass and mundticin CRL1656 production by E. mundtii CRL1656 were obtained in different conditions. Maximal growth was recorded in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or M17 broths, pH 6.5, 5.0% inoculum, 30 °C, with agitation. However, bacteriocin titers were higher in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or de Man-Rogosa-Sharpe (MRS) broths, pH 6.5, 30°C, both with or without agitation. Knowledge of the optimum conditions for growth and bacteriocin production of E. mundtii CRL1656 will allow the obtainment of high levels of biomass and mundticin CRL1656 as bioingredients of potential products to prevent bovine mastitis., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Aim: To evaluate the effect of freeze-drying and storage conditions on the viability and beneficial properties of lactic acid bacteria (LAB) for raniculture., Methods and Results: Lactococcus lactis CRL 1584, L. lactis CRL 1827, Lactococcus garvieae CRL 1828 and Lactobacillus plantarum CRL 1606 viability under different conditions was studied. 10% lactose and 5% skim milk + 5% lactose were excellent lyoprotectants, but 5% skim milk + 5% lactose and whey protein concentrated (WPC) or WPC + sugars were the lower cost lyoprotective options. The effect of temperature depended on both lyoprotectants and storage time. Thus, for Lactococcus, skim milk, skim milk + sucrose and WPC + sucrose were selected for lyophilization and storage at 4°C and skim milk + lactose for 25°C. For Lact. plantarum CRL 1606, the best lyoprotectants for lyophilization and storage at 4°C were milk + sugars and WPS + sucrose and, at 25°C, skim milk + sucrose., Conclusions: Lactic acid bacteria viability after freeze-drying was strain-specific and depended on the lyoprotectant used. Highest viability was obtained when stored at 4°C, and the beneficial properties remained stable for 18 months independently of storage temperature., Significance and Impact of the Study: The studies reported for the first time in this work are of primary interest to obtain dried bacteria to be included in beneficial products for raniculture., (© 2013 The Society for Applied Microbiology.)

The cultivable microbiota of skin and cloaca of captive Lithobates catesbeianus includes microorganisms generally accepted as beneficial and potentially pathogenic bacteria. In order to select a group of potentially probiotic bacteria, 136 isolates were evaluated for their surface properties and production of antagonistic metabolites. Then, 11 lactic acid bacteria (LAB) strains were selected and identified as Lactobacillus plantarum, Lb. brevis, Pediococcus pentosaceus, Lactococcus lactis, L. garvieae and Enterococcus gallinarum. Studies of compatibility indicate that all the strains could be included in a multi-strain probiotic, with the exception of Ent. gallinarum CRL 1826 which inhibited LAB species through a bacteriocin-like metabolite. These results contribute to the design of a probiotic product to improve the sanitary status of bullfrogs in intensive culture systems, to avoid the use of antibiotics and thus to reduce production costs. It could also be an alternative to prevent infectious diseases during the ex situ breeding of amphibian species under threat of extinction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aims: To assess the ability of vaginal lactobacilli to form biofilm under different culture conditions and to determine the relationship between their growth and the capability of biofilm formation by selected strains., Methods and Results: Fifteen Lactobacillus strains from human vagina were tested for biofilm formation by crystal violet staining. Only Lactobacillus rhamnosus Centro de Referencia para Lactobacilos Culture Collection (CRL) 1332, Lact. reuteri CRL 1324 and Lact. delbrueckii CRL 1510 were able to grow and form biofilm in culture media without Tween 80. However, Lact. gasseri CRL 1263 (a non-biofilm-forming strain) did not grow in these media. Scanning electron microscopy showed that Lact. rhamnosus CRL 1332 and Lact. reuteri CRL 1324 formed a highly structured biofilm, but only Lact. reuteri CRL 1324 showed a high amount of extracellular material in medium without Tween., Conclusions: Biofilm formation was significantly influenced by the strain, culture medium, inoculum concentration, microbial growth and chemical nature of the support used for the assay., Significance and Impacts of the Study: The results allow the selection of biofilm-forming vaginal Lactobacillus strains and the conditions and factors that affect this phenomenon., (© 2012 The Society for Applied Microbiology.)

Vaginal probiotics containing lactic acid bacteria with activity towards pathogenic microorganisms that cause urogenital tract infections have been proposed as a valid strategy for their prophylaxis and therapy. A murine experimental model was set up to evaluate the colonization capability of beneficial human lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells. Five Lactobacillus strains were intravaginally inoculated into previously estrogenized BALB/c mice. The significance of the effects observed in the vaginal tract was determined by analysis of variance using the general linear model. The numbers of viable vaginal lactobacilli were significantly higher at proestrous-estrous than those at the metaestrous-diestrous phase and decreased markedly on the days after inoculation. Lactobacilli inoculation did not cause cytological or histological modifications of the murine vaginal tract. Moreover, the intravaginal administration of Lactobacillus salivarius CRL (Centro de Referencia para Lactobacilos culture collection) 1328 and Lactobacillus gasseri CRL 1263 did not affect the amounts of granulocytes and macrophages present in vaginal washings. In conclusion, the results demonstrate that vaginal lactobacilli did not produce adverse effects on the murine vaginal tract. Therefore, they could be proposed as safe probiotic candidates to promote a balanced microbiota in the urogenital tract.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Bovine mastitis produces a wide variety of problems in the dairy farm. The treatment of this disease is based on the use of antibiotics which are not always effective. These drugs are also responsible for the presence of residues in the milk and the increase of antibiotic-resistant strains. Probiotic products were proposed as a valid alternative to antibiotic therapies and are also useful for the prevention of infectious syndromes. With the aim of designing a probiotic product to prevent bovine mastitis, lactic acid bacteria (LAB) were isolated from foremilk samples from different dairy farms in Córdoba-Argentina. One hundred and seventeen LAB were isolated and their beneficial characteristics such as the production of inhibitory substances, surface properties and production of exopolysaccharides (EPS) were assessed. Most of them displayed low degree of hydrophobicity, autoaggregation, EPS negative phenotype and were identified as Enterococcus hirae and Pediococcus pentosaceus. Nine LAB strains inhibited three indicator bacteria. Some isolates were pre-selected and genetically identified according to the results obtained. Antibiotic resistance and virulence factors were studied for the assessment of the safety of the strains. The results obtained were compared to those reported previously from samples obtained in the North-western area of the country and some differences were found., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We report the draft genome sequence of Enterococcus mundtii CRL1656, which was isolated from the stripping milk of a clinically healthy adult Holstein dairy cow from a dairy farm of the northwestern region of Tucumán (Argentina). The 3.10-Mb genome sequence consists of 450 large contigs and contains 2,741 predicted protein-coding genes.

Objectives: To evaluate the production of inhibitory substances against several urogenital pathogens and to determine the compatibility between selected vaginal lactobacilli to be considered as probiotic candidates., Study Design: The inhibitory activity of 38 vaginal Lactobacillus strains against urogenital pathogens was assessed using the agar plate diffusion., Method: The production of hydrogen peroxide was determined by the semi-qualitative tetramethyl-benzidine-peroxidase assay and that of lactic acid by high performance liquid chromatography. Pre-selected lactobacilli were genetically identified by ribosomal 16S-DNA sequencing and used to determine the compatibility of strains. Correlation values between some inhibitory properties were calculated., Results: 45% of the lactobacilli assayed inhibited the growth of various urogenital pathogens, except Candida albicans, mainly by the effect of organic acids. Even though 71% of the Lactobacillus strains produced hydrogen peroxide in the semi-qualitative test, the antimicrobial effect of the oxidative metabolite on pathogens was not detected under the experimental conditions assayed. On the other hand, the inhibition spectrum of salivaricin CRL 1328 was widened with respect to the one previously reported by our research team. With respect to the degree of interaction between the pre-selected lactobacilli, most of them showed inhibition of at least one strain, mainly due to the effects of both organic acids and hydrogen peroxide., Conclusion: Several lactobacilli that showed inhibitory activity against urogenital pathogens or produced hydrogen peroxide are compatible between them. Therefore, different potential combinations of strains are proposed for their inclusion in the design of probiotic products to be vaginally administered for the prevention of urogenital infections in women., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Red-leg syndrome (RLS) is one of the main infectious diseases that cause economic losses in Lithobates catesbeianus hatcheries, Citrobacter freundii being an etiological agent. Treatment or prevention with therapeutics or chemicals results in modifications of the indigenous microbiota, development of antibiotic resistance, presence of their residues in food and enhancement of production costs. Thus, probiotics could be used as an alternative therapy. Lactic acid bacteria are part of the indigenous microbiota of healthy frogs and can prevent pathogen colonization by different mechanisms, including the production of antagonistic substances. In this work, the evaluation and characterization of the inhibition of C. freundii CFb by Lactococcus lactis subsp. lactis CRL 1584, a potentially probiotic candidate, were carried out. This strain produced lactic acid, H(2)O(2) and bacteriocin in static and shaken conditions and inhibited pathogen growth in associative cultures, with an earlier inhibition under agitated conditions. The elimination of each of the antimicrobial metabolites partially abolished the inhibition of the pathogen, suggesting that the inhibitory effect could be attributed to a combined action of the three antagonistic molecules. Electron microphotographs revealed the damage caused by L. lactis CRL 1584 supernatants to C. freundii cells. The addition of pure lactic acid, H(2)O(2) and bacteriocin to the culture media showed that each metabolite caused different morphological modifications in C. freundii, in agreement with the effect on viable cell counts. The results support the possibility that L. lactis CRL 1584 might be considered as a probiotic to be used in the prevention of RLS in raniculture., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Vaginal Lactobacillus salivarius CRL 1328 (Centro de Referencia para Lactobacilos Culture Collection) or the bacteriocin that produces could be included in a urogenital probiotic formula to prevent urogenital infections in women. The objective of this work was to determine the effects of different carbon and nitrogen sources on the growth and bacteriocin production of this microorganism. A fractional factorial design 3(5-1) was applied to evaluate the effects of five nutrients (glucose, lactose, yeast extract, tryptone and meat peptone), at three different concentrations (0, 1 and 2%). Results were statistically analyzed for linear and quadratic effects of nutrients, along with their interactions. All the nutrients tested stimulated the cell growth and bacteriocin production, but lactose had not a significant influence on the last response. The linear effects of higher magnitude on biomass and bacteriocin production were those of yeast extract and tryptone. The results demonstrated that there were significant interactions between the different nutrients, depending on the response evaluated. Maximum bacteriocin production was reached in different growth media with a lower cost than conventional culture media used in the laboratory. These findings will contribute to the design of a pharmaceutical product for the restoration of ecological balance of urogenital tract.

The aim of this work was to formulate a culture medium of lower cost than conventional laboratory media, in order to simultaneously obtain high amounts of both biomass and bacteriocin of vaginal Lactobacillus salivarius CRL 1328. The growth assays under different culture conditions were performed by using a 2(8-2) central composite experimental design, with a central point and sixteen additional points. The factors taken into consideration were glucose, lactose, yeast extract, tryptone, ammonium citrate, sodium acetate, MgSO4 and MnSO4. The simultaneous presence of a carbon source (mainly glucose), a nitrogen source (mainly yeast extract) and salts (mainly MnSO4, MgSO4 and sodium acetate) allowed the highest cell biomass and bacteriocin levels to be reached in the experimental design. Through the application of the desirability function, several optimal medium compositions to achieve efficient production of biomass and bacteriocin were predicted. The optimized growth media allow a cost reduction of around 25 to 40% compared with conventional broths. The results obtained represent an advance in the search of the most suitable strategies for the production of bioactive compounds for pharmaceutical products to prevent or treat female urogenital infections.

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.

The industrial use of lactic acid bacteria as probiotic cultures depends on the preservation techniques employed, which are required to guarantee stable cultures in terms of viability and functional activity. The aim of this study was to evaluate the effects of 12% lactose and 12% sucrose suspended in water or reconstituted skim milk on the survival and expression of beneficial characteristics during freeze-drying and subsequent storage of 6 vaginal lactobacilli strains. A cubic polynomial model was also used for the first time to evaluate the effects of different protectors on survival behavior during storage. Different survival patterns were observed among the strains considered. The presence of both lactose and sucrose in water or in 6% skim milk as the suspension medium proved to be effective in maintaining a high degree of survival and expression of potentially probiotic characteristics (production of antimicrobial substances or auto-aggregation capabilities) of most strains after lyophilization and long-term storage. This study constitutes a valuable step to obtain concentrated cultures with the highest stability of microorganisms for pharmaceutical purposes.

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 H(2)O(2)-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.

The aim of this study was to analyze the influence of vitamins, glycerol, and salts on the growth and bacteriocin production by Lactobacillus salivarius CRL 1328, a human vagina isolate, by using a chemically defined medium to determine the optimal conditions for salivaricin production. The single omission of d-biotin, thiamine, p-aminobenzoic acid, folic acid, or cyanocobalamin did not affect the bacterial growth, whereas the removal of nicotinic acid, riboflavin, and pyridoxal produced a decrease of about 30% in the growth rate. Maximum salivaricin activity was observed after the addition of 5 or 10 g/L of NaCl. On the basis of the nutritional requirements and the levels of salivaricin production, a new optimized and simplified defined medium (SDM-NaCl) for L. salivarius CRL 1328 bacteriocin production was formulated. The kinetics of salivaricin production in SDM-NaCl and in the complex media LAPTg revealed that bacteriocin production was growth linked. A combination of tricine - sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), Lumitein protein gel staining, and a bioassay for antibacterial activity indicated that the molecular mass of salivaricin CRL 1328 is about 4.5 kDa. The partially purified bacteriocin, obtained from SDM-NaCl after concentration, allowed for the design of a relatively simple method for the recovery of a biologically active protein.

Probiotics are frequently used for multiple objectives and the majority of applications are performed both in human beings and in different animals. The specificity of species showed by the members of the indigenous microbiotia led us to isolate lactic acid bacteria (LAB) from their natural bovine ecosystems and to select those with probiotic potentiality. The beneficial probiotic LAB were isolated from the vaginal tract and mammary gland of adult heifers, faeces and oral cavity of newborn calves. Bacterial strains and their numbers varied in each tract under study. Their surface properties, evaluated using the MATH test, revealed that the higher number of low hydrophobic strains were isolated from vaginal tract, mammary gland or faeces. The capability of autoaggregation also differed with higher degree in those isolated from vagina, whereas those that originated in the mammary gland or faeces showed low autoaggregation characteristics. The production of antagonistic substances showed that the production of hydrogen peroxide was higher in the strains isolated from the vaginal tract, while those able to produce organic acid were isolated from all the environments. A few strains were able to produce bacteriocins and were isolated only from the mammary gland and faeces, but not from the vaginal tract. Further studies are being performed to complete the functional characterization of the strains in order to evaluate their survival in the environmental conditions of each tract. Finally, the selected strains could be combined for the design of probiotic products to be used both in adult cows and in neonates, based on the host-specificity, due to their autochthonous origin and their potential use in the tract where they were isolated.

Clofibric acid (CL) is a compound used to control hypertriglyceridemia, and ethacrynic acid (ET) is administered to enhance diuresis. These compounds are structurally analogous to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as they have a chlorinated phenoxy moiety. As these agents are mainly excreted by the renal route, they could potentially coexist with Escherichia coli in the urinary tract of infected patients. Induction of the in vitro resistance of E. coli to hydrophilic antibiotics was determined by increasing the values of the minimum inhibitory concentration (2-40-fold). These results correlated with drastically inhibited expression of the hydrophilic bacterial channel OmpF. In vivo assays were performed in ascending urinary tract infection in female BALB/c mice. Treatment with the hydrophilic antibiotic cephalexin 25 mg kg(-1) day(-1) by the oral route diminished renal infection. The CFU mean values in the kidneys were between 75% and 89% lower than those in animals without treatment. Simultaneous exposure to CL (at a therapeutic dose, 28.6 mg kg(-1) day(-1)) did not change the effect of the treatment. In contrast, ET at 2.9 mg kg(-1) day(-1) or 2,4-D at 70 mg kg(-1) day(-1) inhibited the antibiotic therapeutic effect. Moreover, 2,4-D dramatically increased bacterial infection after 9 days of exposure.

Infectious diseases are the major reproductive complication during postpartum. The bacteria that colonize the postpartum uterus of cattle are specific as well as opportunistic pathogens that include Staphylococcus aureus. The antibiotics and antiseptic agents used in the treatment of postpartum acute infections induce residues in foods, spread of bacterial resistance, increase in financial costs and failures in defense mechanisms of the host. Preventive treatment with probiotic products could decrease the use of antibiotics in dairy farming systems. Lactobacilli are present in the vaginal microflora of healthy cows. They can prevent pathogen colonization by mechanisms such as the production of antagonistic substances as lactic acid, H2O2, or bacteriocins. The aim of the present study was the selection of H2O2 generating lactobacilli from a group of 72 strains isolated from the vagina of cattle. Both Lactobacillus gasseri CRL1421 and Lactobacillus gasseri CRL1412, which share some probiotic properties, produce H2O2, detected by the plate colorimetric method. They were chosen to study the kinetics of H2O2 production under different culture conditions. Both microorganisms produced greater amounts of H2O2 in aerated than in static cultures. As L. gasseri CRL1421 had a greater capacity to generate H2O2, associative cultures with this strain and S. aureus were conducted. A significant decrease in the growth of the pathogen was detected after culture for 6h, this effect being greater under aerated conditions. The addition of catalase to mixed cultures partially abolished the inhibition, an effect that could be attributed to the combined action of H2O2 and other antagonistic metabolites. The simultaneous addition of catalase and NaOH to these cultures restored S. aureus growth. This observation suggests that the inhibition was produced by the combination of H2O2 and lactic acid, both released by the lactobacilli. Electron microphotographs showed the damage caused by the lactobacilli supernatant on the pathogenic cells. The treatment of S. aureus with lactic acid and hydrogen peroxide evidenced that each metabolite produced a different type of morphological damage. The number of viable cells obtained agrees with the electron microscopy observations. The results support the idea that L. gasseri CRL1421 could be successfully included in a probiotic product to prevent S. aureus infection in cows.

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., Methods and Results: Out of 76 Lactobacillus sp. and seven Streptococcus sp. strains, a small number showed high- and medium hydrophobicity when the microbial adhesion to hydrocarbons method (MATH) was applied. In the agar plate diffusion test, a large number of strains inhibited vaginal bovine Escherichia coli 99/14 and human E. coli. This inhibition was due to acid. Only a few strains inhibited Actinomyces pyogenes 96/393, a pathogen isolated from bovine metritis. This inhibition remained after neutralization. The taxonomic identification of the selected strains was carried out by an amplified ribosomal DNA restriction analysis (ARDRA). Most of the strains were identified as Lactobacillus fermentum, a few as Lactobacillus gasseri and one as Lactobacillus rhamnosus., Conclusions: Bovine vaginal lactobacilli strains have differential surface properties. The strains selected are capable of inhibiting specific metritis pathogens., Significance and Impact of the Study: Our results can be applied for future studies to design a probiotic product to prevent metritis in dairy postpartum cows.

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 microg/mL of chloramphenicol, aztreonam, norfloxacin, ciprofloxacin, ceftazidime, ceftriaxone, streptomycin and kanamycin. Four lactobacilli were sensitive to 1 microg/mL vancomycin and all of them were resistant to 1000 microg/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.

This study aims to evaluate potentially beneficial properties of 20 strains of vaginal lactobacilli isolated from women in Tucumán, Argentina, by determining acid and hydrogen peroxide production and auto-aggregation ability. The microorganisms were characterised genetically by amplified ribosomal 16S-DNA restriction analysis (ARDRA). Lactobacillus gasseri and L. rhamnosus were the predominant species identified among the 20 vaginal lactobacilli strains. Most achieved low pH values after 12 h incubation at 37 degrees C and produced hydrogen peroxide in static culture. However, pH decrease and semi-quantitative hydrogen peroxide production of most homofermentative lactobacilli were significantly higher than those of heterofermentative lactobacilli. Of the 20 strains studied, only three demonstrated remarkable auto-aggregation patterns. Four strains were selected for possible use in a probiotic product for vaginal application; however, further in vitro study of other potentially probiotic characteristics is required before attempting clinical trials.

Aims: To evaluate the effects of different physico-chemical factors on the growth and auto-aggregating ability of vaginal Lactobacillus johnsonii CRL 1294., Methods and Results: L. johnsonii CRL 1294 was cultivated in different culture media, initial pH and temperature of incubation. The growth parameters were estimated by the Gompertz model, being optimal (higher final biomass and growth rate, and shorter lag phase) at an initial pH of 6.5 and at a temperature of 37 degrees C, both in LAPTg and MRS. The auto-aggregation ability, which was assessed by a model of exponential association, was evidenced in all the growth phases, being higher at pH 5 or 6.5., Conclusions: The growth of L. johnsonii CRL 1294 was affected in different way by all the physico-chemical factors tested. However, the auto-aggregation ability increased mainly at low initial pH of growth media., Significance and Impact of the Study: The auto-aggregation ability under different culture conditions of a vaginal Lactobacillus strain was systematically and statistically evaluated for the first time. The higher cellular aggregation evidenced at low pH could be a fundamental characteristic in the acidic vaginal environment to promote the protective role of lactobacilli.

Interfering Escherichia coli attachment to the urinary tract, using P-fimbriation inhibitors, can prevent pyelonephritis. Clofibric and ethacrynic acids are organic compounds structurally related, but with different pharmacological uses. These agents are potentially active in the urinary tract due to its elimination in an unaltered form by the renal route. This study described a pyelonephritogenic E. coli strain, grown in the presence of sub-inhibitory concentrations of clofibric or ethacrynic acids (0.1 and 1 mM, respectively), which exhibits inhibition of P1 erythrocytes agglutination and a drastic decrease in fimbriation, using electron microscopy and quantitative analyses of superficial proteins (decrease to a 17-25% in comparison with the control). In vivo assays were performed using ascending urinary tract infection in mice. The treatment with therapeutic doses of the drugs, administered 2 days before the bacterial challenge and daily until the end of the experiment (22 days), abolished renal infection after 7-10 days of drug exposure. Within this period clofibric acid did not produce adverse effects on the renal parenchyma. However, ethacrynic acid caused pyelitis and tubular cellular desquamation. These results suggested that clofibric acid might be useful in the short-term prophylaxis of urinary tract infection.

Ampicillin was selected to further study the effect of this antibiotic on the colonization capability of S. pneumoniae and L. fermentum intranasally inoculated in a mice experimental model. The sensitivity of S. pneumoniae and L. fermentum to antibiotics was evaluated by different "in vitro" techniques. The results showed that both microorganisms have a typical pattern of sensitivity to antibiotics in these assays. The "in vivo" experiments showed that the treatment with Ampicillin increased the number of lactobacilli and neumococci in the groups of mice treated only with one of the microorganisms. In those mice treated with Lactobacillus, challenged later with neumococci and treated with Ampicillin, the pathogen in lung decreased on the 4th day, disappearing completely after on. The histological studies showed that the antibiotic treatment decreased the inflammatory response produced by the pathogen at the lung and trachea levels.

The viability of six different strains of probiotic vaginal Lactobacillus was examined in two different cryoprotective media, during refrigerated versus frozen storage, and using two traditional types of stock cultures for starting the biomass production. Freezing at -20 degrees C and -70 degrees C had much less adverse effect on viability than did storage at 7 degrees C, and the reduction in viability was greater at -20 degrees C than at -70 degrees C. The strains showed variation in the extent of the viability losses during both types of storage. Milk-yeast extract (MYE) was shown to be the more suitable protective medium to maintain viability of the strains during the storage. The vaginal Lactobacillus strains are most stable in MYE at -70 degrees C with only a small decrease of the viability observed under these conditions. The viable cell counts of Lactobacillus paracasei CRL 1251 and CRL 1289, L. crispatus CRL 1266 and L. salivarius CRL 1328 remained around 1 x 10(8) CFU/mL after 24 months of storage at -70 degrees C, or up to 18 months for L. acidophilus CRL 1259.

Respiratory tract infections are among the bacterial infections that affect humans with higher frequency. Those produced by Streptococcus pneumoniae are reported to have the highest incidence in the world, affecting both children and old people. As a 2001 report from the World Health Organization expressed it, the basic fight of children under 5 yr old is to survive. Five different conditions (acute respiratory infections, diarrhea, measles, palludism, and undernutrition) directly produce more than 50% of the deaths in this age group. Respiratory tract infections in the developing countries in the Americas are among the first three causes of death in children under 1 yr and between the first and second cause in children between 1 and 4 yr old. Pneumonia is responsible for 85 and 90% of deaths in children under 5 yr old (approx 150,000 annually), 95% of them occurring in the developing countries in the Americas. There is an increased worldwide tendency to use preventive measures and to consume products that help to maintain the health status of the individual. Thus the use of probiotics has increased systematically during the last decade, and the scientific literature trying to demonstrate the positive effect of such preparations has also increased. The term probiotic has been applied to products that (1) contain live microorganisms, freeze-dried or included in fermented products or (2) improve the health status of humans and animals, exerting effects in the mouth or gastrointestinal tract (included in foods or capsules), in the respiratory tract (as aerosols), or in the urogenital tract (by local application)Having in mind the high incidence of respiratory tract infections, and looking for preventive measures as well as the possible applications of probiotics, the aim of this chapter was to use mice as experimental models to determine whether members of the genus Lactobacillus were able to colonize and give protection from infections after inoculation by the intranasal route. To this end, the following procedures were carried out: 1. Screening of the predominant bacterial species in respiratory organs. 2. Study of the kinetics of colonization of the different groups of microorganisms from 15 d up to adult (2 mo). 3. Screening of the probiotic characteristics of all the isolated strains.

Lactic acid-producing lactobacilli were selected from 134 human vaginal isolates by testing their capability to inhibit the growth of different pathogenic micro-organisms. Lactobacillus acidophilus CRL 1259 (from the CERELA Culture Collection) was selected to study the effects of temperature, pH and culture medium on growth and lactic acid production. Growth parameters were estimated by using the model of Gompertz. Kinetics of inhibition of uropathogenic Escherichia coli were evaluated in mixed cultures of the pathogen and L. acidophilus. Optimal conditions for growth and lactic acid production by L. acidophilus were pH 6.5 or 8.0 and 37 degrees C. Under these conditions, growth was higher in LAPTg (yeast extract/peptone/tryptone/Tween 80/glucose) broth than in MRS (De Man-Rogosa-Sharpe) broth. However, lactic acid production was more efficient in MRS broth. Under optimal conditions for lactic acid production, L. acidophilus inhibited the growth of E. coli. These results suggest that inclusion of L. acidophilus CRL 1259 in probiotic products for vaginal application would be beneficial.

Objective: To assess, using structural and ultrastructural studies of the urinary tract, the effects of the intraurethral inoculation of lactobacilli (probiotic treatment) as lactobacilli are the predominant micro-organisms of the urogenital tract of humans, monkeys and mice., Materials and Methods: Previous work showed the protective effect of Lactobacillus fermentum CRL 1058 intraurethrally inoculated against the challenge of uropathogenic Escherichia coli. There was also an effect of oestrogens and antibiotics in the kinetics of colonization of both micro-organisms in mice. In the present study L. fermentum was inoculated with agarose beads (107 colony-forming units) and the number of micro-organisms determined by plating in selective media, giving a high degree of colonization in all the organs studied. The urinary tract organs were processed by histological and electron microscopy techniques standardized in our laboratory., Results: The intraurethral inoculation of lactobacilli produced no adverse effects or significant changes in any of the organs assessed (kidney, ureter, bladder or urethra), when evaluated by histological and ultrastructural techniques., Conclusion: The use of lactobacilli as a probiotic treatment is probably safe.

Lactobacilli were considered the prime cariogenic microorganisms until phylogenetic related bacteria, mutans streptococci, were associated with caries. Today, lactobacilli are still considered a factor in determining the predisposition to develop cavities. As a first step in colonization, microorganisms adhere to oral tissues. Based on this fact, the purpose of our study was to determine if there was a statistical association or difference related to the state of oral health with the surface characteristics of lactobacilli. Patients were classified as caries-free and caries-active. Interviews were performed to establish the nutritional and oral hygiene habits. The previously reported characteristics of isolated lactobacilli's quantification and association to dental tissues were determined. Although the nutritional habits for caries-free and caries-active patients were similar, the patients' oral hygiene and dental care determined differences in risk indices. The number of lactobacilli was statistically lower in saliva of subjects with good oral health. Certain species of lactobacilli could not be associated to specific areas of the mouth, although some species could be localized. Lactobacilli from caries active (CA) subjects showed a greater ability to adhere to hydrophobic substances, had a greater salt agglutination property, and showed lower production of inhibitory substances. Lactobacilli from caries free (CF) subjects were better able to inhibit oral, potentially pathogenic, microorganisms. These studies prove that preliminary differences between oral lactobacilli in CF and CA patients exist. Non-specific and specific adhesion mechanisms in bacteria should be further demonstrated.

Objective: The purpose of this study was to determine the optimal conditions for the growth and hydrogen peroxide production by vaginal lactobacilli., Study Design: Two vaginal lactobacillus strains were cultured under different growth conditions (temperature, pH, agitation, and growth media) with a fractional factorial experimental design., Results: The optimal growth conditions of Lactobacillus paracasei CRL (Centro de Referencia para Lactobacilus Culture Collection) 1289 and Lactobacillus crispatus CRL 1266 were temperature 37 degrees C and initial pH 6.5, nonagitated cultures, in autolysat de levure; peptone, tryptone, Tween 80 et glucose (LAPTg) broth for L paracasei or in de Man-Rogosa-Sharpe (MRS) and LAPTg broths for L crispatus. The hydrogen peroxide production, detected only in agitated cultures, was higher at 37 degrees C and pH 6.5. The oxidative metabolite produced the self-inhibition of the lactobacilli growth. In mixed cultures of lactobacilli and Staphylococcus aureus under the optimal conditions of hydrogen peroxide production, the pathogen growth was inhibited., Conclusion: The results provided information about the factors that affect the hydrogen peroxide production and about the most favorable conditions with which to obtain the highest biomass in the shortest possible time.

The effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-widely used in the world and mainly excreted by the renal route in exposed humans-were studied on the virulence and surface characteristics of an uropathogenic Escherichia coli strain. When the urine was supplemented with 2,4-D in vitro, the compound significantly reduced the bacterial fimbriation assayed by hemagglutination and surface protein quantification. Protein values decreased from 0.24 mg/g dw to 0.05 or 0.12 mg/g dw by 1 or 0.1 mM 2,4-D treatment, respectively. The effects in vivo were studied in groups of mice challenged intra-urethra with E. coli and exposed by the oral route with three different 2,4-D doses (2.6, 25 or 70 mg/kg bw) during 22 days. Depending on the dose used, the herbicide significantly decreased or removed bacterial cells in mice bladder and kidneys; except in the group treated with the highest dose from the 9th day of treatment. The histological studies showed mononuclear cell infiltration at low doses, and toxic damage in the renal parenchyma at prolonged exposure with higher doses, up to tisular necrosis in the 70 mg/kg bw group after 9 days of treatment. Our investigations performed in an experimental model suggest that short time 2,4-D exposure at low doses could act in prevention of UTI stimulating leukocytic migration and decreasing bacterial fimbriation. On the contrary, high doses and long-term exposure enhanced renal damage resulting in infection recurrence.

Lactobacilli are widely described as probiotic microorganisms used to restore the ecological balance of different animal or human tracts. For their use as probiotics, bacteria must show certain characteristics or properties related to the ability of adherence to mucosae or epithelia or show inhibition against pathogenic microorganisms. It is of primary interest to obtain the highest biomass and viability of the selected microorganisms. In this report, the growth of seven vaginal lactobacilli strains in four different growth media and at several inoculum percentages was compared, and the values of growth parameters (lag phase time, maximum growth rate, maximum optical density) were obtained by applying the Gompertz model to the experimental data. The application and estimation of this model is discussed, and the evaluation of the growth parameters is analyzed to compare the growth conditions of lactobacilli. Thus, these results in lab experiments provide a basis for testing different culture conditions to determine the best conditions in which to grow the probiotic lactobacilli for technological applications.

Aims: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328., Methods and Results: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths., Conclusions: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6.5 and a temperature of 37 degrees C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37 degrees C, and at an initial pH of 6.5 or 8.0., Significance and Impact of the Study: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.

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 and a 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.

The capability of lactobacilli to adhere to vaginal epithelial cells (VEC) has been found to be an important factor in the formation of a barrier to prevent colonization of pathogenic bacteria. The aim of this study was to examine the adhesion ability to VEC, of vaginal Lactobacillus which had been selected for their probiotic properties. A modification of the Mardh and Weströn method was employed for this purpose. Adherent bacteria were determined as CFU-grown using selective media. There was a slight degree of difference in the adhesion properties observed among vaginal and non-vaginal Lactobacillus. Different pH values of 4 and 7 did not affect adhesion (P < 0.70). Scanning and transmission electronic microscopy were performed to illustrate the adhesion of the self-aggregating L. salivarius CRL 1328.

Lactic acid bacteria are increasingly used to restore the ecological equilibrium of different mucosal areas in humans and/or animals. Likewise, they can be used to potentially protect against pathogenic microorganisms. In the present paper, the preventive effect of intranasally inoculated Lactobacillus fermentum against challenge with Streptococcus pneumoniae was studied, using a mouse experimental model. L. fermentum inoculated four times at a dose of 10(7) colony forming units per mouse was able to decrease the number of S. pneumoniae throughout the respiratory tract. The L. fermentum treatment increased the number of activated macrophages in lung slices, and a higher lymphocyte population in the tracheal lamina propria. S. pneumoniae challenge showed a typical response against pathogen with a higher non-specific immune response. Preventive treatment, i.e. L. fermentum administration prior to S. pneumoniae challenge, showed a response close to that of L. fermentum. Anti-S. pneumoniae antibodies increased in lactobacilli-treated animals compared to the non-treated lactobacilli mice. The increase in the antibody levels suggests that the mucosal immune system could be involved in the protective effect, accomplished with competitive exclusion, nutrient competition and production of inhibitory substances. This paper will be the basis for further studies of the protective effect of lactobacilli against S. pneumoniae in the respiratory tract.