Vaginal bacterial diversity from healthy gilts and pregnant sows subjected to natural mating or artificial insemination.

The profitability of commercial pig farms largely depends on the reproductive performance of gilts and sows. The aim of this study was to identify differences in the composition and diversity of vaginal microbiota between gilts (G) and pregnant (P) sows, both artificially inseminated (AI) and natural mating (NM). Samples were collected by scraping the vaginal mucosa of G (n = 10) and P (NM, n = 10 and AI, n = 7) sows. Samples were analysed by culture-dependent techniques and 16S-rRNA gene High-Throughput-Sequencing. The profiles of the cultured microbiota showed two distinctive clusters, one of them grouped four samples of P sows from the AI group. The vaginal microbiota from P had lower richness than G sows (Mann-Whitney/Kruskal-Wallis test, p < 0.01), but all vaginal samples had a similar diversity. The PERMANOVA analyses revealed significant differences (p < 0.01) between the microbial communities' structures from G and P sows. The bacteria phyla with the highest relative abundances were Proteobacteria (33.1%), followed by Firmicutes (32%), Cyanobacteria (13.3%) and Actinobacteria (13.2%). The relative abundance for phyla, families and genera was estimated and Proteobacteria was significantly higher (p = 0.038) in P than in G sows; Firmicutes was significantly lower in AI than G and NM sows. A "core microbiota" included Lactobacillus, Bacillus, Enterococcus, Acinetobacter and Pseudomonas. The results presented highlight the differences in the bacterial composition between G and P sows, as well as the changes in the microbial populations associated with the breeding method.

Bacterial communities associated to the urethra of healthy gilts and pregnant sows undergoing different reproductive protocols.

Nowadays, it is known that the urogenital microbiota plays a key role in the urinary health of mammalians. Despite the urinary infections affect the health and the welfare of breeding sows, the urethral microbiota of healthy sows remains unknown. Therefore, this work evaluates the urethral bacterial communities of healthy gilts and sows to determine the presence of Enterobacteriaceae populations, and the structure of this microbiota in gilts (G) and pregnant (P) sows. Samples were collected by scraping the urethral mucosa of G (n = 9) and P sows, which included natural mating (NM, n = 9) and artificial inseminated (AI, n = 7) sows. Samples were analyzed by culture-dependent techniques and 16S-rRNA gene high-throughput-sequencing. All females were positive for Enterobacteriaceae culture, without significant differences (Kruskal-Wallis) between G and P groups (median values: 2.78 and 3.09 log CFU/mL, respectively; P = 0.497). Also, the rate of Enterobacteriaceae/total mesophilic microorganisms was individually calculated, without significant differences between G and P sows (median values: 0.61 and 0.66, respectively; P = 0.497). When analyzing the bacterial communities, it was found similar richness in G, NM, and AI; however, diversity was lower in P sows than G (Mann Whitney/Kruskal-Wallis test, P < 0.01). The dominating phyla that constituted a "core microbiome" included Firmicutes, Proteobacteria, Cyanobacteria, Actinobacteria, and Bacteroidetes, which were common for all the studied females. The relative abundance for phyla, families, and genera was estimated, and Firmicutes was significantly higher in NM than AI sows (P = 0.02, Mann-Whitney/Kruskal Wallis test for univariate statistical comparisons); Pseudomonadaceae and Enterobacteriaceae were higher in AI than in NM (Mann-Whitney/Kruskal-Wallis, P < 0.05). Lactobacillus and Pseudomonas were among the dominant genera; however, only Pseudomonas sp. was significantly higher in AI than NM (Mann-Whitney/Kruskal-Wallis, P = 0.006). The results represent the first evidence about the existence of a urethral microbiota that includes Enterobacteriaceae, as well as the patterns of this microbiota in G and P sows. The knowledge of this urethral microbiota might allow for future research to develop innovative protocols to restore and/or preserve the healthy ecology of the urinary microbiome to prevent diseases ensuring the welfare of breeding sows.

Antimicrobial resistant Escherichia coli in the reproductive tract microbiota of cows and sows.

Escherichia coli is a natural colonizer of the urogenital mucosa of healthy females; however it is one of the pathogens associated to reproductive failures in cows and sows. A better knowledge about the characteristics of native E. coli will allow us to differentiate them from pathogenic strains. Ninety autochthonous isolates from the reproductive tract of sows and cows were characterized to determine the phylogenetic profile, antibiotic resistance and virulence factors; also, comparisons between different breeding systems were performed. Vaginal colonization of E. coli was statistically higher in cows (57.5%) than sows (23.8%), and most isolates belonged to the phylogenetic group A: 79.69 and 80.77%, respectively; moreover phylo-groups B1 (12.5 and 11.54%) and D (7.81 and 7.69%) were significantly lower; however, none was classified as B2. Positive associations between virulence factors and group D were found. Isolates with antimicrobial susceptibility were associated with group A and the MDR (Multiple Drug Resistance) was related to the porcine source. These results contribute to the knowledge of extra-intestinal E. coli populations; which could affect the reproductive performance of females.

Nisin Z produced by Lactococcus lactis from bullfrog hatchery is active against Citrobacter freundii, a red-leg syndrome related pathogen.

Lactococcus lactis subsp. lactis CRL 1584 isolated from a bullfrog hatchery produces a bacteriocin that inhibits both indigenous Citrobacter freundii (a Red-Leg Syndrome related pathogen) and Lactobacillus plantarum, and Listeria monocytogenes as well. Considering that probiotics requires high cell densities and/or bacteriocin concentrations, the effect of the temperature on L. lactis growth and bacteriocin production was evaluated to find the optimal conditions. Thus, the growth rate was maximal at 36 °C, whereas the highest biomass and bacteriocin activity was achieved between 20 and 30 °C and 20-25 °C, respectively. The bacteriocin synthesis was closely growth associated reaching the maximal values at the end of the exponential phase. Since bacteriocins co-production has been evidenced in bacterial genera, a purification of the bacteriocin/s from L. lactis culture supernatants was carried out. The active fraction was purified by cationic-exchange chromatography and then, a RP-HPLC was carried out. The purified sample was a peptide with a 3353.05 Da, a molecular mass that matches nisin Z, which turned out to be the only bacteriocin produced by L. lactis CRL 1584. Nisin Z showed bactericidal effect on C. freundii and L. monocytogenes, which increased in the presence L-lactic acid + H2O2. This is the first report on nisin Z production by L. lactis from a bullfrog hatchery that resulted active on a Gram-negative pathogen. This peptide has potential probiotic for raniculture and as food biopreservative for bullfrog meat.

Glycerol production by Oenococcus oeni during sequential and simultaneous cultures with wine yeast strains.

Growth and fermentation patterns of Saccharomyces cerevisiae, Kloeckera apiculata, and Oenococcus oeni strains cultured in grape juice medium were studied. In pure, sequential and simultaneous cultures, the strains reached the stationary growth phase between 2 and 3 days. Pure and mixed K. apiculata and S. cerevisiae cultures used mainly glucose, producing ethanol, organic acids, and 4.0 and 0.1 mM glycerol, respectively. In sequential cultures, O. oeni achieved about 1 log unit at 3 days using mainly fructose and L-malic acid. Highest sugars consumption was detected in K. apiculata supernatants, lactic acid being the major end-product. 8.0 mM glycerol was found in 6-day culture supernatants. In simultaneous cultures, total sugars and L-malic acid were used at 3 days and 98% of ethanol and glycerol were detected. This study represents the first report of the population dynamics and metabolic behavior of yeasts and O. oeni in sequential and simultaneous cultures and contributes to the selection of indigenous strains to design starter cultures for winemaking, also considering the inclusion of K. apiculata. The sequential inoculation of yeasts and O. oeni would enhance glycerol production, which confers desirable organoleptic characteristics to wines, while organic acids levels would not affect their sensory profile.

Characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery.

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.

Cultivable microbiota of Lithobates catesbeianus and advances in the selection of lactic acid bacteria as biological control agents in raniculture.

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.

In vitro inhibition of Citrobacter freundii, a red-leg syndrome associated pathogen in raniculture, by indigenous Lactococcus lactis CRL 1584.

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.

Sugar-glycerol cofermentations by Lactobacillus hilgardii isolated from wine.

Glycerol catabolism was studied in Lactobacillus hilgardii X(1)B from wine, growing on glycerol and limiting glucose or fructose concentrations in anaerobiosis and microaerophilia. Glycerol consumption occurred simultaneously with sugar use, and it was higher with fructose as a cofermenting sugar in microaerophilia. Enzymatic activities of the glycerol kinase and glycerol dehydratase pathways were detected in both incubation conditions. In anaerobiosis, the main products were lactate, acetate, ethanol, and the intermediary product of the glycerol dehydratase pathway, 3-hydroxypropionaldehyde. However, in microaerophilia, 1,3-propanediol was also detected. In anaerobic glucose + glycerol and fructose + glycerol cultures as in microaerophilic glucose + glycerol cultures, glycerol was degraded mainly through the reductive pathway. However, when L. hilgardii X(1)B was grown on fructose + glycerol cultures in microaerophilia, glycerol dissimilation occurred mainly via the glycerol kinase way. According to these results, L. hilgardii X(1)B can degrade glycerol by producing 3-hydroxypropionaldehyde and acetic acid, both undesirable products for wine sensorial quality.

Characterization of the beneficial properties of lactobacilli isolated from bullfrog (Rana catesbeiana) hatchery.

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.

Transport of glycerol by Pediococcus pentosaceus isolated from wine.

Pediococcus pentosaceus N(5)p is a strain isolated from wine that uses glycerol as its sole carbon source, mainly via the glycerol kinase pathway. The transport of glycerol was investigated in resting cells of this strain. Glycerol uptake followed a Michaelis-Menten relationship with an observed apparent K(m) of 33 microM and a V(max) of 2.5 nmol/min/mg of cell protein. The transport system was specific for glycerol, which was present in the cells grown either on glycerol or glucose suggesting its constitutive nature. The presence of uptake when resting cells were treated with HgCl(2) and the absence of counterflow indicate that facilitated diffusion is not involved in glycerol transport. On the other hand, glycerol uptake was inhibited by the metabolic poisons that affect ATP availability by acting on either electron transport or ATPase activity, and by the proton-conducting uncouplers without any effect on glycerol kinase activity. The restoration of glycerol uptake in de-energized cells by the addition of glucose and low concentration of cyanide-m-chlorophenyl hydrazone was achieved. These results, the first in the genus Pediococcus, provide evidence for an energy-dependent uptake of glycerol that involves the proton motive force directly or coupled with ATP synthesis.

Glycerol metabolism of Lactobacillus rhamnosus ATCC 7469: cloning and expression of two glycerol kinase genes.

Lactobacillus rhamnosus ATCC 7469 was able to grow in glycerol as the sole source of energy in aerobic conditions, producing lactate, acetate, and diacetyl. A biphasic growth was observed in the presence of glucose. In this condition, glycerol consumption began after glucose was exhausted from the culture medium. Glycerol kinase activity was detected in L. rhamnosus ATCC 7469, a characteristic of microorganisms which catabolize glycerol in aerobic conditions. Genetic analysis revealed that this strain possesses two glycerol kinase genes: gykA and glpK, that encode for two different glycerol kinases GykA and GlpK, respectively. The glpK geneis associated in an operon with alpha-glycerophosphate oxidase (glpO) and glycerol facilitator (glpF) genes. Transcriptional analysis revealed that only glpK is expressed when L. rhamnosus was grown on glycerol.