Core and Accessory Genome Analysis of Vibrio mimicus.

Vibrio mimicus is an emerging pathogen, mainly associated with contaminated seafood consumption. However, little is known about its evolution, biodiversity, and pathogenic potential. This study analyzes the pan-, core, and accessory genomes of nine V. mimicus strains. The core genome yielded 2424 genes in chromosome I (ChI) and 822 genes in chromosome II (ChII), with an accessory genome comprising an average of 10.9% of the whole genome for ChI and 29% for ChII. Core genome phylogenetic trees were obtained, and V. mimicus ATCC-33654 strain was the closest to the outgroup in both chromosomes. Additionally, a phylogenetic study of eight conserved genes (ftsZ, gapA, gyrB, topA, rpoA, recA, mreB, and pyrH), including Vibrio cholerae, Vibrio parilis, Vibrio metoecus, and Vibrio caribbenthicus, clearly showed clade differentiation. The main virulence genes found in ChI corresponded with type I secretion proteins, extracellular components, flagellar proteins, and potential regulators, while, in ChII, the main categories were type-I secretion proteins, chemotaxis proteins, and antibiotic resistance proteins. The accessory genome was characterized by the presence of mobile elements and toxin encoding genes in both chromosomes. Based on the genome atlas, it was possible to characterize differential regions between strains. The pan-genome of V. mimicus encompassed 3539 genes for ChI and 2355 genes for ChII. These results give us an insight into the virulence and gene content of V. mimicus, as well as constitute the first approach to its diversity.

Molecular and Genomic Characterization of Vibrio mimicus Isolated from a Frozen Shrimp Processing Facility in Mexico.

Vibrio mimicus is a gram-negative bacterium responsible for diseases in humans. Three strains of V. mimicus identified as V. mimicus 87, V. mimicus 92 and V. mimicus 93 were isolated from a shrimp processing facility in Guaymas, Sonora, Mexico. The strains were analyzed using several molecular techniques and according to the cluster analysis they were different, their similarities ranged between 51.3% and 71.6%. ERIC-PCR and RAPD (vmh390R) were the most discriminatory molecular techniques for the differentiation of these strains. The complete genomes of two strains (V. mimicus 87, renamed as CAIM 1882, and V. mimicus 92, renamed as CAIM 1883) were sequenced. The sizes of the genomes were 3.9 Mb in both strains, with 2.8 Mb in ChI and 1.1 Mb in ChII. A 12.7% difference was found in the proteome content (BLAST matrix). Several virulence genes were detected (e.g. capsular polysaccharide, an accessory colonization factor and genes involved in quorum-sensing) which were classified in 16 categories. Variations in the gene content between these genomes were observed, mainly in proteins and virulence genes (e.g., hemagglutinin, mobile elements and membrane proteins). According to these results, both strains were different, even when they came from the same source, giving an insight of the diversity of V. mimicus. The identification of various virulence genes, including a not previously reported V. mimicus gene (acfD) in ChI in all sequenced strains, supports the pathogenic potential of this species. Further analysis will help to fully understand their potential virulence, environmental impact and evolution.

Antimicrobial activity and partial characterization of bacteriocin-like inhibitory substances produced by Lactobacillus spp. isolated from artisanal Mexican cheese.

Lactobacillus spp. from Mexican Cocido cheese were shown to produce bacteriocin-like substances (BLS) active against Staphylococcus aureus,Listeria innocua,Escherichia coli, andSalmonella typhimurium by using the disk diffusion method. Crude extracts of Lactobacillus fermentum showed strong inhibitory activity against Staph. aureus, L. innocua, E. coli, and Salmonella cholerae. Complete inactivation of antimicrobial activity was observed after treatment of crude extracts with proteinase K, pronase, papain, trypsin, and lysozyme, confirming their proteinaceous nature. However, antimicrobial activity was partly lost for some of the crude extracts when treated with α-amylase, indicating that carbohydrate moieties were involved. The antimicrobial activity of the crude extracts was stable at 65°C for 30min over a wide pH range (2-8), and addition of potassium chloride, sodium citrate, ethanol, and butanol did not affect antibacterial activity. However, antimicrobial activity was lost after heating at 121°C for 15min, addition of methanol or Tween 80. Fourteen out of 18 Lactobacillus spp. showed antimicrobial activity against different test microorganisms, and 12 presented bacteriocin-like substances. Generation time and growth rate parameters indicated that the antimicrobial activity of crude extracts from 3 different strains was effective against the 4 indicator microorganisms. One of the crude extracts showed inhibition not only against gram-positive but also against gram-negative bacteria. Bacteriocin-like substances produced by this specific Lactobacillus strain showed potential for application as a food biopreservative.

Antioxidant and antimicrobial activity of commercial propolis extract in beef patties.

The objective of this study was to evaluate the efficacy of propolis extract (PE) to reduce lipid oxidation and microbial growth on beef patties during refrigerated storage. Beef patties were manufactured by incorporating PE in 4 different treatments: (1) Control (no PE addition); (2) commercial propolis 1 (2% w/w; CP1); (3) commercial propolis 2 (2% w/w; CP2); and (4) noncommercial propolis (2% w/w; NCP). Raw patties were wrapped with polyvinyl chloride and stored at 2 °C for 8 d. Total phenolic content (TPC), free-radical scavenging activity (FRS), and polyphenolic content of the PE were evaluated using high-performance liquid chromatography (HPLC). Lipid oxidation (thiobarbituric acid-reactive substances (TBARS), conjugated dienes (CnDs), metmyoglobin (MetMb%), pH variation, and color (L*, a*, b*, C*, and h*), and microbial growth (mesophilic and psychrotrophic bacteria) of patty samples were measured. NCP treatment demonstrated the highest FRS (64.8% at 100 µg/mL), which correlated with TPC and the presence of polyphenolic compounds. Lipid oxidation (78.54%, TBARS; 45.53%, CnD; 58.57%, MetMb) and microbial mesophilic and psychrotrophic growth (19.75 and 27.03%, respectively) values were reduced by NCP treatment in refrigerated samples after 8 d. These results indicate that PE has great potential as a natural antioxidant and antimicrobial additive to extend the shelf life of beef patties.

Draft Genome Sequence of Vibrio mimicus Strain CAIM 602T.

Vibrio mimicus is a Gram-negative bacterium associated with gastrointestinal diseases in humans around the world. We report the complete genome sequence of the Vibrio mimicus strain CAIM 602(T) (CDC1721-77, LMG 7896(T), ATCC 33653(T)).

Actividad antibacteriana de lactoferrina bovina y lactoferrina porcina sobre Escherichia Coli K88+ / Antibacterial Activity of Bovine and Porcine Lactoferrins Against Escherichia coli K88+

Se comparó el efecto antibacteriano de las lactoferrinas (Lfs) bovina y porcina sobre Escherichia coli K88+ (E. coli K88+), uno de los principales agentes etiológicos de las diarreas en lechones en el hemisferio norte. Las Lfs se purificaron por cromatografía de intercambio iónico, confirmando su pureza por electroforesis en condiciones desnaturalizantes y reductoras (SDS-PAGE) en geles al 8% y por inmuno-detección con anticuerpos anti- lactoferrina. La actividad bacteriostática se probó utilizando concentraciones de 0,5 y 1,0 mg/mL de Holo (saturada de hierro) y Apo-Lf (libre de hierro). En todos los casos la actividad bacteriostática de las Holo-Lfs fue insignificante, mientras que en ambas concentraciones, la Apo-Lf bovina mostró un mayor efecto en la inhibición del crecimiento de la E. coli K88+ que la Apo-Lf porcina. La actividad bactericida se ensayó utilizando concentraciones de 2,0; 4,0; 6,0 y 8,0 mg/mL de Lfs bovina o porcina. La Lf bovina mostró efecto bactericida a una concentración de 8 mg/mL, mientras que la Lf porcina no presentó este efecto. Los resultados indican que la fuente bovina puede ser útil en la prevención de diarreas en lechones.

The antibacterial effect of bovine lactoferrin (Lf) towards Escherichia coli K88+ (E. coli K88+) was compared with that of porcine Lf. E. coli K88+ is one of the main etiological agents of piglet diarrhea in the northern hemisphere. Lactoferrins (Lfs) were purified by ion exchange chromatography and further analyzed by electrophoresis using 8% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and immuno detection with anti-lactoferrin antibodies. Bacteriostatic effect was assayed using 0.5 and 1.0 mg/mL of Holo-Lf (iron saturated) or Apo-Lf (iron free). In all test the holo-LFs showed negligible bacteriostatic activity while for Apo-Lfs the bovine protein had the highest activity. Bactericide activity was assayed using bovine or porcine Lf concentrations of 2.0, 4.0, 6.0 y 8.0 mg/mL. Bovine Lf had bactericide activity at 8.0 mg/mL while no growth inhibition was observed with porcine Lf. These results suggest that bovine Lf may serve in the prophylaxis of piglets’ diarrhea.

Caracterización polifásica de salmonella spp: Aislada de campos agrícolas de melón (cucumis melo) y cilantro (coriandrum sativum) / Polyphasic typing of salmonella spp: Isolated from cantaloupe melon (cucumis melo) and coriander (coriandrum sativum) fields / Caracterização polifásica de salmonella spp: Isolada de campos agrícolas de melão (cucumis melo) e coentro (coriandrum sativum)

Se evaluó la diversidad serológica y genética de cepas de Salmonella spp. aislada de campos de cultivo de melón Cantaloupe (Cucumis melo) y cilantro (Coriandrum sativum). Para identificar al patógeno se realizaron pruebas bioquímicas, serología, susceptibilidad a antibióticos y caracterización por PFGE. Se encontraron 11 muestras contaminadas de los campos de cilantro y 7 de los de melón. Se aisló Salmonella anatum y S. give, principalmente. Solo una de las cepas presentó resistencia a tobramicina. El PFGE mostró que cepas del mismo serogrupo, produjeron patrones electroforéticos similares y el análisis polifásico desplegó cinco grupos distintos con similitud menor al 35%. Es necesario intensificar las buenas prácticas agrícolas y de producción, ya que los serogrupos identificados han sido involucrados en brotes epidémicos. Por otro lado, el análisis polifásico mostró variabilidad genómica y además permite suponer que las cepas de S. give son clonales, no así para S. anatum, que estuvo presente en diferentes muestras.

The serological and genetic diversity of Salmonella spp. strains isolated from Cantaloupe melon (Cucumis melo) and coriander (Coriandrum sativum) fields were evaluated. Biochemical test, serology, antibiotic resistance and PFGE were performed to identify isolated strains. Eleven contaminated samples from coriander fields were found, and seven from melon fields. The serogroups identified were mainly Salmonella anatum and S. give. Only one strain was resistant to tobramicyn. PFGE showed five electrophoretic profiles, in agreement with the identified serotypes and in the polyphasic analysis five clusters were observed (<35% similarity). It is necessary to implement good agricultural and production practices, since identified serogroups have been involved in epidemic outbreaks. Moreover, the polyphasic analysis showed genomic variability and also suggests that strains of S. give are clonal. It is not the same for S. anatum strains, which were isolated from different samples.

Avaliou-se a diversidade sorológica e genética de cepas de Salmonella spp. isolada de campos de cultivo de melão Cantalupo (Cucumis melo) e coentro (Coriandrum sativum). Para identificar ao patógeno foram realizadas provas bioquímicas, sorologia, suscetibilidade a antibióticos e caracterização por PFGE. Encontraram-se 11 amostras contaminadas dos campos de coentro e 7 dos de melão. Isolou-se Salmonella anatum e S. give, principalmente. Somente uma das cepas apresentou resistência à tobramicina. O PFGE mostrou que cepas do mesmo sorogrupo, produziram padrões eletroforéticos similares e a análise polifásica desprendeu cinco grupos distintos com similitude menor a 35%. É necessário intensificar as boas práticas agrícolas e de produção, já que os sorogrupos identificados têm sido envolvidos em surtos epidêmicos. Por outro lado, a análise polifásica mostrou variabilidade genômica e além disso permite supor que as cepas de S. give são clonais, não sendo assim para S. anatum, que esteve presente em diferentes amostras.

An alternative method for evaluation of resistance to low pH and bile salts in probiotic characterization of Lactobacillus reuteri strains: a technical note

A considerable amount of attention has been focused on the use of lactobacilli as probiotics. The use of microorganisms as probiotics must meet certain criteria, among these; the resistance to low pH and bile salts is of great importance. Different procedures have been designed to evaluate tolerance to gastrointestinal transit. Since resistance to low pH and bile salts is critical in the evaluation of probiotic strains, the aim of this study was to compare resistance to bile salts for 20 strains of porcine Lactobacillus reuteri that had been isolated from healthy pigs, using two different procedures based on exposure to low pH. Statistical analysis revealed no difference in resistance of the strains to bile salts whether pH was low or not. The use of low pH prior to exposure to bile salts therefore more closely represents physiological conditions that should be used for the evaluation of potential porcine probiotic lactobacilli. Further studies using digestive enzymes are required to evaluate resistance to gastrointestinal transit, since it is an important factor in the evaluation of strains with potential probiotic activity.

Existe un interés creciente en el uso de lactobacilos como probióticos. El uso de microorganismos como probióticos, requiere que éstos cubran ciertos requisitos, dentro de los cuales la resistencia a pH ácido y a sales biliares, se consideran de suma importancia. Actualmente existen distintos procedimientos que permiten evaluar la tolerancia al tránsito gastrointestinal. Debido a que la resistencia a pH ácido y a sales biliares es una condición crítica dentro de la evaluación de cepas probióticas, la finalidad del presente trabajo consistió en comparar la resistencia a sales biliares de 20 cepas de Lactobacillus reuteri aisladas de cerdos sanos, mediante dos procedimientos diferentes basados en la exposición a pH ácido. El análisis estadístico no mostró diferencias en la resistencia de las cepas a sales biliares cuando se realizó una exposición previa a pH ácido. Así, la exposición a pH ácido previo a la incubación con sales biliares, asemeja más a las condiciones fisiológicas que el microorganismo encontrará de manera natural y es una buena alternativa en la evaluación probiótica de lactobacilos de origen porcino. Se requieren estudios que incluyan enzimas digestivas, con el fin de evaluar la resistencia al tránsito gastrointestinal, dado que es uno de los factores más importantes en la evaluación de cepas con potencial actividad probiótica.

Resistance of Enterococcus strains isolated from pigs to gastrointestinal tract and antagonistic effect against Escherichia coli K88.

The intestinal flora plays an important role in health and wellbeing of different organisms. Indigenous microflora can be innocuous or pathogenic. Consumption of food supplemented with beneficial microorganisms as probiotics provides a good health state and this can be maintained and recovered. Currently, probiotic strains of Bifidobacterium and Lactobacillus are widely used in humans as well as animals. Swine industry would benefit with the application of probiotics, mainly to overcome diarrheal diseases produced by different causes, as a pathogenic E. coli K88. The aim of this work was to isolate strains of Enterococcus from gastrointestinal tract of pigs to use them as probiotic. Two strains of E. faecalis, 2 of E. mundii and 7 of E. faecium were isolated with characteristics of resistance to acid pH, tolerance to biliary salts and a high antagonistic activity (>80%) against E. coli K88. Based on their characteristics and species affinity, we believe that these strains could be administered to pigs as a probiotic.

Significant differences between Lactobacillus casei subsp. casei ATCC 393T and a commonly used plasmid-cured derivative revealed by a polyphasic study.

Many studies on Lactobacillus casei subsp. casei (L. casei) have been carried out using strain ATCC 393 (pLZ15-). Four strains of L. casei ATCC 393T and three of ATCC 393 (pLZ15-) were compared using phenotypic methods and many of the available genotyping techniques. These tests showed that strains of ATCC 393T obtained from independent public type-culture collections were significantly different from the plasmid-free (pLZ15-) strains of ATCC 393T. These findings were confirmed by sequencing the first 580 nt (domain I) of the 16S and 23S rDNAs of the strains. Complete sequencing of the 16S rDNA of one representative strain from each group revealed that strain ATCC 393T from culture collections was 99% similar to Lactobacillus zeae ATCC 15820T and that the strain so far considered as L. casei ATCC 393 (pLZ15-) was, in turn, 100% similar to L. casei ATCC 334 and Lactobacillus paracasei subsp. paracasei ATCC 4022. All data obtained in this work indicate that the ancestral strain of ATCC 393 (pLZ15-) might never have been the strain that is now available from culture collections.