La doble faceta del género enterococcus, y su importancia en alimentos

El género Enterococcus incluye a un conjunto de microorganismos extremadamente versátiles, capaces de provocar infecciones hospitalarias principalmente en individuos con enfermedades subyacentes o inmunodeprimidos. Determinados componentes celulares pueden actuar como factores de virulencia en las infecciones enterocócicas, lo que junto con la resistencia adquirida a diferentes antimicrobianos hacen más difícil su erradicación. Los enterococos forman parte de la microbiota intestinal normal de individuos sanos, y están presentes en muchos alimentos. Poseen propiedades tecnológicas de interés como sus actividades glicolítica, lipolítica y proteolítica, y la producción de bacteriocinas. Están presentes en numerosos quesos regionales, jugando un papel importante en su maduración. Esta doble faceta despierta una gran inquietud sobre la seguridad de las cepas presentes en alimentos, y aquellas sociadas a ambientes hospitalarios

Resistencia a antimicrobianos en enterococos aislados de alimentos de origen animal, pescado y mariscos

La incidencia de resistencias a agentes antimicrobianos en alimentos es una preocupación de la que ya se ha hecho eco la Autoridad Europea de Seguridad Alimentaria (EFSA). Dado que los enterococos están presentes con gran frecuencia en alimentos y son considerados como un reservorio de elementos genéticos transmisibles implicados en la diseminación de resistencias, hemos estudiado la incidencia de resistencia a diferentes antimicrobianos en una colección de cepas procedentes de queso, carne, pescado, y marisco. Los resultados obtenidos indican una elevada incidencia de resistencias a antimicrobianos en cepas de enterococos procedentes de queso y carne, siendo también frecuente la presencia de cepas multirresistentes. Por el contrario, las cepas de enterococos procedentes de pescado y marisco presentan una frecuencia menor de resistencia a antimicrobianos

In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties

Lactiplantibacillus pentosus CF2-10 N, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. High throughput sequencing and annotation of genome sequences underline the potential of L. pentosus CF2-10 N as excellent probiotic candidate of vegetable origin. In a previous study we could show the probiotic potential of CF2-10 N in vitro, while in this study in silico analysis of its genome revealed new insights into its safety and functionality. Our findings highlight the microorganism’s ecological flexibility and adaptability to a broad range of environmental niches, food matrices and the gastrointestinal tract. These features are shared by both phylogenetically very close L. pentosus strains (CF2-10 N and MP-10) isolated from the same ecological niche with respect to their genome size (≅ 3.6 Mbp), the presence of plasmids (4–5) and several other properties. Nonetheless, additional and unique features are reported in the present study for L. pentosus CF2-10 N. Notably, the safety of L. pentosus CF2-10 N was shown by the absence of virulence determinants and the determination of acquired antibiotic resistance genes, i.e., resistome, which is mostly represented by efflux-pump resistance genes responsible for the intrinsic resistance. On the other hand, defense mechanisms of L. pentosus CF2-10 N include eight prophage regions and a CRISPR/cas system (CRISPR-I and CRISPR-II) as acquired immune system against mobile elements. Finally, the probiotic potential of this strain was further demonstrated by the presence of genes coding for proteins involved in adhesion, exopolysaccharide biosynthesis, tolerance to low pH and bile salts, immunomodulation, and vitamin and enzyme production. Taken together these results, we propose the use of L. pentosus CF2-10 N as a potential and promising probiotic candidate able to colonize several niches and adapt to different lifestyles. The strain can provide attractive functional and probiotic features necessary for its application as starter culture and probiotic

Biocide tolerance, phenotypic and molecular response of Lactic Acid Bacteria isolated from naturally-fermented Aloreña table olives throughout fermentation to induction by different physico-chemical stresses

Lactic acid bacteria isolated from Aloreña table olives throughout fermentation process were resistant at least to three antibiotics (Casado Muñoz et al., 2014), however they were very sensitive to all biocides tested in this study (MIC below the epidemiological cut-off values “ECOFF” determined in the present study) except 2-15% of Lc. pseudomesenteroides which were resistant to hexachlorophene and Lb. pentosus to cetrimide and hexadecylpiridinium. To give new insights of how LAB become resistant in a changing environment, the effect of different physico-chemical stresses -including antimicrobials- on phenotypic and genotypic responses of LAB was analyzed in the present study. The results obtained indicated that a similar phenotypic response was obtained under all stress conditions tested (antimicrobials, chemicals and UV light) producing changes in susceptibility patterns of antibiotics (increased MICs for ampicillin, chloramphenicol, ciprofloxacin, teicoplanin and tetracycline, while decreased MICs were shown for clindamycin, erythromycin, streptomycin and trimethoprim in the majority of strains). By means of statistical analysis, cross resistance between different antibiotics was detected in all stress conditions. However, expression profiles of selected genes involved in stress/resistance (rpsL, recA, uvrB and srtA genes) were different depending on the stress parameter, LAB species and strain, and also the target gene. We can conclude that, in spite of the uniform phenotypic responses to several stresses, the repertoire of induced and repressed genes were different upon the stress parameter and the LAB strain. So, a search for a target to improve stress tolerance of LAB especially those of importance as starter/protective cultures or as probiotics may depend on the individual screening of each strain although, we could predict the antibiotic phenotypic response to all stresses

Integración de herramientas para la anotación global de genomas procariotas: desde la predicción de genes hasta la definición de rutas metabólicas y reacciones

Motivación: Debido al abaratamieno de la secuenciación de genomas, en los últimos años ha aumentado el número de organismos cuyo genoma ha sido completamente secuenciado, especialmente en el caso de organismos procariotas, con genomas más cortos. Pero una vez que se dispone de la secuencia de ADN de un nuevo genoma, es necesario extraer toda la información que encierran y que puede resolver preguntas biológicas relacionadas con el organismo en estudio, como por ejemplo el tipo de compuestos que pueden metabolizar o la relación evolutiva que presentan con respecto a otros organismos cercanos. Para ello, hay que llevar a cabo una anotación tanto estructural como funcional del genoma, tratando de situar sus elementos genómicos y la función que llevan a cabo, todo lo puede llevar a definir el mapa metabólico del organismo y sus relaciones con la información almacenada en las bases de datos actuales.Métodos: La propuesta de este trabajo para la anotación de un genoma bacteriano sigue el siguiente flujo de trabajo: partiendo del genoma secuenciado de una bacteria se emplea el programa Prodigal para la localizar la posición de todos los genes codificantes de proteínas. Para completar el paso de anotación estructural, se localizan también los genes no codificantes de proteínas, utilizando para ello el programa Infernal, entrenado para encontrar genes de ARNr, ARNt y ARNnc. Una vez disponemos de las coordenadas de todos los genes, se pasa a la anotación funcional de aquellos que codifican para proteínas, utilizando para ello el programa Sma3s, el cual ya ha demostrado buenos resultados en genomas bacterianos. Por último, a partir de la anotación generada, se usa el programa Pathway Tools para predecir las rutas metabólicas y reacciones enzimáticas a partir de los “EC number” (códigos de enzimas),  los términos GO (Gene Ontology) y la concordancia con los nombres de genes anotados, lo que nos da como resultado final el mapa metabólico completo de la bacteria, así como un mapa genómico que incluye una predicción de operones. Asimismo, las proteínas de membrana son también predichas, mediante una asignación manual de la reacción de transporte.Resultados y conclusiones: El flujo de trabajo propuesto ha sido ensayado ya con dos genomas bacterianos completos, y ha demostrado que en apenas una semana se puede concluir toda la anotación, teniendo una visión global del genoma analizado la cual puede ayudar en análisis más particulares

New insights into the molecular effects and probiotic properties of Lactobacillus pentosus pre-adapted to edible oils

In this study, the survival and growth of seven probiotic Lactobacillus pentosus strains isolated from Aloreña green table olives in the presence of vegetable-based edible oils (i.e., sunflower, olive, linseed, soy, corn, almond and argan) and mint essential oil were determined for the first time. Slight decreases in bacterial viability were observed depending on the strain and oil exposure, mainly mint essential oil. However, pre-adaptating the strains to the corresponding oils significantly increased their cell viabilities. As such, this study examined whether pre-adaptating probiotic L. pentosus strains with oils will constitute a new strategy to increase stress resistance, e.g., acids (pH 1.5) or bile (up to 3.6%) in food production and/or during digestion, and improve functional probiotic properties. Improvements in stress resistance (acidic and bile conditions) were noticed in some pre-adapted strains with oils; further, pre-adaptations with olive, argan, sunflower and linseed oils induced gene expression (e.g., fus, rpsL, pgm, groEL, enol and prep) for moonlighting proteins involved in several stress responses and other functions. As such, pre-adaptation with vegetable edible oils may represent a novel approach for manufacturing probiotic products by improving the stability of bacteria during industrial processes that would otherwise reduce their viability and functionality

Comparative proteomic analysis of a potentially probiotic Lactobacillus pentosus MP-10 for the identification of key proteins involved in antibiotic resistance and biocide tolerance

Probiotic bacterial cultures require resistance mechanisms to avoid stress-related responses under challenging environmental conditions; however, understanding these traits is required to discern their utility in fermentative food preparations, versus clinical and agricultural risk. Here, we compared the proteomic responses of Lactobacillus pentosus MP-10, a potentially probiotic lactic acid bacteria isolated from brines of naturally fermented Aloreña green table olives, exposed to sub-lethal concentrations of antibiotics (amoxicillin, chloramphenicol and tetracycline) and biocides (benzalkonium chloride and triclosan). Several genes became differentially expressed depending on antimicrobial exposure, such as the up-regulation of protein synthesis, and the down-regulation of carbohydrate metabolism and energy production. The antimicrobials appeared to have altered Lb. pentosus MP-10 physiology to achieve a gain of cellular energy for survival. For example, biocide-adapted Lb. pentosus MP-10 exhibited a down-regulated phosphocarrier protein HPr and an unexpressed oxidoreductase. However, protein synthesis was over-expressed in antibiotic- and biocide-adapted cells (ribosomal proteins and glutamyl-tRNA synthetase), possibly to compensate for damaged proteins targeted by antimicrobials. Furthermore, stress proteins, such as NADH peroxidase (Npx) and a small heat shock protein, were only overexpressed in antibiotic-adapted Lb. pentosus MP-10. Results showed that adaptation to sub-lethal concentrations of antimicrobials could be a good way to achieve desirable robustness of the probiotic Lb. pentosus MP-10 to various environmental and gastrointestinal conditions (e.g., acid and bile stresses)

In silico mapping of microbial communities and stress responses in a porcine slaughterhouse and pork products through its production chain, and the efficacy of HLE disinfectant

The use of shotgun metagenomic sequencing to understand ecological-level spread of microbes and their genes has provided new insights for the prevention, surveillance and control of microbial contaminants in the slaughterhouse environment. Here, microbial samples were collected from products and surrounding areas though a porcine slaughter process; shotgun metagenomic DNA-sequencing of these samples revealed a high community diversity within the porcine slaughterhouse and pork products, in zones originating from animal arrival through to the sale zones. Bacteria were more prevalent in the first zones, such as arrival- and anesthesia-zones, and DNA viruses were prevalent in the scorching-and-whip zone, animal products and sale zone. Data revealed the dominance of Firmicutes and Proteobacteria phyla followed by Actinobacteria, with a clear shift in the relative abundance of lactic acid bacteria (mainly Lactobacillus sp.) from early slaughtering steps to Proteobacteria and then to viruses suggesting site-specific community compositions occur in the slaughterhouse. Porcine-type-C oncovirus was the main virus found in slaughterhouse, which causes malignant diseases in animals and humans. As such, to guarantee food safety in a slaughterhouse, a better decipher of ecology and adaptation strategies of microbes becomes crucial. Analysis of functional genes further revealed high abundance of diverse genes associated with stress, especially in early zones (animal and environmental surfaces of arrival zone with 57,710 and 40,806 genes, respectively); SOS responsive genes represented the most prevalent, possibly associated with genomic changes responsible of biofilm formation, stringent response, heat shock, antimicrobial production and antibiotic response. The presence of several antibiotic resistance genes suggests horizontal gene transfer, thus increasing the likelihood for resistance selection in human pathogens. These findings are of great concern, with the suggestion to focus control measures and establish good disinfection strategies to avoid gene spread and microbial contaminants (bacteria and viruses) from the animal surface into the food chain and environment, which was achieved by applying HLE disinfectant after washing with detergent