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

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

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

Response of Bacillus cereus ATCC 14579 to challenges with sublethal concentrations of enterocin AS-48

Background: Enterocin AS-48 is produced by Enterococcus faecalis S48 to compete with other bacteria in their environment. Due to its activity against various Gram positive and some Gram negative bacteria it has clear potential for use as a food preservative. Here, we studied the effect of enterocin AS-48 challenges on vegetative cells of Bacillus cereus ATCC 14579 by use of transcriptome analysis. Results: Of the 5200 genes analysed, expression of 24 genes was found to change significantly after a 30 min treatment with a subinhibitory bacteriocin concentration of 0.5 μg/ml. Most of upregulated genes encode membrane-associated or secreted proteins with putative transmembrane segments or signal sequences, respectively. One operon involved in arginine metabolism was significantly downregulated. The BC4206-BC4207 operon was found to be the most upregulated target in our experiments. BC4206 codes for a PadR type transcriptional regulator, while BC4207 codes for a hypothetical membrane protein. The operon structure and genes are conserved in B. cereus and B. thuringiensis species, but are not present in B. anthracis and B. subtilis. Using real-time qPCR, we show that these genes are upregulated when we treated the cells with AS-48, but not upon nisin treatment. Upon overexpression of BC4207 in B. cereus, we observed an increased resistance against AS-48. Expression of BC4207 in B. subtilis 168, which lacks this operon also showed increased resistance against AS-48. Conclusion: BC4207 membrane protein is involved in the resistance mechanism of B. cereus cells against AS-48.

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 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

Ensayo de la bacteriocina AS-48 en la bioconservación de zumos de frutas

La enterocina AS-48 es una bacteriocina de amplio espectro producida por E. faecalis. Alicyclobacillus acidoterrestris es una bacteria formadora de endosporas que causa alteraciones en los zumos de fruta. Se ha estudiado el control de esta bacteria por la enterocina AS-48. Esta bacteriocina es activa frente a todas las cepas ensayadas de Alicyclobacillus acidocaldarius y A. acidoterrestris. En zumos naturales de naranja y manzana incubados a 37ºC, las células de A. acidoterrestris DSMZ 2498 eran inactivadas con una concentración de bacteriocina de 2.5 μg/ml hasta los 14 días. En zumos comerciales con AS-48 (2.5 μg/ml) e inoculados con células vegetativas de estas bacterias, incubados a 37ºC, los recuentos se mantuvieron por debajo del límite de detección hasta los 60 días (en zumos de manzana, melocotón y uva) y de los 90 días (en naranja y piña), mientras se observaba crecimiento en los controles. En los cinco tipos de zumos comerciales de frutas ensayados no se detectó presencia de células de A. acidoterrestris en los 90 días de incubación a las temperaturas de 15 y 5ºC y con una concentración de bacteriocina de 2.5 μg/ml. Las células vegetativas y endosporas tratadas mostraban serias alteraciones en su ultraestructura al microscopio electrónico