Estudio de los determinantes genéticos de resistencias a biocidas y su papel en la resistencia cruzada con antibióticos en bacterias de origen alimentario

[ES] Nuestro objetivo fue investigar la incidencia de la resistencia a antibióticos y biocidas en bacterias de origen alimentario. Para ello, hemos analizado mediante procedimientos dependientes e independientes de cultivo dicha resistencia a lo largo de la cadena de producción de la carne, así mismo hemos evaluado fenotípicamente y genotípicamente la resistencia en dos grupos microbianos patógenos tales como Pseudomonas sp. y Enterococcus sp. Nuestros resultados indicaron una alta resistencia a antibóticos de uso común en la clínica y biocidas frecuentemente usados en la industria alimentaria, así mismo hemos revelado los mecanismos moleculares implicados en dicha resistencia (genes específicos y bombas de exporte). Además, mediante estudios estadísticos, hemos determinado la interconexión entre ambos antimicrobianos la cual se ve reflejada por una resistencia cruzada entre algunos biocidas y antibióticos sugiriendo así que tipo de medidas a adoptar para evitar la diseminación de genes de resistencia a lo largo de la cadena alimentaria.[EN] Our objective was to investigate the incidence of antibiotic and biocide resistance in food-borne bacteria. For this, such resistance was analyzed by culture dependent and independent methods throughout the chain of meat production, also phenotypic and genotypic resistance was evaluated in both pathogens microbial groups such as Pseudomonas sp. and Enterococcus sp. Our results indicated a high resistance to antibiotics commonly used in clinical treatments and to biocides frequently used in the food industry, also we have revealed the molecular mechanisms involved in this resistance (specific genes and efflux pumps) . In addition, we have determined by means of statistical studies the interconnection between both antimicrobials which was reflected by a cross-resistance between sorne biocides and antibiotics thus suggesting that such measures to be taken to prevent the spread of resistance genes throughout the food chain.Tesis Univ. Jaén. Departamento de Ciencias de la Salud. Leída el 7 de noviembre de 201

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

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

New insights into the role of plasmids from probiotic Lactobacillus pentosus MP-10 in Aloreña table olive brine fermentation

In silico analysis of Lactobacillus pentosus MP-10 plasmids (pLPE-1 to pLPE-5) suggests that plasmid-borne genes mediate the persistence of lactobacilli during olive fermentation and enhance their probiotic properties and their competitiveness in several ecological niches. The role of plasmids in the probiotic activities of L. pentosus MP-10 was investigated by plasmid-curing process which showed that plasmids contribute in increased metal tolerance and the biosequestration of several metals such as iron, aluminium, cobalt, copper, zinc, cadmium and mercury. Statistically significant differences in mucin adhesion were detected between the uncured and the cured L. pentosus MP-10, which possibly relied on a serine-rich adhesin (sraP) gene detected on the pLPE-2 plasmid. However, plasmid curing did not affect their tolerance to gastro-intestinal conditions, neither their growth ability under pre-determined conditions, nor auto-aggregation and pathogen co-aggregation were changed among the cured and uncured L. pentosus MP-10. These findings suggest that L. pentosus MP-10 plasmids play an important role in gastro-intestinal protection due to their attachment to mucin and, thus, preventing several diseases. Furthermore, L. pentosus MP-10 could be used as a bioquencher of metals in the gut, reducing the amount of these potentially toxic elements in humans and animals, food matrices, and environmental bioremediation

Changes in resistome profile of potential probiotic Lactiplantibacillus pentosus in response to edible oil adaptation

Despite increasing interest to investigate horizontal gene transfer as a leading cause of antibiotic resistance spread, the resistome is not only influenced by the influx and efflux of genes in different environments. Rather, the expression of existing genes under different stress conditions requires special attention. This study determined whether pre-adapting Lactiplantibacillus pentosus strains, isolated from Aloreña green table olives, to vegetable-based edible oils influence their phenotypic and genotypic responses to antibiotics. This has significant diet, food matrix, gut health, and food safety concerns. Pre-adapting L. pentosus strains to oils significantly changed their susceptibility profile to antibiotics. However, results generally differed among the three strains; although changes in the Minimum Inhibitory Concentration (MIC) of antibiotics occurred, it depended on the L. pentosus strain and the oil used for adaptation. The pre-adaptation of L. pentosus strains with olive, sunflower, argan and linseed oils induced gene expressions (e.g., rpsL, recA and uvrB) in several stress responses. Thus, to analyze this fact in-depth, transcriptional changes were reported in the selected potential probiotic L. pentosus CF2-10 adapted with olive or sunflower, rerouting its metabolic pathways to export toxic molecules through efflux pumps and ABC transporters. Pre-adaptation of some lactobacilli with olive or sunflower oils may represent a novel approach for manufacturing probiotic products with improved stability, functionality and robustness

Efficacy of "HLE"—a multidrug efflux-pump inhibitor—as a disinfectant against surface bacteria

We evaluated the efficacy of a new disinfectant product, HLE, to inhibit multiple species of planktonic and biofilm bacterial cultures. The HLE disinfectant comprised of EDTA, lactic acid and hydrogen peroxide, and our data indicated that the disinfectant had effective antimicrobial and anti-biofilm activity even at low concentrations (0.15% to 0.4% HLE, v/v). Furthermore, the HLE disinfectant destabilized biofilm structures eradicated them due to the synergistic effect of EDTA and both antimicrobials (lactic acid and hydrogen peroxide), as revealed by confocal laser scanning microscopy. Additionally, sub-inhibitory concentrations of HLE disinfectant, with EDTA as an efflux pump inhibitor, inhibited the expression of multidrug EfrAB, NorE and MexCD efflux pumps in both planktonic and biofilm cultures. This could provide an alternative way to disinfect surfaces to avoid spreading multi-drug resistant strains in the food chain and the environment by decreasing efflux pump expression and consequently reducing the antibiotic selective pressure caused by systemic antibiotics and disinfectant use

Transcriptomic profile and probiotic properties of Lactiplantibacillus pentosus pre-adapted to edible oils

In this study, we determined whether pre-adapting Lactiplantibacillus pentosus strains, isolated from Aloreña green table olives, to vegetable-based edible oils improved their robustness and functionality; this may have great importance on their stress response during fermentation, storage, and digestion. Pre-adapting the strains to the corresponding oils significantly increased their probiotic functionality (e.g., auto-aggregation, co-aggregation with pathogens, and mucin adhesion), although results depended on the strain and the oil used for pre-adaptation. As such, we selected olive-adapted (TO) L. pentosus AP2-16, which exhibited improved functionality, and subjected it to transcriptomic profiling with the aim to understand the molecular mechanisms involved in the adaptation and the increased functionality. Global transcriptomic analysis of oil-adapted (olive or almond) and non-adapted (control) L. pentosus AP2-16 realized that 3,259 genes were expressed, with 2,779 mapped to the reference database. Comparative transcriptomic analysis showed that 125 genes (olive vs. control) and 108 genes (olive vs. almond) became significantly differentially expressed. TO L. pentosus AP2-16 responded by rerouting its metabolic pathways to balance energy production and storage, cell growth and survivability, host interactions (glycoconjugates), and other physiological features. As such, the pre-adaptation of lactobacilli with olive oil switches their transcriptional network to regulate robustness and functionality, possibly representing a novel approach toward the design and manufacture of probiotic products with improved stability and functionality

Food processing as a risk factor for antimicrobial resistance spread along the food chain

Documento post-printFarms and food industries rely to a large extent on the use ofbiocides as disinfectants and other antimicrobial agents andpreservatives with antimicrobial properties in order to providefood of high microbiological quality and safe for consumers.However, in the last decades it has become apparent that long-term sub-lethal exposure to these antimicrobial agents canexert a selective pressure leading to the emergence and spreadof microbial strains with a reduced susceptibility to the usedantimicrobials, which can persistently colonize food-processing environments and recurrently contaminate food. Inaddition, it may induce resistance to unrelated and clinicallyrelevant antibiotics, in a phenomenon known as cross-resistance. This review aims to provide insights on howantimicrobial resistance emergence and spread can beaffected by certain food processing activities and to discussrecent research focused on different pathways through whichbiocides and other antimicrobials could co-select for bacteriaresistant to clinically relevant antibiotics.S