Metagenomic insights into microbial contamination in critical healthcare environments and the efficacy of a novel "HLE" disinfectant.

BACKGROUND: Bacterial contamination on inanimate clinical surfaces is directly linked to severe health problems, especially those caused by multidrug resistant (MDR) pathogens. Here, we evaluated the microbial burden in these environments and tested the efficacy of a novel HLE disinfectant solution. METHODS: Microbial contamination of healthcare surfaces [Intensive Care Unit (ICU), Long Period Hospitalization Room (LPHR) and Otolaryngology Consultation (OC)] and the efficacy of HLE disinfectant solution were determined analyzing the viable counts on general and selective media, and also by molecular studies focused on metagenomic and specific qPCR. RESULTS: Different contamination loads were detected with LPHR showing the highest contamination. Treatment with the HLE disinfectant solution curbed the spread of well-adapted pathogens on touched surfaces (ICU, LPHR and OC). Metagenomic analysis of microbial diversity of the Patient Table (most contaminated surface in LPHR) revealed the presence of mainly A. johnsonii and P. putida. Furthermore, functional annotation of toxin, virulence and antibiotic resistance sequences showed a high diversity of Acinetobacter spp. and Pseudomonas spp. In this context, specific qPCR analysis confirmed the efficacy of HLE disinfectant solution against the most prevalent and critical pathogens Pseudomonas sp. and Acinetobacter sp. achieving their complete eradication. CONCLUSION: Given the persistence of detrimental resistant pathogens, the application of HLE disinfection solution could be a highly beneficial and effective option -used either alone or in combination-for infection prevention and control with the aim to eliminate microbial pathogens and their genes from contaminated contact-surfaces and thus limit the spread to humans and other ecological niches.

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.

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.

Associations between the Severity of Sarcopenia and Health-Related Quality of Life in Community-Dwelling Middle-Aged and Older Adults.

(1) Background: The aim of this study was to analyze the associations between severity of sarcopenia and health-related quality of life (HRQoL) among community-dwelling middle-aged and older adults. (2) Methods: A cross-sectional study involving 304 older-adult participants was used to assess the severity of sarcopenia by measuring muscle strength (handgrip dynamometer), muscle mass (bioelectrical impedance analysis), and physical performance (Timed Up-and-Go test). The generic 36-item Short-Form Health Survey (SF-36) was used to evaluate HRQoL. Anxiety and depression (Hospital Anxiety and Depression Scale) as well as age were considered as possible confounders. Probable sarcopenia was determined by low muscle strength; confirmed sarcopenia was defined by the presence of both low muscle strength and muscle mass; and severe sarcopenia was defined by low muscle strength and mass along with poor physical performance. (3) Results: The linear regression analysis showed that the presence of probable sarcopenia was associated with the SF-36 domains physical role (adjusted R2 = 0.183), general health (adjusted R2 = 0.290), and social functioning (adjusted R2 = 0.299). As for the SF-36 mental (MCS) and physical (PCS) component summary scores, probable sarcopenia, as well as depression and anxiety, remained associated with MCS (adjusted R2 = 0.518), and these three variables, together with age, were linked to PCS (adjusted R2 = 0.340). (4) Conclusions: Probable sarcopenia, but not confirmed or severe sarcopenia, was independently associated with poor HRQoL. More precisely, it was related to PCS and MCS, as well as to the physical role, general health, and social functioning of SF-36 domains.

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.

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.

Fermented Aloreña Table Olives as a Source of Potential Probiotic Lactobacillus pentosus Strains.

A collection of 31 Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives were screened in depth in the present study for their probiotic potential. Several strains could be considered promising probiotic candidates since they showed good growth capacity and survival under simulated gastro-intestinal conditions (acidic pH of 1.5, up to 4% of bile salts and 5 mM of nitrate), good ability to auto-aggregate which may facilitate their adhesion to host cells as multiple aggregates and the subsequent displacement of pathogens. Moreover, co-aggregation of lactobacilli with pathogenic bacteria was shown with Listeria innocua, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis as good defense strategy against gut and food pathogens. Furthermore, they exhibited adherence to intestinal and vaginal cell lines, such property could be reinforced by their capacity of biofilm formation which is also important in food matrices such as the olive surface. Their antagonistic activity against pathogenic bacteria by means of acids and plantaricins, and also their different functional properties may determine their efficacy not only in the gastro-intestinal tract but also in food matrices. Besides their ability to ferment several prebiotics, the new evidence in the present study was their capacity to ferment lactose which reinforces their use in different food matrices including dairy as a dietary adjunct to improve lactose digestibility. Lactobacillus pentosus CF2-10N was selected to have the best probiotic profile being of great interest in further studies. In conclusion, spontaneous fermented Aloreña table olives are considered a natural source of potential probiotic L. pentosus to be included as adjunct functional cultures in different fermented foods.

Complete Genome Sequence of a Potential Probiotic, Lactobacillus pentosus MP-10, Isolated from Fermented Aloreña Table Olives.

We report here a 3,698,214-bp complete genome sequence of a potential probiotic Lactobacillus pentosus strain, MP-10, isolated from brines of naturally fermented Aloreña green table olives; it is considered the largest sequenced genome among lactobacilli to date. The annotated genome sequence revealed the presence of 3,558 open reading frames (ORFs) and 87 structural RNAs.

Biocide tolerance, phenotypic and molecular response of lactic acid bacteria isolated from naturally-fermented Aloreña table to different physico-chemical stresses.

Lactic acid bacteria (LAB) isolated throughout the fermentation process of Aloreña table olives were found to be resistant at least to three antibiotics (Casado Muñoz et al., 2014); however, most were sensitive to the biocides tested in this study (with minimum inhibitory concentrations [MIC] below the epidemiological cut-off values). 2-15% of the isolates were found to be biocide resistant: Leuconostoc Pseudomesenteroides, which were resistant to hexachlorophene, and Lactobacillus pentosus to cetrimide and hexadecylpiridinium. We analyzed the effect of different physico-chemical stresses, including antimicrobials, on the phenotypic and genotypic responses of LAB, providing new insights on how they become resistant in a changing environment. Results indicated that similar phenotypic responses were obtained under three stress conditions: antimicrobials, chemicals and UV light. Susceptibility patterns to antibiotics changed: increasing MICs for ampicillin, chloramphenicol, ciprofloxacin, teicoplanin and tetracycline, and decreasing the MICs for clindamycin, erythromycin, streptomycin and trimethoprim in most strains. Statistically, cross resistance between different antibiotics was detected in all stress conditions. However, expression profiles of selected genes involved in stress/resistance response (rpsL, recA, uvrB and srtA) differed depending on the stress parameter, LAB species and strain, and the target gene. We conclude that, despite the uniform phenotypic response to stresses, the repertoire of induced and repressed genes differs. So, a search for a target to improve stress tolerance of LAB, especially those of importance as starter/protective cultures or probiotics, may depend on the individual screening of each strain, even though 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 over-expressed 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).

Correlation between antibiotic and biocide resistance in mesophilic and psychrotrophic Pseudomonas spp. isolated from slaughterhouse surfaces throughout meat chain production.

The aim of this study was to evaluate biocide susceptibility in mesophilic and psychrotrophic pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which was representative of the region. To determine biocide resistance in pseudomonads, we determined for the first time the epidemiological cut-off values (ECOFFs) of benzalkonium, cetrimide, chlorhexidine, hexachlorophene, P3 oxonia, polyhexamethylene guanidine hydrochloride (PHMG), topax 66 and triclosan being generally very similar in different Pseudomonas spp. with some exceptions. Thus, resistance of pseudomonads was mainly shown to triclosan, and in lesser extent to cetrimide and benzalkonium chloride depending on the species, however they were highly susceptible to industrial formulations of biocides. By means of statistical analysis, positive correlations between antibiotics, biocides and both antimicrobials in pseudomonads were detected suggesting a co- or cross resistance between different antimicrobials in goat and lamb slaughterhouse environment. Cross-resistance between biocides and antibiotics in pseudomonads were especially detected between PHMG or triclosan and different antibiotics depending on the biocide and the population type. Thus, the use of those biocides as disinfectant in slaughterhouse zones must be carefully evaluated because of the selection pressure effect of antimicrobials on the emergence of resistant bacteria which could be spread to the consumer. It is noteworthy that specific industrial formulations such as topax 66 and oxonia P3 showed few correlations with antibiotics (none or 1-2 antibiotics) which should be taken into consideration for disinfection practices in goat and lamb slaughterhouse.

Diversity, distribution and quantification of antibiotic resistance genes in goat and lamb slaughterhouse surfaces and meat products.

The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated 'hot spots.' The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination.

Role of EfrAB efflux pump in biocide tolerance and antibiotic resistance of Enterococcus faecalis and Enterococcus faecium isolated from traditional fermented foods and the effect of EDTA as EfrAB inhibitor.

Enterococcus faecalis and Enterococcus faecium isolated from various traditional fermented foods of both animal and vegetable origins have shown multidrug resistance to several antibiotics and tolerance to biocides. Reduced susceptibility was intra and inter-species dependent and was due to specific and unspecific mechanisms such as efflux pumps. EfrAB, a heterodimeric ABC transporter efflux pump, was detected in 100% of multidrug resistant (MDR) E. faecalis strains and only in 12% of MDR E. faecium strains. EfrAB expression was induced by half of minimum inhibitory concentration (MIC) of gentamicin, streptomycin and chloramphenicol. However, expression of efrA and efrB genes was highly dependent on the strain tested and on the antimicrobial used. Our results indicated that 3 mM EDTA highly reduced the MICs of almost all drugs tested. Nevertheless, the higher reductions (>8 folds) were obtained with gentamicin, streptomycin, chlorhexidine and triclosan. Reductions of MICs were correlated with down-regulation of EfrAB expression (10-140 folds) in all three MDR enterococci strains. This is the first report describing the role of EfrAB in the efflux of antibiotics and biocides which reflect also the importance of EfrAB in multidrug resistance in enterococci. EDTA used at low concentration as food preservative could be one of the best choices to prevent spread of multidrug resistant enterococci throughout food chain by decreasing EfrAB expression. EfrAB could be an attractive target not only in enterococci present in food matrix but also those causing infections as well by using EDTA as therapeutic agent in combination with low doses of antibiotics.

Antibiotic multiresistance analysis of mesophilic and psychrotrophic Pseudomonas spp. isolated from goat and lamb slaughterhouse surfaces throughout the meat production process.

The aim of this study was to investigate the phenotypic and genotypic antibiotic resistance profiles of pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which were representative of areas that are possible sources of meat contamination. Mesophilic (85 isolates) and psychrotrophic (37 isolates) pseudomonads identified at the species level generally were resistant to sulfamethoxazole, erythromycin, amoxicillin, ampicillin, chloramphenicol, trimethoprim, rifampin, and ceftazidime (especially mesophiles), as well as colistin and tetracycline (especially psychrotrophes). However, they generally were sensitive to ciprofloxacin, gentamicin, imipenem, and kanamycin regardless of species identity. Worryingly, in the present study, we found multidrug resistance (MDR) to up to 13 antibiotics, which was related to intrinsic and acquired resistance mechanisms. Furthermore, a link between various antimicrobial resistance genes was shown for beta-lactams and tetracycline, trimethoprim, and sulfonamides. The distribution and resistome-based analysis of MDR pseudomonads in different slaughterhouse zones indicated that the main sources of the identical or related pseudomonad strains were the animals (feet and wool) and the slaughterhouse environment, being disseminated from the beginning, or entrance environment, to the environment of the finished meat products. Those facts must be taken into consideration to avoid cross-contamination with the subsequent flow of mobile resistance determinants throughout all slaughterhouse zones and then to humans and the environment by the application of adequate practices of hygiene and disinfection measures, including those for animal wool and feet and also the entrance environment.

Prevalence of bacteria resistant to antibiotics and/or biocides on meat processing plant surfaces throughout meat chain production.

In order to investigate the prevalence of resistant bacteria to biocides and/or antibiotics throughout meat chain production from sacrifice till end of production line, samples from various surfaces of a goat and lamb slaughterhouse representative of the region were analyzed by the culture dependent approach. Resistant Psychrotrophs (n=255 strains), Pseudomonas sp. (n=166 strains), E. coli (n=23 strains), Staphylococcus sp. (n=17 strains) and LAB (n=82 represented mainly by Lactobacillus sp.) were isolated. Resistant psychrotrophs and pseudomonads (47 and 29%, respectively) to different antimicrobials were frequently detected in almost all areas of meat processing plant regardless the antimicrobial used, although there was a clear shift in the spectrum of other bacterial groups and for this aim such resistance was determined according to several parameters: antimicrobial tested, sampling zone and the bacterial group. Correlation of different parameters was done using a statistical tool "Principal component analysis" (PCA) which determined that quaternary ammonium compounds and hexadecylpyridinium were the most relevant biocides for resistance in Pseudomonas sp., while ciprofloxacin and hexachlorophene were more relevant for psychrotrophs, LAB, and in lesser extent Staphylococcus sp. and Escherichia coli. On the other hand, PCA of sampling zones determined that sacrifice room (SR) and cutting room (CR) considered as main source of antibiotic and/or biocide resistant bacteria showed an opposite behaviour concerning relevance of antimicrobials to determine resistance being hexadecylpyridinium, cetrimide and chlorhexidine the most relevant in CR, while hexachlorophene, oxonia 6P and PHMG the most relevant in SR. In conclusion, rotational use of the relevant biocides as disinfectants in CR and SR is recommended in an environment which is frequently disinfected.

Phenotypic and molecular antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolated from different traditional fermented foods.

A collection of 55 enterococci (41 Enterococcus faecium and 14 E. faecalis strains) isolated from various traditional fermented foodstuffs of both animal and vegetable origins, and water was evaluated for resistance against 15 antibiotics. Lower incidence of resistance was observed with gentamicin, ampicillin, penicillin and teicoplanin. However, a high incidence of antibiotic resistance was detected for rifampicin (12 out of 14 of isolates), ciprofloxacin (9/14), and quinupristin/dalfopristin (8/14) in E. faecalis strains. Enterococcus faecium isolates were resistant to rifampicin (25/41), ciprofloxacin (23/41), erythromycin (18/41), levofloxacin (16/41), and nitrofurantoin (15/41). One Enterococcus faecalis and two E. faecium strains were resistant to vancomycin (MIC>16 µg/mL). Among 55 isolates, 27 (19 E. faecium and eight E. faecalis) were resistant to at least three antibiotics. High level of multidrug resistance to clinically important antibiotics was detected in E. faecalis strains (57% of E. faecalis versus 46% of E. faecium), which showed resistance to six to seven antibiotics, especially those isolated from foods of animal origin. So, it is necessary to re-evaluate the use of therapeutic antibiotics in stock farms at both regional and international levels due to the high number of multiple resistant (MR) bacteria. Fifty-six MR E. faecalis and E. faecium strains selected from this and previous studies (Valenzuela et al., 2008, 2010) were screened by polymerase chain reaction for antibiotic resistance genes, revealing the presence of tet(L), tet(M), ermB, cat, efrA, efrB, mphA, or msrA/B genes. The ABC Multidrug Efflux Pump EfrAB was detected in 96% of E. faecalis strains and also in 13% of E. faecium strains; this is the first report describing EfrAB in this enterococcal species. The efflux pump-associated msrA/B gene was detected in 66.66% of E. faecium strains, but not in E. faecalis strains.