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.

Biocide and copper tolerance in enterococci from different sources.

Antimicrobial resistance in enterococci is a matter of concern. A collection of 272 strains (including 107 Enterococcus faecalis and 165 Enterococcus faecium strains) isolated from meat and dairy products, seafood, vegetable foods, wildflowers, animal feces (ewe, goat, horse, mule), and hospitals were tested for sensitivity to biocides of different classes (quaternary ammonium compounds, a bisphenol, and a biguanide) and copper sulfate. Most isolates were inhibited at 25 mg of benzalkonium chloride or cetrimide per liter or at 2.5 mg of hexadecylpyridinium chloride per liter. Few isolates had MICs higher than 25 mg/liter for benzalkonium chloride (2.2%), cetrimide (0.74%), or hexadecylpyridinium chloride (0.37%), although they were all inhibited at 250 mg/liter. The population response to triclosan was very homogeneous, and most isolates (98.16%) were inhibited at 250 mg of triclosan per liter. Chlorhexidine showed the greatest variability, with MICs in a range from 2.5 to 2,500 mg/liter. Remarkably, 74.57% of isolates from clinical samples required 2,500 mg of chlorhexidine per liter for inhibition, compared to much-lower concentrations required for most isolates from other sources. Enterococci were inhibited by copper sulfate in a concentration range from 4 to 16 mM, with no bimodal distribution. However, most isolates required 12 mM (41.91%) or 16 mM (47.43%) for inhibition. The highest percentages of isolates requiring 16 mM CuSO4 were from vegetable foods, seafood, and wildflowers. The results from the present study suggest intermediate levels of copper tolerance and a low incidence of biocide tolerance in the enterococci investigated, except for chlorhexidine in clinical isolates.

Isolation and identification of Enterococcus faecium from seafoods: antimicrobial resistance and production of bacteriocin-like substances.

A collection of isolates from uncooked seafoods (molluscs, fish, and fish fillets) were identified as Enterococcus faecium species and studied in further detail. Isolates were clustered in well-defined genomic groups according to food origin after ERIC-PCR analysis. Four isolates (FR 1-2, FB 1-3-B, FB 3-1, FTA 1-2) decarboxylated lysine, ornithine, and tyrosine. Isolate FR 1-2 also decarboxylated histidine. Most isolates were sensitive to antibiotics of clinical use, but resistance was detected more frequently towards nitrofurantoin (50%), erythromycin (33.33%) or rifampicin (33.33%) to quinupristin/dalfopristin (12.5%). Resistance to beta-lactams or vancomycin was not detected. The enterococcal antigen A was the presumed virulence trait detected most frequently. None of isolates carried haemolysin/cytolysin genes. Twelve isolates produced anti-listerial activity. Among them, seven isolates also produced bacteriocin-like inhibitory substances against other enterococci, and one isolate was also able to inhibit Staphylococcus aureus. Three isolates only were active against Listeria monocytogenes, and two only were active against enterococci. One bacteriocinogenic isolate carried the enterocin A structural gene, but genes corresponding to other enterocins (EntB, EntP, EntQ, Ent1071, EntL50A/EntL50B, and Ent31) were not detected. Bacteriocin-producing enterococci lacking undesirable traits (such as antibiotic resistance or biogenic amine production) or their produced bacteriocins could be potential candidates to aid in preservation of seafoods and other food products as well.

Risk factors in enterococci isolated from foods in Morocco: determination of antimicrobial resistance and incidence of virulence traits.

A collection of enterococci isolated from meat, dairy and vegetable foods from Morocco including 23 Enterococus faecalis and 15 Enterococcus faecium isolates was studied. All isolates were sensitive to ampicillin, penicillin, and gentamicin. Many E. faecalis isolates were resistant to tetracycline (86.95%), followed by rifampicin (78.26% ciprofloxacin (60.87%), quinupristin/dalfopristin (56.52%), nitrofurantoin (43.47%), levofloxacin (39.13%), erythromycin (21.73%), streptomycin (17.39%), chloramphenicol (8.69%), vancomycin (8.69%), and teicoplanin (4.34%). E. faecium isolates showed a different antibiotic resistance profile: a high percentage were resistant to nitrofurantoin (73.33%), followed by erythromycin (66.60%), ciprofloxacin (66.66%), levofloxacin (60.00%), and rifampicin (26.66%), and only a very low percentage were resistant to tetracycline (6.66%). One isolate was resistant to vancomycin and teicoplanin. The incidence of virulence factors was much higher among E. faecalis isolates, especially for genes encoding for sex pheromones, collagen adhesin, enterococcal endocarditis antigen, and enterococcal surface protein. Isolates with multiple factors (both antibiotic resistance and virulence traits) were also more frequent among E. faecalis isolates, in which one isolate cumulated up to 15 traits. By contrast, several isolates of E. faecium had only very few unwanted traits as compared to only two isolates in E. faecalis. The high abundance of isolates carrying virulence factors and antibiotic resistance traits suggests that the sanitary quality of foods should be improved in order to decrease the incidence of enterococci.