Antonio Sánchez Valenzuela

A classification system for plasmids from enterococci and other Gram-positive bacteria.

A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiating genes (rep), alignment of these sequences and using a cutoff value of 80% identity on both protein and DNA level, 19 replicon families (rep-families) were defined together with several unique sequences. The prevalence of these rep-families was tested on 79 enterococcal isolates from a collection of isolates of animal and human origin. Difference in prevalence of the designed rep-families were detected with rep(9) being most prevalent in Enterococcus faecalis and rep(2) in Enterococcus faecium. In 33% of the tested E. faecium and 32% of the tested E. faecalis no positive amplicons were detected. Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup.

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.

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.

Bacteriocin-producing Lactobacillus strains isolated from poto poto, a Congolese fermented maize product, and genetic fingerprinting of their plantaricin operons.

Thirty one bacteriocin-producing Lactobacillus isolates were identified among 135 lactobacilli isolated from the Congolese fermented maize product poto poto, during the preparation and from the finished product. Using species-specific PCR and 16S rRNA gene sequencing, 28 and 3 isolates were identified as L. plantarum and L. fermentum, respectively. Cluster analysis of RAPD-PCR fingerprints revealed two main groups (G1 and G2) plus the L. fermentum isolate C4-13. Group G1 contained 23 isolates with a similarity coefficient > 74.5%, and could be divided in two subgroups (G1-1, G1-2) each with several branches, plus the L. plantarum isolate C11. Group G2 contained 8 isolates with a similarity coefficient > 86%, with two main branches. Using PCR amplification with specific primers, several genes of the plantaricin cluster found in L. plantarum C11 were identified in the isolates. The number of genes that were detected varied between the strains. The L. fermentum isolate EC11 also contained the plnDEFG genes. PCR amplification of DNA from isolates with primers directed to the upstream and downstream region of the plantaricin cluster generated an amplicon identical to that obtained with DNA from the control strain L. plantarum WCFS1. Amplification products from the positive strains were used for restriction analysis with HindIII, EcoRI and KpnI in separate reactions. Cluster analysis of restriction profiles revealed high similarities for EcoRI and HindII digest profiles, and an identical profile for all KpnI digests. The L. fermentum EC11 isolate clustered with L. plantarum strains in a group with a high correlation coefficient. The results suggest a low degree of diversity in the plantarincin gene cluster. However, other strains that tested positive for individual plantaricin genes may present great heterogeneity in the plantaricin operons. Because of their broad spectra of inhibition (including Escherichia coli, Salmonella enterica, Enterobacter aerogenes, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis), isolates from the present study could be used to improve the safety and storage stability of poto poto.

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.

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.

Characterization of Enterococcus faecalis and Enterococcus faecium from wild flowers.

Wild flowers in the South of Spain were screened for Enterococcus faecalis and Enterococcus faecium. Enterococci were frequently associated with prickypear and fieldpoppy flowers. Forty-six isolates, from 8 different flower species, were identified as E. faecalis (28 isolates) or E. faecium (18 isolates) and clustered in well-defined groups by ERIC-PCR fingerprinting. A high incidence of antibiotic resistance was detected among the E. faecalis isolates, especially to quinupristin/dalfopristin (75%), rifampicin (68%) and ciprofloxacin (57%), and to a lesser extent to levofloxacin (35.7%), erythromycin (28.5%), tetracycline (3.5%), chloramphenicol (3.5%) and streptomycin (3.5%). Similar results were observed for E. faecium isolates, except for a higher incidence of resistance to tetracycline (17%) and lower to erythromycin (11%) or quinupristin/dalfopristin (22%). Vancomycin or teicoplanin resistances were not detected. Most isolates (especially E. faecalis) were proteolytic and carried the gelatinase gene gelE. Genes encoding other potential virulence factors (ace, efaAfs, ccf and cpd) were frequently detected. Cytolysin genes were mainly detected in a few haemolytic E. faecium isolates, three of which also carried the collagen adhesin acm gene. Hyaluronidase gene (hylEfm) was detected in two isolates. Many isolates produced bacteriocins and carried genes for enterocins A, B, and L50 mainly. The similarities found between enterococci from wild flowers and those from animal and food sources raise new questions about the puzzling lifestyle of these commensals and opportunistic pathogens.