Sónia Ramos

Clonal Diversity of ESBL-Producing Escherichia coli in Pigs at Slaughter Level in Portugal

We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy pigs, and to evaluate their clonality and associated resistance. Forty-nine percent of pigs sampled (n=35/71) in a slaughterhouse in Portugal revealed ESBL-producing E. coli isolates. Most isolates produced CTX-M-1 enzyme (71.4%; n=25/35), followed by CTX-M-9 (11.4%; n=4/35), CTX-M-14 (5.7%; n=2/35), SHV-12 (5.7%; n=2/35), and CTX-M-32 (5.7%; n=2/35). Ninety-four percent of the isolates presented a phenotype of multi-resistance. Most isolates belonged to phylogroups B1 (42.8%; n=15/35) and A (40%; n=14/35). Multilocus sequence typing (MLST) analysis revealed nine sequence types (STs) under six clonal complexes (CCs) and nine singletons, including overrepresentation of CC10 and three new STs (ST2524, ST2525, ST2528). We observed the frequent presence of CTX-M-producing E. coli in pigs at slaughter level, most of them belonging to CC10, commonly recovered from clinical samples.

Genetic characterisation of antibiotic resistance and virulence factors in vanA-containing enterococci from cattle, sheep and pigs subsequent to the discontinuation of the use of avoparcin.

The prevalence of vancomycin resistant-enterococci (VRE) in faecal samples from cattle, sheep and pigs slaughtered for human consumption was evaluated. Enterococci containing the vanA gene were detected in 25.3% and 2.7% of the porcine and ovine samples, respectively, and were identified as Enterococcus faecium. No vanA-containing enterococcal strains were detected in bovine samples. Enterococcal strains with intrinsic vancomycin resistance were detected in seven (9.9%) faecal samples from pigs and in two samples from both cattle and sheep (3.7% and 2.7%, respectively). All vanA-positive isolates from pigs were resistant to tetracycline and erythromycin, and the mobile element Tn916/Tn1545-like transposon was detected in 90.5% of the tetracycline-resistant isolates that contained the tet(M) gene. Although gelatinase and haemolytic activity were not detected, the hyl and cylB virulence genes were found within the VRE strains isolated.

High prevalence of antimicrobial-resistant Escherichia coli from animals at slaughter: a food safety risk

BACKGROUND There has been concern about the increase of antimicrobial resistant bacteria and protection of animal and public health, along with food safety. In the present study, we evaluate the incidence of antimicrobial resistance among 192 strains of Escherichia coli isolated from faecal samples of healthy food-producing animals at slaughter in Portugal. RESULTS Ninety-seven % of the pig isolates, 74% from sheep and 55% from cattle were resistant to one or more antimicrobial agents, with the resistances to ampicillin, streptomycin, tetracycline and trimethoprim-sulfamethoxazole the most common phenotype detected. Genes encoding resistance to antimicrobial agents were detected in most of the resistant isolates. Ninety-three % of the resistant isolates were included in the A or B1 phylogenetic groups, and the virulence gene fimA (alone or in association with papC or aer genes) was detected in 137 of the resistant isolates. Five isolates from pigs belonging to phylogroup B2 and D were resistant to five different antimicrobial agents. CONCLUSION Our data shows a high percentage of antibiotic resistance in E. coli isolates from food animals, and raises important questions in the potential impact of antibiotic use in animals and the possible transmission of resistant bacteria to humans through the food chain.

Effect of vancomycin on the proteome of the multiresistant Enterococcus faecium SU18 strain

UNLABELLED Enterococci are not highly pathogenic bacteria, but the incidence of vancomycin resistance among clinical isolates of this microbial group is steadily increasing, posing a threat to public health. Vancomycin-resistant enterococci are currently some of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. To understand the molecular mechanisms of bacterial resistance to glycopeptides, we obtained proteomic profiles of the vancomycin-resistant Enterococcus faecium SU18 strain treated with and without vancomycin. Fourteen proteins were differentially expressed in SU18, seven of which were up-regulated and seven down-regulated. Proteins involved in the vancomycin resistance mechanism, such as the VanA protein, VanA ligase, VanR and D-Ala-D-Ala dipeptidase, were up-regulated in the presence of vancomycin, while metabolism-related proteins, such as triosephosphate isomerase, guanine monophosphate synthase and glyceraldehyde-3-phosphate dehydrogenase were down-regulated. Overall the compensatory response of SU18 to antibiotics is to alter expression of proteins related to antibiotic resistance, cell wall formation and energy metabolism. Some of the differentially expressed proteins might enhance antimicrobial activity and are now being investigated as potential therapeutic drug targets in other pathogenic bacteria. BIOLOGICAL SIGNIFICANCE This study highlights the power of proteomics in the study of differential protein expression in a multiresistant Enterococcus faecium strain when subjected to vancomycin stress.

Antibiotic resistance and mechanisms implicated in fecal enterococci recovered from pigs, cattle and sheep in a Portuguese slaughterhouse

A total of 194 enterococcal isolates were recovered from 198 fecal samples of pigs, cattle, and sheep obtained in a Portuguese slaughterhouse. The enterococcal species most prevalent were Enterococcus faecium and E. hirae. High percentages of resistance were detected for tetracycline in pig isolates (95.7%), sheep isolates (76.7%), and cattle isolates (49%); erythromycin resistance was higher in pig isolates than in cattle or sheep isolates. Intermediate level of resistance was obtained to quinupristin/dalfopristin in all animal isolates (15.1–23.5%). High-level resistance to aminoglycosides was detected, HLR-S and -K was higher in pig isolates (44.3 and 32.9%, respectively) compared with cattle or sheep isolates, and modest percentages of HLR-G were obtained in pig and cattle isolates (7.1 and 3.9%, respectively). The aac(6`)-aph(2”), aph(3`)-IIIa, ant(6)-Ia, cat(A), erm(B), and tet(M) genes were demonstrated in most of the gentamycin-, kanamycin-, streptomycin-, chloramphenicol-, erythromycin- and tetracycline-resistant isolates, respectively. The association between the tet(M) gene and Tn916/Tn1545-like or Tn5397-like transposons was detected in 30.8 and 11.2% of the isolates, respectively. Food animals could be a reservoir for antibiotic resistance genes, and slaughterhouse cross-contamination of animals carcasses may be a food safety risk.

Multiresistant extended-spectrum β-lactamase producing Escherichia coli in human urine samples in Portugal

The introduction of antimicrobial drugs in clinical practice is usually followed by the emergence of resistant strains. The inadequate use, auto-medication, exclusive use of extended-spectrum drugs, or the premature break of antimicrobial treatments are factors to take in consideration. Multiresistant extended-spectrum b-lactamase (ESBL) producing-Escherichia coli are among the top six most worrying human pathogens. This organism is a common inhabitant of the human intestinal tract but, in certain cases, it may cause infectious diseases, such as enteric, lung, nervous, and urinary tract infections. In recent years, E. coli strains have been developing resistance to some antimicrobial agents used to treat infections and are associated with therapeutic failure, causing great concern. Sixty-five ESBL-producing E. coli isolates, obtained from individual human urine samples,were selected for study. The isolates were obtained monthly from urine samples of patients at the Hospital Center of Trás-os-Montes and Alto Douro, fromNovember2009 to January 2011. Susceptibility to 16 antibiotics (ampicillin, amoxicillin þ clavulanic acid, cefoxitin, cefotaxime, ceftazidime, aztreonam, imipenem, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulfamethoxazole/trimethoprim, tetracyclineandchloramphenicol)was testedby thedisk-diffusion method in all recovered isolates and ESBL-phenotypic detection was confirmed out by double-disk test according to the CLSI criteria. The presence of resistance genes as well the classification in phylogenetic groups (A, B1, B2, or D) were performed by PCR (Table 1).

First report on MRSA CC398 recovered from wild boars in the north of Portugal. Are we facing a problem

The aim of the present study was to evaluate the resistance of Staphylococcus aureus recovered from wild boars, to analyze their genetic lineages, and to investigate the susceptibility to oxacillin. Samples from mouth and nose of 45 wild boars (Sus scrofa) were collected during hunt activity from November 2012 to January 2013 in the North of Portugal. S. aureus isolates were recovered from 30 of these samples (33%); one isolate/sample was further studied. The susceptibility of the isolates was tested by disk-diffusion test against 14 antimicrobial agents and minimal inhibitory concentration was used to test oxacillin according to EUCAST guidelines. The genetic lineages of S. aureus were characterized by agr-typing, spa-typing and MLST. From the 30 isolates, 18 S. aureus were susceptible to all antibiotics tested and 7 presented resistance to one or more of the following antibiotics: penicillin (n=3), oxacillin (n=4), cefoxitin (n=1), clindamycin (n=2), gentamicin (n=1), fusidic acid (n=1), ciprofloxacin (n=2), tetracycline (n=1) and linezolid (n=1). One MRSA CC398 (spa-type t899) isolate was detected (oxacillin MIC=32mg/L and mecA-positive), which presented resistance to penicillin, tetracycline, and ciprofloxacin and contained the genes of immune evasion cluster (IEC) system (type B). The 29 methicillin-susceptible isolates were typed as ST1 (t1533), ST133 (t3583), ST1643 (t10712), ST2328 (t3750) and the new STs (3220, 3222, 3223, 3224) associated to new spa-types t14311 and t14312. The agr-types I, II, III and IV were identified. It is a matter of concern when MRSA and some specific lineages of S. aureus are taken as commensal habitants of the skin and nose of wild animals and are characterized with resistance to various antimicrobial agents in clinical use.