Rui Pacheco

Molecular characterization of vancomycin-resistant enterococci and extended-spectrum β-lactamase-containing Escherichia coli isolates in wild birds from the Azores Archipelago

To study the prevalence of vancomycin-resistant enterococci (VRE) and extended-spectrum β-lactamase (ESBL)-containing Escherichia coli isolates, and the mechanisms of resistance implicated, 220 faecal samples from wild birds were collected between 2006 and 2010 in the Azores Archipelago. Samples were spread on Slanetz–Bartley agar plates supplemented with 4 mg/l vancomycin and on Levine agar plates supplemented with 2 mg/l cefotaxime for VRE and ESBL-containing E. coli isolation, respectively. vanA-containing enterococcal isolates (four Enterococcus faecium and two Enterococcus durans) and vanC-1 Enterococcus gallinarum isolates were detected in six and seven faecal samples, respectively. VRE isolates showed ampicillin (n=11), ciprofloxacin (n=9), tetracycline (n=6), erythromycin (n=5), quinupristin/dalfopristin (n=3) and high-level kanamycin resistance (n=1). The tet(L) and/or tet(M) gene was found in all tetracycline-resistant isolates and the erm(B) gene in all erythromycin-resistant isolates. Three vanA-containing E. faecium and two E. gallinarum presented specific sequences of the Tn5397 transposon. Four VRE isolates harboured the ace virulence gene. One faecal sample revealed one ESBL-containing E. coli isolate that belongs to the A phylogenetic group, showed a phenotype of resistance to β-lactams and tetracycline, and harboured the bla CTX-M-14, bla SHV-12 and the tet(A) genes. To our knowledge, this is the first study to focus on defining the prevalence of VRE and/or ESBL-containing E. coli strains in wild birds from the Azores. The data recovered are essential to improve knowledge about the dissemination of resistant strains through wild ecosystems and their possible implications by transferring these resistances to other animals or to humans.

Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf.

The aim of this study was to report the antimicrobial resistance, the molecular mechanisms associated and the detection of virulence determinants within faecal Enterococcus spp. and Escherichia coli isolates of Iberian wolf. Enterococci (n = 227) and E. coli (n = 195) isolates were obtained from faecal samples of Iberian wolf (Canis lupus signatus). High rates of resistance were detected for tetracycline and erythromycin among the enterococci isolates, and most of resistant isolates harboured the tet(M) and/or tet(L) and erm(B) genes, respectively. The blaTEM, tet(A) and/or tet(B), and aadA or strA‐strB genes were detected among most ampicillin‐, tetracycline‐ or streptomycin‐resistant E. coli isolates, respectively. E. coli isolates were ascribed to phylogroups A (n = 56), B1 (91), B2 (13) and D (35). The occurrence of resistant enterococci and E. coli isolates in the faecal flora of Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was showed in this study. Iberian wolf might act as reservoir of certain resistance genes that could be spread throughout the environment.

Molecular characterization of vanA-containing Enterococcus from migratory birds: song thrush (Turdus philomelos)

Vancomycin-resistant enterococci (VRE) were detected in two faecal samples (1.3%) of song thrush in Portugal. vanA isolates showed high level vancomycin/teicoplanin resistance, as well as resistance to ciprofloxacin, quinupristin-dalfopristin and cloranfenicol. Thrush can be a reservoir of VRE and transmit these resistant bacteria to other animals including humans.

Iberian wolf as a reservoir of extended-spectrum β-lactamase-producing Escherichia coli of the TEM, SHV, and CTX-M groups.

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment. Moreover, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The association between antibiotic resistance and virulence determinants should be monitored, as it constitutes a serious public health problem.

High prevalence of ESBL-producing Escherichia coli isolates among hemodialysis patients in Portugal: appearance of ST410 with the blaCTX-M-14 gene ☆

Ten extended-spectrum β-lactamase-producing Escherichia coli isolates were detected among 121 fecal samples (8.3%) recovered from hemodialysis patients in Portugal. The isolates harbored the bla(CTX-M-15), bla(CTX-M-14a), and/or bla(CTX-M-1) genes. A new sequence type, ST2229, was detected, and this study also reports, for the first time, ST410 CTX-M-14-producing isolates.

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).

The Role of Proteomics in Elucidating Multiple Antibiotic Resistance in Salmonella and in Novel Antibacterial Discovery

Rui Pacheco1-4**, Susana Correia1-4**, Patricia Poeta3,4, Luis Pinto1-4 and Gilberto Igrejas1,2, 1Institute for Biotechnology and Bioengineering, Center of Genomics and Biotechnology University of Tras-os-Montes and Alto Douro 2Department of Genetics and Biotechnology, University of Tras-os-Montes and Alto Douro 3Center of Studies of Animal and Veterinary Sciences 4Veterinary Science Department, University of Tras-os-Montes and Alto Douro Portugal