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Objectives: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. Methods: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. Results: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). Conclusions: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

The extended-spectrum β-lactamase (ESBL) PER-1 initially disseminated among Pseudomonas aeruginosa strains in Turkey. Despite reports from other European countries, such strains have not been detected in Greece until now. We describe the first bla PER-1-positive P. aeruginosa isolates from Greece and their genetic environment. Methods: From January 2008 to December 2009, 287 consecutive non-duplicate P. aeruginosa isolates with reduced susceptibility or resistance to ceftazidime (MIC >8 mg/L) were screened for ESBL production with a modified boronic acid-based double-disc synergy test. Phenotypically ESBL-positive isolates were subjected to agar dilution, PFGE and multilocus sequence typing (MLST). Broad-spectrum bla genes were identified by PCR and sequencing. Plasmid analysis and conjugation experiments were performed. The location of the bla PER-1 gene was detected by Southern blotting and its genetic environment was characterized using inverse PCR. Results: Five isolates were phenotypically positive for ESBL production, exhibited resistance to cefepime, ceftazidime, aztreonam and meropenem, and carried the bla PER-1 gene. MLST showed that they belonged to sequence type (ST) 235, which belongs to the international clonal complex 11. Four isolates had the same PFGE pattern. Southern blotting revealed the chromosomal location of the bla PER-1 gene. Analysis of the bla PER-1 flanking regions showed identity to transposon Tn1213 downstream and 1406 bp upstream of bla PER-1. Further upstream, an orfA gene and ISPa12 were identified; both were truncated by the insertion of IS6100. Conclusions: This study confirmed the presence of PER-1-producing P. aeruginosa strains in Greece. The chromosomal location of bla PER-1, as part of a truncated transposon, suggests clonal expansion rather than horizontal gene transfer. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

Background: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) often display resistance to almost all classes of antimicrobial agents used in veterinary medicine. In the present study, we investigated the emergence of rifampicin resistance in MRSP, the persistence of these isolates and identified the corresponding mutations in the rpoB gene. Methods: In addition to two rifampicin-resistant MRSP isolates from a multicentre study, consecutive MRSP isolates collected prior to and after rifampicin therapy from nine dogs at five Dutch veterinary hospitals were included in this study. The isolates were tested for resistance to rifampicin and other antimicrobial agents. The rifampicin resistance-determining region (RRDR) within the rpoB gene of the rifampicin-resistant and -susceptible isolates was amplified by PCR and sequenced. PFGE served to determine the genetic relationships of the MRSP isolates. Results: Two MRSP isolates of the multicentre study showed mutations at position 513 or 522 in the RRDR of the rpoB gene. In contrast to the rifampicin-susceptible isolates, all rifampicin-resistant MRSP isolates showed mutations at one or two of the amino acid positions 508, 509, 513, 516, 522, 526 and 531. In most strains, a single amino acid exchange was observed. PFGE analysis confirmed that the rifampicin-resistant MRSP isolates were indistinguishable from or closely related to the rifampicin-susceptible isolate obtained from the same dog prior to rifampicin application. Conclusions: Therapy of MRSP infections with rifampicin results in the rapid emergence of rifampicin resistance and these isolates can persist for months. As a consequence, single therapy with rifampicin is not recommended.

Citation for published version: Kadlec, K, Schwarz, S, Perreten, V, Andersson, UG, Finn, M, Greko, C, Moodley, A, Kania, SA, Frank, LA, Bemis, DA, Franco, A, Iurescia, M, Battisti, A, Duim, B, Wagenaar, JA, van Duijkeren, E, Weese, JS, Fitzgerald, JR, Rossano, A & Guardabassi, L 2010, 'Molecular analysis of methicillin-resistant Staphylococcus pseudintermedius of feline origin from different European countries and North America' Journal of Antimicrobial Chemotherapy, vol 65, no. 8, pp. 1826-1828., 10.1093/jac/dkq203