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. 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. 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 β-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%). 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.

A new gene, sul3 , which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized. Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E. coli . Two copies of the insertion element IS 15 Δ /26 flanked the region containing sul3 . The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E. coli isolates from pigs in Switzerland.

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis , Enterococcus faecium , and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

bla_OXA-48, bla_NDM-1 and bla_CTX-M-3 are clinically relevant resistance genes, frequently associated with the broad-host range plasmids of the IncL/M group. The L and M plasmids belong to two compatible groups, which were incorrectly classified together by molecular methods. In order to understand their evolution, we fully sequenced four IncL/M plasmids, including the reference plasmids R471 and R69, the recently described bla_OXA-48-carrying plasmid pKPN-El.Nr7 from a Klebsiella pneumoniae isolated in Bern (Switzerland), and the bla_SHV-5 carrying plasmid p202c from a Salmonella enterica from Tirana (Albania). Sequencing was performed using 454 Junior Genome Sequencer (Roche). Annotation was performed using Sequin and Artemis software. Plasmid sequences were compared with 13 fully sequenced plasmids belonging to the IncL/M group available in GenBank. Comparative analysis of plasmid genomes revealed two distinct genetic lineages, each containing one of the R471 (IncL) and R69 (IncM) reference plasmids. Conjugation experiments demonstrated that plasmids representative of the IncL and IncM groups were compatible with each other. The IncL group is constituted by the bla_OXA-48-carrying plasmids and R471. The IncM group contains two sub-types of plasmids named IncM1 and IncM2 that are each incompatible. This work re-defines the structure of the IncL and IncM families and ascribes a definitive designation to the fully sequenced IncL/M plasmids available in GenBank.

Because of the frequency of multiple antibiotic resistance,Staphylococcusspecies often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genesmecAandmph(C), respectively. After 3 days, the percentage ofStaphylococcusisolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment.Staphylococcusspecies displaying multiple resistance were found to harbor mainly genes conferring resistance to β-lactams (mecAandblaZ), aminoglycosides [strandaac(6′)-Ie-aph(2′)-Ia], and trimethoprim [dfr(A) anddfr(D)]. Additional genes conferring resistance to macrolides [mph(C),erm(C), anderm(B)], tetracycline [tet(K) andtet(M)], chloramphenicol [cat(pC221) andcat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.