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ABSTRACTStreptococcus suisis an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia colidespite strong expression repression. Finally, site-directed insertion of smrAand satABin the amylocus of the Bacillus subtilischromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillusis a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.

ABSTRACTThe 16S rRNA methyltransferase ArmA is a worldwide emerging determinant that confers high-level resistance to most clinically relevant aminoglycosides. We report here the identification and characterization of a multidrug-resistant Salmonella entericasubspecies I.4,12:i:− isolate recovered from chicken meat sampled in a supermarket on February 2009 in La Reunion, a French island in the Indian Ocean. Susceptibility testing showed an unusually high-level resistance to gentamicin, as well as to ampicillin, expanded-spectrum cephalosporins and amoxicillin-clavulanate. Molecular analysis of the 16S rRNA methyltransferases revealed presence of the armAgene, together with blaTEM-1, blaCMY-2, and blaCTX-M-3. All of these genes could be transferred en blocthrough conjugation into Escherichia coliat a frequency of 10−5CFU/donor. Replicon typing and S1 pulsed-field gel electrophoresis revealed that the armAgene was borne on an ∼150-kb broad-host-range IncP plasmid, pB1010. To elucidate how armAhad integrated in pB1010, a PCR mapping strategy was developed for Tn1548, the genetic platform for armA.The gene was embedded in a Tn1548-like structure, albeit with a deletion of the macrolide resistance genes, and an IS26was inserted within the melgene. To our knowledge, this is the first report of ArmA methyltransferase in food, showing a novel route of transmission for this resistance determinant. Further surveillance in food-borne bacteria will be crucial to determine the role of food in the spread of 16S rRNA methyltransferase genes worldwide.

Plasmid pB1000 is a mobilizable replicon bearing the blaROB-1β-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, ≥64 µg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the PASTEURELLACEAE:

ABSTRACTPlasmid pB1000 is a mobilizable replicon bearing the blaROB-1β-lactamase gene that we have recently described in Haemophilus parasuisand Pasteurella multocidaanimal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000′, in four Haemophilus influenzaeclinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzaeisolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, ≥64 μg/ml). Stability experiments showed that pB1000 is stable in H. influenzaewithout antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzaestrains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceaeact as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocidais the only member of this family that can colonize both humans and animals, we propose that P. multocidais the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

ABSTRACTIn most gram-negative bacteria, acquired multiresistance is conferred by large plasmids compiling numerous antimicrobial resistance genes. Here, we show an evolutionary alternative strategy used by Pasteurella multocidato become resistant to multiple clinically relevant antibiotics. Thirteen β-lactam-resistant clinical isolates, concomitantly resistant to tetracyclines and/or streptomycin as well as to sulfonamides, were studied. Pulsed-field gel electrophoresis analysis revealed different profiles among the isolates, showing that clonal dissemination was not the sole event responsible for the spread of multiresistance. Each P. multocidastrain carried two or three small plasmids between 4 and 6 kb in size. A direct association between resistance profile and plasmid content was found. Complete nucleotide sequencing of all plasmids revealed seven different replicons, six of them belonging to the ColE1 superfamily. All plasmids carried one, or a maximum of two, antimicrobial resistance determinants. Plasmids pB1000 and pB1002 bore blaROB-1, pB1001 carried tet(B), pB1003 and pB1005 carried sul2and strA, pB1006 harbored tet(O), and p9956 bore the tet(H) gene. All plasmids except pB1002 and pB1006 were successfully transformed into Escherichia coli. pB1000, also involved in β-lactam resistance in Haemophilus parasuis(A. San Millan et al., Antimicrob. Agents Chemother. 51:2260-2264, 2007), was mobilized in E. coliusing the conjugation machinery of an IncP plasmid. Stability experiments proved that pB1000 was stable in P. multocidabut highly unstable in E. coli. In conclusion, blaROB-1is responsible for β-lactam resistance in P. multocidain Spain. Coexistence and the spread of small plasmids are used by P. multocidato become multiresistant.

{szligbeta}-Lactam resistance in Haemophilus parasuis is an emerging phenomenon that has not yet been characterized from a molecular perspective. Clinical high-level {szligbeta}-lactam-resistant isolates from Spain bore a novel plasmid, pB1000, expressing a functionally active ROB-1 {szligbeta}-lactamase. Pulsed-field gel electrophoresis was applied for the first time to H. parasuis and showed that {szligbeta}-lactam resistance is due to clonal spread of a resistant strain, BB1018, bearing pB1000.

ABSTRACTβ-Lactam resistance in Haemophilus parasuisis an emerging phenomenon that has not yet been characterized from a molecular perspective. Clinical high-level β-lactam-resistant isolates from Spain bore a novel plasmid, pB1000, expressing a functionally active ROB-1 β-lactamase. Pulsed-field gel electrophoresis was applied for the first time to H. parasuisand showed that β-lactam resistance is due to clonal spread of a resistant strain, BB1018, bearing pB1000.

ABSTRACTIn this study, we characterized two tigecycline-resistant Klebsiella pneumoniaeisolates from dog urine samples. The isolates were genetically unrelated, belonging to sequence type 11 (ST11) and ST147, both classically related to human isolates. To the best of our knowledge, this is the first identification of tigecycline-resistant isolates from animals. We unveil here the worrisome circulation among animals of bacterial clones resistant to this last-resort antibiotic.