Multiresistance in Pasteurella multocidaIs Mediated by Coexistence of Small Plasmids

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.