Multiresistance in Pasteurella multocida is mediated by coexistence of small plasmids

In 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 multocida to become resistant to multiple clinically relevant antibiotics. Thirteen beta-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. multocida strain 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 bla(ROB-1), pB1001 carried tet(B), pB1003 and pB1005 carried sul2 and 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 beta-lactam resistance in Haemophilus parasuis (A. San Millan et al., Antimicrob. Agents Chemother. 51:2260-2264, 2007), was mobilized in E. coli using the conjugation machinery of an IncP plasmid. Stability experiments proved that pB1000 was stable in P. multocida but highly unstable in E. coli. In conclusion, bla(ROB-1) is responsible for beta-lactam resistance in P. multocida in Spain. Coexistence and the spread of small plasmids are used by P. multocida to become multiresistant.

Single-nucleotide polymorphism in two representative multidrug-resistant Mycobacterium bovis isolates collected from patients in a Spanish hospital harboring a human infection outbreak

Mycobacterium bovis is the etiological agent of tuberculosis in domestic and wild animals. Its involvement as a human pathogen has been highlighted again with the recent descriptions of transmission through dairy products (18), reactivation or primary infection in human immunodeficiency virus-infected patients (5), and association with meat industry workers, animal keepers, or hunters (3). Strains resistant to antituberculous drugs (M. bovis is naturally resistant to pyrazinamide) pose an additional risk (2). Several studies have demonstrated that mutations in target genes are associated with resistance to antituberculous drugs (4, 7, 10, 11, 16). However, most of them have been developed in Mycobacterium tuberculosis strains and limited data are available regarding M. bovis isolates. The aim of this study was to characterize by sequencing the main genes involved in antibiotic resistance in two multidrug-resistant (MDR) M. bovis isolates in a human outbreak detected in a hospital in Madrid that subsequently spread to several countries (5, 6, 15). The isolates were resistant to 11 drugs, but only their rpoB and katG genes have been analyzed so far (1, 14). We studied the first (93/R1) and last (95/R4) M. bovis isolates of this nosocomial outbreak, characterized by spoligotyping as SB0426 (hexacode 63-5F-5E-7F-FF-60 in the database at www.mbovis.org) (1, 13). Several genes involved in resistance to isoniazid (katG, ahpC, inhA, and the oxyR-ahpC intergenic region), rifampin (rpoB), streptomycin (rrs, rpsL), ethambutol (embB), and quinolones (gyrA) were studied. These genes, or fragments of genes, were amplified and sequenced as previously described (12). The sequence analysis revealed polymorphisms in five (ahpC, rpoB, rpsL, embB, and gyrA) out of nine analyzed genes (Table 1). Nucleotide substitutions in four genes cause a change in the encoded amino acid. Two additional synonymous mutations in ahpC and rpsL differentiated the first and last isolates from the outbreak.