Multiple-antibiotic resistance mediated by structurally related IncL/M plasmids carrying an extended-spectrum beta-lactamase gene and a class 1 integron

Abstract

A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extended-spectrum beta-lactamase gene, bla(SHV-5), was identified 3. 5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb bla(SHV-5)-In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expanded-spectrum cephalosporins and aminoglycosides.

FIG. 1

(A) agarose gel electrophoresis of XbaI- and EcoRI-digested pSEM extracted from E. coli 202-24. HindIII-digested lambda DNA (λ-HindIII) and 1-kb marker (KiloBase DNA marker; Pharmacia Biotech, Milan, Italy) were used as standards. (B) Southern blot hybridization (25) of EcoRI-digested pSEM (electrophoresed as in panel A) with [α-³²P]dCTP-labeled bla_SHV-5 and intI1 probes. The bla_SHV-5 probe was obtained by PCR amplification with the OS5-OS6 primer pair. The intI1 probe was obtained by agarose elution of the 596-bp PvuII-RsaI restriction fragment from the plasmid pACYC184::Tn21 (4).

FIG. 2

Consensus map of the antibiotic resistance region of pSEM. Thin arrows show the orientation of genes. Δ5′CS and 3′CS are the 5′ and 3′ conserved region of the In-t3 integron. The short boldface arrows indicate the position and direction of primers used in PCR amplifications. The aacA4R-OS6 amplicon was obtained by the Expand 20 kb PCR system (Boehringer GmbH, Mannheim, Germany). The DNA sequence of the aacA4R primer is: 5′-TGTGACGGAATCGTTGC-3′. Thin bars below the map represent cloned inserts used for sequencing, with the plasmid designation given on the right of each construct. Dashed lines show the In-t3 and In-t3/bla_SHV-5 intervening sequences released under EMBL accession no. AJ009820 and AJ245670, respectively. Black thick bars represent pSEM regions homologous to pPAG-KE, pPa-1, pZMP1, and pACM1 plasmids; hatched thick bars are gaps between homologous regions; white thick bars are nonhomologous regions. The numbering is relative to positions of nucleotides in the released plasmid sequences.