Evolution of an incompatibility group IncA/C plasmid harboring blaCMY-16 and qnrA6 genes and its transfer through three clones of Providencia stuartii during a two-year outbreak in a Tunisian burn unit

Abstract

During a 2-year period in 2005 and 2006, 64 multidrug-resistant Providencia stuartii isolates, including 58 strains from 58 patients and 6 strains obtained from the same tracheal aspirator, were collected in a burn unit of a Tunisian hospital. They divided into four antibiotypes (ATB1 to ATB4) and three SmaI pulsotypes (PsA to PsC), including 49 strains belonging to clone PsA (48 of ATB1 and 1 of ATB4), 11 strains to clone PsB (7 of ATB2 and 4 of ATB3), and 4 strains to clone PsC (ATB3). All strains, except for the PsA/ATB4 isolate, were highly resistant to broad-spectrum cephalosporins due to the production of the plasmid-mediated CMY-16 β-lactamase. In addition, the 15 strains of ATB2 and ATB3 exhibited decreased quinolone susceptibility associated with QnrA6. Most strains (ATB1 and ATB3) were gentamicin resistant, related to an AAC(6')-Ib' enzyme. All these genes were located on a conjugative plasmid belonging to the incompatibility group IncA/C(2) of 195, 175, or 100 kb. Despite differences in size and in number of resistance determinants, they derived from the same plasmid, as demonstrated by similar profiles in plasmid restriction analysis and strictly homologous sequences of repAIncA/C(2), unusual antibiotic resistance genes (e.g., aphA-6), and their genetic environments. Further investigation suggested that deletions, acquisition of the ISCR1 insertion sequence, and integron cassette mobility accounted for these variations. Thus, this outbreak was due to both the spread of three clonal strains and the dissemination of a single IncA/C(2) plasmid which underwent a remarkable evolution during the epidemic period.

Fig 1

Temporal relationship among the 64 P. stuartii isolates of the burn unit. At the top, the blue full arrows indicate the 48 isolates belonging to the clone PsA/ATB1, and the orange dotted line arrow indicates strain PsA/4. At the bottom, the red full arrows indicate the seven isolates of clone PsB/ATB2, the black double-headed arrows represent the four strains of clone PsB/ATB3, and the green dotted line arrows represent the four isolates of clone PsC/ATB2. The open star-like symbol indicates the six environmental strains. In June 2005, three strains were collected from reservoirs 1 and 2 and from the tube of the tracheal aspirator. In February 2006, two strains were isolated from reservoirs 1 and 2. The closed diamonds indicate the representative strains.

Fig 2

S1-PFGE with hybridization with the repAIncA/C₂ and qnrA6 probes. Lanes 1 to 5, TcPsB/2, TcPsB/3, TcPsC/3, TcPsA/1, and PsA/4, respectively; lanes 6, E. coli K-12; lanes M, PFGE marker. (a) S1-PFGE; (b) Southern hybridization with the repAIncA/C₂ probe; (c) Southern hybridization with the qnrA6 probe.

Fig 3

Characterization of multidrug resistance regions identified in the IncA/C2 plasmids carrying bla_CMY-16 (A), the class 1 integron with the same gene cassette array and differing by presence or not of qnrA6 (B), and the class 1 integron harboring aac(6′)-Ib′ (C). Open reading frames (ORFs) correspond to arrow-shaped boxes; the arrows express the direction of transcription of the corresponding ORFs. Boxes bordered by a dotted line indicate ORFs detected by PCR amplification but not sequenced. The names of the ORFs are indicated below the arrows. Black boxes show the genes of interest, i.e., bla_CMY16 (A), qnrA6 (B), and aac(6′)-Ib′ (C). (A and B) Dark gray boxes, genes implicated in transposition and/or gene mobilization mechanisms; crosshatched boxes, resistance genes; horizontal dashed lines, sequence of the cloned fragment from quinolone-resistant clone C6 (B); horizontal dotted lines framed by triangles, PCR mapping strategy used to characterize the multidrug resistance region; arrowheads, position of primers used for the PCR amplifications; black and white circles, attI and attC sites implicated in gene cassette mobilization by the integrase IntI1, respectively; horizontal bold lines, identity with similar regions available in the GenBank database; arrow, transcription promoter upstream from qnrA6 (pOUT). (C) Arrows and squared rectangles, direct repeat sequence framed by the aac(6′)-Ib′ gene (DR1 and DR2).