Comparative genomics of the IncA/C multidrug resistance plasmid family

Abstract

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

FIG. 1.

Circular representation of pRA1 and pRAx compared to previously sequenced IncA/C plasmids. Circles from inside to outside: 1, pRAx (A. hydrophila); 2, pRA1 (A. hydrophila); 3, pYR1 (Y. ruckeri); 4, pIP1202 (Y. pestis); 5, pP99-018 (P. damselae subsp. piscicida); 6, pP91278 (P. damselae subsp. piscicida); 7, pSN254 (S. enterica serovar Newport). Genes were color coded, depending on functional annotations, as follows: transposition/recombination, gold; plasmid maintenance, blue; conjugative plasmid transfer, green; antimicrobial and heavy metal resistance, red; other functions, black; and hypothetical proteins, gray. Sequence fragments present in pRA1 but absent from pRAx are shaded in green. Major differences between pRA1 and pYR1, pIP1202, pP99-018, pP91278, and pSN254 such as additional or different sequence fragments are shown with their specific locations and shaded in blue. Locations of minor differences between pRA1 and all other plasmids (except pRAx) or between pRA1 and any of the other plasmids (except pRAx) are indicated by black and gray arrows, respectively.

FIG. 2.

Phylogenetic relationships of conjugative transfer systems found on IncA/C plasmids and integrating conjugative elements, based on maximum likelihood and Bayesian methods. The tree was created based on the alignment of concatenated protein sequences of TraD, TraB, and TraF. Bootstrap values and posterior probabilities are indicated on branches (see Materials and Methods for details).