Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources

Abstract

Incompatibility group A/C (IncA/C) plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR) phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the bla(CMY-2) gene encoding for extended spectrum β-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the bla(CMY-2) plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.

Figure 1. Comparison of the sul2 regions of the plasmids sequenced in this study.

Colored boxes represent predicted open reading frames as follows: grey = unknown function, yellow = mobile genetic element. The arrows indicate transcription direction. Dotted lines indicate the positions where the plasmids differ. See Table S1 for a full list of sequenced gene annotations.

Figure 2. Comparison of the conjugative transfer and the bla _CMY-2 containing regions of the plasmids sequenced in this study.

Colored boxes represent predicted function as follows: red = antibiotic resistance, grey = unknown function, yellow = mobile genetic element, blue = transfer, green = known function. The arrows indicate transcription direction. Dotted lines and red lines indicate the positions where the plasmids differ.

Figure 3. Amino acid sequence alignment of the predicted H-NS- and HU-like proteins of IncA/C plasmids.

Evolutionary history was inferred using the Neighbor-Joining method. The percentage of trees in which associated taxa clustered together in the bootstrap test (500 replicates) greater than 60% are shown next to branches. Evolutionary distances were computed using the Poisson correction method. The H-NS-like protein alignment used 107 positions and the HU-like protein alignment used 90 positions. Phylogenetic analyses were conducted in MEGA4 .

Figure 4. Linear maps of sequenced IncA/C plasmids lacking bla _CMY-2.

Core sequences are colored red (IncA/C replicon and hypothetical genes), blue (Tra1 region), and green (hypothetical genes and Tra2 region). Pink boxes depict the sul2-containing regions. Blue box depicts the Tn21-like region containing a class 1 integron. Scale is depicted in kb.

Figure 5. Linear maps of sequenced IncA/C plasmids possessing bla _CMY-2.

Core sequences are colored red (IncA/C replicon and hypothetical genes), blue (Tra1 region), and green (hypothetical genes and Tra2 region). Pink boxes depict the sul2-containing regions. Green boxes depict bla _CMY-2 insertions. Blue boxes depict the Tn21-like region containing a class 1 integron. Scale is depicted in kb.