A Whole-Genome Sequencing Approach To Study Cefoxitin-Resistant Salmonella enterica Serovar Heidelberg Isolates from Various Sources

Abstract

This study characterized cefoxitin-resistant and -susceptible Salmonella enterica serovar Heidelberg strains from humans, abattoir poultry, and retail poultry to assess the molecular relationships of isolates from these sources in Québec in 2012. Isolates were collected as part of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). All isolates were subjected to antimicrobial susceptibility testing, PCR for CMY-2, pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). A total of 113 S Heidelberg isolates from humans (n = 51), abattoir poultry (n = 18), and retail poultry (n = 44) were studied. All cefoxitin-resistant isolates (n = 65) were also resistant to amoxicillin-clavulanic acid, ampicillin, ceftiofur, and ceftriaxone, and all contained the CMY-2 gene. PFGE analysis showed that 111/113 (98.2%) isolates clustered together with ≥90% similarity. Core genome analysis using WGS identified 13 small clusters of isolates with 0 to 4 single nucleotide variations (SNVs), consisting of cefoxitin-resistant and -susceptible human, abattoir poultry, and retail poultry isolates. CMY-2 plasmids from cefoxitin-resistant isolates all belonged to incompatibility group I1. Analysis of IncI1 plasmid sequences revealed high identity (95 to 99%) to a previously described plasmid (pCVM29188_101) found in Salmonella Kentucky. When compared to pCVM29188_101, all sequenced cefoxitin-resistant isolates were found to carry 1 of 10 possible variant plasmids. Transmission of S Heidelberg may be occurring between human, abattoir poultry, and retail poultry sources, and transmission of a common CMY-2 plasmid may be occurring among S Heidelberg strains with variable genetic backgrounds.

Keywords: Québec; Salmonella Heidelberg; beta-lactamase; cefoxitin; plasmid analysis; whole-genome sequencing.

FIG 1

Minimum spanning phylogenetic tree of the core genome of 113 sequenced Salmonella Heidelberg isolates with reference S. Heidelberg 12-4374, generated using Phyloviz. Colors for sources of isolates are as follows: red, human; yellow, abattoir chicken; blue, retail chicken; green, retail turkey. Isolates outlined in dark black are resistant to cefoxitin. Isolates in the same circle have 0 hqSNVs, and the size of each circle is proportionate to the number of isolates in the circle. Numbers on branches between 2 isolates represent the number of hqSNV differences. Clusters of isolates outlined in gray indicate those having 0 to 4 hqSNVs and are labeled 1 to 13. The 2 isolates with arrows indicate strains with a portion of a bla_CMY-2 plasmid integrated into the chromosome.

FIG 2

Schematic diagram of plasmid insertion into chromosome for strains 12-6245 and N13-01320 (top). The previously closed IncI1 bla_CMY-2 Salmonella Kentucky pCVM29188_101 plasmid was used as a scaffold to identify the insertions (bottom). Blue arrows represent chromosomal open reading frames (ORFs); red arrows represent plasmid ORFs; h, hypothetical protein.

FIG 3

Nucleotide sequence alignments of the previously characterized S. Kentucky pCVM29188_101 plasmid against the 10 variant bla_CMY-2-containing plasmids found in all S. Heidelberg isolates in this study, generated using GView Server. The reference plasmid is represented by the black boxes that denote open reading frames. For the remaining plasmids, solid colored boxes indicate that DNA is present. The gray line toward the bottom represents the GC content of the reference. Proteins listed above the alignment indicate those that are not present in certain plasmids, except for CMY-2, which was found in all plasmids. A representative sequence of a group A plasmid has been previously published (16).