Molecular Epidemiology of Dairy Cattle-Associated Escherichia coli Carrying blaCTX-M Genes in Washington State

Abstract

An increase in the prevalence of commensal Escherichia coli carrying bla_CTX-M genes among dairy cattle was observed between 2008 and 2012 in Washington State. To study the molecular epidemiology of this change, we selected 126 bla_CTX-M-positive and 126 bla_CTX-M-negative isolates for determinations of the multilocus sequence types (MLSTs) and antibiotic resistance phenotypes from E. coli obtained during a previous study. For 99 isolates, we also determined the bla_CTX-M alleles using PCR and sequencing and identified the replicon types of bla_CTX-M-carrying plasmids. The bla_CTX-M-negative E. coli isolates comprised 76 sequence types (STs) compared with 32 STs in bla_CTX-M-positive E. coli isolates. The bla_CTX-M-positive E. coli isolates formed three MLST clonal complexes, accounting for 83% of these isolates; 52% of bla_CTX-M-negative E. coli isolates clustered into 10 clonal complexes, and the remainder were singletons. Overall, bla_CTX-M-negative E. coli isolates had more diverse genotypes that were distinct to farms, whereas bla_CTX-M-positive E. coli isolates had a clonal population structure and were widely disseminated on farms in both regions included in the study. Plasmid replicon types included IncI1 which predominated, followed by IncFIB and IncFIA/FIB. bla_CTX-M-15 was the predominant CTX-M gene allele, followed by bla_CTX-M-27 and bla_CTX-M-14 There was no significant association between plasmid replicon types and bacterial STs, and neither clonal complexes nor major plasmid groups were associated with two discrete dairy-farming regions of Washington State.IMPORTANCE Infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli occur globally and present treatment challenges because of their resistance to multiple antimicrobial drugs. Cattle are potential reservoirs of ESBL-producing Enterobacteriaceae, and so understanding the causes of successful dissemination of bla_CTX-M genes in commensal bacteria will inform future approaches for the prevention of antibiotic-resistant pathogen emergence.

Keywords: Escherichia coli; dairy cattle; extended-spectrum β-lactamases; molecular epidemiology; plasmids; sequence types.

FIG 1

Minimum spanning tree analysis of 126 bla_CTX-M-positive Escherichia coli isolates based on MLST genotypes. Each circle represents a genotype, and the partitions within a circle are individual isolates. There are three main clonal complexes, and each is surrounded by a gray zone. Thick lines connect genotypes that differ at one locus, and thin lines connect genotypes that differ by two or more loci. Shading indicates the region from which isolates were obtained: pale gray indicates eastern Washington (n = 78) and dark gray indicates northwestern Washington (n = 48).

FIG 2

Minimum spanning tree analysis of 126 bla_CTX-M-negative Escherichia coli isolates based on MLST genotypes. Each circle or node represents a genotype, and the partitions within a node are individual isolates. Each clonal complex is surrounded by a gray zone. Thick lines connect genotypes that differ by one locus, and thin lines connect genotypes that differ by two or more loci. Shading indicates the region from which isolates were obtained: pale gray indicates eastern Washington (n = 78) and dark gray indicates northwestern Washington (n = 48).

FIG 3

Minimum spanning tree analysis of 252 Escherichia coli isolates based on MLST genotypes. Each circle represents a sequence type, and the partitions within each circle represent individual isolates. Clonal complexes are surrounded by gray zones. Within each circle, dark gray represents bla_CTX-M-positive Escherichia coli isolates (n = 126) and light gray represents bla_CTX-M-negative Escherichia coli isolates (n = 126).

FIG 4

Distributions of bla_CTX-M genes among the different MLST genotypes in bla_CTX-M-positive E. coli isolates (n = 99) from dairy cattle in Washington State. NT, nontypeable.