Molecular characterization of extended-spectrum cephalosporin-resistance in Escherichia coli in pigs on-farm and from clinical cases throughout Quebec, Canada during 16 years

Development of multidrug resistance in Escherichia coli is an important problem in animal and human medicine. Further, emergence and spread of determinants for resistance to third generation extended-spectrum cephalosporins (ESCs) such as the critically important cephalosporins in human medicine (e.g. ceftriaxone and ceftiofur) among isolates is a public health concern. Thus, the objectives of the present thesis were (1) to study the dynamic of antimicrobial resistance (AMR) phenotypes as well as virulence and AMR gene profiles in E. coli from pigs receiving a feed medicated with chlortetracycline and penicillin G following weaning and to study the effect of feed supplementation with a clay mineral, clinoptilolite, on this dynamic; (2) To investigate the mechanisms leading to an increase in the prevalence of blaCMY-2 conferring resistance to ceftiofur in pigs receiving medicated feed but which had not received ceftiofur, and to examine the effect of feed supplementation with a clay mineral on this phenomenon; and (3) to investigate the temporal characterization of clinical isolates of ceftiofur-resistant E.coli from diseased pigs in Quebec, Canada from 1997, when ceftiofur resistance was first reported, to 2012.  In the initial study, prevalence of resistance to 10 antimicrobial agents, including ceftiofur, increased over time in E.coli isolates from weaned pigs. A late increase in the frequency of blaCMY-2, the gene encoding resistance to ceftiofur, and the presence of virulence genes iucD and tsh were observed in the isolates. Feed supplementation with clinoptilolite was associated with an early increase but later decrease in the frequency of the blaCMY-2 gene in isolates. Concurrently, a later increase in the frequency of the blaCMY-2 and the virulence genes iucD and tsh was observed in the control pig isolates, being significantly greater than in the supplemented pigs at day 28. Diversity among the blaCMY-2-positive E. coli isolates with respect to AMR patterns was observed. Certain clonal lineages of E. coli became predominant with time. The clonal lineage of phylotype A predominated in the supplemented group, whereas the clonal lineages of phylotype B1 which often possessed the ExPEC-associated virulence gene iucD, predominated in the control group. The blaCMY-2-carrying plasmids of incompatibility (Inc) groups, I1, A/C, and ColE were observed in transformants. In ESC-resistant E.coli clinical isolates from diseased pigs in Quebec, from 1997 to 2012, blaCMY-2 was the most frequently detected β-lactamase gene, followed by blaTEM and blaCTX-M and clonal analysis showed high diversity. E. coli isolates with identical PFGE patterns were found in multiple farms in the same year and also in different years. Resistance to gentamicin, kanamycin, chloramphenicol, and the frequency of blaTEM and IncA/C significantly decreased over the study time, whereas the frequency of IncI1 and multidrug-resistance to seven antimicrobial categories significantly increased over time. Emergence of blaCTX-M-positive, extended-spectrum β-lactamase (ESBL)-producing E. coli isolates was observed in 2011 and 2012 from distinct clonal lineages and multiple farms.  Taken together, this work gives some insight into the spread of ceftiofur resistance in E.coli isolates from pigs. Results reveal that in weaned pigs receiving medicated feed on one farm in which an increase in ceftiofur resistance was observed, the blaCMY-2-positive E.coli resistant to ESCs belonged to several different clonal lineages with diverse AMR patterns. The blaCMY-2 gene spread both horizontally and clonally in E. coli. Feed supplementation with clinoptilolite and time period after weaning influenced the clonality and the prevalence of blaCMY-2 gene in E. coli. In ceftiofur-resistant E.coli strains isolated from diseased pigs in farms throughout Quebec over a 16 year period, several different pathogenic clonal lineages were observed, although none was persistent or predominant over the study time. The results suggest that blaCMY-2 gene spreads both horizontally and clonally on and between farms. Furthermore, blaCMY-2 was the major β-lactamase gene in these isolates. From 2011, we report the emergence of blaCTX-M in distinct clonal lineages.

Keyphrases: E. coli, Pig, Virulence gene, Antimicrobial resistance, Plasmid, blaCMY-2 gene, blaCTX-M gene, ESC-resistance, Clay mineral (clinoptilolite)