Recent trends in virulence and antimicrobial resistance of E. coli in pigs

Antimicrobial use in animal production has been monitored over the past two decades because of potential adverse effects on animal and human health related to antimicrobial resistance. Administration of antimicrobials in animal production began early after their initial discovery, primarily for treatment of diseases, but also for promoting growth and for disease prevention. The latter is particularly important because animals are commonly housed at high densities that can facilitate the spread of disease. Nevertheless, administration of antimicrobials may select, or co-select for the presence of antimicrobial resistance (AMR) genes in the commensal or pathogenic bacterial populations in animals and lead to a breakdown in the treatment of associated diseases or transfer of resistant pathogenic bacteria to humans by contaminated meat. AMR genes may also be transferred to human pathogens and lead to problems in the treatment of disease in human patients.

In weaning pigs, antimicrobials are used in feed primarily as a medication to prevent disease and thus reduce mortality and morbidity. Among the most frequently used in-feed antimicrobials are penicillin and tetracycline, often in combination. Use of in-feed antimicrobials in pigs has been associated with increased resistance of fecal E. coli within and between classes of antimicrobials.

The emergence and prevalence of extended-spectrum cephalosporin (ESC)-resistant E. coli in food-producing animals is a global public health concern. Cephalosporins are used in both animals and humans to treat various bacterial infections. Ceftiofur, a third-generation cephalosporin, is used in pigs to treat respiratory disease, lameness and enteric disease, the regulation of use varying from country to country. Third-generation cephalosporins are considered to be a critically important class of antimicrobial as they are of very high importance to human medicine. For example, the third-generation cephalosporin ceftriaxone is one of the drugs of choice to treat invasive pediatric salmonellosis. Resistance to ceftiofur in food-producing animals is a potential public health issue because ceftiofur resistance determinants also confer cross-resistance to ceftriaxone and other third-generation cephalosporins. Ceftiofur has been used therapeutically in food-producing animals since 1989 and ceftiofur resistance mediated via the blaCMY-2 gene was first reported in 1998. Since 1997, prevalence of ceftiofur-resistance in E. coli isolates from clinically ill pigs has increased in Quebec, reaching about 22% in 2014. In Enterobacteriaceae, ESC-resistance has been associated with production of AmpC β-lactamases (e.g. CMY-2) and extended spectrum β- lactamases (ESBLs) (e.g. CTX-M) encoded by genes on transferable plasmids. The CTX-M and CMY families of β- lactamase producing gram negative bacteria are a major public health threat due to the limited therapeutic options to treat infections with these bacteria as a result of co-association of resistance to that of other classes of antimicrobials, including aminoglycosides, sulphonamides, phenicols and tetracyclines, giving rise to multidrugresistant (MDR) strains.

AMR bacteria are shed into the environment where they may persist for long periods. Ceftiofur resistance genes are located commonly on transferable elements (integrons, transposons, insertion sequences) carried by plasmids that facilitate the horizontal spread of resistance and most strains carrying these may also carry resistance genes for other antimicrobials. Hence, certain resistance genes can be conserved due to a link with the genes encoding resistance to antimicrobials that are registered for use in animal production.

Common replicon types of plasmids encoding beta-lactamase genes have been observed in E. coli isolates from humans and food producing animals and have been found to circulate in E. coli from human and animal sources. They may also be transmitted between E. coli and Salmonella among humans and animals.

 

In one study, we looked at the changes in AMR phenotype and virulence and AMR gene profiles in E. coli from one batch of healthy pigs following weaning1. This batch of pigs was fed a diet containing chlortetracycline and penicillin G in therapeutic doses for protection of respiratory problems and to prevent Streptococcus suis infections but had not been treated with other antimicrobials, including ceftiofur.

Overall, an increased resistance to 10 antimicrobials was observed with time. At weaning, resistance to streptomycin, trimethoprim-sulfamethoxazole, sulfisoxazole, ampicillin, amoxicillin/clavulanic acid, chloramphenicol, and tetracycline was observed in up to 60% of the isolates. The resistance prevalence increased significantly from day 0 to day 7 for most of these antimicrobials, remaining high until day 28 at the end of the study or decreasing slightly
in the case of streptomycin, sulfisoxazole, and trimethoprim-sulfamethoxazole. On the other hand, resistance remained low for kanamycin, gentamicin, ciprofloxacin and nalidixic acid. In contrast, no strains resistant to ceftiofur, ceftriaxone, or cefoxitin were observed at weaning, and the prevalence of resistance to these antimicrobials increased from 7 days after weaning, the increase being significant at day 28. One AMR pattern was predominant in extended-spectrum cephalosporin (ESC)-resistant E.coli strains, with co-resistance to streptomycin, cefoxitin, trimethoprim-sulfamethoxazole, sulfisoxazole,  amoxicillin/clavulanic acid, ampicillin, chloramphenicol, and tetracycline. Concurrently, an increase in the frequency of the gene blaCMY-2, encoding for ceftiofur resistance, was observed in the pig isolates.

The increased frequency of AMR in E. coli isolates from pigs with time could be a result of continuous administration of chlortetracycline and penicillin G in the feed. Interestingly, in our study, whereas prevalence of resistance to antimicrobials in the same classes as the medicated feed, such as tetracycline and ampicillin, increased and remained high throughout the study, during which time pigs continued to be medicated, prevalence of resistance to antimicrobials of other classes, such as streptomycin, trimethoprim-sulfamethoxazole and sulfisoxazole, initially increased and then decreased with time. An exception was chloramphenicol, for which prevalence of resistance increased and remained high until the end of the study.

We then investigated the possible mechanisms of spread of the blaCMY-2 gene in these pigs2. Pulsed-field gel electrophoresis and antimicrobial susceptibility testing results revealed that the blaCMY-2-positive E.coli isolates were polyclonal with diverse antimicrobial resistance patterns. Moreover, blaCMY-2-carrying plasmids of incompatibility (Inc) groups, A/C, I1, and ColE were observed in transformants as detected by PCR. In addition, Enterobacter cloacae possessing blaCMY-2-carrying IncA/C plasmids were found in the pens before introduction of this batch of pigs.

Thus, the blaCMY-2 gene appears to have spread both horizontally and clonally in this batch of pigs and may have spread from previous batches of pigs via plasmids carried by E. cloacae and expanded in animals of the present batch in the presence of the selection pressure due to administration of chlortetracycline and penicillin G in the feed. 

Overall, our results suggest that implementation of improved hygiene measures and decreased administration of certain antimicrobials on farm may limit antimicrobial resistance spread between and within batches of animals.

We investigated the evolution with time of ceftiofur-resistant clinical E. coli isolates from pigs in Québec, Canada, between 1997 and 2012 with respect to E. coli pathotypes, clones, and antimicrobial resistance.3 The most prevalent pathovirotypes were enterotoxigenic E. coli (ETEC):F4 (40%), extraintestinal pathogenic E. coli (ExPEC) (16.5%) and shiga toxinproducing E. coli (STEC):F18 (8.2%). We observed a high prevalence of resistance to 13 of 15 tested antimicrobials, all isolates being multidrug-resistant. blaCMY-2 (96.5%) was the most frequently detected β-lactamase gene. Pulsed-field gel electrophoresis revealed that resistance to ceftiofur is spread both horizontally and clonally in E. coli isolates. In addition, the emergence of ESBL-producing E. coli isolates carrying- blaCTX-M was observed in 2011 and 2012 in distinct clones. The most predominant plasmid incompatibility (Inc) groups were IncFIB, IncI1, IncA/C and IncFIC. Resistance to gentamicin, kanamycin, chloramphenicol, and the frequency of IncA/C significantly decreased over the study time, whereas the frequency of IncI1 significantly increased over time. Our findings reveal that extendedspectrum cephalosporin-resistant porcine E. coli isolates in Québec belong to several different clones with diverse AMR patterns and plasmids.

Identical ETEC:F4 ceftiofur-resistant clones were found in different years on the same farm suggesting persistence of certain clones between batches. In addition, some clones were observed from multiple farms which suggested potential circulation among the farms. Also, these clones may have originated from a single source (e.g. same breeding pig farm). Transmission may have occurred via imported and exported pigs, and also by the movement of water and wild animals and/or through contaminated feed. It is interesting to note that presently only 44% of farms in Quebec are «farrow-to-fattening», that is, on a single site, as compared to 33% fattening and 23% farrowing farms which together constitute multisite farms. Transport of pigs between these sites would result in a more widespread spreading of clones. Likewise, E. coli may be transmitted from farm to farm by vehicles such as transport trucks and visitors on farms (truck drivers, veterinarians, producers, technicians) or by farm workers

Heterogeneity among ESC-resistant isolates suggests that their spread is not only due to the dispersion of successful E. coli clones, and horizontal gene transfer via plasmids may be contributing to the emergence of ESC-resistance in pigs. Interestingly, the plasmid IncA/C was mostly seen prior to 2006 in ETEC:F4 O149 clones although it re-emerged in 2009-2012 clones on one particular farm. The presence of IncA/C was significantly associated with resistance to gentamicin, kanamycin, streptomycin, trimethoprim-sulfamethoxazole, sulfisoxazole, chloramphenicol, tetracycline. The decrease in resistance to gentamicin, kanamycin and chloramphenicol in ESC-resistant E. coli could be a result of less administration of these antimicrobials in pig production and less pressure for the persistence of certain plasmids. The ban on the use of chloramphenicol in food animals in Canada since the 1980s could contribute to the decrease in resistance to chloramphenicol over time.

Similarly, based on anecdotal evidence from local authorities and veterinarians, a tendency to less prescribing of gentamicin has recently been seen. On the other hand, kanamycin is not used in pig production in Canada. The emergence and prevalence of antimicrobial resistance in bacteria are likely to be linked to administration of antimicrobials in animals through cross-resistance or co-resistance. It can also be associated with disinfectant use and
administration of metals like copper and zinc as feed additives.

In the beginning of 2014, an increase in the frequency and severity of diarrhea cases from pre-weaning and nursery farms was observed in Québec. A significant increase in prevalence of ETEC:F4 cases was observed during the second quarter of 2014. This increase was due to two different virotypes of ETEC:F4: an emerging LT:STb:STa:F4 (3TF4) virotype and the previously predominant LT:STb:F4 (2TF4) virotype. Subsequently, in 2014 and 2015, 3TF4 diarrhea cases remained the most prevalent whereas 2TF4 cases were still frequently observed but to a lesser extent. 3TF4 isolates demonstrated an interesting AMR pattern, most being non-susceptible to enrofloxacin. Overall, multi-drug resistant ETEC:F4 isolates, especially 3TF4 isolates, became increasingly more frequent between 2013 and 2016 and potentially ultra-resistant isolates were observed. Our results suggest that a potentially new clone of fluoroquinolone-non-susceptible ETEC:F4 could be emerging in pig production farms in Québec.

 

 

In conclusion, our findings reveal that ESC-resistance was mostly associated with the blaCMY- 2 gene and that the blaCTX-M gene has recently emerged. ESC-resistant E. coli belonged to several different clones with diverse AMR patterns and often carried plasmids of several Inc groups. However, there was no persistent and predominant clone over the study time. Further, we confirmed that some clonal ESC-resistant isolates were present at multiple farms in the same and different years. Our results suggest that the blaCMY-2 gene spreads both horizontally and clonally and the spread of blaCTX-M gene may have occurred by horizontal transmission. Our findings underline the importance of continued monitoring E. coli isolates from pigs for antimicrobial resistance, as well as virulence and antimicrobial resistance genes, and plasmid and clonal types.

 

 

 

1. Jahanbakhsh, S., Kabore, K. P., Fravalo, P., Letellier, A., Fairbrother, J. M. Impact of medicated feed along with clay mineral supplementation on Escherichia coli resistance to antimicrobial agents in pigs after weaning in field conditions. Research in Veterinary Science, 2015, 102, 72-79.

2. Jahanbakhsh, S., Letellier, A., Fairbrother, J. M. Circulating of CMY-2 beta-Lactamase gene in weaned pigs and their environment in a commercial farm and the effect of feed supplementation with a clay mineral. Journal of Applied Microbiology, 2016, doi:10.1111/jam.13166.

3. Jahanbakhsh S., Smith MG., Kohan-Ghadr HR., L etellier A., Abraham S., Trott DJ., Fairbrother JM. Dynamics of extended-spectrum cephalosporin-resistance in pathogenic Escherichia coli isolated from diseased pigs in Quebec-Canada. International Journal of Antimicrobial Agents, 2016, doi:10.1016/j.ijantimicag.2016.05.001.