Extended-Spectrum β-Lactamase and AmpC β-Lactamase-Producing Escherichia coli Isolates from Chickens Raised in Small Flocks in Ontario,Canada

Abstract

Food-producing animals are recognized to play a role in the epidemiology of antimicrobial resistance in Canada. However, the presence of resistant organisms in particular groups of animals, such as chickens raised in small-holder flocks, has not been studied. The purpose of this study was, therefore, to identify and characterize Escherichia coli possessing broad-spectrum β-lactamase genes among a collection of third-generation cephalosporin-resistant isolates recovered from 205 small flocks in southern Ontario. Extended-spectrum β-lactamase (ESBL; CTX-M-1) positive strains were isolated from 26 out of 205 flocks (12.7%), whereas 39 strains possessing AmpC (CMY-2) were grown from 31 out of 205 flocks (15.1%). Pulsed-field gel electrophoresis (PFGE) revealed that the isolates were genetically heterogeneous. Further testing by multi-locus sequence typing confirmed that none of the PFGE-defined clusters belonged to ST131. Our results suggest that the dissemination of this resistance in bacteria isolated from chickens in small-holder flocks may be associated with the spread of plasmids rather than particular E. coli clones and that these isolates do not possess the ESBL types most commonly associated with human infections (CTX-M-15).

Introduction

Antimicrobial Resistance (AMR) has been identified as an important public health priority worldwide due to the emergence of multidrug-resistant bacteria. The United States Centers for Disease Control and Prevention has listed extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae as a serious threat to the United States.¹ β-Lactamases are the bacterial enzymes that hydrolyze the β-lactam ring structure, inactivating these antibiotics. The β-lactams are a broad class of antibiotics that are important for treating infections caused by Escherichia coli.² E. coli is commonly associated with urinary tract infection in humans and animals, and isolates producing these broad-spectrum β-lactamases are becoming increasingly encountered in clinical settings.^3,4 Extra-intestinal E. coli infections in chickens are also highly problematic; colibacillosis, which describes a constellation of syndromes including airsacculitis, cellulitis, pericarditis, perihepatitis, and respiratory distress, is a critical production-limiting disease for the poultry industry.⁵

In Canada, data from surveillance programs, such as the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS), suggest that broad-spectrum β-lactamase-producing E. coli are present in Canada. However, the CIPARS abattoir program includes only chickens processed in federally inspected abattoirs and does not capture birds that are slaughtered outside of the federal system (provincially inspected abattoirs).⁶ Chickens processed in federally inspected plants originate from large producers that supply uniform chickens (age, weight, and breed) raised by using standard husbandry practices. Animals processed within provincially inspected abattoirs may largely vary in terms of age, weight, and breed and although these animals constitute a smaller share of the market, they may pose a unique set of risks in terms of antimicrobial-resistant bacteria. The intestinal microbiota changes with age, including the distribution of pathotypes and resistance among E. coli; therefore, chickens processed in provincially inspected abattoirs, who differ in age, may be colonized with E. coli populations with resistance profiles other than those captured by CIPARS.⁵

Although the use of antimicrobials on a given chicken farm varies, use by small-holder producers who may have less knowledge of antimicrobials, poorer bio-security practices (including housing other species in close proximity to chickens), and, consequently, a higher burden of disease is even less predictable.⁷ Further, the increasing popularity of “local food,” farmer's markets, and farm gate sales may result in an increase in the overall proportion of chicken production arising from small holders and slaughtered in provincially inspected abattoirs over time. Previous work by our group in Ontario demonstrated that E. coli isolated from chickens raised in small-holder flocks were significantly less likely than those sampled by CIPARS to be resistant to a number of antimicrobials, including ampicillin.⁸ Interestingly, this study did not find significant differences in the prevalence of resistance to third-generation cephalosporins (3GCs).

In Canada, CIPARS does not report the genotypes associated with resistance in E. coli, including the identification of β-lactamases limiting our ability to compare these data with other regions of the world. Studies from other countries indicate that CTX-M type β-lactamases are increasing, and they are the most prevalent ESBL type. In European countries, chicken E. coli isolates have been extensively studied; CTX-M-14, CTX-M-32, and TEM-52 type ESBLs were detected in cefotaxime-resistant E. coli obtained from broiler chickens in Portugal,⁹ whereas CTX-M-1, CTX-M-32, and SHV-12 were found in Italy.¹⁰ In Switzerland, CTX-M-1, SHV-12, and TEM-52 ESBLs were detected in E. coli isolates recovered from fecal samples from healthy chickens.¹¹ In the United States, a recent study also described the presence of CTX-M-1 positive E. coli from retail chicken meat.¹²

A potential hazard resulting from the selection and spread of AMR in food animal production is the transmission of resistant bacteria, or plasmids carrying resistance genes to humans via contact with animals or contaminated food.¹³ The relationship between in vitro resistance to β-lactams and ceftiofur usage has been observed in a number of food animal species, including chickens.¹⁴ A positive temporal association between the incidence of ceftiofur-resistant organisms in humans and in chickens and the use of ceftiofur in hatcheries was previously observed in Canada.¹⁵ As the broad-spectrum β-lactamases are increasingly common among E. coli isolated from community settings, determining the presence of these among chicken E. coli isolates is important to better understand the epidemiology of resistance. Overall, the objective of this study was to determine and characterize the broad-spectrum β-lactamase-producing E. coli isolated from chickens originating from small flocks, processing in provincially inspected abattoirs in Ontario, Canada.

Materials and Methods

Samples for molecular characterization

Cecal samples were collected at slaughter from 1,025 chickens originating from 205 small flocks (five per flock) at provincially inspected abattoirs in Ontario between May and September in 2014 and 2015. Small flocks were defined as <300 birds produced per year. Samples were selectively cultured for E. coli on MacConkey and CHROMagar ESBL. Isolates were preferentially selected from CHROMagar ESBL, and their antimicrobial susceptibility was determined by broth micro-dilution. All isolates resistant to the 3GC (n = 99) and a random subset of 3GC-susceptible isolates (n = 162) were selected for molecular characterization.

Detection of ESBLs, AmpC β-lactamase, and class I integrons using PCR

All 3GC-resistant isolates were screened for CTX-M and SHV type ESBLs and CMY-2 type AmpC β-lactamases and class 1 integrons by using previously published primers.^16–19 In each set of reactions, negative controls (no template) and positive controls (in house strains previously confirmed by sequencing) were included. PCR products were purified by using the EZ-10 Spin Column PCR purification kit (Bio Basic Canada, Inc., Canada), and DNA was sequenced by a commercial laboratory (Macrogen, Inc., Seoul, Republic of Korea). The identity of contigs generated from sequence analysis was determined by comparison to a set of reference sequences listed on Lahey database using CLC sequence viewer version 7 (Qiagen, Inc., CA).^a Resistance gene cassettes within class 1 integrons were identified by a BLAST comparison of sequenced amplicons with NCBI GenBank and using the integrall curated database.^b

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was performed on all 261 isolates as described by PulseNet protocol using the CHEF-DRIII system (Bio-Rad Laboratories Ltd., Canada).²⁰ DNA fragments were visualized with an AlphaImager^® HP (Fisher Scientific, Canada) and photographed. Cluster analysis was done by using the Dice co-efficient with an unweighted pair-group method using arithmetic averages and band tolerance 1.00%. The relatedness of all isolates was determined by using Gel Compare II (Applied Maths, Inc., TX); clusters were defined as groups of at least three isolates with ≥85% band similarity.

Multi-locus sequence typing

Multi-locus sequence typing (MLST) was performed to identify strains within PFGE clusters. For MLST, 15 isolates were randomly selected to represent each PFGE cluster as well as 9 non-clustering isolates that were evenly distributed throughout the PFGE-derived dendrogram. A published MLST scheme including seven housekeeping genes: adk, fumC, gyr, icd, mdh, purA, and recA was used.²¹ With the exception of recA, the amplification and sequencing were carried out by using the primers listed on the E. coli MLST website.^20,c For recA, new primers (Forward—5′-GTGCGTTTATCGATGCTGAA-3′ and reverse—5′-TCTTTTACGCCCAGGTCAAC-3′) were designed in-house to amplify the region included in the MLST scheme. Allelic profiles and sequence types were determined via the online MLST database.^d

Results

Identification of β-lactamase genes

Of the 99 3GC-resistant isolates, 65 were originally cultivated by using CHROMagar ESBL whereas 34 were grown on MacConkey agar. Of the 205 flocks sampled, E. coli was isolated from all 205 and broad-spectrum β-lactamase-producing E. coli were isolated from 56, including: 25 producing only ESBLs, 30 producing AmpC type enzymes, and 1 in which both ESBLs and AmpC were identified together (Table 1). The CTX-M genes identified were homogeneous; sequences from all 55 isolates had amino acid sequences that were 100% identical to CTX-M-1 over the amplified length (Table 2). AmpC genes were also homogeneous; sequences from all 39 isolates were 100% similar to CMY-2 over the sequence amplified. The majority of ESBL producers were detected from isolates grown on CHROMagar ESBL, whereas the majority of AmpC-producing isolates were recovered from MacConkey agar (Table 2).

Table 1.

Number of Flocks Positive for Extended-Spectrum β-Lactamase and AmpC β-Lactamase-Producing Escherichia coli

	AmpC positive flocks		Total
	(+)	(−)	Total
ESBL positive flocks
(+)	1	25	26
(−)	30	149	179
Total	31	174	205

Number of flocks from which ESBL and AmpC positive E. coli were isolated.

ESBL, extended-spectrum β-lactamase.

Table 2.

Frequency of β-Lactamases Genes in Escherichia coli Isolated from Chickens from Small Flocks (<300 Birds/Year)

Samples isolated from	No. of samples resistant to 3GC	No. of samples positive for β-lactamases resistance genes
Samples isolated from	No. of samples resistant to 3GC	CTX-M type ESBL	CMY type AmpC	Both ESBL and AmpC
CHROMagar ESBL	65^a	CTX-M-1 (n = 53)	CMY-2 (n = 8)	CTX-M-1 and CMY-2 (n = 4)
MacConckey	34^b	CTX-M-1 (n = 2)	CMY-2 (n = 31)	—
Total	99	55	39	4

Two isolates from the same sample were positive for ESBL and AmpC separately, so we included both isolates in our study.

Isolates from one sample out of 34 were not positive for either ESBL or AmpC.

3GC, third-generation cephalosporin.

Identification of class 1 integrons

All 99 3GC-resistant isolates were screened for class I integrons, and 22 were found to possess 1 kb elements. Sequencing revealed that all integrons carried the aadA1 cassette, which confers resistance to streptomycin and spectinomycin. Of the integron positive isolates, 11 carried CTX-M-1 and 11 possessed CMY-2.

Pulsed-field gel electrophoresis

Of the 261 isolates characterized by PFGE, 226 (87%) yielded analyzable banding patterns, including 80/99 β-lactamase-producing isolates and 146/162 non-resistant isolates. A repeat test was performed on the isolates that were not analyzable, but the results were similar with the previous run and showed no analyzable banding patterns. When the banding patterns from all 226 isolates were analyzed together, a heterogeneous population structure was observed. Only 9 clusters comprising 32 isolates were found, and none of these clusters contained more than 6 isolates (Fig. 1). A total of 23 broad-spectrum β-lactamase producers were found within 7 clusters, only 3 of which contained isolates from multiple flocks. Isolates with and without β-lactamase were found together in one cluster. One cluster comprised isolates from three flocks harboring both class I integrons and the CTX-M-1 ESBL.

FIG. 1.

Relatedness of isolates as determined by pulsed-field gel electrophoresis. Only the cluster-forming isolates (32 out of 226) are represented in this dendrogram. Cluster number is indicated by the dark gray numbers on the left. Restriction banding patterns (DNA fingerprints) are displayed in the middle. The presence of broad-spectrum β-lactamases is indicated for each isolate by the extended-spectrum β-lactamase and AmpC columns. Cluster analysis was done by using GelCompare II, and similarities were determined by using the Dice co-efficient with the unweighted pair-group method using arithmetic averages and band tolerance 1.00%.

Multi-locus sequence typing

To characterize the PFGE clusters, 24 isolates were selected. Fifteen isolates from 9 clusters defined by PFGE and 10 non-clustering isolates were included. A sequence type was not assigned for one isolate, where on repeated attempts sequenceable purA amplicons were not generated. Novel allelic combinations and, therefore, new sequence types were identified in six isolates (Table 3). Among the isolates that clustered together by PFGE that were tested by MLST, they were the same sequence type except for cluster number 3, which contained closely related double-locus variants. Isolates not belonging to PFGE-defined clusters were heterogeneous.

Table 3.

Distribution of Escherichia coli Sequence Types in Chickens from Small-Scale Farms

Cluster number	Sample ID	adk-fumC-gyr-icd-mdh-purA-recA	Sequence type	Clonal complex	β-lactamase genes
1	EC-008	6-31-5-28-1-1-2	ST57	ST350 complex	CTX-M-1
1	EC-087	6-31-5-28-1-1-2	ST57	ST350 complex	CTX-M-1
1	EC-068	6-31-5-28-1-1-2	ST57	ST350 complex	CTX-M-1
2	EC-033	6-4-4-16-24-8-14	ST58	ST155 complex	CTX-M-1 and CMY-2
3	EC-001	87-7-231-140-1-187-14	ST2792-2^a		CTX-M-1
3	EC-024	87-53-231-140-1-187-2	ST2792		CTX-M-1
4	EC-050	6-11-4-8-8-8-6	ST3270		CTX-M-1
5	EC-093	6-4-159-44-112-1-17	ST1011		ND
5	EC-097	6-4-159-44-112-1-17	ST1011		ND
6	EC-064	6-95-4-18-11-7-14	ST1304		CTX-M-1
7	EC-080	6-65-344-1-11-13-6	ST3580		CTX-M-1
8	EC-003	6-4-14-16-24-8-14	ST155	ST155 complex	CTX-M-1
8	EC-180	6-4-14-16-24-8-14	ST155	ST155 complex	ND
9	EC-020	6-11-4-8-8-8-2	ST48	ST10 complex	CTX-M-1
9	EC-108	6-11-4-8-8-8-2	ST48	ST10 complex	CTX-M-1
NC	EC-038	6-11-4-8-8-N-2	ST48-1^a		CTX-M-1
NC	EC-055	6-65-32-26-9-8-2	ST297		CTX-M-1
NC	EC-107	6-4-15-1-22-1-7	ST5617-1^a		CMY-2
NC	EC-120	10-53-4-8-12-8-2	ST746-1^a		CMY-2
NC	EC-165	6-4-5-26-20-8-14	ST40	ST40 complex	ND
NC	EC-192	6-11-4-8-8-8-2	ST48	ST10 complex	ND
NC	EC-199	111-7-4-8-12-8-2	ST813-1^a		ND
NC	EC-203	6-11-15-1-22-8-7	ST366-1^a		ND
NC	EC-209	10-4-4-8-8-8-2	ST178	ST10 Complex	ND

Indicates a novel allelic combination; the most similar sequence type and the number of loci differing between them are indicated.

NC, non-cluster; ND, not detected; N, poor quality sequence that was not assigned an allelic number.

Discussion

In Canada, chickens originating from small-holder flocks that are processed in provincially inspected abattoirs are not included in the current national AMR surveillance program, CIPARS.⁶ There are very little data describing the antimicrobial susceptibility of E. coli from these chickens, although we have previously shown that the prevalence of resistance among birds raised in small flocks is lower than in those captured by CIPARS.⁸ To further address this gap, we conducted this study to characterize the β-lactamases present among E. coli resistant to 3GC from this understudied bird population.

We found that among E. coli resistant to 3GC isolated from chickens raised in small-holder flocks in southern Ontario, ESBL-producing E. coli were isolated from 12.7% flocks tested. CTX-M-1 and CMY-2 were the most frequently detected CTX-M and AmpC alleles. Our findings are consistent with those of a French study where CTX-M and CMY-2 were the predominant broad-spectrum β-lactamases in 3GC-resistant E. coli in poultry.²² Other studies in Europe have also reported a high frequency of CTX-M-1 among E. coli from chickens.^23,24

Although our study sampled small-holder flocks exclusively, differences in the frequency of ESBL-producing E. coli between conventionally raised and organic or small-flock chicken productions have been previously identified. Researchers in India detected ESBL-producing E. coli isolates from 29.4% of commercial flocks but failed to identify any ESBLs among birds raised in free-range systems.²⁵ Similarly, and consistent with our findings published elsewhere,⁸ low levels of AMR were found among E. coli from backyard poultry in Australia compared with intensively farmed birds.²⁶ Both the Indian and Australian studies reported very limited or occasional use of β-lactam drugs in those backyard chicken populations; β-lactam use was previously reported to be associated with the presence of resistance to these drugs.^14,25,26 In Canada, β-lactam drugs, including amino-penicillins and ceftiofur, have been reported as commonly used drugs to treat E. coli infection in chickens.⁷ This may explain the seemingly high frequency of ESBL-producing E. coli identified in this study; however, as detailed antimicrobial use data were not available for this study population, this association is speculative.

To better understand the relatedness of the resistant isolates in our current study, PFGE was performed and 86.6% (226/261) isolates yielded analyzable banding patterns. Failure to obtain analyzable data from all strains is consistent with previous studies that found that some strains are non-typeable by PFGE.^27,28 Heterogeneous fingerprint patterns were found among resistant and susceptible isolates, whereas relatively few isolates were genetically clustered. Of the nine isolate clusters, four contained isolates from a single flock, whereas five contained isolates from multiple flocks. The presence of the same β-lactamase gene in unrelated isolates suggests that in the population studied the dissemination of these genes in E. coli was associated with the spread of plasmids rather than particular E. coli clones. These findings are consistent with previous studies indicating that the spread of CTX-M ESBLs was associated with plasmid transfer rather than clonal spread.²⁴

As expected, MLST revealed that the majority of the isolates that clustered together by PFGE were the same sequence type. Although a small sample of isolates in this study were characterized by MLST, our findings are in agreement with other studies where ST10, ST57, ST58, and ST155 were predominant among ESBL-producing strains from chickens.^29,30 In Canada, E. coli within the ST10 clonal complex have been previously isolated from human infections as well as from chickens and Franklin's gulls (Leucophaeus pipixcan).^31,32

Interestingly, none of the isolates tested were found to be ST131, the human pandemic E. coli clone that is frequently isolated from community-onset urinary tract infections globally, including in Canada.^33,34 Among human E. coli isolates, including ST131, CTX-M-15 has been reported to be the most commonly encountered ESBL type globally and in Canada.³⁵ Interestingly, CTX-M-15 was not identified in this study; CTX-M-1, which has been reported among E. coli of animal origin but is less common among human isolates globally, predominated in our isolate collection.^23,36 The difference in the predominant ESBLs from human infections and the genes found here suggests that the contribution of chicken-origin ESBL strains to human infections may be lower than previously suggested.¹⁵

Finally, our results highlight the effects of test methodology on the identification of resistance genes. ESBL-producing E. coli were more commonly isolated from CHROMagar ESBL, whereas AmpC producers were more commonly grown on MacConkey agar. As CHROMagar ESBL is particularly designed to screen ESBLs by inhibiting the growth of AmpC producers, finding a lower number of AmpC producers on CHROMagar ESBL was, therefore, an expected outcome.

Conclusion

Although organisms with identical resistance profiles may be found in humans and animals, genotypic characterization is clearly required to strengthen the evidence in support of transmission. Among the chickens sampled in this study, CTX-M-1 ESBLs predominated, which is in contrast to the situation in human infections, where CTX-M-15 is more common. The genetic heterogeneity of study isolates possessing ESBL genes suggests that the dissemination of CTX-M-1 was associated with plasmid transfer rather than the elaboration of a successful clone. Our study also identified that selective media could help in identifying ESBL-producing E. coli and including this type of selective media in surveillance studies could assist in tracking important resistance patterns. Finally, as this research was only done in Ontario, similar investigations at a national level would be useful to more completely assess the public health impact of ESBLs originating in poultry E. coli.

Footnotes

Acknowledgments

The authors would like to thank Michelle Sniatynski, Andrea Deruisseau, Chad Gill, Hillary Esdon, Jenny Kennedy, Phyllis Lam, and Krystin Pusztai for technical assistance. This work was supported by the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) (grant number FS2013-1866).

Disclosure Statement

No competing financial interests exist.

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