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. 2022 Sep 27;66(10):e00677-22. doi: 10.1128/aac.00677-22

A One Health Genomic Investigation of Gentamicin Resistance in Escherichia coli from Human and Chicken Sources in Canada, 2014 to 2017

Graham W Cox a,b, Brent P Avery c, E Jane Parmley d, Rebecca J Irwin c, Richard J Reid-Smith c, Anne E Deckert c, Rita L Finley c, Danielle Daignault e, George G Zhanel b, Michael R Mulvey a,b, Amrita Bharat a,b,✉
PMCID: PMC9578425  PMID: 36165686

ABSTRACT

We investigated whether gentamicin resistance (Genr) in Escherichia coli isolates from human infections was related to Genr E. coli in chicken and whether resistance may be due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on 483 Genr E. coli isolates isolated between 2014 and 2017. These included 205 human-source isolates collected by the Canadian Ward (CANWARD) program and 278 chicken-source isolates: 167 from live/recently slaughtered chickens (animals) and 111 from retail chicken meat collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). The predominant Genr gene was different in human and chicken sources; however, both sources carried aac(3)-IId, aac(3)-VIa, and aac(3)-IVa. Forty-one percent of human clinical isolates of Genr E. coli contained a blaCTX-M extended-spectrum beta-lactamase (ESBL) gene (84/205), and 53% of these were sequence type 131 (ST131). Phylogenomic analysis revealed a high diversity of Genr isolates; however, there were three small clusters of closely related isolates from human and chicken sources. Genr and spectinomycin resistance (Specr) genes were colocated in 148/167 (89%) chicken animal isolates, 94/111 (85%) chicken retail meat isolates, and 137/205 (67%) human-source isolates. Long-read sequencing of 23 isolates showed linkage of the Genr and Specr genes on the same plasmid in 14/15 (93%) isolates from chicken(s) and 6/8 (75%) isolates from humans. The use of lincomycin-spectinomycin on farms may be coselecting for gentamicin-resistant plasmids in E. coli in broiler chickens; however, Genr isolates and plasmids were mostly different in chickens and humans.

KEYWORDS: aminoglycosides, antimicrobial resistance, genomics, gentamicin, poultry, whole-genome sequencing

INTRODUCTION

Escherichia coli is the second most common cause of infections in Canadian hospitals including infections of the urinary tract, respiratory system, wounds, and bloodstream (1). In addition, infections caused by E. coli are the fifth most common cause of foodborne illness in Canada, with approximately 122 cases per 100,000 population (2). Antimicrobials may be utilized for serious infections in immunocompromised patients, in infants or elderly patients, and for extraintestinal infections (3).

Gentamicin is a broad-spectrum bactericidal aminoglycoside that inhibits protein synthesis (4–6). The Health Canada Veterinary Drugs Directorate categorizes antimicrobials into categories I to IV based on their importance to human medicine. Gentamicin is defined as a category II antimicrobial (high importance to human medicine) (7). It may be used to treat systemic infections caused by some Enterobacterales (8, 9), and the World Health Organization recommends combination therapy with gentamicin and ampicillin for neonatal sepsis in low- and middle-income countries (10). Due to increasing resistance to frontline antimicrobial agents in Gram-negative organisms, there is renewed interest in gentamicin and other aminoglycosides (5). Aminoglycoside-modifying enzymes (AMEs) are the most common mechanism of gentamicin resistance (Genr) and include the aminoglycoside acetyltransferase (AAC), O-nucleotidyltransferase (ANT), and O-phosphotransferase (APH) enzymes. Each of these classes contains numerous subclasses of enzymes that modify gentamicin and other medically relevant aminoglycosides at different positions (11). These resistance genes can be carried on mobile genetic elements which facilitate transfer between bacteria (12).

Antimicrobial use and antimicrobial resistance (AMR) in food production animals have been a recent public health focus, with concern about the potential transfer of resistant organisms from animals to humans via the food chain. In 2016, approximately 80% of antimicrobials sold in Canada were for use in production animals and were mostly used for disease prevention (13). In response to increasing levels of cephalosporin resistance in broiler chickens, in 2014, the Chicken Farmers of Canada implemented an industry-wide ban on the preventative use of category I antimicrobials (very high importance to human medicine), which included ceftiofur and other third-/fourth-generation cephalosporins (13). By the end of 2018, a veterinary prescription was also required for use of category II antimicrobials (high importance to human medicine) in animals, which included gentamicin, lincomycin, and spectinomycin (13). Following the ban on ceftiofur, increasing occurrence of Genr was observed in E. coli and Salmonella isolates from broiler chickens. It was hypothesized that the increased use of the combination of lincomycin-spectinomycin on chicken farms may be coselecting for Genr (14, 15). Gentamicin (aminoglycoside class), spectinomycin (aminocyclitol class), and lincomycin (lincosamide class) all inhibit protein synthesis through different mechanisms of action and generally have different resistance genes (4–6). Chalmers et al. described the linkage of Genr and spectinomycin resistance (Specr) genes on the same plasmid in E. coli isolates from broiler chicken in Quebec in 2017 (15).

In this report, we carried out a genomic study of gentamicin-resistant E. coli from human and chicken sources in Canada. We compared the genetic backgrounds, resistance genes, and resistance plasmids between isolates from humans, chickens (animals), and retail chicken (meat) sources that were collected from 2014 to 2017. We also investigated whether there was plasmid linkage of Genr and Specr genes present in the E. coli isolates.

RESULTS

Changes in proportions of Genr E. coli.

From 2009 to 2017, Canadian Ward (CANWARD) collected human-source isolates from hospitals and conducted antimicrobial susceptibility testing on 6,354 E. coli isolates. Of these, 576 (9.0%) were Genr. In the same period, Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) collected and conducted antimicrobial susceptibility testing for 14,573 E. coli isolates from chickens (animal) and chicken retail meat from across Canada. Of these, 18.4% (1,590/8,643) of animal isolates and 16.6% (986/5,930) of retail meat isolates were Genr. Comparing the two time periods of 2009 to 2013 and 2014 to 2017, the proportions of Genr E. coli were 9.4% (369/3,916) and 8.5% (207/2,438) in human isolates (P value of 0.225), 18.3% (612/3,338) and 18.4% (978/5,305) in chicken animal isolates (P value of 0.932), and 14.0% (539/3,846) and 21.4% (447/2,084) in retail chicken meat isolates (P value of <0.0001), respectively (Fig. 1). Thus, the proportion of Genr in retail chicken meat increased significantly by 50% from the years 2009 to 2013 to the years 2014 to 2017.

FIG 1.

FIG 1

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Proportion of gentamicin-resistant E. coli isolates from human and chicken sources (animal and retail meat) collected from 2009 to 2017. Shown are the proportions of Genr E. coli isolates within the CIPARS chicken animal (open circles) and retail meat (closed circles) isolate and CANWARD human clinical isolate (closed squares) surveillance programs.

STs and phylogenomic analyses.

Short-read whole-genome sequencing (WGS) was performed on Genr isolates collected from 2014 to 2017 (n = 483), including 205 isolates from humans and 278 from chicken sources (167 from animal and 111 from retail sources). A minimum spanning tree (MST) was created using multilocus sequence type (MLST) profiles (Fig. 2). The most common STs in the human isolates were ST131 (n = 109, 53.2%), ST69 (n = 14, 6.8%), ST1193 (n = 11, 5.4%), and ST410 (n = 10, 4.9%), with the remaining 29.8% (n = 61) comprised of 26 other STs, and four isolates with unknown STs. The most common STs in chicken-source isolates were ST117 (n = 23, 8.3%), ST10 (n = 17, 6.1%), ST101 (n = 17, 6.1%), and ST155 (n = 15, 5.4%), with the remaining 74.1% (n = 200) comprised of 102 other STs, and six isolates with unknown STs. Thus, chicken-source isolates were very diverse, comprising at least 107 different STs with little overlap with STs from human isolates. The globally disseminated ST131 was the predominant ST in human isolates and present in seven chicken-source isolates.

FIG 2.

FIG 2

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Minimum spanning trees (MSTs) with isolates grouped according to multilocus sequence types. Trees were constructed using the PHYLOViZ 2.0 online software (goeBURST full MLST algorithm) with all study isolates (n = 483). Panel A depicts the source of isolates within each node, and panel B depicts the ST of each node. Node sizes represent the proportion of isolates (log scale), and linked nodes are connected by lines. MLST clusters labeled 1 to 5 (shaded in gray) as well as the ST131 node (labeled 6) were further analyzed with SNV-based phylogenetic dendrograms in Fig. S2.

We carried out phylogenomic analysis based on single nucleotide variants (SNVs) in the core genome for all isolates (see Fig. S1 in the supplemental material). In the phylogenomic dendrogram of all isolates, there were three small clusters of closely related human and chicken-source isolates (either human and animal or human and meat). In a cluster of ST23 isolates, a human-source isolate (collected in Eastern Canada in 2016) differed by 4 to 5 SNVs from two retail meat isolates (collected from Central Canada in 2015 and 2017). In a cluster of ST349 isolates, a human-source isolate (collected from Central Canada in 2017) differed by 6 SNVs from two retail meat isolates (collected from Central Canada in 2015 and 2017). Finally, within a pair of ST648 isolates, a human-source isolate (collected from Eastern Canada in 2015) differed by 5 SNVs from an animal isolate (collected from Central Canada in 2017). In all 3 of these cases, the Genr gene aac(3)-VIa was common to all isolates.

In order to better visualize potential genomic relatedness of isolates from human sources and chicken sources, six smaller phylogenomic dendrograms were created for each MLST cluster that contained isolates from all three sources: human, animal, and retail. An MLST cluster was defined as isolates/isolate groups that differed at only one locus. Five dendrograms included samples from MLST clusters marked 1 to 5 in Fig. 2 and Fig. S2. There were multiple instances of closely related isolates (≤10 SNVs) within each source; however, there were no instances of closely related human and chicken isolates within these five smaller dendrograms. The sixth phylogenomic tree was constructed for ST131 isolates, since this sequence type is of interest due to high rates of extended-spectrum beta-lactam resistance and higher virulence in human infections (16). The most closely related ST131 isolates from human and chicken sources differed by 115 SNVs (Fig. S2f).

Antimicrobial resistance.

Six antimicrobials were tested by both surveillance programs (amoxicillin-clavulanic acid, cefoxitin, ceftriaxone, ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole). Among 205 Genr E. coli isolates from human sources, coresistance with gentamicin was observed with ciprofloxacin (n = 141, 68.8%), trimethoprim-sulfamethoxazole (n = 28, 62.4%), ceftriaxone (n = 89, 43.4%), amoxicillin-clavulanic acid (n = 26, 12.7%), and cefoxitin (n = 6, 2.9%). Among 278 chicken-source isolates, coresistance with gentamicin was observed with trimethoprim-sulfamethoxazole (n = 45, 16.2%), ceftriaxone (n = 40, 14.4%), amoxicillin-clavulanic acid (n = 39, 14.0%), cefoxitin (n = 38, 13.7%), and ciprofloxacin (n = 3, 1.1%). The three ciprofloxacin-resistant chicken isolates were from animal (n = 2) and retail meat (n = 1). Thus, chicken-source isolates were more frequently coresistant to cefoxitin while human-source isolates were more frequently coresistant to ciprofloxacin, trimethoprim-sulfamethoxazole, and ceftriaxone.

Detection of antimicrobial resistance genes with Staramr revealed that 87/205 (42.4%) Genr E. coli isolates from humans carried an extended-spectrum beta-lactamase (ESBL) allele: 62 isolates carried blaCTX-M-15, 14 carried blaCTX-M-14, five carried blaCTX-M-55, two carried blaSHV-12, and one each carried blaSHV-2, blaCTX-M-9, blaCTX-M-27, and blaCTX-M-88. Eight isolates (2.9%) from chicken animal/retail meat carried an ESBL allele: six with blaSHV-2 and one each with blaCTX-M-1 and blaCTX-M-14. The AmpC-type beta-lactamase blaCMY-2, which also confers resistance to ceftriaxone, was detected in isolates from humans (n = 5, 2.4%), chickens (n = 25, 15%), and chicken meat (n = 13, 11.7%).

We analyzed the proportions of multiclass resistance for the six antimicrobials, which included four classes (aminoglycosides, beta-lactams, fluoroquinolones, and sulfonamides). Among human-source isolates, 32.7% (n = 67) were resistant to two classes, 34.1% (n = 70) were resistant to three classes, and 25.9% (n = 53) were resistant to all four classes. For chicken isolates, 14.7% (n = 41) were resistant to two classes, and 14.4% (n = 40) were resistant to three classes. Thus, 60.0% of human-source isolates and 14.4% of chicken-source isolates were multidrug resistant (MDR), with resistance to three or more antimicrobial classes.

Gentamicin and spectinomycin resistance genes.

The Genr and Specr genes detected in human and chicken (animal and retail) isolates are shown in Tables 1 and 2. The Genr genes found in human isolates were as follows: aac(3)-IId (n = 108, 52.7%), aac(3)-IIa (n = 76, 37.1%), aac(6′)-Ib-cr (n = 57, 27.8%), and aac(3)-VIa (n = 12, 5.9%), with six genes [aac(3)-IVa, aac(6′)-Ib3, aac(6′)-IIc, aac(6′)-Ib-Hangzhou, and ant(2″)-Ia] occurring eight times combined (3.9%), while no known Genr genes were detected in 3.4% (n = 7) of isolates. Additionally, one 16S rRNA methyltransferase, rmtB, was found. Sixty-eight (33.2%) human isolates contained more than one Genr gene. In chicken isolates, the four Genr genes identified were as follows: aac(3)-VIa (n = 235, 84.5%), aac(3)-IId (n = 41, 14.7%), aac(3)-IVa (n = 9, 3.2%), and ant(2″)-Ia (n = 1, 0.4%). Seven chicken isolates (2.5%, 6 animal and 1 retail) contained more than one Genr gene.

TABLE 1.

Genes conferring resistance to gentamicin in E. coli from human and chicken sources

Genr gene No. (%) by source:
 Total gene occurrence
Human (n = 205 isolates)a Chicken—animal (n = 167 isolates)a Chicken—retail (n = 111 isolates)a
aac(3)-VIa 12 (5.9) 144 (86.2) 91 (82.0) 247
aac(3)-IId 108 (52.7) 22 (13.2) 19 (17.1) 149
aac(3)-IIa 76 (37.1) 0 0 76
aac(6′)-Ib-cr 57 (27.8) 0 0 57
aac(3)-IVa 2 (1.0) 7 (4.2) 2 (1.8) 11
No gene detected 7 (3.4) 0 0 7
aac(6′)-Ib3 3 (1.5) 0 0 3
aac(6′)-IIc 2 (1.0) 0 0 2
aac(6′)-Ib-Hangzhou 1 (0.5) 0 0 1
ant(2″)-Ia 0 1 (0.6) 0 1
rmtB 1 (0.5) 0 0 1
555
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a

Multiple Genr genes were detected in isolates from humans (n = 68), chicken (animal) (n = 6), and retail chicken (n = 1).

TABLE 2.

Genes conferring resistance to spectinomycin in E. coli from human and chicken sources

Specr gene No. (%) by source:
 Total gene occurrence
Human (n = 205 isolates)a Chicken—animal (n = 167 isolates)a Chicken—retail (n = 111 isolates)a
ant(3″)-Ia 11 (5.4) 130 (77.8) 83 (74.8) 224
None 68 (33.2) 19 (11.4) 17 (15.3) 104
aadA5 95 (46.3) 0 0 95
aadA1 13 (6.3) 16 (9.6) 10 (9.0) 39
aadA2 22 (10.7) 5 (3.0) 4 (3.6) 31
aph(3″)-Ib 26 (12.7) 0 0 26
strA 2 (1.0) 0 0 2
aadA12 0 1 (0.6) 0 1
aadA15 0 1 (0.6) 0 1
523
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a

Multiple Specr genes were detected in isolates from humans (n = 29), animals (n = 5), and retail meat (n = 3).

Of the lincomycin and/or spectinomycin resistance (Lincor/Specr) genes detected, all were predicted to confer resistance to spectinomycin alone. The Specr genes in human-source isolates were aadA5 (n = 95, 46.3%), followed by aph(3″)-Ib (n = 26, 12.7%), aadA2 (n = 22, 10.7%), aadA1 (n = 13, 6.3%), ant(3″)-Ia (n = 11, 5.4%), and strA (n = 2, 1.0%), with none detected in 68 (33.2%) isolates. Multiple Specr genes were found in 29 (14.1%) human isolates. The Specr genes found in chicken isolates were ant(3″)-Ia (n = 213, 76.6%), aadA1 (n = 26, 9.4%), aadA2 (n = 9, 3.2%), aadA15 (n = 1, 0.4%), and aadA12 (n = 1, 0.4%), with none detected in n = 36 (12.9%) isolates. Multiple Specr genes were found in eight isolates (2.9%, five from animal and three from retail meat).

In human isolates, the most common cooccurrence of Genr and Specr genes within the same isolate was aac(3)-IId (Genr) and aadA5 (Specr), where aac(3)-IId was found in 60.0% of isolates carrying aadA5, and vice versa, aadA5 was found in 52.8% of isolates carrying aac(3)-IId (Table 3). In chicken isolates, the most common cooccurrence was aac(3)-VIa (Genr) and ant(3″)-Ia (Specr), where aac(3)-VIa was found in 98.6% of isolates carrying ant(3″)-Ia, and, vice versa, ant(3″)-Ia was found in 89.4% of isolates carrying aac(3)-VIa (Table 3).

TABLE 3.

Cooccurrence of the most frequently observed Genr and Specr genes in E. coli isolates from human and chicken sources

Resistance type Human source
 Chicken source
Most frequent gene No. of occurrences (%) Most frequent gene No. of occurrences (%)
Genr aac(3)-IId 108 aac(3)-VIa 235
Specr aadA5 95 ant(3″)-Ia 213
Genr with Specr aac(3)-IId with aadA5 57/108 (52.8) aac(3)-VIa with ant(3″)-Ia 210/235 (89.4)
Specr with Genr aadA5 with aac(3)-IId 57/95 (60) ant(3″)-Ia with aac(3)-VIa 210/213 (98.6)
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Of the 483 isolates, short-read sequencing was able to link only 14.9% (n = 72) of Genr genes to a known Inc group on the same assembled contig. Of those 72, the majority were located on IncI1 plasmids (68.1%, n = 49) with the remainder on IncF (12.5%, n = 9), IncA/C2 (6.9%, n = 5), p0111 (5.5%, n = 5), and other plasmids (8.3%, n = 6 [Table S1]). Of the 483 isolates, 74 (15.3%) had Specr genes linked to known Inc groups on the same contig, with the majority located on IncI1 plasmids (66.2%, n = 49) and the remainder on IncA/C2 (10.8%, n = 8) and other plasmids (28.4%, n = 21). Thus, both Genr and Specr were most commonly linked to IncI1 plasmids whenever linkage was possible with the short-read data.

Plasmid analysis.

Supplemental long-read sequencing was performed on 23 isolates to facilitate complete assembly of the Genr plasmids and evaluate linkage of Genr and Specr. Three isolates had Genr genes on two separate plasmids within the same isolate. The 23 isolates included eight human isolates and 15 chicken isolates comprising 11 unique STs and two isolates lacking a defined ST. Of the 23 Genr isolates, 22 also contained a Specr gene.

The pangenome feature of the GView tool was used to align annotated plasmids of the same Inc type (Fig. 3). Plasmids within each of the groups (IncI1, p0111, other Inc types, and no Inc) showed diversity in length and content. Seven plasmids were IncI1 (all chicken, Fig. 3A), and three were p0111 (all chicken, Fig. 3B). For the longest homologous region between any pair of plasmids within the IncI1 and p0111 groups, there was a minimum of 98.5%, and 99.8% nucleotide identity, respectively. Nine plasmids were various other Inc types (four human and five chicken) including IncA/C2, IncH, IncF, IncP1, and IncQ1, while six plasmids (four human and two chicken) had no defined Inc type. Plasmids within the “other Inc type” group and the “no Inc type” group contained plasmids from both human and chicken sources; however, there was very little overlap in sequences, and thus, these plasmid alignments are not shown. In the “other Inc type” group, the longest homologous region of the four human-source plasmids accounted for only <5% of the total length of the plasmid from chicken. The chicken-source isolate was an IncH plasmid while the human-source plasmids were all IncF/IncP. In the “no Inc type” group, plasmids from human and chicken sources were highly dissimilar, with homologous regions accounting for <6% of the total plasmid length when comparing plasmids from both sources. In general, there was little homology between Genr plasmids from human and chicken sources.

FIG 3.

FIG 3

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Pangenome sequence alignments of closed plasmids obtained through long-read sequencing. Genr plasmids of similar incompatibility (Inc) groups were aligned in GView, which displays a pangenome at the bottom of each alignment containing all of the genetic content of all plasmids. Genetic maps are shown for IncI1 (A) and p0111 (B) plasmids. Shown below each AMR gene are the antimicrobials that the gene is predicted to confer resistance to and the number of plasmids in the long-read data set that the gene is located on. AMC, amoxicillin-clavulanic acid; AMP, ampicillin; AZM, azithromycin; FOX, cefoxitin; CRO, ceftriaxone; CHL, chloramphenicol; CIP, ciprofloxacin; ERY, erythromycin; HYG, hygromycin; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; SSS, sulfisoxazole; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole; SPT, spectinomycin; TIO, ceftiofur.

DISCUSSION

While no significant increase of Genr was observed in human and chicken animal E. coli from the years 2009 to 2013 to the years 2014 to 2017, we found an increase in Genr in retail meat isolates during this time period (P value of <0.0001). Meat may become contaminated with the contents of the gastrointestinal tract of the animal during processing and packaging. There was apparent increased contamination of chicken meat with Genr E. coli in 2014 to 2017 compared to 2009 to 2013 in our study. Other studies have reported Genr increases in E. coli in both human and chicken sources (17–19). Kronvall (17) and Mendoza-Palomar et al. (19) described Genr increases in Swedish hospital isolates over a 30-year period and in neonatal isolates collected from a tertiary-care hospital in Barcelona, Spain, over a 20-year period, respectively. Tadesse et al. (18) noted a significant upward trend in gentamicin resistance in isolates from various farm animal sources (chicken, cattle, and pig) throughout a 24-year period, with the highest increase observed in chicken.

Genome sequencing showed that Genr E. coli isolates from human and chicken sources were mostly unrelated, but there were a small number of examples of similar genetic backgrounds and resistance genes. The most frequently identified STs were ST131, ST69, ST1193, and ST410 in human-source isolates and ST117, ST10, ST101, and ST155 in chicken-source isolates. All of these STs have been found before in these respective sources (20–24), with some STs (ST410 [24] and ST1193 [25] in human isolates, ST10 in chickens [20], and ST131 in both sources [20, 23]) frequently carrying ESBL genes. In humans, many of these are also known to cause urinary tract and bloodstream infections (20–22). Phylogenomic analyses revealed three small clusters of closely related (≤10 SNVs) (26, 27) isolates that included at least one isolate each from human and chicken, suggesting that chickens may be a minor reservoir of Genr E. coli causing human infections.

A large proportion of isolates were multidrug resistant, with over one-third of all isolates displaying resistance to three or more antimicrobial classes. The proportion of MDR was overall much higher in human isolates. High proportions of Genr human isolates were coresistant to antimicrobials that are classified by Health Canada as category 1 (very high importance to human medicine), including third-generation cephalosporins and fluoroquinolones. Regarding third-generation cephalosporins, ceftriaxone resistance was observed more frequently in human isolates (43.4%) than in chicken isolates (14.4%), which is consistent with recent Canadian surveillance reports for human (1) and chicken (13, 28), specifically the observation that ceftriaxone resistance in chickens has been declining during this study period as per CIPARS annual reports. Some 42.4% of Genr E. coli isolates from humans contained an ESBL allele, usually blaCTX-M-15 or blaCTX-M-14. This is consistent with the observation that more than half of Genr E. coli isolates from humans were ST131, a globally disseminated strain that is associated with high rates of antimicrobial resistance (AMR) and virulence (16). The ESBL allele blaCTX-M-14 has been found to coexist with other important resistance genes such as mcr-1 (29). It is also notable that eight isolates (2.9%) from chicken sources contained an ESBL gene, since preventative use of cephalosporins in the poultry industry was discontinued in 2014 and third-generation cephalosporins remain an important drug in human medicine (30). However, the ESBL genes were very different between humans and chickens, whereby blaCTX-M was predominant in humans (96.5% of ESBL genes) while blaSHV was predominant (75.0% of ESBL genes) in chicken sources. Many of the ESBLs found here have also been frequently found in other studies of E. coli from chicken (31, 32). Falgenhauer et al. (31) found that in E. coli isolates, blaCTX-M genes were very common in humans (as well as in chickens) while blaSHV was found only in chickens. Saliu et al. (32) also found both blaSHV-2 and blaCTX-M-1 in chicken isolates in a 2016 European study. The AmpC-type beta-lactamase blaCMY-2 was found almost exclusively in chicken sources and was over seven times more frequent in chickens than in humans. Most of the ceftriaxone resistance in chicken-source isolates could be explained by the presence of the AmpC-type beta-lactamase blaCMY-2. Of the 11 isolates containing an ESBL or AmpC-type beta-lactamase on which long-read sequencing was performed, these genes were most frequently carried on IncA/C2 plasmids. Ciprofloxacin resistance was observed almost exclusively in human isolates, which likely reflects differences in fluoroquinolone usage, as this class of antibiotics was reported by CIPARS in 2016 to be the third most frequently sold class of antibiotics in humans while it was rarely used in production animals (13). Three chicken-source isolates were resistant to ciprofloxacin, a phenomenon noted to have risen within the past decade in Enterobacterales in several countries (33). Resistance was also frequently observed for category II antimicrobials (high importance), including trimethoprim-sulfamethoxazole, in both sources (7).

Aminoglycoside-modifying enzymes are the most common mechanism of resistance to aminoglycosides, and within this group, aminoglycoside acetyltransferases (AACs) are the most common class (12). In this study, aac(3)-IId was the most frequently detected Genr gene in human isolates while aac(3)VIa was the most frequent in chicken isolates. Although the most common Genr gene was different in human and chicken sources, both sources carried aac(3)-IId, aac(3)-VIa, and aac(3)-IVa. Indeed, the predominant Genr gene in human-source isolates, aac(3)-IId, was also identified in 14.7% of chicken-source isolates. The observation that aac(3)-VIa was the most frequently detected gene in E. coli from chickens is consistent with a previous study by Chalmers et al. where aac(3)-VIa was also frequently identified in E. coli from broiler chickens in Quebec, Canada (15). In addition, we also recently reported that aac(3)-VIa was commonly found in human and chicken-source Salmonella isolates (34). Some AMEs identified here are known to confer resistance to other aminoglycosides such as tobramycin and kanamycin (11). All genes encoding Lincor/Specr detected in this study conferred resistance specifically to spectinomycin. These genes, including ant(3″)-Ia and several aadA genes, code for the enzyme ANT(3″)-I. The cooccurrence of Genr and Specr genes was more common in isolates from chicken sources than in those from human sources. Further, long-read sequencing showed that Genr and Specr genes were frequently linked on the same plasmid. These observations suggest that spectinomycin use in broiler chickens on farms may coselect for Genr.

One limitation of this study is that the human-source isolates were not obtained from gastrointestinal illness, the isolates from which are more likely to represent foodborne illness than are hospital isolates. There are no national surveillance programs of antimicrobial resistance in E. coli from foodborne illness in Canada; thus, we used E. coli isolates from the CANWARD hospital surveillance program.

There were a few instances of similar Genr strains in human and chicken sources, suggesting that chicken may be a minor reservoir of Genr E. coli causing human infection. Human infections with Genr E. coli may be acquired from other sources, including domestic person-to-person transmission, travel, or domestic and imported food commodities that were not included in this study.

MATERIALS AND METHODS

Bacterial isolates.

Isolates in the study were collected from 2014 to 2017 by two national surveillance programs. The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) collected E. coli isolates from chicken sources (clinical, manure, and cecal) and retail chicken products. Further information on the sampling and testing methods used by CIPARS can be found in the annual report (35). Within the Canadian Antimicrobial Resistance Alliance (CARA; www.can-r.ca), human E. coli isolates were collected for the Canadian Ward (CANWARD) surveillance study, a national, multiyear study of pathogens isolated from patients in Canadian hospitals. CANWARD focuses on specific wards (medical, surgical, intensive care units [ICUs], emergency rooms, and clinics) and specific infection sites (urine, blood, respiratory tract, and wound) (36). Isolates in this study were categorized into one of the following three sources: human clinical isolates, animal (live or recently slaughtered broiler chickens collected from farms and abattoirs and veterinary clinical samples), and retail chicken meat purchased from stores.

AST.

CIPARS carried out antimicrobial susceptibility testing (AST) by broth microdilution with the Sensititre Complete Automated AST system using the CMV3AGNF panel in 2014 to 2015 and the CMV4AGNF panel in 2016 to 2017. CANWARD carried out AST by broth microdilution using panels prepared in-house with antimicrobials obtained as laboratory-grade powders from the respective manufacturers or from Sigma-Aldrich (Oakville, Canada). Six antimicrobials were tested by both programs (amoxicillin-clavulanic acid, cefoxitin, ceftriaxone, ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole). MICs were interpreted according to breakpoints from the Clinical and Laboratory Standards Institute (37) for all drugs. Susceptible, intermediate, and resistant breakpoints for gentamicin were ≤4 μg/mL, 8 μg/mL, and ≥16 μg/mL, respectively.

WGS.

Short-read whole-genome sequencing (WGS) was performed on Genr isolates collected from 2014 to 2017 (n = 483), including 205 isolates from humans and 278 from chicken sources (167 from animal and 111 from retail meat). The 278 chicken isolates comprised a random selection of approximately 70 Genr isolates per year for each of the 4 years from 2014 to 2017 using the RAND function in Excel for a simple random sample. DNA was extracted using the DNeasy 96 blood and tissue kit (Qiagen, Hilden, Germany). DNA libraries were prepared using Nextera XT (Illumina, CA, USA), and WGS was performed on the NextSeq platform (Illumina, CA, USA) using NextSeq 500/550 150 cycle kits. If necessary, isolates were resequenced to obtain a minimum coverage of ≥40×.

For long-read sequencing, a convenience sample of isolates was chosen (n = 23) to include diverse sources, sequence types (STs), resistance profiles, and plasmid Inc types. Eight of these isolates were from human sources, 10 were from chickens, and five were from retail chicken meat. DNA extractions for long-read sequencing were carried out using the MasterPure complete DNA and RNA purification kit (Epicentre, Madison, WI, USA). Libraries were prepared using the Rapid Barcoding Sequencing kit (SQK-RBK004) (Oxford Nanopore Technologies, Oxford, UK), and sequencing was performed on the MinION platform (Oxford Nanopore Technologies, Oxford, UK).

Genomic analysis.

Phylogenomic analyses based on single nucleotide variants (SNVs) in the core genome were carried out with the SNVPhyl v1.0.1b (38) pipeline (minimum coverage, 15; minimum mean mapping quality, 30; SNV abundance ratio, 0.75; SNV density filter, 2 or more SNVs per 20-base window). Minimum spanning trees (MSTs) were constructed using PHYLOViZ 2.0 online software (http://online.phyloviz.net/index; goeBURST MLST algorithm) (39) based on multilocus sequence type (MLST) profiles generated using the Achtman scheme (40). Genomes were assembled from short reads using SPAdes v3.11.1 with FLASH v1.2.11.3 (41), and resistance genes were identified with Staramr v0.6.0 (42) (https://github.com/phac-nml/staramr; percent length overlap of BLAST hit for ResFinder, 60; percent nucleotide identity for BLAST hit, 98). For the subset of isolates with long-read sequence data, genomes were also constructed by hybrid assembly of short reads and long reads using Unicycler v0.4.7 (43), and assemblies were annotated using Prokka v1.13 (44). Plasmids were visualized with the GView v1.7 web server (45) (https://server.gview.ca/) using pangenome analyses with blastn (parameters: minimum expected value, 1 × 10−10; minimum alignment length, 150; minimum percent identity, 98). To quantify plasmid similarity, nucleotide sequences were aligned with MegaBLAST (46).

Data availability.

Sequence read data for all 483 E. coli isolates were submitted to the National Center for Biotechnology Information (NCBI) under BioProject accession no. PRJNA756559. BioSample identifiers (IDs) for all isolates are listed in Table S1 in the supplemental material.

ACKNOWLEDGMENTS

We thank the National Microbiology Laboratory’s Genomics Core Facility and Bioinformatics Core Facility for support with whole-genome sequencing and bioinformatics. We also thank the Technicians at the NML Winnipeg, NML Guelph, and NML Saint-Hyacinthe laboratories and Winnipeg Health Science Centre for performing antimicrobial susceptibility testing.

This research was supported by a grant from the Canadian Institutes for Health Research (CFC-150770).

We declare no conflicts of interest.

Footnotes

Supplemental material is available online only.

Supplemental file 1
Fig. S1. Download aac.00677-22-s0001.svg, SVG file, 0.1 MB (113.6KB, svg)
Supplemental file 2
Fig. S2. Download aac.00677-22-s0002.svg, SVG file, 0.1 MB (133.8KB, svg)
Supplemental file 3
Isolate line list. Download aac.00677-22-s0003.xlsx, XLSX file, 0.1 MB (94.7KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Fig. S1. Download aac.00677-22-s0001.svg, SVG file, 0.1 MB (113.6KB, svg)

Supplemental file 2

Fig. S2. Download aac.00677-22-s0002.svg, SVG file, 0.1 MB (133.8KB, svg)

Supplemental file 3

Isolate line list. Download aac.00677-22-s0003.xlsx, XLSX file, 0.1 MB (94.7KB, xlsx)

Data Availability Statement

Sequence read data for all 483 E. coli isolates were submitted to the National Center for Biotechnology Information (NCBI) under BioProject accession no. PRJNA756559. BioSample identifiers (IDs) for all isolates are listed in Table S1 in the supplemental material.

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