PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0221429 PONE-D-19-16109 Research Article Biology and life sciencesMicrobiologyMicrobial controlAntimicrobial resistanceMedicine and health sciencesPharmacologyAntimicrobial resistanceBiology and life sciencesOrganismsBacteriaCampylobacterBiology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensBacterial pathogensCampylobacterMedicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensBacterial pathogensCampylobacterBiology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesBirdsPoultryBiology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesBirdsFowlGamefowlChickensBiology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesBirdsPoultryChickensMedicine and health sciencesPharmacologyDrugsAntimicrobialsAntibioticsTetracyclinesBiology and life sciencesMicrobiologyMicrobial controlAntimicrobialsAntibioticsTetracyclinesBiology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesBirdsBiology and life sciencesOrganismsBacteriaCampylobacterCampylobacter jejuniBiology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensBacterial pathogensCampylobacterCampylobacter jejuniMedicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensBacterial pathogensCampylobacterCampylobacter jejuniBiology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesBirdsFowlGamefowlTurkeysBiology and life sciencesOrganismsEukaryotaAnimalsVertebratesAmniotesBirdsPoultryTurkeys Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from small poultry flocks in Ontario, Canada: A two-year surveillance study Antimicrobial resistance in Campylobacter isolated from small poultry flocks in Ontario, Canada http://orcid.org/0000-0003-2751-3677 Varga Csaba Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing – original draft Writing – review & editing 1 * Guerin Michele T. Conceptualization Data curation Investigation Methodology 2 Brash Marina L. Conceptualization Investigation Methodology Resources 3 Slavic Durda Project administration Resources 3 Boerlin Patrick Investigation Methodology 4 Susta Leonardo Conceptualization Funding acquisition Investigation Methodology Resources 4 Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario, Canada Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada Animal Health Laboratory, University of Guelph, Guelph, Ontario, Canada Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada Karunasagar Iddya Editor Nitte University, INDIA

The authors have declared that no competing interests exist.

 * E-mail: cvarga@uoguelph.ca 29 8 2019 2019 14 8 e0221429 6 6 2019 6 8 2019 2019 Varga et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Antimicrobial resistance in Campylobacter, common in poultry, is a global public health issue. The emergence and spread of antimicrobial resistant Campylobacter has been linked to the use of antimicrobials in food animals. Small poultry flocks are becoming increasingly popular not only as a source of food but also as pets, yet not all small flock owners are aware of proper antimicrobial use practices and safe food handling protocols. This trend could contribute to antimicrobial resistance. In order to determine the prevalence of antimicrobial resistance in Campylobacter in small poultry flocks, we analyzed data from birds that had been submitted to a diagnostic laboratory in Ontario between October 2015 and September 2017. A pooled cecal sample was obtained from each submission and cultured for Campylobacter jejuni and Campylobacter coli. Three isolates were recovered from each positive sample and tested for susceptibility to nine antimicrobials using a broth microdilution method. Overall, 176 isolates were recovered (141 chicken, 21 turkey, 6 duck, and 8 game bird). A high frequency of resistance to tetracycline was observed in the C. jejuni isolates from chickens (77%) and turkeys (100%), and in the C. coli isolates from turkeys (50%) and game birds (40%). Campylobacter jejuni isolates had higher odds of resistance to tetracycline (OR = 3.54, P ≤ 0.01) compared to C. coli isolates. Overall, there was a low frequency of resistance to quinolones and a very low frequency of resistance to macrolides. Multidrug resistance was uncommon. The high prevalence of tetracycline resistance emphasizes the importance of prudent antimicrobial use in small flocks. Although low, the presence of resistance to macrolides and quinolones, which are used to treat campylobacteriosis in humans, highlights the need for proper food safety and infection control practices by small flock owners to prevent exposure to antimicrobial resistant Campylobacter.

 Ministry of Agriculture, Food and Rural Affairs (KR) UofG 2015-2282 Susta Leonardo Ontario Agri-Food Innovation Alliance, under the Disease Surveillance Plan xx http://orcid.org/0000-0003-2751-3677 Varga Csaba The surveillance project led by Dr. L. Susta, was funded by the OMAFRA-University of Guelph Strategic Partnership (grant UofG 2015-2282), under the Disease Surveillance Plan, which was a joint federal-provincial Growing Forward 2 project. The antimicrobial susceptibility testing project led by Dr. C. Varga, was funded by the Ontario Agri-Food Innovation Alliance, under the Disease Surveillance Plan (Project #: 009098). Data Availability All relevant data are within the manuscript. Introduction

Small poultry flocks are becoming increasingly popular in Canada [1, 2]. Poultry are reservoirs of thermophilic Campylobacter species, especially Campylobacter jejuni and Campylobacter coli, and they shed the bacteria in their feces [3]. Campylobacter can be transmitted from backyard poultry to humans through activities that might expose a flock owner to their birds’ feces, such as cleaning the birds’ housing and removing soiled bedding, bird handling, petting, and kissing [4], or through handling and consumption of contaminated eggs and meat [3]. Campylobacter are a significant cause of enteric disease in humans [5–7]. Antimicrobial resistance (AMR) poses an additional risk [8, 9] because infections caused by antimicrobial resistant Campylobacter lead to longer hospitalizations, higher treatment failures, and increased morbidity and mortality [10].

The transmission of antimicrobial resistant Campylobacter from commercial broiler and free-range chickens to humans has been described previously [11]. In Canada, Campylobacter isolates from retail poultry meat were genetically linked to isolates from human clinical cases, and a high proportion of those isolates were resistant to tetracycline [12]. Moreover, other Canadian research studies and surveillance programs have reported a high prevalence of tetracycline and fluoroquinolone resistance among Campylobacter isolates from on-farm and abattoir chicken samples in Ontario, and from retail chicken meat samples in British Columbia, Saskatchewan, and Ontario [13–15]. A high prevalence of tetracycline and fluoroquinolone resistance in Campylobacter isolates from commercial poultry has also been reported in Poland [16], China [17], and Italy [18].

Antimicrobial use, including overuse and misuse in food animals, is considered a contributing factor for the selection and emergence of antimicrobial resistant enteric bacteria [19, 20]. Until recently, small flock owners in Canada could purchase antimicrobials over the counter without a veterinary prescription [21]. This might have favoured improper antimicrobial use.

There are few research studies on AMR in enteric bacteria from small flocks [22, 23]. To address this knowledge gap, we evaluated AMR patterns in C. jejuni and C. coli isolates from small flock chickens, turkeys, waterfowl, and game birds submitted to a veterinary diagnostic laboratory in Ontario.

 Methods Study design

Samples were obtained through a 2-year prospective surveillance study conducted in Ontario from October 2015 to September 2017 [24]. In brief, small flock owners, through their veterinarian, submitted cases suffering production issues, clinical illness, or mortality to the Animal Health Laboratory, University of Guelph. A submission consisted of not more than 5 sick and/or dead birds of one species from the same flock. Small poultry flocks were defined as flocks consisting of not more than 299 broiler chickens, 99 layer chickens, 49 turkeys, 300 waterfowl, or 300 game birds.

 Sample collection and <italic>Campylobacter</italic> isolation

Campylobacter isolation and antimicrobial susceptibility testing were performed at the Animal Health Laboratory in Guelph, Ontario. One pooled cecal sample was collected from each submission and cultured for Campylobacter. The cecal material was directly plated onto Campylobacter Blood Free media (Bio-Media Unlimited Ltd., Toronto, Ontario, Canada) and incubated in a microaerophilic environment at 37°C for 72 h. Presumptive Campylobacter colonies (i.e., yellowish-gray, translucent, round, 1–2 mm diameter, smooth to slightly mucoid) were selected and identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Bruker Ltd., Billerica, Massachusetts, USA).

 Antimicrobial susceptibility testing and classification

Three isolates were selected from each positive sample and tested for susceptibility to nine antimicrobials (NARMS CAMPY plates) using an automated broth microdilution technique (Sensititre®; Trek Diagnostic Systems Inc., Westlake, Ohio, USA). The minimum inhibitory concentration interpretive standards of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (for most antimicrobials) [15] or the Centers for Disease Control and Prevention (for telithromycin) [25] were used to classify Campylobacter isolates as susceptible or resistant (resistant plus intermediate). The antimicrobials, and their antimicrobial class, concentration range, and established susceptibility breakpoints are presented in Table 1. In addition, an isolate was defined as multidrug resistant if it was non-susceptible to one or more antimicrobials in ≥ 3 different antimicrobial classes [26].

10.1371/journal.pone.0221429.t001 Antimicrobial classes, antimicrobial agents, concentration ranges, and susceptibility breakpoints for <italic>Campylobacter</italic> isolates.
Antimicrobial Class Antimicrobial Agent Concentration Range (μg/mL) MIC Interpretive Standard (μg/mL)A
Susceptible Resistant
Aminoglycosides Gentamicin 0.12–32 ≤2 ≥4
Ketolides Telithromycin 0.015–8 ≤4 ≥8
Lincosamides Clindamycin 0.03–16 ≤2 ≥4
Macrolides Azithromycin 0.015–64 ≤2 ≥4
Erythromycin 0.03–64 ≤8 ≥16
Phenicols Florfenicol 0.03–64 ≤4 N/A
Quinolones Ciprofloxacin 0.015–64 ≤1 ≥2
Nalidixic acid 4–64 ≤16 ≥32
Tetracyclines Tetracycline 0.06–64 ≤4 ≥8

AIsolates were tested for antimicrobial susceptibility using an automated broth microdilution technique (Sensititre®). Minimum inhibitory concentration (MIC) interpretive standards of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (for most antimicrobials) [15] or the Centers for Disease Control and Prevention (for telithromycin) [25] were used to classify isolates as susceptible or resistant (resistant plus intermediate).

 Data analysis

All analyses were conducted using Stata 14.2 (Stata Corp, College Station, Texas, USA).

Descriptive statistics

Overall, and for each poultry species (chicken, turkey, other), prevalence estimates for resistance of C. jejuni and C. coli isolates to each of the nine tested antimicrobials were calculated by dividing the number of isolates resistant to an antimicrobial by the total number of isolates tested for the antimicrobial. An exact binomial 95% confidence interval was computed for each prevalence estimate.

In addition, to account for sample-level clustering, adjusted prevalence estimates were computed using a generalized estimating equation (GEE) with a binary outcome, logit-link function, and exchangeable correlation structure. The intercepts (β0) obtained from null binomial models were used to calculate population-averaged prevalence estimates using the following formula [27]: P=[1+exp(‐β0)]‐1.

To achieve model convergence, adjusted prevalence estimates were only calculated for Campylobacter isolated from chicken samples. The 95% confidence interval of the intercept was used for each adjusted prevalence estimate.

Further, prevalence estimates for multidrug resistance were calculated by dividing the number of multidrug resistant Campylobacter isolates by the total number of isolates tested.

 Cluster analyses

To compare individual antimicrobials in terms of their similarity in Campylobacter resistance, cluster analysis, using the Jaccard binary similarity coefficient, was performed for C. jejuni and C. coli isolates from chicken samples. Cluster analysis was not performed for isolates from turkeys or other poultry species due to the small number of isolates. The number of isolates that are resistant to both antimicrobials, and the number that are resistant to one and susceptible to the other are utilized in the calculation of the coefficient. Dendrograms were created using the single-linkage clustering technique with the Jaccard distance. The Jaccard distance measures dissimilarity between antimicrobials and is calculated by subtracting the Jaccard similarity coefficient from one [28].

To explore relationships within the set of nine selected antimicrobials in terms of their similarity in Campylobacter resistance, multiple correspondence analysis, using the Burt method with principal normalization [29, 30], was conducted for C. jejuni and C. coli isolates from chicken samples. Multiple correspondence analysis was not conducted for isolates from turkeys or other poultry species due to the small number of isolates and lack of variation. Dimensions that explained at least two-thirds of the variation in the data were included for further analysis. Observation scores were calculated and plotted to visualize the distribution of antimicrobial resistance patterns along the first two dimensions.

 Logistic regression

To identify differences in Campylobacter resistance between poultry species, logistic regression was used. Only antimicrobials for which ≥ 5% of the isolates were resistant were assessed. Therefore, 3 of 9 antimicrobials were analyzed: ciprofloxacin, nalidixic acid, and tetracycline. Three population-averaged models were fit for each antimicrobial using the GEE described previously. In these univariable models, the dependent variable represented the prevalence of resistance to the antimicrobial, while the independent dichotomous variable was poultry species (model 1: chickens compared to all the other poultry species; model 2: turkeys compared to all the other poultry species; model 3: ducks and game birds compared to all the other poultry species). One additional population-averaged model was fit for each antimicrobial (ciprofloxacin, nalidixic acid, tetracycline) to identify differences between species of Campylobacter. In these models, the dependent variable represented the prevalence of resistance to the antimicrobial, while the independent dichotomous variable was Campylobacter species (C. jejuni compared to C. coli). A P-value ≤ 0.05 on the Wald χ2 test implied a statistically significant association.

 Results Description of submissions

In total, the Animal Health Laboratory received 160 small flock submissions over the 2-year study period. The number of birds per submission ranged from 1–5 (median 1). Flock sizes ranged from 1–299 birds (median 25) and birds ranged from 6 days to 7 years of age (median 7 months). Most of the submissions were chickens (134), although a few were turkeys (10), ducks (8), and game birds (8) [24].

 Descriptive statistics

Of 158 submissions tested for Campylobacter, a total of 176 isolates were recovered: 141 isolates from chicken submissions (47 pooled samples, 3 isolates recovered from each pooled sample); 21 isolates from turkey submissions (7 pooled samples, 3 isolates recovered from each pooled sample); 6 isolates from duck submissions (2 pooled samples, 3 isolates recovered from each pooled sample); and 8 isolates from game bird (pheasant and quail) submissions (3 pooled samples in total; 3 isolates recovered from each of 2 pooled samples and 2 isolates recovered from 1 of the pooled samples). Of 176 isolates, 86 were C. jejuni (77 chicken, 3 turkey, 6 duck), 87 were C. coli (64 chicken, 18 turkey, and 5 game bird), and 3 were not speciated (3 game bird). Of the 176 isolates, 33.3% of the chicken (47/141), 42.9% of the turkey (9/21), and 42.9% of the other poultry species (6/14) were pan-susceptible.

Overall (i.e., all poultry species and all Campylobacter spp. combined), at the isolate-level, there was a high prevalence of resistance (≥ 40% of isolates) to tetracycline, a low prevalence of resistance (5–14% of isolates) to ciprofloxacin and nalidixic acid, and a very low prevalence of resistance (< 5% of isolates) to gentamicin, telithromycin, clindamycin, azithromycin, erythromycin, and florfenicol (Table 2). In the chicken C. jejuni isolates, there was a high frequency of resistance to tetracycline. In the chicken C. coli isolates, there was a moderate frequency of resistance (15–39% of isolates) to tetracycline. In the turkey C. jejuni isolates, there was a high frequency of resistance to tetracycline. In the turkey C. coli isolates, there was a high frequency of resistance to tetracycline, and a moderate frequency of resistance to ciprofloxacin and nalidixic acid. In the C. jejuni isolates of other poultry species, there was a high frequency of resistance to ciprofloxacin. In the C. coli isolates of other poultry species, there was a high frequency of resistance to tetracycline and a moderate frequency of resistance to nalidixic acid. There was no resistance detected for most of the antimicrobials (6 of 9) in the turkey, waterfowl, and game bird isolates.

10.1371/journal.pone.0221429.t002 Percentage of <italic>Campylobacter jejuni</italic> and <italic>Campylobacter coli</italic> isolates from Ontario small poultry flocks that were resistant to nine selected antimicrobials, as determined by a broth microdilution technique, by poultry species.
All poultry species Chicken Turkey Other poultry speciesA
Antimicrobial Campylobacter (N = 176)B C. jejuni (N = 77) C. coli (N = 64) C. jejuni (N = 3) C. coli (N = 18) C. jejuni (N = 6) C. coli (N = 5)
Class AgentC %D [CI]E % [CI] % [CI] % [CI] % [CI] % [CI] % [CI]
Aminoglycosides GEN 0.57 [0.01–3.12] 0 1.56 [0.04–8.40] 0 0 0 0
Ketolides TEL 3.98 [1.61–8.02] 0 10.94 [4.51–21.25] 0 0 0 0
Lincosamides CLI 3.98 [1.61–8.02] 0 10.94 [4.51–21.25] 0 0 0 0
Macrolides AZT 4.55 [1.98–8.76] 1.30 [0.04–7.02] 10.94 [4.51–21.25] 0 0 0 0
ERY 3.98 [1.61–8.02] 0 10.94 [4.51–21.25] 0 0 0 0
Phenicols FLO 0.57 [0.01–3.12] 0 1.56 [0.04–8.40] 0 0 0 0
Quinolones CIP 8.52 [4.84–13.67] 3.90 [0.81–10.97] 9.38 [3.52–19.30] 0 16.67 [3.58–41.42] 50.00 [11.81–88.19] 0
NAL 7.39 [3.99–12.30] 3.90 [0.81–10.97] 9.38 [3.52–19.30] 0 16.67 [3.58–41.42] 0 20.00 [0.50–71.64]
Tetracyclines TET 56.25 [48.58–63.70] 76.62 [65.59–85.52] 35.94 [24.32–48.90] 100 [29.24–100] 50.00 [26.02–73.98] 0 40.00 [5.27–85.34]

AWaterfowl (ducks) and game birds (pheasant and quail).

BAntimicrobial susceptibility testing was conducted on all 176 isolates, including the three game bird isolates that were not speciated.

CAZT = azithromycin; CIP = ciprofloxacin; CLI = clindamycin; ERY = erythromycin; FLO = florfenicol; GEN = gentamicin; NAL = nalidixic acid; TEL = telithromycin; TET = tetracycline.

DPercentage of isolates resistant to the antimicrobial. Prevalence estimates were calculated by dividing the number of isolates resistant to an antimicrobial by the total number of isolates tested for the antimicrobial.

ECI = Exact binomial 95% confidence interval for the percentage of isolates resistant to the antimicrobial.

The adjusted prevalence estimates of AMR in Campylobacter spp. isolated from chicken samples are presented in Table 3. At the sample-level, there was a high prevalence of resistance to tetracycline, a low prevalence of resistance to telithromycin, clindamycin, azithromycin, erythromycin, ciprofloxacin, and nalidixic acid, and a very low prevalence of resistance to gentamicin and florfenicol.

10.1371/journal.pone.0221429.t003 Adjusted prevalence of antimicrobial resistance, accounting for sample-level clustering, in <italic>Campylobacter</italic> isolates from chicken cecal samples from small poultry flocks in Ontario between October 2015 and September 2017 using population-averaged logistic regression models.
Antimicrobial Campylobacter spp. (N = 141)
Class Agent Percentage resistant [95% Confidence Interval]
Aminoglycosides Gentamicin 0.71 [0.10–4.81]
Ketolides Telithromycin 5.28 [1.67–15.49]
Lincosamides Clindamycin 5.28 [1.67–15.49]
Macrolides Azithromycin 6.02 [2.14–15.79]
Erythromycin 5.28 [1.67–15.49]
Phenicols Florfenicol 0.71 [0.10–4.81]
Quinolones Ciprofloxacin 6.81 [2.34–18.18]
Nalidixic acid 6.81 [2.34–18.18]
Tetracyclines Tetracycline 58.69 [44.73–71.39]

The most common antimicrobial resistance patterns in Campylobacter spp. isolated from chicken samples are presented in Table 4. Multidrug resistance was detected in 4.26% (6/141) of isolates (all C. coli) from chicken samples; however, it was not detected in C. jejuni chicken isolates, or in turkey, duck, or game bird Campylobacter isolates.

10.1371/journal.pone.0221429.t004 Most common antimicrobial resistance patterns of <italic>Campylobacter</italic> isolates (N = 141) from chicken cecal samples from small poultry flocks in Ontario between October 2015 and September 2017.
Antimicrobial resistance patternA Number of antimicrobial classes in pattern (multidrug resistant)B n (%)C
TET 1 (no) 77 (54.61)
AZT-CLI-ERY-TEL 3 (yes) 6 (4.26)
CIP-NAL 1 (no) 5 (3.55)
CIP-NAL-TET 2 (no) 3 (2.13)

AResistance to nine selected antimicrobials (including gentamicin and florfenicol) as determined by a broth microdilution technique. AZT = azithromycin; CIP = ciprofloxacin; CLI = clindamycin; ERY = erythromycin; NAL = nalidixic acid; TEL = telithromycin; TET = tetracycline.

BAn isolate was defined as multidrug resistant if it was non-susceptible to one or more antimicrobials in ≥ 3 different antimicrobial classes (Ketolides: TEL; Lincosamides: CLI; Macrolides: AZT, ERY; Quinolones: CIP, NAL; Tetracyclines: TET).

CNumber and percentage of isolates with each antimicrobial resistance pattern. Only patterns with ≥ 3 isolates are shown.

 Cluster analyses

Single-linkage clustering dendrograms with Jaccard distances for C. jejuni and C. coli isolates from chicken samples are presented in Figs 1 and 2, respectively. A low, non-zero dissimilarity measure implies that a relatively high proportion of isolates were resistant to both antimicrobials, whereas a high dissimilarity measure implies that relatively few isolates were resistant to both antimicrobials, and a dissimilarity measure of zero implies that all isolates were susceptible to both antimicrobials. For C. jejuni, there was one cluster of isolates susceptible to ciprofloxacin and nalidixic acid, and a second cluster of isolates susceptible to gentamicin, telithromycin, clindamycin, erythromycin, and florfenicol. For C. coli, a relatively high proportion (i.e., a cluster) of isolates were resistant to telithromycin, clindamycin, azithromycin, and erythromycin. In addition, there was one cluster of isolates susceptible to ciprofloxacin and nalidixic acid, a second cluster susceptible to gentamicin and florfenicol, and a third cluster susceptible to clindamycin, azithromycin, and erythromycin.

10.1371/journal.pone.0221429.g001 Single-linkage clustering dendrogram for resistance to nine antimicrobials in <italic>Campylobacter jejuni</italic> isolates from chicken cecal samples from small poultry flocks in Ontario between October 2015 and September 2017 (n = 77).

AZT = azithromycin; CIP = ciprofloxacin; CLI = clindamycin; ERY = erythromycin; FLO = florfenicol; GEN = gentamicin; NAL = nalidixic acid; TEL = telithromycin; TET = tetracycline.

 10.1371/journal.pone.0221429.g002 Single-linkage clustering dendrogram for resistance to nine antimicrobials in <italic>Campylobacter coli</italic> isolates from chicken cecal samples from small poultry flocks in Ontario between October 2015 and September 2017 (n = 64).

AZT = azithromycin; CIP = ciprofloxacin; CLI = clindamycin; ERY = erythromycin; FLO = florfenicol; GEN = gentamicin; NAL = nalidixic acid; TEL = telithromycin; TET = tetracycline.

Multiple correspondence analysis coordinate plots for the first two dimensions for resistance in C. jejuni and C. coli isolates from chicken samples are presented in Figs 3 and 4, respectively. Five antimicrobials (gentamicin, telithromycin, clindamycin, erythromycin, and florfenicol) were omitted from the C. jejuni analysis because they completely predicted the presence or absence of resistance (i.e., there was no variation). For C. jejuni, the first two dimensions explained 68.2% of the variation in antimicrobial resistance (Fig 3). When observation scores were plotted along dimensions 1 and 2, it was observed that the antimicrobial susceptibility testing results (resistant and susceptible denoted as 1 and 0, respectively) for ciprofloxacin and nalidixic acid formed a pattern (i.e., were clustered together). For C. coli, the first two dimensions explained 82.1% of the variation in antimicrobial resistance (Fig 4). Three patterns were identified: the antimicrobial susceptibility testing results for ciprofloxacin and nalidixic acid clustered together; results for gentamicin and florfenicol clustered together; and results for azithromycin, clindamycin, and erythromycin clustered together.

10.1371/journal.pone.0221429.g003 Multiple correspondence analysis coordinate plot displaying the presence (1) and absence (0) of resistance to four antimicrobials in <italic>Campylobacter jejuni</italic> isolates from chicken cecal samples from small poultry flocks in Ontario between October 2015 and September 2017 for the first two dimensions (n = 77).

AZT = azithromycin; CIP = ciprofloxacin; NAL = nalidixic acid; TET = tetracycline. Five antimicrobials (gentamicin, telithromycin, clindamycin, erythromycin, and florfenicol) were omitted from the analysis because they completely predicted the presence or absence of resistance (i.e., there was no variation).

 10.1371/journal.pone.0221429.g004 Multiple correspondence analysis coordinate plot displaying the presence (1) and absence (0) of resistance to nine antimicrobials in <italic>Campylobacter coli</italic> isolates from chicken cecal samples from small poultry flocks in Ontario between October 2015 and September 2017 for the first two dimensions (n = 64).

AZT = azithromycin; CIP = ciprofloxacin; CLI = clindamycin; ERY = erythromycin; FLO = florfenicol; GEN = gentamicin; NAL = nalidixic acid; TEL = telithromycin; TET = tetracycline.

 Logistic regression

Poultry species were not significantly associated with the prevalence of resistance to individual antimicrobials among Campylobacter isolates. The odds of resistance to tetracycline were significantly higher in C. jejuni isolates (OR = 3.54, 95% CI: 2.00–6.26, P ≤ 0.01) compared to C. coli isolates.

 Discussion

In this study, we assessed the AMR patterns in C. jejuni and C. coli isolates from Ontario small poultry flocks that were submitted by veterinarians to a diagnostic laboratory because of mortality, morbidity, or production problems. A high proportion of the Campylobacter isolates (56%) were resistant to tetracycline, particularly the C. jejuni isolates. This finding is in agreement with a study in Kenya where a high proportion of C. jejuni and C. coli isolates from small scale and backyard chicken flocks were resistant to tetracycline [31], and with a study in Finland where a high proportion of C. jejuni isolates from backyard chicken flocks were resistant to tetracycline [32]. High frequencies of tetracycline resistance have also been identified in Campylobacter isolates (45–96%) from retail meat and commercial poultry flock samples in research studies in Canada [12], China [17], Poland [16], and Italy [18].

It is recognized that antimicrobial use, including overuse and misuse in food animals, is a contributing factor to AMR emergence in commensal and zoonotic enteric bacteria [33, 34]. Previous studies have shown that tetracycline resistance in Campylobacter is generally determined by a plasmid-encoded tet(O) gene [35], and that this resistance gene can be transferred horizontally between C. jejuni and C. coli isolates in the intestinal tract of food animals and humans [36, 37]. Tetracycline is commonly used to treat bacterial diseases of poultry and it was readily available to small flock owners over the counter at livestock medicine outlets during the period of study. In our study, 61% of the flock owners administered medication to their flock within the last 12 months, and 61% of those gave antibiotics, including tetracycline, penicillin, and tylosin (unpublished data). This easy access and potential preferential use could have been a source of selection pressure, resulting in the comparatively higher prevalence of tetracycline resistance in our study than in Canadian commercial chicken flocks (20%) [15].

To reduce the development and spread of AMR in enteric bacteria of food animals, an updated antimicrobial use regulation was implemented in Canada on December 1, 2018 [21]. Under this regulation, a veterinary prescription is required [21] for all medically important antimicrobials in human medicine [38]. As our study was conducted before the updated regulation came into effect, our findings will provide a benchmark for AMR in Campylobacter isolates from small poultry flocks.

Our single-linkage clustering and multiple correspondence analyses revealed a high degree of relatedness between resistance to ciprofloxacin (a fluoroquinolone) and nalidixic acid (a quinolone) in the C. jejuni and C. coli isolates from chicken samples. Fluoroquinolone-resistance in Campylobacter is generally mediated by mutations in the gyrA gene at the Thr-86 position [16]; these mutations lead to resistance to both ciprofloxacin and nalidixic acid [16], and resistant strains may have a competitive advantage over susceptible strains in colonizing the intestinal tract of poultry, even without antimicrobial use selection pressure [39]. Therefore, although the prevalence of quinolone resistance was low, small flock owners should follow good husbandry, biosecurity, and food safety practices given the health risk associated with fluoroquinolone-resistant Campylobacter infection acquired from consumption of contaminated meat [12] or direct contact with live poultry that are actively shedding the bacteria [40]. On the other hand, the lack of relatedness between resistance to the quinolones and other antimicrobial classes is an encouraging finding.

In our study, although we could not assess this statistically, a relatively higher number of C. coli isolates from chicken samples were resistant to azithromycin and erythromycin (macrolides) compared to C. jejuni isolates. Macrolides are first choice antibiotics for the treatment of campylobacteriosis in humans [41]. The emergence of macrolide resistance is supported by substitutions in the 23S rRNA gene, specifically A2075G, and less frequently A2074C/G [42]. Interestingly, this mutation in C. jejuni is associated with a decreased ability to colonize chickens; however, this reduced colonization ability has not been observed in C. coli [42], which might explain the lower frequency of macrolide resistance in the C. jejuni isolates.

Although multidrug resistance was uncommon, the azithromycin—clindamycin—erythromycin—telithromycin pattern was identified in our study. Despite belonging to three different antimicrobial classes (macrolides, lincosamides, and ketolides) and having chemically distinct structures, these four antimicrobials are related molecules that have similar modes of antibacterial action and a single mechanism that encodes resistance for all at once [43–45].

Overall, we found a very low frequency of resistance to ketolides and aminoglycosides, which are categorized in Canada as being of very high and high importance in human medicine, respectively [38]. Further, only a few C. coli chicken isolates were multidrug resistant, and a moderate to high percentage of isolates were pan-susceptible, which is a promising finding from a human health perspective. The high degree of relatedness between gentamicin (an aminoglycoside) and florfenicol (a phenicol) in the isolates from chicken samples was unexpected and could be an incidental finding given the very low prevalence of resistance to these antimicrobials.

Interestingly, there were no differences between poultry species in terms of resistance to individual antimicrobials. However, because the majority of the isolates in our study were from chicken samples, future studies that include a larger number of isolates from other poultry species are needed to identify factors that influence the development of AMR in Campylobacter in small flocks.

Limitations of our study should be considered when interpreting our findings. Our study might overestimate the frequency of AMR in Campylobacter isolates because isolates were obtained from diagnostic submissions of diseased birds that might have been treated with antimicrobials. Also, samples were not obtained randomly, and submissions from areas closer to the diagnostic laboratory were overrepresented [24].

In conclusion, a high proportion of isolates were resistant to tetracycline, an antimicrobial commonly used to treat bacterial diseases of poultry, which emphasizes the importance of prudent antimicrobial use in small flocks. Although low, the presence of resistance to macrolides and quinolones, which are used to treat campylobacteriosis in humans, highlights the need for proper food safety and infection control practices by small flock owners to prevent the transmission of antimicrobial resistant Campylobacter through consumption of contaminated poultry products or direct contact with infected birds. The very low prevalence of resistance to ketolides and aminoglycosides is an encouraging finding from a public health standpoint. As our study was conducted before the antimicrobial use regulation was updated, the results can be used by governmental agencies and researchers as a benchmark to measure changes in AMR patterns in Campylobacter isolates of small poultry flocks.

The authors thank the veterinarians and small flock owners who submitted birds and participated in this study. We also thank the laboratory staff of the Animal Health Laboratory for coordinating and processing all submissions and performing antimicrobial susceptibility testing.

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The epidemiology of antibiotic resistance in Campylobacter. Microbes Infect. 2006; 8(7):1955–66. doi: 10.1016/j.micinf.2005.12.030 16716632 Thakur S, Zhao S, McDermott PF, Harbottle H, Abbott J, English L, Gebreyes WA, White DG. Antimicrobial resistance, virulence, and genotypic profile comparison of Campylobacter jejuni and Campylobacter coli isolated from humans and retail meats. Foodborne Pathog Dis. 2010; 7(7):835–44. doi: 10.1089/fpd.2009.0487 20367499 Doyle ME. Multidrug-resistant pathogens in the food supply. Foodborne Pathog Dis. 2015; 12(4):261–79. doi: 10.1089/fpd.2014.1865 25621383 de Vries SPW, Vurayai M, Holmes M, Gupta S, Bateman M, Goldfarb D, Maskell DJ, Matsheka MI, Grant AJ. Phylogenetic analyses and antimicrobial resistance profiles of Campylobacter spp. from diarrhoeal patients and chickens in Botswana. PLoS One. 2018; 13(3): e0194481. doi: 10.1371/journal.pone.0194481 29561903 Narvaez-Bravo C, Taboada EN, Mutschall SK, Aslam M. 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Antimicrobial resistance in fecal Escherichia coli and Salmonella enterica isolates: A two-year prospective study of small poultry flocks in Ontario, Canada. BMC Vet Res (under review). Brochu NM, Guerin MT, Varga C, Lillie BN, Brash ML, Susta L. A two-year prospective study of small poultry flocks in Ontario, Canada, part 1: prevalence of viral and bacterial pathogens. J Vet Diagn Invest. 2019; 31(3):327–35. doi: 10.1177/1040638719843577 30973091 Centers for Disease Control and Prevention (CDC). National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria. Available from: https://www.cdc.gov/narms/antibiotics-tested.html. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi: 10.1111/j.1469-0691.2011.03570.x 21793988 Dohoo I, Martin W, Stryhn H: Veterinary Epidemiology Research. 2nd Edition. Charlottetown, Prince Edward Island, Canada: AVC Inc.; 2009. Kaufman L, Rousseeuw PJ. Finding groups in data: An introduction to cluster analysis. New York: Wiley; 1990. Greenacre MJ. Multiple and Joint Correspondence Analysis. In: Greenacre MJ and Blasius J, eds., Correspondence Analysis in the Social Sciences, London: Academic Press, 1994. Conover WJ. Practical nonparametric statistics. 3rd ed. John Wiley & Sons Inc. Nova Iorque; 1999. Nguyen TN, Hotzel H, Njeru J, Mwituria J, El-Adawy H, Tomaso H, Neubauer H, Hafez HM. Antimicrobial resistance of Campylobacter isolates from small scale and backyard chicken in Kenya. Gut Pathog. 2016; 8(1):39. doi: 10.1186/s13099-016-0121-5 27570543 Pohjola L, Nykäsenoja S, Kivistö R, Soveri T, Huovilainen A, Hänninen ML, Fredriksson-Ahomaa M. Zoonotic public health hazards in backyard chickens. Zoonoses Public Health. 2016; 63(5):420–30. doi: 10.1111/zph.12247 26752227 Varga C, Rajić A, McFall ME, Reid-Smith RJ, McEwen SA. Associations among antimicrobial use and antimicrobial resistance of Salmonella spp. isolates from 60 Alberta finishing swine farms. Foodborne Pathog Dis. 2009; 6(1):23–31. doi: 10.1089/fpd.2008.0118 18991537 Angulo FJ, Nargund VN, Chiller TC. Evidence of an association between use of anti-microbial agents in food animals and anti-microbial resistance among bacteria isolated from humans and the human health consequences of such resistance. J Vet Med B Infect Dis Vet Public Health. 2004; 51(8–9):374–9. doi: 10.1111/j.1439-0450.2004.00789.x 15525369 Gibreel A, Tracz DM, Nonaka L, Ngo TM, Connell SR, Taylor DE. Incidence of antibiotic resistance in Campylobacter jejuni isolated in Alberta, Canada, from 1999 to 2002, with special reference to tet(O)-mediated tetracycline resistance. Antimicrob Agents Chemother. 2004; 48:3442–50. doi: 10.1128/AAC.48.9.3442-3450.2004 15328109 Elhadidy M, Miller WG, Arguello H, Álvarez-Ordóñez A, Dierick K, Botteldoorn N. Molecular epidemiology and antimicrobial resistance mechanisms of Campylobacter coli from diarrhoeal patients and broiler carcasses in Belgium. Transbound Emerg Dis. 2019; 66(1):463–75. doi: 10.1111/tbed.13046 30346650 Kim JM, Hong J, Bae W, Koo HC, Kim SH, Park YH. Prevalence, antibiograms, and transferable tet(O) plasmid of Campylobacter jejuni and Campylobacter coli isolated from raw chicken, pork, and human clinical cases in Korea. J Food Prot. 2010; 73(8):1430–7. doi: 10.4315/0362-028x-73.8.1430 20819352 Government of Canada. Categorization of Antimicrobial Drugs Based on Importance in Human Medicine. 2009. Available from: https://www.canada.ca/en/health-canada/services/drugs-health-products/veterinary-drugs/antimicrobial-resistance/categorization-antimicrobial-drugs-based-importance-human-medicine.html Luo N, Pereira S, Sahin O, Lin J, Huang S, Michel L, Zhang Q. Enhanced in vivo fitness of fluoroquinolone-resistant Campylobacter jejuni in the absence of antibiotic selection pressure. Proc. Natl. Acad. Sci. U. S. A. 2005; 102:541–6. doi: 10.1073/pnas.0408966102 15634738 El-Tras WF, Holt HR, Tayel AA, El-Kady NN. Campylobacter infections in children exposed to infected backyard poultry in Egypt. Epidemiol Infect. 2015; 143(2):308–15. doi: 10.1017/S095026881400096X 24774694 Reddy S, Zishiri OT. Detection and prevalence of antimicrobial resistance genes in Campylobacter spp. isolated from chickens and humans. Onderstepoort J Vet Res. 2017; 84(1): e1–e6. doi: 10.4102/ojvr.v84i1.1411 28582978 Bolinger H, Kathariou S. The current state of macrolide resistance in Campylobacter spp.: Trends and impacts of resistance mechanisms. Appl Environ Microbiol. 2017; 83(12). doi: 10.1128/AEM.00416-17 28411226 Leclercq R. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin Infect Dis. 2002; 15;34(4):482–92. doi: 10.1086/324626 11797175 Mamelli L, Prouzet-Mauléon V, Pagès JM, Mégraud F, Bolla JM. Molecular basis of macrolide resistance in Campylobacter: role of efflux pumps and target mutations. J Antimicrob Chemother. 2005; 56(3):491–7. doi: 10.1093/jac/dki253 16055509 Gibreel A, Taylor DE. Macrolide resistance in Campylobacter jejuni and Campylobacter coli. J Antimicrob Chemother. 2006; 58(2):243–55. doi: 10.1093/jac/dkl210 16735431 10.1371/journal.pone.0221429.r001 Decision Letter 0 Karunasagar Iddya Academic Editor 2019 Iddya Karunasagar This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

10 Jul 2019

PONE-D-19-16109

Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from small poultry flocks in Ontario, Canada: A two-year surveillance study

PLOS ONE

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Two reviewers have commented on the manuscript. Some minor revisions are required, particularly related to medium used and clarifications on number of isolates. Please address all points raised by the referees.

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Reviewer #1: The work presented in this manuscript is very relevant to our understanding of AMR in Campylobacter. The study is technically sound and the statistical analysis appropriate for the work. The study is particularly valuable as it was conducted before a change of regulation, which has now been implemented, and will represent a baseline for future work in the area. The authors have not overstated their conclusions and categorically indicate the limitations of the study.

Reviewer #2: The authors’ describe antimicrobial resistance (AMR) in Campylobacter from small poultry flocks in Ontario, Canada. The manuscript is well written. With a few revisions, it should be acceptable for publication.

Specific comments:

Line 49: This sentence need a reference(s).

Lines 78-79: I searched for Campy-Charcoal media at Bio-Media, but could not find it. Please verify the media you used and add this to the revised manuscript.

Line 82: What does a presumptive Campylobacter colony look like? Please indicate this in the revised manuscript.

Lines 153-162: The numbers presented here are confusing and don’t add up, especially on line 157 with the game birds. Can this be rewritten to clarify the number of isolates?

Lines 230-233: Most thermophillic Campylobacter are naturally competent. However, Campylobacter restricts most foreign DNA based on methylation (https://www.ncbi.nlm.nih.gov/pubmed/28855338). I don’t agree tetO can be transferred and expressed in Campylobacter from commensals. Please think about this sentence and revise.

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 10.1371/journal.pone.0221429.r002 Author response to Decision Letter 0 Submission Version 1

1 Aug 2019

PONE-D-19-16109

Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from small poultry flocks in Ontario, Canada: A two-year surveillance study

Reviewer #1: The work presented in this manuscript is very relevant to our understanding of AMR in Campylobacter. The study is technically sound and the statistical analysis appropriate for the work. The study is particularly valuable as it was conducted before a change of regulation, which has now been implemented, and will represent a baseline for future work in the area. The authors have not overstated their conclusions and categorically indicate the limitations of the study.

Reviewer #2: The authors’ describe antimicrobial resistance (AMR) in Campylobacter from small poultry flocks in Ontario, Canada. The manuscript is well written. With a few revisions, it should be acceptable for publication.

Specific comments:

Line 49: This sentence needs a reference(s).

A reference (de Vries et al., ~11) was added as requested (Lines 49 and 360 – 363). Note that the citations and Reference List had to be updated due to the addition of this reference.

We also rewrote the sentence to make it more specific: “The transmission of antimicrobial resistant Campylobacter from commercial broiler and free-range chickens to humans has been described previously [11]” (Lines 48-49).

Lines 78-79: I searched for Campy-Charcoal media at Bio-Media, but could not find it. Please verify the media you used and add this to the revised manuscript.

The information on Campylobacter media was verified and updated as requested:

“The cecal material was directly plated onto Campylobacter Blood Free media (Bio-Media Unlimited Ltd., Toronto, Ontario, Canada)” (Lines 78-79).

Please find the list of products available at Bio Media, as a reference:

https://www.bio-media.ca/Bio_Media/Bio_Media_Products.htm

Line 82: What does a presumptive Campylobacter colony look like? Please indicate this in the revised manuscript.

The description of a presumptive Campylobacter colony was added as requested:

“Presumptive Campylobacter colonies (i.e., yellowish-gray, translucent, round, 1-2 mm diameter, smooth to slightly mucoid) were selected (Lines 80-82).

Lines 153-162: The numbers presented here are confusing and don’t add up, especially on line 157 with the game birds. Can this be rewritten to clarify the number of isolates?

We verified that the numbers are correct and rewrote the sentence for clarification:

“Of 158 submissions tested for Campylobacter, a total of 176 isolates were recovered: 141 isolates from chicken submissions (47 pooled samples, 3 isolates recovered from each pooled sample); 21 isolates from turkey submissions (7 pooled samples, 3 isolates recovered from each pooled sample); 6 isolates from duck submissions (2 pooled samples, 3 isolates recovered from each pooled sample); and 8 isolates from game bird (pheasant and quail) submissions (3 pooled samples in total; 3 isolates recovered from each of 2 pooled samples and 2 isolates recovered from 1 of the pooled samples).” (Lines 154-160)

Lines 230-233: Most thermophillic Campylobacter are naturally competent. However, Campylobacter restricts most foreign DNA based on methylation (https://www.ncbi.nlm.nih.gov/pubmed/28855338). I don’t agree tetO can be transferred and expressed in Campylobacter from commensals. Please think about this sentence and revise.

The reference (Stevenson et al., ~37) was replaced with the reference (Kim et al., ~37) (Line 235) and the sentence was revised as requested:

“Previous studies have shown that tetracycline resistance in Campylobacter is generally determined by a plasmid-encoded tet(O) gene [35], and that this resistance gene can be transferred horizontally between C. jejuni and C. coli isolates in the intestinal tract of food animals and humans [36, 37]”. (Lines 231-235).

Submitted filename: Response to Reviewers.docx

 10.1371/journal.pone.0221429.r003 Decision Letter 1 Karunasagar Iddya Academic Editor 2019 Iddya Karunasagar This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

7 Aug 2019

Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from small poultry flocks in Ontario, Canada: A two-year surveillance study

PONE-D-19-16109R1

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Additional Editor Comments (optional):

The revisions made are satisfactory

Reviewers' comments:

 10.1371/journal.pone.0221429.r004 Acceptance letter Karunasagar Iddya Academic Editor 2019 Iddya Karunasagar This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

20 Aug 2019

PONE-D-19-16109R1

Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from small poultry flocks in Ontario, Canada: A two-year surveillance study

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