- Email: [email protected]
Epidemiology of antimicrobial resistant Campylobacter spp. isolated from retail meats in Canada
International Journal of Food Microbiology 253 (2017) 43–47
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Epidemiology of antimicrobial resistant Campylobacter spp. isolated from retail meats in Canada
MARK
Claudia Narvaez-Bravoa, Eduardo N. Taboadab, Steven K. Mutschallb, Mueen Aslamc,⁎ a b c
Department of Food Science, University of Manitoba, Winnipeg, Manitoba, Canada Public Health Agency of Canada, Lethbridge, Alberta, Canada Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords: Campylobacter Retail meat Antimicrobial resistance Comparative genomic fingerprinting
Campylobacter is an important zoonotic pathogen found in livestock and can cause illness in humans following consumption of raw and undercooked meat products. The objectives of this study were to determine the prevalence of Campylobacter spp. in retail meat (poultry, turkey, pork and beef) purchased in Alberta, Canada and to assess antimicrobial resistance and genetic relatedness of recovered Campylobacter strains with previously isolated strains from clinical and environmental sources. A Comparative Genomic Fingerprinting (CGF) method was used for assessing genetic relatedness of isolates. A total of 606 samples comprising 204, 110, 145 and 147 samples of retail chicken, turkey, ground beef and pork, respectively, were obtained. Campylobacter was isolated from 23.5% (48/204) of chicken samples and 14.2% (8/110) of turkey samples. Pork and beef samples were negative for Campylobacter. Campylobacter jejuni was the most common (94.6%) spp. found followed by C. coli (5.4%). Resistance to tetracycline was found in 48.1% of isolates, followed by resistance to ciprofloxacin (5.5%), nalidixic acid (5.5%), azithromycin (1.78%), and erythromycin (1.78%). All isolates were susceptible to clindamycin, florfenicol, gentamicin and telithromycin. Tetracycline resistance was attributable to the presence of the tetO gene. CGF analysis showed that Campylobacter isolated from poultry meat in this study were genetically related to clinical isolates recovered from human infections and to those isolated from animals and the environment.
1. Introduction The National Enteric Surveillance Program (NESP) (PHAC, 2014) in Canada reported that Campylobacter isolates represented 11.8% of the enteric pathogens isolated from humans and was the top foodborne pathogen reported. Data from the U.S. suggested that campylobacteriosis is the second most prevalent cause of gastroenteritis and is responsible for a significant number of foodborne illnesses and deaths each year (CDC, 2016; Nyachuba, 2010). The majority of human infections are caused by C. jejuni with most of the remaining infections being attributed to C. coli. Most Campylobacter infections in humans are associated with the ingestion of contaminated raw/undercooked poultry products (EFSA, 2010; Suzuki and Yamamoto, 2009); however other types of meats, such as pork and ground beef have also been associated with Campylobacter species (Korsak et al., 2015; Trokhymchuk et al., 2014). Although most Campylobacter infections are self-limiting and do not require treatment with antimicrobials, severe and prolonged cases of campylobacteriosis and infections in immuno-compromised, vulnerable
⁎
populations and children may require antimicrobial therapy. In these instances, erythromycin and fluoroquinolone (i.e. ciprofloxacin) are the drugs of choice (Allos, 2001; Engberg et al., 2001). In addition to Campylobacter infection with the risk to develop long term sequelae, the development of resistance to antimicrobial drugs by Campylobacter strains is also a concern. Antimicrobial resistance in C. jejuni is increasing globally (Alfredson and Korolik, 2007; Bohaychuk et al., 2006; Kos et al., 2006a). Quinolone resistance is relatively common in C. jejuni (Iovine and Blaser, 2004) and resistance mostly arises from a mutation in the quinolone resistance determining region (QRDR) of the gyrA gene (Alfredson and Korolik, 2007). Occasionally, a mutation in the parC gene encoding for topoisomerase IV may also result in reduced susceptibility to quinolones. Another important antibiotic used for the treatment Campylobacter infections is erythromycin, a macrolide, whose resistance in Campylobacter is chromosomally mediated and mainly due to mutation in domain V of 23S rRNA (Lehtopolku et al., 2011). Generally, tetracycline is rarely used to treat campylobacteriosis in humans and resistance in C. jejuni and C. coli is primarily mediated by a plasmid-encoded tet(O) gene (Kozak et al., 2009). In addition a
Corresponding author at: Lacombe Research and Development Centre, 6000 C & E Trail Lacombe, Alberta T4L 1W1, Canada. E-mail address: [email protected] (M. Aslam).
http://dx.doi.org/10.1016/j.ijfoodmicro.2017.04.019 Received 22 July 2016; Received in revised form 21 April 2017; Accepted 28 April 2017 Available online 29 April 2017 0168-1605/ © 2017 Published by Elsevier B.V.
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
with species-specific primers (Inglis and Kalischuk, 2003; Kos et al., 2006a). Up to three confirmed Campylobacter isolates per positive sample were stored in brain heart infusion broth supplemented with 10% blood and with 25% glycerol at −80 °C for further analysis. Only one isolate per positive samples was used for further analyses.
kanamycin resistance marker may also be present on the same plasmid (Iovine, 2013). Therefore it is important to monitor trends of antimicrobial resistance for commonly used antimicrobials. An understanding of foodborne pathogen sources of contamination and dissemination in the food chain is key to develop effective mitigation strategies for Campylobacter reduction in livestock as well as prudent antimicrobial use in order to decrease the likelihood for antimicrobial resistance. In order to investigate the epidemiology of Campylobacter spp., molecular subtyping methods with enhanced discriminatory power are required. In recent years the Comparative Genomic Fingerprinting (CGF) technique has been used to subtype Campylobacter jejuni and C. coli (Taboada et al., 2012). This technique represents a high resolution subtyping approach which assesses genetic variability in the accessory genome. The objectives of this study were i) to determine Campylobacter prevalence in retail meat samples purchased in Alberta, Canada, ii) to determine the antimicrobial resistance profiles of recovered Campylobacter strains, and iii) to assess the genetic relatedness of retail meat Campylobacter strains with previously recovered clinical, animal and environmental isolates by using CGF and comparing the profiles to the Canadian CGF database in order to gain a better understanding of Campylobacter epidemiology.
2.3. Antimicrobial susceptibility testing Antimicrobial susceptibility of Campylobacter isolates was tested with a panel of 9 antimicrobials (Campy plates) using an automated microbroth dilution method (Sensititre®; Trek Diagnostic Systems Inc., Westlake, OH, USA) as described previously (CIPARS, 2014). Briefly, colonies were streaked onto Mueller Hinton agar plates with 5% sheep blood and incubated in a microaerophilic atmosphere at 42 ± 1 °C for 24 h. A 0.5 McFarland suspension of bacterial growth was prepared by transferring selected bacterial colonies into a tube containing 5 mL of Mueller Hinton Broth (MHB). Afterward, 10 μL of the MHB were transferred to 11 mL of MHB with laked horse blood. The mixture was dispensed onto CAMPY plates at 100 μL per well. The plates were sealed with perforated adhesive plastic sheets. After a 24 h incubation in microaerophilic atmosphere at 42 ± 1 °C, plates were read using the Sensititre Vizion System40. Campylobacter jejuni ATCC 33560 was used as quality control organism. The results were interpreted by following the guidelines of the Clinical and Laboratory Standard Institute (CLSI, 2012). Where Clinical and Laboratory Standards Institute interpretive criteria were not available for Enterobacteriaceae, the antimicrobial, breakpoints were based on the distribution of minimal inhibitory concentrations and were harmonized with those of the National Antimicrobial Resistance Monitoring System (NARMS), United States. Antimicrobial susceptibility was performed for the following antimicrobial agents (antimicrobial abbreviations and breakpoints are shown in parentheses): ciprofloxacin (CIP; 0.015–64 μg/mL), telithromycin (TEL; 0.015–8 μg/mL), azithromycin (AZT; 0.015–64 μg/mL), clindamycin (CLI; 0.03–16 μg/mL), erythromycin (ERY; 0.03–64 μg/mL), gentamicin (GEN; 0.12–32 μg/ mL), nalidixic acid (NAL; 4–64 μg/mL), florfenicol (FLO; 0.03–64 μg/ mL), and tetracycline (TET; 0.06–64 μg/mL).
2. Materials and methods 2.1. Sampling Retail meat samples were obtained according to protocols of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS, 2014). Retail meat samples consisted of ground beef, chicken, turkey, and pork. For ground beef, a systematic collection of extra-lean, lean, medium, and regular ground beef was performed. It was intended to collect chicken leg or thigh samples, however, when these parts were not available breasts or wings were collected. For turkey, leg samples were preferred, but if they were not available, samples of wings and/or ground turkey were obtained. For pork, samples of chops were obtained. 2.2. Sample preparation, processing and microbiological procedures
2.4. Genetic determinants of antimicrobial resistance A total of 606 samples consisting of chicken (n = 204), turkey (n = 110), pork (147) and ground beef (n = 145) were purchased from retail stores in 2013. Upon arrival at the laboratory, samples were processed following standard protocols (CIPARS, 2014) described in the Compendium of Analytical Methods, Methods of Microbiological Analysis of Food, Health Protection Branch, Government of Canada for the isolation and identification of Campylobacter spp. This procedure has been validated and is routinely used by CIPARS for Campylobacter isolation. Briefly, one chicken leg, one turkey leg/wing, one pork chop or 25 g of ground meat (beef) were transferred to a stomacher bag containing 225 mL buffered peptone water (BPW, Oxoid Ltd., Basingstoke, UK) and mixed for 15 min. 50 mL of the sample/BPW mixture was transferred to 50 mL of double strength Bolton broth (2xBB, Oxoid Ltd.). The 2xBB was incubated under microaerophilic conditions (5% O2, 10% CO2 and balance N2) (CampyPak Plus jar and CampyPak gas generator, Becton-Dickinson Microbiology Systems [BDMS], Cockeysville, MD) at 42 ± 1 °C for 48 ± 4 h. After incubation, the 2xBB was streaked for isolated colonies onto modified cefoperazone charcoal deoxycholate agar (mCCDA, Oxoid Ltd.). The mCCDA plates were incubated under microaerophilic conditions at 42 ± 1 °C for 24 to 72 h and then examined for typical colonies. These were purified using Mueller Hinton agar (MHA Oxoid Ltd.) with 5% citrated sheep blood. Colonies were tested for catalase, oxidase and Gram stain. A maximum of three colonies exhibiting reactions typical for Campylobacter spp. were further characterized by determining growth at 25 °C, and for cephalothin sensitivity, hippurate and indoxyl acetate hydrolysis. Additional confirmation of Campylobacter spp. was done using PCR
Confirmed Campylobacter isolates were screened for the presence of genetic determinants conferring antimicrobial resistance for tetracycline (tet O gene). DNA was obtained using a Qiagen DNA purification kit with the QIAcube® automated DNA isolation system (Qiagen Inc. Mississauga, ON, Canada), following manufacturer recommendations. PCR to detect the tetO gene was performed by using the primer set and PCR conditions as described previously (Gibreel et al., 2004). 2.5. Comparative genomic fingerprinting (CGF) analysis CGF is a subtyping approach recently described that is based on assessing the presence/absence status of a set of 40 accessory genes distributed throughout the various plasticity regions in the C. jejuni genome (Taboada et al., 2012). To generate Campylobacter CGF profiles, the 40 CGF target genes were analyzed in a series of 8 multiplex PCR assays each containing 5 sets of primers along with other PCR components as previously described by (Taboada et al., 2012). The PCR results were converted to binary values with “0” representing the absence of a specific marker and “1” representing the presence of a marker. Clustering analysis was performed with Bionumerics software (v. 7.0; Applied Maths, Austin, TX, US). CGF profiles were compared to those found in the national reference CGF database, which comprises data on over 22,000 C. jejuni and C. coli isolates obtained from human clinical and non-human (environmental, animal, retail) sources collected from 1998 to date through several national and regional surveillance programs and a range of ad hoc sampling activities. 44
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
multiple provinces and in multiple sampling years. Our data also showed that 22 recurring subtypes have been previously observed in human clinical cases. Reference clusters 83.1.2 and 169.1.2 were the major clusters associated with human cases in Alberta, and matched with 16 strains recovered from chicken and turkey meats in this research. Other strains recovered in this study showed unique fingerprints (did not belong to any cluster) but did match clusters associated with human clinical cases (Table 2). Comparison of cluster information against the national reference CGF database, which comprises data on a pan-Canadian collection of C. jejuni and C. coli isolates (n = 22,904) collected from 1998 to date from a range of food, animal, environmental, and human clinical sources, can provide clues on potential epidemiological and ecological associations of isolates analyzed in this study. The CGF analysis in this study grouped 56 Campylobacter isolates into 32 clusters with one subtype (83.1.2) representing 11 isolates (9 from chicken and two from turkey). When this cluster was compared with the Canadian national reference CGF database, it was found to be the fifth most prevalent subtype (n = 449). This subtype is highly associated with poultry (n = 274; 61%) and it has been observed consistently in multiple years. This subtype also has a significant association with human clinical cases (n = 93; 27%). This CGF subtype is also associated with the ST-48 Clonal Complex (CC), the fifth most commonly encountered CC in the MLST database, which has also been strongly associated with human clinical cases, chicken and cattle. The second largest cluster among the isolates in this study (169.1.2 with 5 isolates from this study) is the most frequently observed subtype in the reference database. This subtype has been observed in multiple Canadian provinces and has been observed in every sampling year. It is also the leading subtype observed among human clinical cases (n = 162). Unlike the 83.1.2 subtype, which is chicken associated, 169.1.2 has cattle as its primary source (n = 347; 58%), with chicken as a relatively minor source (n = 38; 6%). This CGF subtype is also associated with the ST-21 Clonal Complex (CC), the most commonly encountered CC in the MLST database, which has also been strongly associated with human clinical cases, chicken and cattle.
Table 1 Prevalence of Campylobacter spp. in retail meat samples purchased in Alberta, Canada. Sample origin
Sample number
Positive (%)
Campylobacter spp.
Chicken
204
48 (23.5)
Turkey
110
8 (14.2)
Beef Pork Total
145 147 606
0 (0) 0 (0) 56 (9.2)
C. jejuni 97.9% (47/48) C. coli 2.0% (1/48) C. jejuni 75% (6/8) C. coli 25% (2/8) N/A N/A N/A
Additional epidemiological and ecological context for CGF subtypes was based on the association between specific CGF subtypes and known MLST subtypes (Taboada et al., 2012) by examining source associations in the C. jejuni MLST database (https://pubmlst.org/campylobacter/). 3. Results Overall prevalence of Campylobacter was 9.2% (56/606). A total of 56 isolates were obtained from retail chicken and turkey samples, but pork and ground beef samples were Campylobacter negative (Table 1). Within the poultry products, the prevalence of Campylobacter for chicken was 23.5%, while for turkey 14.2% of the samples were Campylobacter positive. Campylobacter jejuni was the most common (94.6%) species found, followed by C. coli (5.4%). Two of the isolates were lost during storage; therefore, antimicrobial resistance was tested for 54 of 56 Campylobacter isolates. Resistance to tetracycline was found in 48.1% (26/54) of the Campylobacter strains recovered; resistance to azithromycin and erythromycin was found in only 1.78% (1/54) of isolates. Three isolates (5.5%) showed resistance to ciprofloxacin and nalidixic acid (Fig. 1). All strains were susceptible to clindamycin, florfenicol, gentamicin and telithromycin. When antimicrobial resistance was examined by meat type, 55.5% (25/45) of chicken samples and 12.5% (1/8) of turkey samples showed antimicrobial resistance (Data not shown). The tet(O) gene was detected in 49% of C. jejuni isolates and 33% of C. coli isolates. CGF40 analysis revealed 32 distinct profiles among the 56 Campylobacter isolates examined, including nine multi-strain clusters, the largest of which was composed of 11 isolates with 100% similarity (Table 2). Twenty three isolates did not belong to any cluster and showed unique CGF40 fingerprints. Comparison to the national reference database showed that the 32 CGF40 subtypes observed among the isolates collected in this study included 2 subtypes that represent novel fingerprints. Among the 30 subtypes that have been previously observed, 27 represent recurring subtypes that have been observed in
4. Discussion Campylobacter has been recognized as one of the most common causes of bacterial enteric infections worldwide. Evidence suggests that there has been a rise in the global incidence of campylobacteriosis in the past decade (Kaakoush et al., 2015). Campylobacter spp. are often associated with handling and consumption of meat products, especially raw poultry products (Suzuki and Yamamoto, 2009). Domestic poultry (chickens, turkeys) is frequently associated with thermophilic Campylobacter, primarily C. jejuni and C. coli (Giombelli and Gloria, 2014; Golz et al., 2014). Thus monitoring Campylobacter prevalence in retail meat products and determining the possible link between strains isolated from retail meat and clinical cases is important to assess a potential human health risk, and to explore possible interventions to reduce it. In this study overall Campylobacter spp. prevalence in retail meat (chicken and turkey) was 9.2% (56/606). Campylobacter was not recovered from ground beef and pork samples. A previous report from the province of Saskatchewan showed that Campylobacter prevalence in retail ground beef was 16.2% (Trokhymchuk et al., 2014). Information from its 2008 annual report FoodNet Canada, formerly known as C-EnterNet, the Canadian sentinel-site surveillance network for enteric disease suggested a Campylobacter prevalence of 0% on pork chops, 1% in ground beef and 43% in chicken breast (PHAC, 2010). Similarly, it was reported that C. jejuni was not found in ground beef samples by cultural methods, but one sample (0.6%) was positive for C. jejuni by PCR (Llarena et al., 2014). The variation in Campylobacter prevalence can be attributed to the differences in prevalence at pre-harvest level (animal reservoirs), isolation procedures, retail meat source, and geographical regions.
60
48.1
50
40
30
20
5.5
10
1.78
5.5 1.78
0
AZM
CIP
ERY
NAL
TET
Fig. 1. Prevalence (%) of antimicrobial resistance in Campylobacter recovered from retail meat samples. TET = tetracycline, AZM = azithromycin, NAL = nalidixic acid, CIP = ciprofloxacin, ERY = erythromycin. All strains were susceptible to clindamycin, florfenicol, gentamicin and telithromycin (not shown).
45
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
Table 2 Recovery of Campylobacter isolates from retail meat in this study and their relatedness to isolates previously observed in the reference CGF database, which contains data on isolates recovered from clinical, animal or water samples from across Canada (n = 22,121). CGF40 Subtype
83.1.2 169.1.2 904.1.3 120.2.1 253.4.1 173.10.2 957.4.1 894.1.2 260.7.3 926.2.1 811.9.2 882.5.1 960.7.1 269.4.1 77.1.3 960.1.2 173.2.3 633.14.2 123.2.1 104.1.3 842.2.2 253.4.6 933.7.3 636.1.4 544.1.2 65.4.3 574.1.2 Novel fingerprints a b c d e
Number of isolates in the clustera
11 5 4 3 2 2 2 2 2
Chicken
9 4 4 3 2 1 2 2 2 1 1 1 1 1 1 1
a
Turkeya
2 1 – – – 1 – – –
1 1 1 1 1 1 1 1 1 1 4
1 1
Source association in reference CGF databaseb
MLST association in reference CGF databasec
Clinical (%)
Animal (%)
Water (%)
Dominant CCd
Dominant STe
27 28 – 100 21 18 15 27 – 11 3 6 13 25 31 5 38 11 46 50 – 33 – – – 50 – –
72 60 – – 70 47 69 73 100 55 11 77 79 75 69 – 62 71 54 50 – 67 100 100 – 50 – –
– 11 100 – 9 – 15 – – 34 86 18 9 – – 95 – 18 – – 100 – – – 100 – 100 –
ST_48 ST_21 ST_45 ST_443 ST_21
48 982 137 443 50
ST_45 ST_283 ST_45 ST_21 ST_206 ST_45 ST_21 ST_828 ST_443
45 693 267 45 8 222 583 21 1219 51
ST_828
2507
Campylobacter recovered during this study. Source association based on pan-Canadian CGF reference database (n = 22,121 isolates). Multilocus Sequence Typing (MLST). Predominant Clonal Complex (CC) for CGF subtype based on subset of isolates from pan-Canadian CGF reference database with MLST data (n = 625). Predominant Sequence Type (ST) for CGF subtype based on subset of isolates from pan-Canadian CGF reference database with MLST data (n = 625).
(Veterinary Drugs Directorate, 2002). Other research has reported a significant association between Campylobacter strains showing resistance to tetracycline and resistance to ciprofloxacin (Agunos et al., 2013). Investigation on the relationship between human cases and potential sources of contamination including meat products has been difficult, partly due to the lack of suitable molecular sub-typing methods. However, a comparative genomics-based method of subtyping with a high resolution has proved to be a reliable approach that enhanced our ability to identify genotypic clusters of Campylobacter (Taboada et al., 2012) and comparison to the reference CGF database can provide insights on the epidemiology and ecology of the subtypes identified in this study. The CGF analysis in this study grouped 56 Campylobacter isolates into 32 clusters with one subtype (83.1.2) representing 11 isolates (9 from chicken and two from turkey). This subtype is highly associated with poultry (n = 274; 61% of isolates with this fingerprint) and although it has been observed consistently in multiple years, 54% of the poultry isolates in the reference database were collected in 2013 during a year-long poultry baseline survey by the Canadian Food Inspection Agency, which suggests that this subtype is in wide circulation in Canadian poultry production. Overall, of the 22 CGF subtypes observed among the isolates analyzed in this study that have been observed in human clinical cases of campylobacteriosis, 19 have been observed either uniquely or primarily in chicken, suggesting that meat products, mainly poultry products, play a significant role in transmission of this pathogen to humans. Campylobacter strains found in this study grouped with cluster 169.1.2, which also contained Campylobacter recovered from water sources. This fingerprint is unusual and one isolate was recovered from water reservoirs in Ontario. Interestingly three isolates from this study
Regarding Campylobacter species, C. jejuni was the most common species (94.6%) found in this study followed by C. coli (5.4%). These results are similar to other investigations where C. jejuni and C. coli were the most frequently isolated species in domestic poultry (Giombelli and Gloria, 2014; Sahin et al., 2015). Baseline surveillance data collected by Bohaychuk et al. (2009) in provincially inspected poultry slaughterhouses in Alberta, showed that 75% of the samples tested positive for Campylobacter (Bohaychuk et al., 2009). The available data has well established that poultry products play an important role as a main vehicle in the transmission of Campylobacter spp. to humans. All isolates recovered in this study were susceptible to gentamicin, clindamycin and florfenicol. However, 49% of C. jejuni and 33% C. coli were resistant to tetracycline and the tet (O) gene was detected in all the isolates that showed phenotypic resistance to tetracycline. Four percent of C. jejuni and 33% C. coli isolates showing resistance to tetracycline were also resistant to ciprofloxacin and nalidixic acid. Others have reported higher incidences of ciprofloxacin resistance (8% to 94%) (Kos et al., 2006b; Sierra-Arguello et al., 2016). Only one C. jejuni isolate (2%) was resistant to azithromycin and erythromycin. During 2005–2010 CIPARS reported an increase in the emergence of Campylobacter isolates resistant to ciprofloxacin, where 4% (4/111) of the Campylobacter strains recovered from chicken ceca were resistant to ciprofloxacin (Agunos et al., 2013). In the present research 5.5% (3/54) of the isolates showed resistance to ciprofloxacin which is similar to the data previously reported by CIPARS. Our data demonstrated a higher prevalence of Campylobacter spp. in poultry products when compared with other meat types, and also suggested that some of these isolates were resistant to antimicrobials commonly used in broiler chicken production such as erythromycin 46
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
CDC, 2016. Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2014 (Final Report). U.S. Department of Health and Human Services, CDC, Atlanta, Georgia. http://www.cdc.gov/foodnet/reports/annualreports-2014.html. CIPARS, 2014. Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) 2012 Annual Report – Chapter 1. Design and Methods. Public Health Agency of Canada. http://publications.gc.ca/collections/collection_2014/aspc-phac/ HP2-4-2012-1-eng.pdf. CLSI, 2012. Performance Standard for Antimicrobial Ssusceptibility Testing; TwentySecond Informatonal Supplement. In: CLSI document M100-S22. Clinical and Laboratory Standards Institute, Wayne, PA. EFSA, 2010. Analysis of the Baseline Survey on the Prevalence of Campylobacter in Broiler Batches and of Campylobacter and Salmonella on Broiler Carcasses in the EU, 2008. pp. 1503. http://www.efsa.europa.eu/en/efsajournal/pub/1503. Engberg, J., Aarestrup, F.M., Taylor, D.E., Gerner-Smidt, P., Nachamkin, I., 2001. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerg. Infect. Dis. 7, 24–34. Gibreel, A., Tracz, D.M., Nonaka, L., Ngo, T.M., Connell, S.R., Taylor, D.E., 2004. 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. 48, 3442–3450. Giombelli, A., Gloria, M.B., 2014. Prevalence of Salmonella and Campylobacter on broiler chickens from farm to slaughter and efficiency of methods to remove visible fecal contamination. J. Food Prot. 77, 1851–1859. Golz, G., Rosner, B., Hofreuter, D., Josenhans, C., Kreienbrock, L., Lowenstein, A., Schielke, A., Stark, K., Suerbaum, S., Wieler, L.H., Alter, T., 2014. Relevance of Campylobacter to public health–the need for a one health approach. Int. J. Med. Microbiol. 304, 817–823. Inglis, G.D., Kalischuk, L.D., 2003. Use of PCR for direct detection of Campylobacter species in bovine feces. Appl. Environ. Microbiol. 69, 3435–3447. Iovine, N.M., 2013. Resistance mechanisms in Campylobacter jejuni. Virulence 4, 230–240. Iovine, N.M., Blaser, M.J., 2004. Antimicrobial resistance in Campylobacter. Emerg. Infect. Dis. 10, 1346. Kaakoush, N.O., Castano-Rodriguez, N., Mitchell, H.M., Man, S.M., 2015. Global epidemiology of Campylobacter infection. Clin. Microbiol. Rev. 28, 687–720. Korsak, D., Mackiw, E., Rozynek, E., Zylowska, M., 2015. Prevalence of Campylobacter spp. in retail chicken, Turkey, pork, and beef meat in Poland between 2009 and 2013. J. Food Prot. 78, 1024–1028. Kos, V.N., Gibreel, A., Keelan, M., Taylor, D.E., 2006a. Species identification of erythromycin-resistant Campylobacter isolates and optimization of a duplex PCR for rapid detection. Res. Microbiol. 157, 503–507. Kos, V.N., Keelan, M., Taylor, D.E., 2006b. Antimicrobial susceptibilities of Campylobacter jejuni isolates from poultry from Alberta, Canada. Antimicrob. Agents Chemother. 50, 778–780. Kozak, G.K., Boerlin, P., Janecko, N., Reid-Smith, R.J., Jardine, C., 2009. Antimicrobial resistance in Escherichia coli isolates from swine and wild small mammals in the proximity of swine farms and in natural environments in Ontario, Canada. Appl. Environ. Microbiol. 75, 559–566. Lehtopolku, M., Kotilainen, P., Haanpera-Heikkinen, M., Nakari, U.M., Hanninen, M.L., Huovinen, P., Siitonen, A., Eerola, E., Jalava, J., Hakanen, A.J., 2011. Ribosomal mutations as the main cause of macrolide resistance in Campylobacter jejuni and Campylobacter coli. Antimicrob. Agents Chemother. 55, 5939–5941. Llarena, A.K., Sivonen, K., Hanninen, M.L., 2014. Campylobacter jejuni prevalence and hygienic quality of retail bovine ground meat in Finland. Lett. Appl. Microbiol. 58, 408–413. Nyachuba, D.G., 2010. Foodborne illness: is it on the rise? Nutr. Rev. 68, 257–269. PHAC, 2010. ARCHIVED - C-EnterNet 2008 Annual Report. PHAC. http://www.phacaspc.gc.ca/foodnetcanada/pubs/2008/campylobacter-eng.php. PHAC, 2014. National Enteric Surveillance Program (NESP). Annual Summary. 2012. Sahin, O., Kassem, I.I., Shen, Z., Lin, J., Rajashekara, G., Zhang, Q., 2015. Campylobacter in poultry: ecology and potential interventions. Avian Dis. 59, 185–200. Sierra-Arguello, Y.M., Perdoncini, G., Morgan, R.B., Salle, C.T., Moraes, H.L., Gomes, M.J., do Nascimento, V.P., 2016. Fluoroquinolone and macrolide resistance in Campylobacter jejuni isolated from broiler slaughterhouses in southern Brazil. Avian Pathol. 45, 66–72. Suzuki, H., Yamamoto, S., 2009. Campylobacter contamination in retail poultry meats and by-products in the world: a literature survey. J. Vet. Med. Sci. 71, 255–261. Taboada, E.N., Ross, S.L., Mutschall, S.K., Mackinnon, J.M., Roberts, M.J., Buchanan, C.J., Kruczkiewicz, P., Jokinen, C.C., Thomas, J.E., Nash, J.H., Gannon, V.P., Marshall, B., Pollari, F., Clark, C.G., 2012. Development and validation of a comparative genomic fingerprinting method for high-resolution genotyping of Campylobacter jejuni. J. Clin. Microbiol. 50, 788–797. Trokhymchuk, A., Waldner, C., Chaban, B., Gow, S., Hill, J.E., 2014. Prevalence and diversity of Campylobacter species in Saskatchewan retail ground beef. J. Food Prot. 77, 2106–2110. Veterinary Drugs Directorate, H.C., 2002. Release of the Final Report of the Advisory Committee on Animal Uses of Antimicrobials and Impact on Resistance and Human Health. Health Canada. http://www.hc-sc.gc.ca/dhp-mps/pubs/vet/amr-ram_final_ report-rapport_06-27_cp-pc-eng.php.
were grouped in cluster 120.2.1 which also contained Campylobacter strains recovered from clinical cases. This cluster has not been related to animal or water sources. Eighteen of the Campylobacter isolates that were unique in this study correspond to subtypes that have been previously observed in the reference CGF database. Among these, 13 represent subtypes that have matches among human clinical cases and have also been observed among isolates recovered from animal samples. Interestingly, some of these correspond to CGF40 subtypes 123.2.1, 104.1.3, 65.4.3, that are more strongly associated with human isolates (46%, 50% and 50%, respectively) than other subtypes that were more frequently observed in this study. It might be expected that Campylobacter strains that are more prevalent and can be recovered more frequently will have a higher likelihood to survive the various processing hurdles and reach the final consumer than strains that are recovered less frequently. These same subtypes were also observed in high frequency among animal isolates and were not observed among isolates recovered from water, which could indicate that these strains may have an increased capacity to colonize human and animal hosts. 5. Conclusions The presence of Campylobacter in raw poultry products poses a risk to human health and the presence of antimicrobial resistance along with resistance genes enhances the potential risk of transfer to humans. A small number of isolates showed resistance to antimicrobials important in human medicine; this could be the result of industry and government efforts towards reducing antimicrobial use in Canadian animal production. Continuous monitoring is warranted to proactively manage risks associated with the presence of resistant strains of pathogens in retail meat consumed by humans. The CGF analysis suggested that Campylobacter strains isolated from poultry meat have an epidemiological link with clinical isolates as well as with isolates from animals and environmental samples. Funding source The funding for this project was provided by Agriculture and AgriFood Canada through peer-reviewed project number 2528. Acknowledgements The authors thank Cara Service technical support for retail meat sample collection, isolation and confirmation of isolates. We also thank Dr. Danielle Daignault, Public Health Agency of Canada, National Microbiology Laboratory, Saint Hyacinthe QC for performing antimicrobial resistance testing of Campylobacter isolates. References Agunos, A., Leger, D., Avery, B.P., Parmley, E.J., Deckert, A., Carson, C.A., Dutil, L., 2013. Ciprofloxacin-resistant Campylobacter spp. in retail chicken, western Canada. Emerg. Infect. Dis. 19, 1121–1124. Alfredson, D.A., Korolik, V., 2007. Antibiotic resistance and resistance mechanisms in Campylobacter jejuni and Campylobacter coli. FEMS Microbiol. Lett. 277, 123–132. Allos, B.M., 2001. Campylobacter jejuni infections: update on emerging issues and trends. Clin. Infect. Dis. 32, 1201–1206. Bohaychuk, V.M., Checkley, S.L., Gensler, G.E., Barrios, P.R., 2009. Microbiological baseline study of poultry slaughtered in provincially inspected abattoirs in Alberta, Canada. Can. Vet. J. 50, 173–178. Bohaychuk, V.M., Gensler, G.E., King, R.K., Manninen, K.I., Sorensen, O., Wu, J.T., Stiles, M.E., McMullen, L.M., 2006. Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada. J. Food Prot. 69, 2176–2182.
47
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Epidemiology of antimicrobial resistant Campylobacter spp. isolated from retail meats in Canada
MARK
Claudia Narvaez-Bravoa, Eduardo N. Taboadab, Steven K. Mutschallb, Mueen Aslamc,⁎ a b c
Department of Food Science, University of Manitoba, Winnipeg, Manitoba, Canada Public Health Agency of Canada, Lethbridge, Alberta, Canada Agriculture and Agri-Food Canada, Lacombe, Alberta, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords: Campylobacter Retail meat Antimicrobial resistance Comparative genomic fingerprinting
Campylobacter is an important zoonotic pathogen found in livestock and can cause illness in humans following consumption of raw and undercooked meat products. The objectives of this study were to determine the prevalence of Campylobacter spp. in retail meat (poultry, turkey, pork and beef) purchased in Alberta, Canada and to assess antimicrobial resistance and genetic relatedness of recovered Campylobacter strains with previously isolated strains from clinical and environmental sources. A Comparative Genomic Fingerprinting (CGF) method was used for assessing genetic relatedness of isolates. A total of 606 samples comprising 204, 110, 145 and 147 samples of retail chicken, turkey, ground beef and pork, respectively, were obtained. Campylobacter was isolated from 23.5% (48/204) of chicken samples and 14.2% (8/110) of turkey samples. Pork and beef samples were negative for Campylobacter. Campylobacter jejuni was the most common (94.6%) spp. found followed by C. coli (5.4%). Resistance to tetracycline was found in 48.1% of isolates, followed by resistance to ciprofloxacin (5.5%), nalidixic acid (5.5%), azithromycin (1.78%), and erythromycin (1.78%). All isolates were susceptible to clindamycin, florfenicol, gentamicin and telithromycin. Tetracycline resistance was attributable to the presence of the tetO gene. CGF analysis showed that Campylobacter isolated from poultry meat in this study were genetically related to clinical isolates recovered from human infections and to those isolated from animals and the environment.
1. Introduction The National Enteric Surveillance Program (NESP) (PHAC, 2014) in Canada reported that Campylobacter isolates represented 11.8% of the enteric pathogens isolated from humans and was the top foodborne pathogen reported. Data from the U.S. suggested that campylobacteriosis is the second most prevalent cause of gastroenteritis and is responsible for a significant number of foodborne illnesses and deaths each year (CDC, 2016; Nyachuba, 2010). The majority of human infections are caused by C. jejuni with most of the remaining infections being attributed to C. coli. Most Campylobacter infections in humans are associated with the ingestion of contaminated raw/undercooked poultry products (EFSA, 2010; Suzuki and Yamamoto, 2009); however other types of meats, such as pork and ground beef have also been associated with Campylobacter species (Korsak et al., 2015; Trokhymchuk et al., 2014). Although most Campylobacter infections are self-limiting and do not require treatment with antimicrobials, severe and prolonged cases of campylobacteriosis and infections in immuno-compromised, vulnerable
⁎
populations and children may require antimicrobial therapy. In these instances, erythromycin and fluoroquinolone (i.e. ciprofloxacin) are the drugs of choice (Allos, 2001; Engberg et al., 2001). In addition to Campylobacter infection with the risk to develop long term sequelae, the development of resistance to antimicrobial drugs by Campylobacter strains is also a concern. Antimicrobial resistance in C. jejuni is increasing globally (Alfredson and Korolik, 2007; Bohaychuk et al., 2006; Kos et al., 2006a). Quinolone resistance is relatively common in C. jejuni (Iovine and Blaser, 2004) and resistance mostly arises from a mutation in the quinolone resistance determining region (QRDR) of the gyrA gene (Alfredson and Korolik, 2007). Occasionally, a mutation in the parC gene encoding for topoisomerase IV may also result in reduced susceptibility to quinolones. Another important antibiotic used for the treatment Campylobacter infections is erythromycin, a macrolide, whose resistance in Campylobacter is chromosomally mediated and mainly due to mutation in domain V of 23S rRNA (Lehtopolku et al., 2011). Generally, tetracycline is rarely used to treat campylobacteriosis in humans and resistance in C. jejuni and C. coli is primarily mediated by a plasmid-encoded tet(O) gene (Kozak et al., 2009). In addition a
Corresponding author at: Lacombe Research and Development Centre, 6000 C & E Trail Lacombe, Alberta T4L 1W1, Canada. E-mail address: [email protected] (M. Aslam).
http://dx.doi.org/10.1016/j.ijfoodmicro.2017.04.019 Received 22 July 2016; Received in revised form 21 April 2017; Accepted 28 April 2017 Available online 29 April 2017 0168-1605/ © 2017 Published by Elsevier B.V.
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
with species-specific primers (Inglis and Kalischuk, 2003; Kos et al., 2006a). Up to three confirmed Campylobacter isolates per positive sample were stored in brain heart infusion broth supplemented with 10% blood and with 25% glycerol at −80 °C for further analysis. Only one isolate per positive samples was used for further analyses.
kanamycin resistance marker may also be present on the same plasmid (Iovine, 2013). Therefore it is important to monitor trends of antimicrobial resistance for commonly used antimicrobials. An understanding of foodborne pathogen sources of contamination and dissemination in the food chain is key to develop effective mitigation strategies for Campylobacter reduction in livestock as well as prudent antimicrobial use in order to decrease the likelihood for antimicrobial resistance. In order to investigate the epidemiology of Campylobacter spp., molecular subtyping methods with enhanced discriminatory power are required. In recent years the Comparative Genomic Fingerprinting (CGF) technique has been used to subtype Campylobacter jejuni and C. coli (Taboada et al., 2012). This technique represents a high resolution subtyping approach which assesses genetic variability in the accessory genome. The objectives of this study were i) to determine Campylobacter prevalence in retail meat samples purchased in Alberta, Canada, ii) to determine the antimicrobial resistance profiles of recovered Campylobacter strains, and iii) to assess the genetic relatedness of retail meat Campylobacter strains with previously recovered clinical, animal and environmental isolates by using CGF and comparing the profiles to the Canadian CGF database in order to gain a better understanding of Campylobacter epidemiology.
2.3. Antimicrobial susceptibility testing Antimicrobial susceptibility of Campylobacter isolates was tested with a panel of 9 antimicrobials (Campy plates) using an automated microbroth dilution method (Sensititre®; Trek Diagnostic Systems Inc., Westlake, OH, USA) as described previously (CIPARS, 2014). Briefly, colonies were streaked onto Mueller Hinton agar plates with 5% sheep blood and incubated in a microaerophilic atmosphere at 42 ± 1 °C for 24 h. A 0.5 McFarland suspension of bacterial growth was prepared by transferring selected bacterial colonies into a tube containing 5 mL of Mueller Hinton Broth (MHB). Afterward, 10 μL of the MHB were transferred to 11 mL of MHB with laked horse blood. The mixture was dispensed onto CAMPY plates at 100 μL per well. The plates were sealed with perforated adhesive plastic sheets. After a 24 h incubation in microaerophilic atmosphere at 42 ± 1 °C, plates were read using the Sensititre Vizion System40. Campylobacter jejuni ATCC 33560 was used as quality control organism. The results were interpreted by following the guidelines of the Clinical and Laboratory Standard Institute (CLSI, 2012). Where Clinical and Laboratory Standards Institute interpretive criteria were not available for Enterobacteriaceae, the antimicrobial, breakpoints were based on the distribution of minimal inhibitory concentrations and were harmonized with those of the National Antimicrobial Resistance Monitoring System (NARMS), United States. Antimicrobial susceptibility was performed for the following antimicrobial agents (antimicrobial abbreviations and breakpoints are shown in parentheses): ciprofloxacin (CIP; 0.015–64 μg/mL), telithromycin (TEL; 0.015–8 μg/mL), azithromycin (AZT; 0.015–64 μg/mL), clindamycin (CLI; 0.03–16 μg/mL), erythromycin (ERY; 0.03–64 μg/mL), gentamicin (GEN; 0.12–32 μg/ mL), nalidixic acid (NAL; 4–64 μg/mL), florfenicol (FLO; 0.03–64 μg/ mL), and tetracycline (TET; 0.06–64 μg/mL).
2. Materials and methods 2.1. Sampling Retail meat samples were obtained according to protocols of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS, 2014). Retail meat samples consisted of ground beef, chicken, turkey, and pork. For ground beef, a systematic collection of extra-lean, lean, medium, and regular ground beef was performed. It was intended to collect chicken leg or thigh samples, however, when these parts were not available breasts or wings were collected. For turkey, leg samples were preferred, but if they were not available, samples of wings and/or ground turkey were obtained. For pork, samples of chops were obtained. 2.2. Sample preparation, processing and microbiological procedures
2.4. Genetic determinants of antimicrobial resistance A total of 606 samples consisting of chicken (n = 204), turkey (n = 110), pork (147) and ground beef (n = 145) were purchased from retail stores in 2013. Upon arrival at the laboratory, samples were processed following standard protocols (CIPARS, 2014) described in the Compendium of Analytical Methods, Methods of Microbiological Analysis of Food, Health Protection Branch, Government of Canada for the isolation and identification of Campylobacter spp. This procedure has been validated and is routinely used by CIPARS for Campylobacter isolation. Briefly, one chicken leg, one turkey leg/wing, one pork chop or 25 g of ground meat (beef) were transferred to a stomacher bag containing 225 mL buffered peptone water (BPW, Oxoid Ltd., Basingstoke, UK) and mixed for 15 min. 50 mL of the sample/BPW mixture was transferred to 50 mL of double strength Bolton broth (2xBB, Oxoid Ltd.). The 2xBB was incubated under microaerophilic conditions (5% O2, 10% CO2 and balance N2) (CampyPak Plus jar and CampyPak gas generator, Becton-Dickinson Microbiology Systems [BDMS], Cockeysville, MD) at 42 ± 1 °C for 48 ± 4 h. After incubation, the 2xBB was streaked for isolated colonies onto modified cefoperazone charcoal deoxycholate agar (mCCDA, Oxoid Ltd.). The mCCDA plates were incubated under microaerophilic conditions at 42 ± 1 °C for 24 to 72 h and then examined for typical colonies. These were purified using Mueller Hinton agar (MHA Oxoid Ltd.) with 5% citrated sheep blood. Colonies were tested for catalase, oxidase and Gram stain. A maximum of three colonies exhibiting reactions typical for Campylobacter spp. were further characterized by determining growth at 25 °C, and for cephalothin sensitivity, hippurate and indoxyl acetate hydrolysis. Additional confirmation of Campylobacter spp. was done using PCR
Confirmed Campylobacter isolates were screened for the presence of genetic determinants conferring antimicrobial resistance for tetracycline (tet O gene). DNA was obtained using a Qiagen DNA purification kit with the QIAcube® automated DNA isolation system (Qiagen Inc. Mississauga, ON, Canada), following manufacturer recommendations. PCR to detect the tetO gene was performed by using the primer set and PCR conditions as described previously (Gibreel et al., 2004). 2.5. Comparative genomic fingerprinting (CGF) analysis CGF is a subtyping approach recently described that is based on assessing the presence/absence status of a set of 40 accessory genes distributed throughout the various plasticity regions in the C. jejuni genome (Taboada et al., 2012). To generate Campylobacter CGF profiles, the 40 CGF target genes were analyzed in a series of 8 multiplex PCR assays each containing 5 sets of primers along with other PCR components as previously described by (Taboada et al., 2012). The PCR results were converted to binary values with “0” representing the absence of a specific marker and “1” representing the presence of a marker. Clustering analysis was performed with Bionumerics software (v. 7.0; Applied Maths, Austin, TX, US). CGF profiles were compared to those found in the national reference CGF database, which comprises data on over 22,000 C. jejuni and C. coli isolates obtained from human clinical and non-human (environmental, animal, retail) sources collected from 1998 to date through several national and regional surveillance programs and a range of ad hoc sampling activities. 44
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
multiple provinces and in multiple sampling years. Our data also showed that 22 recurring subtypes have been previously observed in human clinical cases. Reference clusters 83.1.2 and 169.1.2 were the major clusters associated with human cases in Alberta, and matched with 16 strains recovered from chicken and turkey meats in this research. Other strains recovered in this study showed unique fingerprints (did not belong to any cluster) but did match clusters associated with human clinical cases (Table 2). Comparison of cluster information against the national reference CGF database, which comprises data on a pan-Canadian collection of C. jejuni and C. coli isolates (n = 22,904) collected from 1998 to date from a range of food, animal, environmental, and human clinical sources, can provide clues on potential epidemiological and ecological associations of isolates analyzed in this study. The CGF analysis in this study grouped 56 Campylobacter isolates into 32 clusters with one subtype (83.1.2) representing 11 isolates (9 from chicken and two from turkey). When this cluster was compared with the Canadian national reference CGF database, it was found to be the fifth most prevalent subtype (n = 449). This subtype is highly associated with poultry (n = 274; 61%) and it has been observed consistently in multiple years. This subtype also has a significant association with human clinical cases (n = 93; 27%). This CGF subtype is also associated with the ST-48 Clonal Complex (CC), the fifth most commonly encountered CC in the MLST database, which has also been strongly associated with human clinical cases, chicken and cattle. The second largest cluster among the isolates in this study (169.1.2 with 5 isolates from this study) is the most frequently observed subtype in the reference database. This subtype has been observed in multiple Canadian provinces and has been observed in every sampling year. It is also the leading subtype observed among human clinical cases (n = 162). Unlike the 83.1.2 subtype, which is chicken associated, 169.1.2 has cattle as its primary source (n = 347; 58%), with chicken as a relatively minor source (n = 38; 6%). This CGF subtype is also associated with the ST-21 Clonal Complex (CC), the most commonly encountered CC in the MLST database, which has also been strongly associated with human clinical cases, chicken and cattle.
Table 1 Prevalence of Campylobacter spp. in retail meat samples purchased in Alberta, Canada. Sample origin
Sample number
Positive (%)
Campylobacter spp.
Chicken
204
48 (23.5)
Turkey
110
8 (14.2)
Beef Pork Total
145 147 606
0 (0) 0 (0) 56 (9.2)
C. jejuni 97.9% (47/48) C. coli 2.0% (1/48) C. jejuni 75% (6/8) C. coli 25% (2/8) N/A N/A N/A
Additional epidemiological and ecological context for CGF subtypes was based on the association between specific CGF subtypes and known MLST subtypes (Taboada et al., 2012) by examining source associations in the C. jejuni MLST database (https://pubmlst.org/campylobacter/). 3. Results Overall prevalence of Campylobacter was 9.2% (56/606). A total of 56 isolates were obtained from retail chicken and turkey samples, but pork and ground beef samples were Campylobacter negative (Table 1). Within the poultry products, the prevalence of Campylobacter for chicken was 23.5%, while for turkey 14.2% of the samples were Campylobacter positive. Campylobacter jejuni was the most common (94.6%) species found, followed by C. coli (5.4%). Two of the isolates were lost during storage; therefore, antimicrobial resistance was tested for 54 of 56 Campylobacter isolates. Resistance to tetracycline was found in 48.1% (26/54) of the Campylobacter strains recovered; resistance to azithromycin and erythromycin was found in only 1.78% (1/54) of isolates. Three isolates (5.5%) showed resistance to ciprofloxacin and nalidixic acid (Fig. 1). All strains were susceptible to clindamycin, florfenicol, gentamicin and telithromycin. When antimicrobial resistance was examined by meat type, 55.5% (25/45) of chicken samples and 12.5% (1/8) of turkey samples showed antimicrobial resistance (Data not shown). The tet(O) gene was detected in 49% of C. jejuni isolates and 33% of C. coli isolates. CGF40 analysis revealed 32 distinct profiles among the 56 Campylobacter isolates examined, including nine multi-strain clusters, the largest of which was composed of 11 isolates with 100% similarity (Table 2). Twenty three isolates did not belong to any cluster and showed unique CGF40 fingerprints. Comparison to the national reference database showed that the 32 CGF40 subtypes observed among the isolates collected in this study included 2 subtypes that represent novel fingerprints. Among the 30 subtypes that have been previously observed, 27 represent recurring subtypes that have been observed in
4. Discussion Campylobacter has been recognized as one of the most common causes of bacterial enteric infections worldwide. Evidence suggests that there has been a rise in the global incidence of campylobacteriosis in the past decade (Kaakoush et al., 2015). Campylobacter spp. are often associated with handling and consumption of meat products, especially raw poultry products (Suzuki and Yamamoto, 2009). Domestic poultry (chickens, turkeys) is frequently associated with thermophilic Campylobacter, primarily C. jejuni and C. coli (Giombelli and Gloria, 2014; Golz et al., 2014). Thus monitoring Campylobacter prevalence in retail meat products and determining the possible link between strains isolated from retail meat and clinical cases is important to assess a potential human health risk, and to explore possible interventions to reduce it. In this study overall Campylobacter spp. prevalence in retail meat (chicken and turkey) was 9.2% (56/606). Campylobacter was not recovered from ground beef and pork samples. A previous report from the province of Saskatchewan showed that Campylobacter prevalence in retail ground beef was 16.2% (Trokhymchuk et al., 2014). Information from its 2008 annual report FoodNet Canada, formerly known as C-EnterNet, the Canadian sentinel-site surveillance network for enteric disease suggested a Campylobacter prevalence of 0% on pork chops, 1% in ground beef and 43% in chicken breast (PHAC, 2010). Similarly, it was reported that C. jejuni was not found in ground beef samples by cultural methods, but one sample (0.6%) was positive for C. jejuni by PCR (Llarena et al., 2014). The variation in Campylobacter prevalence can be attributed to the differences in prevalence at pre-harvest level (animal reservoirs), isolation procedures, retail meat source, and geographical regions.
60
48.1
50
40
30
20
5.5
10
1.78
5.5 1.78
0
AZM
CIP
ERY
NAL
TET
Fig. 1. Prevalence (%) of antimicrobial resistance in Campylobacter recovered from retail meat samples. TET = tetracycline, AZM = azithromycin, NAL = nalidixic acid, CIP = ciprofloxacin, ERY = erythromycin. All strains were susceptible to clindamycin, florfenicol, gentamicin and telithromycin (not shown).
45
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
Table 2 Recovery of Campylobacter isolates from retail meat in this study and their relatedness to isolates previously observed in the reference CGF database, which contains data on isolates recovered from clinical, animal or water samples from across Canada (n = 22,121). CGF40 Subtype
83.1.2 169.1.2 904.1.3 120.2.1 253.4.1 173.10.2 957.4.1 894.1.2 260.7.3 926.2.1 811.9.2 882.5.1 960.7.1 269.4.1 77.1.3 960.1.2 173.2.3 633.14.2 123.2.1 104.1.3 842.2.2 253.4.6 933.7.3 636.1.4 544.1.2 65.4.3 574.1.2 Novel fingerprints a b c d e
Number of isolates in the clustera
11 5 4 3 2 2 2 2 2
Chicken
9 4 4 3 2 1 2 2 2 1 1 1 1 1 1 1
a
Turkeya
2 1 – – – 1 – – –
1 1 1 1 1 1 1 1 1 1 4
1 1
Source association in reference CGF databaseb
MLST association in reference CGF databasec
Clinical (%)
Animal (%)
Water (%)
Dominant CCd
Dominant STe
27 28 – 100 21 18 15 27 – 11 3 6 13 25 31 5 38 11 46 50 – 33 – – – 50 – –
72 60 – – 70 47 69 73 100 55 11 77 79 75 69 – 62 71 54 50 – 67 100 100 – 50 – –
– 11 100 – 9 – 15 – – 34 86 18 9 – – 95 – 18 – – 100 – – – 100 – 100 –
ST_48 ST_21 ST_45 ST_443 ST_21
48 982 137 443 50
ST_45 ST_283 ST_45 ST_21 ST_206 ST_45 ST_21 ST_828 ST_443
45 693 267 45 8 222 583 21 1219 51
ST_828
2507
Campylobacter recovered during this study. Source association based on pan-Canadian CGF reference database (n = 22,121 isolates). Multilocus Sequence Typing (MLST). Predominant Clonal Complex (CC) for CGF subtype based on subset of isolates from pan-Canadian CGF reference database with MLST data (n = 625). Predominant Sequence Type (ST) for CGF subtype based on subset of isolates from pan-Canadian CGF reference database with MLST data (n = 625).
(Veterinary Drugs Directorate, 2002). Other research has reported a significant association between Campylobacter strains showing resistance to tetracycline and resistance to ciprofloxacin (Agunos et al., 2013). Investigation on the relationship between human cases and potential sources of contamination including meat products has been difficult, partly due to the lack of suitable molecular sub-typing methods. However, a comparative genomics-based method of subtyping with a high resolution has proved to be a reliable approach that enhanced our ability to identify genotypic clusters of Campylobacter (Taboada et al., 2012) and comparison to the reference CGF database can provide insights on the epidemiology and ecology of the subtypes identified in this study. The CGF analysis in this study grouped 56 Campylobacter isolates into 32 clusters with one subtype (83.1.2) representing 11 isolates (9 from chicken and two from turkey). This subtype is highly associated with poultry (n = 274; 61% of isolates with this fingerprint) and although it has been observed consistently in multiple years, 54% of the poultry isolates in the reference database were collected in 2013 during a year-long poultry baseline survey by the Canadian Food Inspection Agency, which suggests that this subtype is in wide circulation in Canadian poultry production. Overall, of the 22 CGF subtypes observed among the isolates analyzed in this study that have been observed in human clinical cases of campylobacteriosis, 19 have been observed either uniquely or primarily in chicken, suggesting that meat products, mainly poultry products, play a significant role in transmission of this pathogen to humans. Campylobacter strains found in this study grouped with cluster 169.1.2, which also contained Campylobacter recovered from water sources. This fingerprint is unusual and one isolate was recovered from water reservoirs in Ontario. Interestingly three isolates from this study
Regarding Campylobacter species, C. jejuni was the most common species (94.6%) found in this study followed by C. coli (5.4%). These results are similar to other investigations where C. jejuni and C. coli were the most frequently isolated species in domestic poultry (Giombelli and Gloria, 2014; Sahin et al., 2015). Baseline surveillance data collected by Bohaychuk et al. (2009) in provincially inspected poultry slaughterhouses in Alberta, showed that 75% of the samples tested positive for Campylobacter (Bohaychuk et al., 2009). The available data has well established that poultry products play an important role as a main vehicle in the transmission of Campylobacter spp. to humans. All isolates recovered in this study were susceptible to gentamicin, clindamycin and florfenicol. However, 49% of C. jejuni and 33% C. coli were resistant to tetracycline and the tet (O) gene was detected in all the isolates that showed phenotypic resistance to tetracycline. Four percent of C. jejuni and 33% C. coli isolates showing resistance to tetracycline were also resistant to ciprofloxacin and nalidixic acid. Others have reported higher incidences of ciprofloxacin resistance (8% to 94%) (Kos et al., 2006b; Sierra-Arguello et al., 2016). Only one C. jejuni isolate (2%) was resistant to azithromycin and erythromycin. During 2005–2010 CIPARS reported an increase in the emergence of Campylobacter isolates resistant to ciprofloxacin, where 4% (4/111) of the Campylobacter strains recovered from chicken ceca were resistant to ciprofloxacin (Agunos et al., 2013). In the present research 5.5% (3/54) of the isolates showed resistance to ciprofloxacin which is similar to the data previously reported by CIPARS. Our data demonstrated a higher prevalence of Campylobacter spp. in poultry products when compared with other meat types, and also suggested that some of these isolates were resistant to antimicrobials commonly used in broiler chicken production such as erythromycin 46
International Journal of Food Microbiology 253 (2017) 43–47
C. Narvaez-Bravo et al.
CDC, 2016. Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2014 (Final Report). U.S. Department of Health and Human Services, CDC, Atlanta, Georgia. http://www.cdc.gov/foodnet/reports/annualreports-2014.html. CIPARS, 2014. Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) 2012 Annual Report – Chapter 1. Design and Methods. Public Health Agency of Canada. http://publications.gc.ca/collections/collection_2014/aspc-phac/ HP2-4-2012-1-eng.pdf. CLSI, 2012. Performance Standard for Antimicrobial Ssusceptibility Testing; TwentySecond Informatonal Supplement. In: CLSI document M100-S22. Clinical and Laboratory Standards Institute, Wayne, PA. EFSA, 2010. Analysis of the Baseline Survey on the Prevalence of Campylobacter in Broiler Batches and of Campylobacter and Salmonella on Broiler Carcasses in the EU, 2008. pp. 1503. http://www.efsa.europa.eu/en/efsajournal/pub/1503. Engberg, J., Aarestrup, F.M., Taylor, D.E., Gerner-Smidt, P., Nachamkin, I., 2001. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerg. Infect. Dis. 7, 24–34. Gibreel, A., Tracz, D.M., Nonaka, L., Ngo, T.M., Connell, S.R., Taylor, D.E., 2004. 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. 48, 3442–3450. Giombelli, A., Gloria, M.B., 2014. Prevalence of Salmonella and Campylobacter on broiler chickens from farm to slaughter and efficiency of methods to remove visible fecal contamination. J. Food Prot. 77, 1851–1859. Golz, G., Rosner, B., Hofreuter, D., Josenhans, C., Kreienbrock, L., Lowenstein, A., Schielke, A., Stark, K., Suerbaum, S., Wieler, L.H., Alter, T., 2014. Relevance of Campylobacter to public health–the need for a one health approach. Int. J. Med. Microbiol. 304, 817–823. Inglis, G.D., Kalischuk, L.D., 2003. Use of PCR for direct detection of Campylobacter species in bovine feces. Appl. Environ. Microbiol. 69, 3435–3447. Iovine, N.M., 2013. Resistance mechanisms in Campylobacter jejuni. Virulence 4, 230–240. Iovine, N.M., Blaser, M.J., 2004. Antimicrobial resistance in Campylobacter. Emerg. Infect. Dis. 10, 1346. Kaakoush, N.O., Castano-Rodriguez, N., Mitchell, H.M., Man, S.M., 2015. Global epidemiology of Campylobacter infection. Clin. Microbiol. Rev. 28, 687–720. Korsak, D., Mackiw, E., Rozynek, E., Zylowska, M., 2015. Prevalence of Campylobacter spp. in retail chicken, Turkey, pork, and beef meat in Poland between 2009 and 2013. J. Food Prot. 78, 1024–1028. Kos, V.N., Gibreel, A., Keelan, M., Taylor, D.E., 2006a. Species identification of erythromycin-resistant Campylobacter isolates and optimization of a duplex PCR for rapid detection. Res. Microbiol. 157, 503–507. Kos, V.N., Keelan, M., Taylor, D.E., 2006b. Antimicrobial susceptibilities of Campylobacter jejuni isolates from poultry from Alberta, Canada. Antimicrob. Agents Chemother. 50, 778–780. Kozak, G.K., Boerlin, P., Janecko, N., Reid-Smith, R.J., Jardine, C., 2009. Antimicrobial resistance in Escherichia coli isolates from swine and wild small mammals in the proximity of swine farms and in natural environments in Ontario, Canada. Appl. Environ. Microbiol. 75, 559–566. Lehtopolku, M., Kotilainen, P., Haanpera-Heikkinen, M., Nakari, U.M., Hanninen, M.L., Huovinen, P., Siitonen, A., Eerola, E., Jalava, J., Hakanen, A.J., 2011. Ribosomal mutations as the main cause of macrolide resistance in Campylobacter jejuni and Campylobacter coli. Antimicrob. Agents Chemother. 55, 5939–5941. Llarena, A.K., Sivonen, K., Hanninen, M.L., 2014. Campylobacter jejuni prevalence and hygienic quality of retail bovine ground meat in Finland. Lett. Appl. Microbiol. 58, 408–413. Nyachuba, D.G., 2010. Foodborne illness: is it on the rise? Nutr. Rev. 68, 257–269. PHAC, 2010. ARCHIVED - C-EnterNet 2008 Annual Report. PHAC. http://www.phacaspc.gc.ca/foodnetcanada/pubs/2008/campylobacter-eng.php. PHAC, 2014. National Enteric Surveillance Program (NESP). Annual Summary. 2012. Sahin, O., Kassem, I.I., Shen, Z., Lin, J., Rajashekara, G., Zhang, Q., 2015. Campylobacter in poultry: ecology and potential interventions. Avian Dis. 59, 185–200. Sierra-Arguello, Y.M., Perdoncini, G., Morgan, R.B., Salle, C.T., Moraes, H.L., Gomes, M.J., do Nascimento, V.P., 2016. Fluoroquinolone and macrolide resistance in Campylobacter jejuni isolated from broiler slaughterhouses in southern Brazil. Avian Pathol. 45, 66–72. Suzuki, H., Yamamoto, S., 2009. Campylobacter contamination in retail poultry meats and by-products in the world: a literature survey. J. Vet. Med. Sci. 71, 255–261. Taboada, E.N., Ross, S.L., Mutschall, S.K., Mackinnon, J.M., Roberts, M.J., Buchanan, C.J., Kruczkiewicz, P., Jokinen, C.C., Thomas, J.E., Nash, J.H., Gannon, V.P., Marshall, B., Pollari, F., Clark, C.G., 2012. Development and validation of a comparative genomic fingerprinting method for high-resolution genotyping of Campylobacter jejuni. J. Clin. Microbiol. 50, 788–797. Trokhymchuk, A., Waldner, C., Chaban, B., Gow, S., Hill, J.E., 2014. Prevalence and diversity of Campylobacter species in Saskatchewan retail ground beef. J. Food Prot. 77, 2106–2110. Veterinary Drugs Directorate, H.C., 2002. Release of the Final Report of the Advisory Committee on Animal Uses of Antimicrobials and Impact on Resistance and Human Health. Health Canada. http://www.hc-sc.gc.ca/dhp-mps/pubs/vet/amr-ram_final_ report-rapport_06-27_cp-pc-eng.php.
were grouped in cluster 120.2.1 which also contained Campylobacter strains recovered from clinical cases. This cluster has not been related to animal or water sources. Eighteen of the Campylobacter isolates that were unique in this study correspond to subtypes that have been previously observed in the reference CGF database. Among these, 13 represent subtypes that have matches among human clinical cases and have also been observed among isolates recovered from animal samples. Interestingly, some of these correspond to CGF40 subtypes 123.2.1, 104.1.3, 65.4.3, that are more strongly associated with human isolates (46%, 50% and 50%, respectively) than other subtypes that were more frequently observed in this study. It might be expected that Campylobacter strains that are more prevalent and can be recovered more frequently will have a higher likelihood to survive the various processing hurdles and reach the final consumer than strains that are recovered less frequently. These same subtypes were also observed in high frequency among animal isolates and were not observed among isolates recovered from water, which could indicate that these strains may have an increased capacity to colonize human and animal hosts. 5. Conclusions The presence of Campylobacter in raw poultry products poses a risk to human health and the presence of antimicrobial resistance along with resistance genes enhances the potential risk of transfer to humans. A small number of isolates showed resistance to antimicrobials important in human medicine; this could be the result of industry and government efforts towards reducing antimicrobial use in Canadian animal production. Continuous monitoring is warranted to proactively manage risks associated with the presence of resistant strains of pathogens in retail meat consumed by humans. The CGF analysis suggested that Campylobacter strains isolated from poultry meat have an epidemiological link with clinical isolates as well as with isolates from animals and environmental samples. Funding source The funding for this project was provided by Agriculture and AgriFood Canada through peer-reviewed project number 2528. Acknowledgements The authors thank Cara Service technical support for retail meat sample collection, isolation and confirmation of isolates. We also thank Dr. Danielle Daignault, Public Health Agency of Canada, National Microbiology Laboratory, Saint Hyacinthe QC for performing antimicrobial resistance testing of Campylobacter isolates. References Agunos, A., Leger, D., Avery, B.P., Parmley, E.J., Deckert, A., Carson, C.A., Dutil, L., 2013. Ciprofloxacin-resistant Campylobacter spp. in retail chicken, western Canada. Emerg. Infect. Dis. 19, 1121–1124. Alfredson, D.A., Korolik, V., 2007. Antibiotic resistance and resistance mechanisms in Campylobacter jejuni and Campylobacter coli. FEMS Microbiol. Lett. 277, 123–132. Allos, B.M., 2001. Campylobacter jejuni infections: update on emerging issues and trends. Clin. Infect. Dis. 32, 1201–1206. Bohaychuk, V.M., Checkley, S.L., Gensler, G.E., Barrios, P.R., 2009. Microbiological baseline study of poultry slaughtered in provincially inspected abattoirs in Alberta, Canada. Can. Vet. J. 50, 173–178. Bohaychuk, V.M., Gensler, G.E., King, R.K., Manninen, K.I., Sorensen, O., Wu, J.T., Stiles, M.E., McMullen, L.M., 2006. Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada. J. Food Prot. 69, 2176–2182.
47