International Journal of Food Microbiology 177 (2014) 49–56

Contents lists available at ScienceDirect

International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Characterization of Extraintestinal Pathogenic Escherichia coli isolated from retail poultry meats from Alberta, Canada Mueen Aslam a, Mehdi Toufeer a, Claudia Narvaez Bravo b, Vita Lai c, Heidi Rempel c, Amee Manges d,e, Moussa Sory Diarra c,⁎ a

Agriculture and Agri-Food Canada, Lacombe Research Centre, Lacombe, Alberta, Canada Department of Food Science, University of Manitoba, Winnipeg, Manitoba, Canada Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Agassiz, British Columbia, Canada d University of British Columbia, School of Population and Public Health, Vancouver, British Columbia, Canada e B.C. Centre for Disease Control, Vancouver, British Columbia, Canada b c

a r t i c l e

i n f o

Article history: Received 22 October 2013 Received in revised form 6 February 2014 Accepted 14 February 2014 Available online 21 February 2014 Keywords: ExPEC Virulence genes Retail poultry meat Multiplex PCR PFGE

a b s t r a c t Extraintestinal Pathogenic Escherichia coli (ExPEC) have the potential to spread through fecal waste resulting in the contamination of both farm workers and retail poultry meat in the processing plants or environment. The objective of this study was to characterize ExPEC from retail poultry meats purchased from Alberta, Canada and to compare them with 12 human ExPEC representatives from major ExPEC lineages. Fifty-four virulence genes were screened by a set of multiplex PCRs in 700 E. coli from retail poultry meat samples. ExPEC was defined as the detection of at least two of the following virulence genes: papA/papC, sfa, kpsMT II and iutA. Genetic relationships between isolates were determined using pulsed field gel electrophoresis (PFGE). Fifty-nine (8.4%) of the 700 poultry meat isolates were identified as ExPEC and were equally distributed among the phylogenetic groups A, B1, B2 and D. Isolates of phylogenetic group A possessed up to 12 virulence genes compared to 24 and 18 genes in phylogenetic groups B2 and D, respectively. E. coli identified as ExPEC and recovered from poultry harbored as many virulence genes as those of human isolates. In addition to the iutA gene, siderophore-related iroN and fyuA were detected in combination with other virulence genes including those genes encoding for adhesion, protectin and toxin while the fimH, ompT, traT, uidA and vat were commonly detected in poultry ExPEC. The hemF, iss and cvaC genes were found in 40% of poultry ExPEC. All human ExPEC isolates harbored concnf (cytotoxic necrotizing factor 1 altering cytoskeleton and causing necrosis) and hlyD (hemolysin transport) genes which were not found in poultry ExPEC. PFGE analysis showed that a few poultry ExPEC isolates clustered with human ExPEC isolates at 55–70% similarity level. Comparing ExPEC isolated from retail poultry meats provides insight into their virulence potential and suggests that poultry associated ExPEC may be important for retail meat safety. Investigations into the ability of our poultry ExPEC to cause human infections are warranted. Crown Copyright © 2014 Published by Elsevier B.V.

1. Introduction Escherichia coli is a commensal bacterium generally found in the gastrointestinal tracts of humans and animals. Some strains can cause nosocomial and community-acquired infections including urinary tract, enteric and systemic post-surgical infections based on their virulence gene content (Pitout, 2012). Of particular concern are Extraintestinal Pathogenic E. coli (ExPEC). ExPEC are associated with human and animal infections that occur outside of the intestinal tract such as urinary tract and bloodstream infections. ExPEC ability to develop resistance to various antimicrobial agents contributes to an increase in human health risk and greater health care costs.

⁎ Corresponding author. Tel.: +1 604 796 1715; fax: +1 604 796 0359. E-mail address: [email protected] (M.S. Diarra).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.006 0168-1605/Crown Copyright © 2014 Published by Elsevier B.V.

ExPEC isolated from avian and human sources contain overlapping features such as virulence factors and phylogenetic groupings (A, B1, B2, and D) suggesting the zoonotic pathogenic potential of avian ExPECs (Giufrè et al., 2012). However, zoonotic potential of these ExPEC strains is still controversial (Bélanger et al., 2011). The Avian Pathogenic E. coli (APEC) strains belonging to the ExPEC groups and causing colibacillosis in birds are reported to carry similar virulence attributes as human ExPEC. This suggests a possible route of ExPEC dissemination in the community through poultry and poultry products that may serve as a potential reservoir of ExPEC (Bergeron et al., 2012; Johnson et al., 2005b; Literak et al., 2013; Moulin-Schouleur et al., 2006, 2007; Tóth et al., 2012). A number of reports have suggested a higher prevalence of ExPEC on retail chicken, beef and pork meat, however, the recovery of ExPEC has been greatest from chicken meat (Jakobsen et al., 2010; Johnson et al., 2005a, 2005b). These studies suggest that poultry meat could play a

50

M. Aslam et al. / International Journal of Food Microbiology 177 (2014) 49–56

role in human infections (Johnson et al., 2005a, 2005c, 2009, 2003; Manges et al., 2007). A detailed comparison of ExPEC strains isolated from poultry is needed to assess possible transmission (Jakobsen et al., 2010). Previously we have demonstrated that chicken is a reservoir for ExPEC carrying virulence and antibiotic resistance genes of animal and human importance (Bonnet et al., 2009; Lefebvre et al., 2009). These bacteria have the potential to spread through fecal waste potentially contaminating both farm workers and poultry carcasses in the processing plants or environment. Obviously, monitoring of verotoxin producing E. coli is crucial for the meat industry but attention should also be paid to ExPEC, which cause serious extraintestinal infections in humans. In this study we analyzed and compared the phylogenetic grouping and the prevalence of ExPEC associated virulence genes in E. coli isolates recovered from retail poultry meats (turkey and chicken) and those from humans by using a set of multiplex PCR assays, serotyping and Pulsed Field Gel Electrophoresis (PFGE). The specific objective of the present study was to provide insight into the properties of ExPEC isolates recovered from retail poultry meats in order to understand their zoonotic potential. Detailed genomic analysis of ExPEC isolates of all possible sources including human and meat would help understanding the ecology of these pathogens. 2. Material and methods 2.1. Bacterial strains Ten E. coli strains (J96/JJ079, 2H25/BUTI3-1-4, L31/Low31, V27/BUTI 1-5-1, PM9/BUTI 1-7-6, 2H15?16?/BUTI 3-1-2, 31A/JJ1166, 1A/JJ1167, 536/JJ425 and JJ055) kindly provided by Dr. James R. Johnson (Department of Medicine, University of Minnesota, USA) were used as controls for the detection of virulence genes. Three APEC strains (D0602195 Barn 3 Pool, D0602195 Barn 4, and 0606519) provided by M. Ngeleka (University of Saskatchewan, SK, Canada) as well as the nonpathogenic E. coli K12 were included (Forgetta et al., 2012). Twelve E. coli isolates from human infections (two strains from stool: GMS002A and GMS009B, two from blood: S20323 and H15 and eight from UTI: UTI PI 141 and UTI PI 500, MSHS 95, MSHS 161, MSHS 258, MSHS 472, MSHS 769 and MSHS 1014A) previously described by Bergeron et al. (2012) were used for comparisons. 2.2. Sampling and E. coli isolation The sampling plan used by Canadian Integrated Program for Antimicrobial Resistance Surveillance was followed which involved continuous weekly sampling from randomly selected census divisions, weighted by population (Sheikh et al., 2012). A total of 297 retail poultry (206 chickens and 91 turkeys) samples were purchased and 700 E. coli isolates recovered from retail chicken (502 isolates) and turkey (198 isolates) meats were characterized. After initial processing, samples were transferred to double strength EC broth and incubated at 44 °C for 24 h. Then a loop full from EC broth was inoculated onto the MacConkey agar plate followed by incubation at 35 °C. Typical lactose fermenting, pink E. coli colonies (up to n = 3) were selected from each sample for further analysis. E. coli were confirmed using standard biochemical methods and by PCR (Aslam et al., 2004). 2.3. Analysis of virulence genes and phylogenetic groups Confirmed E. coli isolates were tested for the presence of 54 virulence genes using specific primer sets (Life Technologies Inc., Burlington, ON) in multiplex PCRs as previously described (Johnson and Stell, 2000). Classification of isolates as ExPEC was based on the presence of two or more of the following virulence genes: pap (P fimbriae), sfa or foc (S/F1C fimbriae), afa or dra (binding, adhesions), iutA (aerobactin receptor), and kpsM II (group II capsule synthesis) as

described (Johnson et al., 2003, 2005a, 2008, 2009). The ExPEC strains were assigned to one of the four designated phylogenetic group (A, B1, B2, or D) based on the pattern of chuA, yjaA, and TSPE4.C2 genes presence as described by Clermont et al. (2000). 2.4. Serotyping and pulsed-field gel electrophoresis All isolates classified as ExPEC were serotyped at the Laboratory for Foodborne Zoonoses, Guelph, Ontario, Canada. Identification of somatic (O) and flagellar (H) antigens were performed by standard agglutination methods that identified O1 to O173 and H1 to H56 by following the procedures described previously (Ewing, 1986). Confirmed ExPEC isolates were subtyped to assess their genetic relatedness using PFGE technique by following the standard protocols as described by Ribot et al. (2006). Briefly, agarose plugs were prepared with E. coli cell suspension and lysed with proteinase K. Genomic DNA in gel matrix was digested using XbaI restriction enzyme and DNA fragments were separated on 1.5% agarose gel by following the conditions as described by Ribot et al. (2006). 2.5. Statistical analyses All data were entered into Excel spreadsheets and frequencies of genes, serotypes and ExPEC were calculated. Statistical analysis of the data was performed using SAS software 9.2 (SAS Institute, Inc., Cary, NC). The association test of Cochran–Mantel–Haenszel was used to determine the relationship between meat types and genotype using the FREQ procedures as well as associations between ExPEC pathotype and genotype (Bonnet et al., 2009). A P value of 0.05 was used to declare significance. 3. Results 3.1. ExPEC prevalence Overall 8.4% of the isolates (59 isolates) from retail poultry including chicken and turkey were classified as ExPEC based on the criteria established by Johnson et al. (2003). Among the detected ExPEC, 8.7% (44/502) of the isolates were from retail chicken meat and 7.5% (15/198) of isolates were from retail turkey meat (data not shown). Fifty-five (93.2%), three (5.1%) and one (1.7%) of the 59 defined ExPEC isolates harbored two, three, and four of the ExPEC-defining markers, respectively. The remaining 641 of the 700 (91.6%) E. coli isolates were defined as non-ExPEC. However, 236 of these 641 (36.8%) isolates harbored one of the ExPEC-defining markers, and 405 out of 641 (63.8%) isolates harbored none. The 236 isolates harboring one ExPECdefining marker may be other ExPEC such as avian pathogenic E. coli. This study focused only on the 59 isolates classified as ExPEC based on the two gene criterion. 3.2. Serotypes and phylogenetic groups of ExPEC One chicken meat and one turkey meat isolate could not be serotyped. Among the remaining 57 ExPEC isolates, 28 different serotypes were identified with 18 and 9 serotypes being found in chicken and turkey meats, respectively. Statistically significant (P b 0.05) association between a serotype and a source (chicken or turkey meats) of ExPEC was observed. In chicken ExPEC isolates, the serotypes O71:H10 (8 isolates), O6:H16 (7 isolates), O7:H18 (4 isolates), O22:H2 (3 isolates), O11:H25 (3 isolates) and O2:H42 (3 isolates) were common (Table 1). None of these serotypes were found in turkey isolates where serotype O?:H7 was more common (Table 2). The serotypes O21:H16, O25:H4, O2:H1 and O2:H6 were found in both chicken (Table 1) and turkey (Table 2). Serotype O25:H4 was found in chicken, turkey and human isolates (Tables 1–3).

M. Aslam et al. / International Journal of Food Microbiology 177 (2014) 49–56

51

Table 1 Characteristics of the 44 potential ExPEC isolates recovered from retail chicken meat purchased in Alberta, Canada. MISC = miscellaneous. Strain #

Serotype

Phylogroup

MISC

Adhesion

Iron

Toxin

Protectin

No. of genes

4701 4702 4703 4941 5006 4747 5001 4896 4897 4898 4928 5133 5134 5135 4618 4619 4767 4859 4860 5082 5083 5084 4906 4487 4960 4961 4875 4876 4877 4954 4956 4958 4470 4627 4628 4926 4940 4821 5101 4987 4604 4819 4822 4803

O11:H25 O11:H25 O11:H25 O21:H16 O21:H4 O21:NM O25:NM O6:H16 O6:H16 O6:H16 O6:H16 O6:H16 O6:H16 O6:H16 O71:H10 O71:H10 O71:H10 O71:H10 O71:H10 O71:H10 O71:H10 O71:H10 O15:H5 O15:H6 O16:H34 O19:H34 O2:H42 O2:H42 O2:H42 O22:H2 O22:H2 O22:H2 O39:H18 O7:H18 O7:H18 O7:H18 O7:H18 O139:NM O2:H1 O2:H6 O25:H4 O25:H4 O25:H4 ND

A A A A A A A A A A A A A A A A A A A A A A D D D D D D D D D D D D D D D B2 B2 B2 B2 B2 B2 B2

2 2 2 2 2 1 2 3 3 3 2 3 3 3 2 2 2 2 2 2 2 2 1 3 3 3 3 3 3 2 2 2 2 2 2 1 2 5 5 7 5 5 5 6

2 2 2 3 2 2 2 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 3 6 3 3 6 6 6 3 3 3 2 2 2 2 3 2 2 6 2 2 2 5

2 2 2 1 1 1 2 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 1 4 1 1 2 2 2 1 1 1 2 2 2 1 1 3 2 2 3 3 3 1

2 2 2 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 1 0 1 1 0 0 0 1 1 1 0 0 0 1 1 1 3 0 1 1 0 1

1 1 1 2 2 1 3 2 2 2 2 2 2 2 4 4 4 4 4 4 4 4 2 5 2 2 4 4 4 1 1 1 3 3 3 2 2 5 4 4 5 4 5 4

9 9 9 9 8 6 10 10 10 10 9 10 10 10 11 11 11 11 11 11 11 11 8 18 10 10 15 15 15 8 8 8 9 9 9 7 9 16 16 19 16 15 15 17

rule out the possibility that phylogroup B1 occur in our “other ExPEC” chicken isolates. The phylogenetic groups B2, A, B1 and D were found in 53.3% (8 isolates), 20.0% (3 isolates), 13.3% (2 isolates) and 13.3% (2) of ExPEC isolates from turkey, respectively (Table 2). Out of the 12 human isolates, five ExPEC isolates belonged to phylogenetic group B2 and five to phylogroup D (Table 3). All eight isolates of serotype O25: H4 (4, 3 and 1 isolates of chicken, turkey and human, respectively)

The four screened phylogenetic groups (A, B1, B2 and D) were detected among the 59 poultry ExPEC isolates. However, no isolate of phylogroup B1 was found in the 44 chicken ExPEC isolates, while 50.0% (22 isolates), 34.1% (15 isolates) and 15.9% (7 isolates) isolates belonged to phylogenetic group A, D and B2, respectively (Table 1). In this study phylogenetic grouping of the 236 potential “other ExPEC” including chicken isolates was not performed therefore we cannot

Table 2 Characteristics of the 15 potential ExPEC isolates recovered from retail turkey meat collected in Alberta, Canada. MISC = miscellaneous. Strain #

Serotype

Phylogroup

MISC

Adhesion

Iron

Toxin

Protectin

No. of genes

5143 5022 4694 5142 5144 4491 5045 4724 4972 4557 4566 4971 4973 4839 4559

O?:H7 O11:H16 O21:H16 O?:H7 O?:H7 O166:H38 ND O2:H1 O2:H6 O25:H4 O25:H4 O25:H4 O25:H4 O39:H7 O83:H1

A A A B1 B1 D D B2 B2 B2 B2 B2 B2 B2 B2

3 2 2 3 3 2 3 5 5 5 5 5 5 6 4

2 3 3 1 1 2 3 2 6 3 2 6 6 9 2

2 2 1 2 2 1 3 3 2 3 3 2 2 4 3

1 2 1 1 1 0 2 2 1 1 0 1 1 3 1

4 3 2 4 4 2 2 5 4 4 4 4 4 2 4

12 12 9 11 11 7 13 17 18 16 14 18 18 24 14

52

M. Aslam et al. / International Journal of Food Microbiology 177 (2014) 49–56

Table 3 Characteristics of the 12 potential ExPEC isolates recovered from human urinary tract infections. MISC = miscellaneous. Strain#

Strain ID

Origin

Serotype

Phylogroup

MISC

Adhesion

Iron

Toxin

Protectin

No. of genes

5298 5299 5300 5301 5302 5303 5304 5305 5306 5307 3708 5309

GMS002A GMS009B UTI PI 141 UTI PI 500 S20323 H15 MSHS 95 MSHS 161 MSHS 258 MSHS 472 MSHS 769 1014A

Stool Stool Pyelo Pyelo Blood Blood UTI UTI UTI UTI UTI UTI

ND ND X19 O1 O73 O153 O2:H7 O25:H4 ND O82:NM O4:H5 O114:H4

A B2 B2 D D D B2 B2 D B1 B2 D

1 7 6 2 2 2 7 5 2 3 5 3

3 4 3 8 8 9 5 2 1 1 10 1

2 2 3 2 2 2 2 2 2 1 2 4

0 4 1 0 0 0 1 1 1 0 4 3

3 2 2 2 2 2 3 5 2 2 3 3

9 19 15 14 14 15 18 15 8 7 24 14

were of phylogenetic group B2, while three isolates of serotypes O22:H2 and four isolates of serotype O7:H18 from chicken belonged to phylogroup D. Four serotypes from human isolates (O2:H7, O4:H5, O82:NM and X19) were of phylogroup B2 (Table 3). 3.3. Prevalence of virulence genes Out of 54 tested genes, 37 virulence-related genes were detected and their presence was significantly different (P b 0.05) between chicken, turkey and human ExPEC isolates (Table 4). Among 59 poultry

ExPEC isolates, 1.7% carried N 20 virulence genes, 18.6% carried 16–20 virulence gene, 3.9% carried 11–15 virulence genes and 45.8% carried 6–10 virulence genes. Chicken ExPEC isolates harbored fewer virulence genes than those of turkey or human ExPEC (P b 0.05). There was no significant difference for the presence of virulence gene number between turkey and human ExPEC isolates (P N 0.05). In general, isolates of phylogenetic group B2 carried the highest numbers of virulence genes with the serotype O39:H7, which was found only in turkey meat, displaying 24 virulence genes. E. coli serotype O25:H4 belonging to phylogenetic group B2, an important and newly emerging ExPEC

Table 4 Distribution of virulence genes among ExPEC isolates recovered from retail chicken, turkey and human and significant statistical associations between isolates. Functions

Adhesion

Toxins

Protectins

Iron acquisition or transport systems

Miscellaneous, various functions

Virulence genes

fimH focG hra iha kii papA papC papEF papG allele 2&3 papG allele II papG allele III sfa sfaS cdts EAST11 pic tsh vat iss kpsMT K1 kpsMT KII kpsMT K5 traT ireA iroN iutA fyuA clbB clbN cvaC H7 fliC hemF ibe10 ompT malX (PAI) uidA usp

Number of isolates (%) Chicken (n = 44)

Turkey (n = 15)

Human (n = 12)

Total (n = 71)

P valuea

36 (81.8) 0 (0.0) 17 (38.8) 0 (0.0) 43 (97.7) 3 (6.8) 4 (9.1) 6 (13.6) 1 (2.3) 4 (9.1) 0 (0.0) 2 (4.6) 2 (4.6) 0 (0.0) 30 (68.2) 1 (2.3) 13 (29.55) 4 (66.7) 23 (52.3) 8 (18.2) 43 (97.7) 0 (0.0) 35 (79.6) 5 (2.3) 23 (52.3) 43 (97.73) 8 (18.18) 1 (2.3) 1 (2.3) 17 (38.6) 1 (2.3) 35 (79.6) 6 (13.6) 25 (56.8) 4 (9.1) 41 (93.2) 7 (15.9)

15 (100) 1 (6.7) 7 (46.7) 2 (13.3) 11 (73.3) 2 (13.3) 5 (33.3) 3 (20.0) 1 (6.7) 1 (6.7) 3 (20.0) 1 (6.7) 0 (0.0) 5 (33.3) 2 (13.3) 1 (6.7) 6 (40.00) 21 (95.5) 11 (73.3) 1 (6.7) 14 (93.3) 1 (6.7) 14 (93.3) 1 (6.7) 13 (86.7) 15 (100.0) 6 (40.0) 1 (6.7) 1 (6.7) 8 (53.3) 3 (20.0) 11 (73.4) 7 (46.7) 11 (73.3) 2 (13.3) 14 (93.3) 8 (53.3)

12 (100) 1 (8.3) 3 (25.0) 4 (33.3) 8 (66.7) 5 (41.7) 5 (41.7) 5 (41.7) 4 (33.3) 5 (41.7) 1 (8.3) 2 (16.7) 0 (0.0) 0 (0.0) 2 (16.7) 2 (16.7) 1 (8.33) 15 (75.0) 4 (33.3) 4 (33.3) 8 (66.7) 1 (8.3) 10 (83.3) 3 (25.0) 6 (50.0) 6 (50.0) 11 (92.0) 4 (33.3) 4(33.3) 2(16.7) 2 (16.7) 3 (25.0) 2 (16.7) 11 (91.7) 2 (16.7) 12 (100) 5 (41.7)

63 (88.7) 2 (2.8) 27 (38.0) 6 (8.4) 62 (87.3) 10 (14.0) 14 (19.7) 14 (19.7) 6 (8.4) 10 (14.0) 4 (5.6) 5 (7.0) 2 (2.8) 5 (7.04) 34 (47.9) 4 (5.6) 20 (28.1) 64 (90.1) 38 (53.5) 13 (18.3) 65 (91.5) 2 (2.8) 59 (83.1) 5 (7.0) 42 (59.1) 64 (90.1) 25 (35.2) 6 (8.4) 6 (8.41) 27 (38.0) 6 (8.4) 49 (69.0) 15(21.1) 47 (66.2) 8 (11.3) 67 (94.4) 20 (28.2)

0.06 0.18 0.51 b0.01 b0.01 b0.01 0.01 0.09 b0.01 0.66 0.01 0.35 0.53 b0.01 b0.01 0.16 0.19 0.15 0.11 0.20 b0.01 0.18 0.47 0.02 0.05 b0.01 b0.01 b0.01 b0.01 0.15 0.05 b0.01 0.02 0.06 0.73 0.65 0.01

a P value obtained by Cochran–Mantel–Haenszel test (P-value ≤0.05 was used to declare statistically difference between sources of isolates). The afa/dra, concnf, hlyD, rfc and kpsMT III genes were detected only in human isolates.

M. Aslam et al. / International Journal of Food Microbiology 177 (2014) 49–56

strain was found in both turkey (16.5 ± 0.4 virulence genes) and chicken (15.3 ± 0.4 virulence genes) and these isolates exhibited high numbers of virulence genes (Table 1–2). All 700 isolates were found to carry at least two of the screened genes with fimH, ironec, iutA, ompT, TraT, hemF and iss being the most frequently detected genes. The most common virulence genes found in the ExPEC isolates were fimH, kii, vat, kpsMT KII, traT, iutA, hemF, ompT and uidA (Table 4). The virulence genes afa/dra, concnf, hlyD, rfc and kpsMT III were detected only in the human isolates. In addition to the iutA gene, other common iron related genes found included siderophore-related iroN fyuA, and ireA detected mostly in combination with other virulence genes. The virulence genes focG, hra, papG allele II,

53

sfa, sfaS, pic, tsh, vat, Iss, kpsMT K1, kpsMT K5, traT, malX (PAI) and uidA were not significantly associated with any particular type of sample source (Table 4). Data obtained in this research showed that ExPEC isolates of human and poultry origin can share a number of virulence genes. The OmpT gene was frequently associated with serotypes O22: H2, O25:H4, O2:H1 and O2:H42. All isolates of phylogenetic group B2 and 72% of isolates of phylogenetic group D were positive for ompT. 3.4. Subtyping of ExPEC isolates The poultry ExPEC isolates and 12 human isolates were analyzed using XbaI-PFGE in order to determine their genetic relationship

Fig. 1. Comparison of human, chicken and turkey ExPEC strains (white = negative; black = positive). At a similarity level (dice) of 55 to 74% the PFGE profile showed that some poultry ExPEC isolates clustered with human ExPEC.

54

M. Aslam et al. / International Journal of Food Microbiology 177 (2014) 49–56

(Fig. 1). Our PFGE data showed that a heterogeneous ExPEC population was present in the poultry and human samples. Isolates exhibiting similar phylogenetic group and virulence gene profiles tended to cluster together but the ExPEC strains found in this study were not closely related. The ExPEC isolates from poultry shared genetic relatedness at 55–74% based on the dice similarity coefficient. A few PFGE profiles of ExPEC isolates from poultry and human clustered at N65% similarity. 4. Discussion With increasing demand for poultry meat and poultry products and the growing poultry industry around the world, the importance of poultry meat safety is a critical public health issue. The ExPEC isolated from retail poultry meats have been associated with potential infections in humans in Europe, North America and Australia (Dezfulian et al., 2003; Girardeau et al., 2003; Maynard et al., 2004). Little information is available about the epidemiology of ExPEC in retail poultry from Canada. Chicken has been reported to be contaminated with ExPEC more frequently than other types of retail meats (Smith et al., 2007; Xia et al., 2011). Data from our study showed a lower prevalence of ExPEC isolates in poultry meats than those reported by Johnson et al. (2003) who reported that about 21% of poultry samples were positive for ExPEC. Another study from the same research group reported an even higher proportion (46%) of retail poultry samples carrying ExPEC isolates (Johnson et al., 2005a). This difference in ExPEC prevalence in poultry meat may be attributed to several factors including initial colonization status of broilers before processing and the degree of fecal contamination of carcasses during the slaughter operation at the processing facility. This contamination is possible particularly if the growth of these E. coli is favored via specific food processing steps such as scalding, evisceration, washing and deboning resulting in the contamination of the retail poultry products (Altekruse et al., 2002; De Brito et al., 2003; Rodriguez-Siek et al., 2005b). Furthermore it has been suggested that clinical E. coli isolates from sick chickens carried similar sets of virulence genes as have been identified in ExPEC isolates from humans and animals (Maryvonne et al., 2007). It is possible that entry of diseased birds into the poultry slaughter plant might have contributed to the contamination of retail meats. The low prevalence observed in the present study requires further confirmation in larger studies in Canada. Rapid evolution of commensal strains, genetic mutations, specifically horizontal gene transfer that may occur between commensal and pathogenic enterobacteriaceae in close contact leads to acquisition of virulence genes required to cause severe infections in humans (Leimbach et al., 2013). A combination of virulence factors may favor the development of potential infection. Sets of several virulence factors (54 VFs) were used to determine if ExPEC of avian origin share virulence gene profiles with ones which cause infection in humans. Human ExPEC isolates used in this study were not epidemiologically related to our poultry isolates because they were not isolated from same geographical regions. Therefore data presented in this study should be interpreted with caution. However, we found that isolates from chicken, turkey and human shared a number of virulence genes. The presence of specific virulence-associated genes may not be sufficient to cause disease, rather the levels of expression of these factors, which can vary between isolates, are also important for virulence in vivo. More investigation will be needed to establish the virulence potential of poultry associated ExPEC in animal extraintestinal disease model. ExPEC isolates belonging to phylogenetic group B1 have been previously described in chicken (Johnson et al., 2009). In our study none of the ExPEC isolated from chicken belonged to phylogroup B1, but two turkey isolates belonged to this group. It should be noted here that screening of the 236 potential “other ExPEC” that also included chicken meat isolates may have produced isolates belonging to phylogroup group B1. In the present study, 25.4% of the chicken ExPEC isolates and 53.3% of the turkey ExPEC belonged to phylogroup B2, whereas

phylogroup D comprised of 34% of chicken ExPEC and 13% of turkey ExPEC. Our phylogenetic data is consistent with a previous study conducted on retail poultry where 21% of the strains belonged to phylogroup B2 and about 29% to phylogroup D (Kobayashi et al., 2011). Based on the assumption that phylogenetic group B2 is more pathogenic (Jakobsen et al., 2011), our phylogroup distribution suggests that isolates from chicken origin are more likely to be pathogenic than those of turkey origin. In our study the majority of ExPEC isolates belonged to phylogenetic groups A, B2 and D. It has been shown that avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) as well neonatal meningitis E. coli (NMEC) belonged to the phylogroup B2 (Johnson et al., 2008). These strains generally belong to serogroups O1, O2 or O18 and carry a higher number of virulence genes as compared to other ExPEC strains. In the present study, serotype O1 or O18 was not detected, while seven isolates of serotype O2 having various “H” types were found both in chicken and turkey meats. This O2 serotype belonged to phylogenetic group D or B2 and carried various combinations of virulence genes. Seven isolates of serotype O25:H4 (3 from chicken and 4 from turkey) from phylogroup B2 were shown to carry between 14 and 18 virulence related genes. The PFGE analysis showed that an isolate #4724 (O25:H4 from turkey) clustered with an isolate #5305 (O25: H4 from human) at 74.8% similarity suggesting existence of a genetic relation between these isolates. The recently emerged E. coli sequence type ST131, which has been associated with world-wide urinary tract infections, and is multidrug resistant, belongs to serotype O25:H4 (Johnson et al., 2012) suggesting that extensive surveillance in Canadian food animals and retail meat for this specific ExPEC is warranted. Three chicken ExPEC belonging to serotype O25:H4 clustered at 65% similarly level with one turkey ExPEC of serotype O25:H5 as well as with one chicken isolate of serotype O139 suggesting existence of a genetic relationship between these isolates. Little is known about E. coli O39:H7, however, this serotype has been isolated as a non-O157 Shiga-toxin producing E. coli from animals (Constantiniu, 2002) and from feces and urinary tract infections in humans (Bettelheim, 1978). Given the very high number (up to 24) of virulence factors detected in these isolates in the present study, more investigation will be required for its full characterization and to determine its prevalence in poultry. Nevertheless our data suggest the existence of potential subsets of E. coli in retail poultry meats that belong to serotypes or pathotypes similar to those associated with either human or avian extra-intestinal infections. Of the adhesion-encoding virulence factors included in our study, the fimH and kii genes were the most frequent. The kii gene was suggested to play a potential role in the pathogenicity of ExPEC (Abdallah et al., 2011). Other virulence genes detected in the present study included sfaS, focG, afa/dra, bmaE, gafD, cnf1, cdtB and kpsMT II which were also found in human ExPEC suggesting an overlap in virulence genes among poultry and human ExPEC isolates. The higher proportion of fimH, fyuA, iutA, traT and sfa/foc genes found in poultry ExPEC is consistent with previously reported studies in UPEC and NMEC in humans (BingenBidois et al., 2002; Sannes et al., 2004). Whether the higher prevalence of these genes among ExPEC of poultry origin makes them more infectious to humans is not clear. Two virulence genes, cvaC and iss, which are known to occur widely in APEC were only detected in ExPEC isolates of poultry origin and not in ExPEC isolates of human origin (Ewers et al., 2007; Rodriguez-Siek et al., 2005a, 2005b), although our sample of human isolates is limited. In conclusion, our results confirmed that ExPEC can be isolated from retail poultry meats. Furthermore, our results showed that E. coli strains harbored various combinations of virulence genes which may potentially be involved in human infections. This study provided for the first time insight into the virulence potential of ExPEC isolated from retail poultry meats in Alberta, Canada and allows assessment of their meat safety risks. It also describes methods that could be used to screen and compare E. coli of various origins and to better understand the evolution of this bacterium from the commensal to the pathogenic. Further

M. Aslam et al. / International Journal of Food Microbiology 177 (2014) 49–56

investigations into the ability of our poultry ExPEC isolates to cause diseases in animal models are warranted. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this report. Acknowledgements The grant for this study was provided by Agriculture and Agri-Food Canada through the risk mitigation strategies initiative. The authors thank Dr. James Johnson (Department of Medicine, University of Minnesota, USA) for providing control E. coli strains and primers sequences used in multiplex PCR. The authors thank Cara Service for providing laboratory support for sample collection and isolation of E. coli and Kim Ziebell for serotyping of ExPEC isolates. References Abdallah, K.S., Cao, Y., Wei, D.-J., 2011. Epidemiologic investigation of extra-intestinal pathogenic E. coli (ExPEC) based on PCR phylogenetic group and fimH single nucleotide polymorphisms (SNPs) in China. Int. J. Mol. Epidemiol. Genet. 2, 339–353. Altekruse, S.F., Elvinger, F., DebRoy, C., Pierson, F.W., Eifert, J.D., Sriranganathan, N., 2002. Pathogenic and fecal Escherichia coli strains from turkeys in a commercial operation. Avian Dis. 46, 562–569. Aslam, M., Greer, G.G., Nattress, F.M., Gill, C.O., McMullen, L.M., 2004. Genotypic analysis of Escherichia coli recovered from product and equipment at a beef-packing plant. J. Appl. Microbiol. 97, 78–86. Bélanger, L., Garenaux, A., Harel, J., Boulianne, M., Nadeau, E., Dozois, C.M., 2011. Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E. coli. FEMS Immunol. Med. Microbiol. 62, 1–10. Bergeron, C.R., Prussing, C., Boerlin, P., Daignault, D., Dutil, L., Reid-Smith, R.J., Zhanel, G.G., Manges, A.R., 2012. Chicken as reservoir for extraintestinal pathogenic Escherichia coli in humans, Canada. Emerg. Infect. Dis. 18, 415–421. Bettelheim, K.A., 1978. The sources of “OH” serotypes of Escherichia coli. J. Hyg. (Lond.) 80, 83–113. Bingen-Bidois, M., Clermont, O., Bonacorsi, S., Terki, M., Brahimi, N., Loukil, C., Barraud, D., Bingen, E., 2002. Phylogenetic analysis and prevalence of urosepsis strains of Escherichia coli bearing pathogenicity island-like domains. Infect. Immun. 70, 3216–3226. Bonnet, C., Diarrassouba, F., Brousseau, R., Masson, L., Topp, E., Diarra, M.S., 2009. Pathotype and antibiotic resistance gene distributions of Escherichia coli isolates from broiler chickens raised on antimicrobial-supplemented diets. Appl. Environ. Microbiol. 75, 6955–6962. Clermont, O., Bonacorsi, S., Bingen, E., 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66, 4555–4558. Constantiniu, S., 2002. Escherichia coli enterohemoragic — an emerged pathogen of human infections Part II. non-o157 Escherichia coli enterohaemorrhagic. J. Prev. Med. 10, 57–73. De Brito, B.G., Gaziri, L.C.J., Vidotto, M.C., 2003. Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis. Infect. Immun. 71, 4175–4177. Dezfulian, H., Batisson, I., Fairbrother, J.M., Lau, P.C.K., Nassar, A., Szatmari, G., Harel, J., 2003. Presence and characterization of extraintestinal pathogenic Escherichia coli virulence genes in F165-positive E. coli strains isolated from diseased calves and pigs. J. Clin. Microbiol. 41, 1375–1385. Ewers, C., Li, G., Wilking, H., Kiessling, S., Alt, K., Antáo, E.-M., Laturnus, C., Diehl, I., Glodde, S., Homeier, T., Böhnke, U., Steinrück, H., Philipp, H.-C., Wieler, L.H., 2007. Avian pathogenic, uropathogenic, and newborn meningitis-causing Escherichia coli: how closely related are they? Int. J. Med. Microbiol. 297, 163–176. Ewing, W.H., 1986. In: Edwards and Ewing's Identification of Enterobacteriaceae, 4th edition. ed. Elsevier. Forgetta, V., Rempel, H., Malouin, F., Vaillancourt Jr., R., Topp, E., Dewar, K., Diarra, M.S., 2012. Pathogenic and multidrug-resistant Escherichia fergusonii from broiler chicken. Poult. Sci. 91, 512–525. Girardeau, J.P., Lalioui, L., Said, A.M.O., De Champs, C., Le Bouguenec, C., 2003. Extended virulence genotype of pathogenic Escherichia coli isolates carrying the afa-8 Operon: evidence of similarities between isolates from humans and animals with extraintestinal infections. J. Clin. Microbiol. 41, 218–226. Giufrè, M., Graziani, C., Accogli, M., Luzzi, I., Busani, L., Cerquetti, M., 2012. Escherichia coli of human and avian origin: detection of clonal groups associated with fluoroquinolone and multidrug resistance in Italy. J. Antimicrob. Chemother. 67, 860–867. Jakobsen, L., Spangholm, D.J., Pedersen, K., Jensen, L.B., Emborg, H.-D., Agersø, Y., Aarestrup, F.M., Hammerum, A.M., Frimodt-Møller, N., 2010. Broiler chickens, broiler chicken meat, pigs and pork as sources of ExPEC related virulence genes and resistance in Escherichia coli isolates from community-dwelling humans and UTI patients. Int. J. Food Microbiol. 142, 264–272.

55

Jakobsen, L., Garneau, P., Kurbasic, A., Bruant, G., Stegger, M., Harel, J., Jensen, K.S., Brousseau, R., Hammerum, A.M., Frimodt-Møller, N., 2011. Microarray-based detection of extended virulence and antimicrobial resistance gene profiles in phylogroup B2 Escherichia coli of human, meat and animal origin. J. Med. Microbiol. 60, 1502–1511. Johnson, J.R., Stell, A.L., 2000. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J. Infect. Dis. 181, 261–272. Johnson, J.R., Murray, A.C., Gajewski, A., Sullivan, M., Snippes, P., Kuskowski, M.A., Smith, K.E., 2003. Isolation and molecular characterization of nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from retail chicken products. Antimicrob. Agents Chemother. 47, 2161–2168. Johnson, J.R., Delavari, P., O'Bryan, T.T., Smith, K.E., Tatini, S., 2005a. Contamination of retail foods, particularly turkey, from community markets (Minnesota, 1999–2000) with antimicrobial-resistant and extraintestinal pathogenic Escherichia coli. Foodborne Pathog. Dis. 2, 38–49. Johnson, J.R., Kuskowski, M.A., Smith, K., O'Bryan, T.T., Tatini, S., 2005b. Antimicrobialresistant and extraintestinal pathogenic Escherichia coli in retail foods. J. Infect. Dis. 191, 1040–1049. Johnson, J.R., Kuskowski, M.A., Smith, K., O'Bryan, T.T., Tatini, S., 2005c. Antimicrobialresistant and extraintestinal pathogenic Escherichia coli in retail foods. J. Infect. Dis. 191, 1040–1049. Johnson, T.J., Wannemuehler, Y., Johnson, S.J., Stell, A.L., Doetkott, C., Johnson, J.R., Kim, K.S., Spanjaard, L., Nolan, L.K., 2008. Comparison of extraintestinal pathogenic Escherichia coli strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens. Appl. Environ. Microbiol. 74, 7043–7050. Johnson, T.J., Logue, C.M., Wannemuehler, Y., Kariyawasam, S., Doetkott, C., DebRoy, C., White, D.G., Nolan, L.K., 2009. Examination of the source and extended virulence genotypes of Escherichia coli contaminating retail poultry meat. Foodborne Pathog. Dis. 6, 657–667. Johnson, J.R., Urban, C., Weissman, S.J., Jorgensen, J.H., Lewis II, J.S., Hansen, G., Edelstein, P.H., Robicsek, A., Cleary, T., Adachi, J., Paterson, D., Quinn, J., Hanson, N.D., Johnston, B.D., Clabots, C., Kuskowski, M.A., AMERECUS Investigators, 2012. Molecular epidemiological analysis of Escherichia coli sequence type ST131 (O25:H4) and blaCTX-M-15 among extended-spectrum-β-lactamase-producing E. coli from the United States, 2000 to 2009. Antimicrob. Agents Chemother. 56, 2364–2370. Kobayashi, R.K., Aquino, I., Ferreira, A.L. da S., Vidotto, M.C., 2011. EcoR phylogenetic analysis and virulence genotyping of avian pathogenic Escherichia coli strains and Escherichia coli isolates from commercial chicken carcasses in southern Brazil. Foodborne Pathog. Dis. 8, 631–634. Lefebvre, B., Gattuso, M., Moisan, H., Malouin, F., Diarra, M.S., 2009. Genotype comparison of sorbitol-negative Escherichia coli isolates from healthy broiler chickens from different commercial farms. Poult. Sci. 88, 1474–1484. Leimbach, A., Hacker, J., Dobrindt, U., 2013. E. coli as an all-rounder: the thin line between commensalism and pathogenicity. Curr. Top. Microbiol. Immunol. 358, 3–32. Literak, I., Reitschmied, T., Bujnakova, D., Dolejska, M., Cizek, A., Bardon, J., Pokludova, L., Alexa, P., Halova, D., Jamborova, I., 2013. Broilers as a source of quinolone-resistant and extraintestinal pathogenic Escherichia coli in the Czech Republic. Microb. Drug Resist. 19, 57–63. Manges, A.R., Smith, S.P., Lau, B.J., Nuval, C.J., Eisenberg, J.N.S., Dietrich, P.S., Riley, L.W., 2007. Retail meat consumption and the acquisition of antimicrobial resistant Escherichia coli causing urinary tract infections: a case–control study. Foodborne Pathog. Dis. 4, 419–431. Maryvonne, M., Maryline, R., Sylvie, L., Annie, B., Sandrine, M., Pierre, G., Denis, R., Catherine, S., 2007. Extraintestinal pathogenic Escherichia coli strains of avian and human origin: link between phylogenetic relationships and common virulence patterns. J. Clin. Microbiol. 45, 3366–3376. Maynard, C., Bekal, S., Sanschagrin, F., Levesque, R.C., Brousseau, R., Masson, L., Lariviere, S., Harel, J., 2004. Heterogeneity among virulence and antimicrobial resistance gene profiles of extraintestinal Escherichia coli isolates of animal and human origin. J. Clin. Microbiol. 42, 5444–5452. Moulin-Schouleur, M., Schouler, C., Tailliez, P., Kao, M.-R., Brée, A., Germon, P., Oswald, E., Mainil, J., Blanco, M., Blanco, J., 2006. Common virulence factors and genetic relationships between O18:K1:H7 Escherichia coli isolates of human and avian origin. J. Clin. Microbiol. 44, 3484–3492. Moulin-Schouleur, M., Répérant, M., Laurent, S., Brée, A., Mignon-Grasteau, S., Germon, P., Rasschaert, D., Schouler, C., 2007. Extraintestinal pathogenic Escherichia coli strains of avian and human origin: link between phylogenetic relationships and common virulence patterns. J. Clin. Microbiol. 45, 3366–3376. Pitout, J.D.D., 2012. Extraintestinal pathogenic Escherichia coli: an update on antimicrobial resistance, laboratory diagnosis and treatment. Expert Rev. Anti Infect. Ther. 10, 1165–1176. Ribot, E.M., Fair, M.A., Gautom, R., Cameron, D.N., Hunter, S.B., Swaminathan, B., Barrett, T.J., 2006. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog. Dis. 3, 59–67. Rodriguez-Siek, K.E., Giddings, C.W., Doetkott, C., Johnson, T.J., Fakhr, M.K., Nolan, L.K., 2005a. Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis. Microbiology (Reading, Engl.) 151, 2097–2110. Rodriguez-Siek, K.E., Giddings, C.W., Doetkott, C., Johnson, T.J., Nolan, L.K., 2005b. Characterizing the APEC pathotype. Vet. Res. 36, 241–256. Sannes, M.R., Kuskowski, M.A., Owens, K., Gajewski, A., Johnson, J.R., 2004. Virulence factor profiles and phylogenetic background of Escherichia coli isolates from veterans with bacteremia and uninfected control subjects. J. Infect. Dis. 190, 2121–2128.

56

M. Aslam et al. / International Journal of Food Microbiology 177 (2014) 49–56

Sheikh, A.A., Checkley, S., Avery, B., Chalmers, G., Bohaychuk, V., Boerlin, P., Reid-Smith, R., Aslam, M., 2012. Antimicrobial resistance and resistance genes in Escherichia coli isolated from retail meat purchased in Alberta, Canada. Foodborne Pathog. Dis. 9, 625–631. Smith, J.L., Fratamico, P.M., Gunther, N.W., 2007. Extraintestinal pathogenic Escherichia coli. Foodborne Pathog. Dis. 4, 134–163.

Tóth, I., Dobrindt, U., Koscsó, B., Kósa, A., Herpay, M., Nagy, B., 2012. Genetic and phylogenetic analysis of avian extraintestinal and intestinal Escherichia coli. Acta Microbiol. Immunol. Hung. 59, 393–409. Xia, X., Meng, J., Zhao, S., Bodeis-Jones, S., Gaines, S.A., Ayers, S.L., McDermott, P.F., 2011. Identification and antimicrobial resistance of extraintestinal pathogenic Escherichia coli from retail meats. J. Food Prot. 74, 38–44.