Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada

International Journal of Food Microbiology 156 (2012) 222–230

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Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada Mueen Aslam a,⁎, Moussa S. Diarra b, Sylvia Checkley c, Valerie Bohaychuk d, Luke Masson e a

Lacombe Research Centre, Lacombe, Agriculture and Agri-Food Canada, Alberta, Canada T4L 1 W1 Pacific Agri-Food Research Centre, Agriculture and Agri-Food Canada, Agassiz, British Columbia, Canada Department of Ecosystem and Public Health, University of Calgary, Calgary, Alberta, Canada d Agri-Food Laboratories Branch, Alberta Agriculture and Rural Development, Edmonton, Alberta, Canada e National Research Council of Canada, Biotechnology Research Institute, Montreal, Quebec, Canada b c

a r t i c l e

i n f o

Article history: Received 6 September 2011 Received in revised form 13 March 2012 Accepted 22 March 2012 Available online 29 March 2012 Keywords: Antimicrobial resistance Resistance genes Virulence genes Enterococcus spp. retail meats

a b s t r a c t The objective of this study was to characterize antimicrobial resistance (AMR) and virulence genotypes of Enterococcus spp. particularly Enterococcus faecalis isolated from retail meats purchased (2007–2008) in Alberta, Canada. Unconditional statistical associations between AMR pheno- and genotypes and virulence genotypes were determined. A total of 532 enterococci comprising one isolate from each positive sample were analyzed for antimicrobial susceptibility. A customized enterococcal microarray was used for species identification and the detection of AMR and virulence genes. E. faecalis was found in > 94% of poultry samples and in about 73% of beef and 86% of pork samples. Enterococcus faecium was not found in turkey meat and its prevalence was 2% in beef and pork and 4% in chicken samples. None of the enterococci isolates were resistant to the clinically important drugs ciprofloxacin, daptomycin, linezolid and vancomycin. Multiresistance (≥ 3 antimicrobials) was more common in E. faecalis (91%) isolated from chicken and turkey (91%) than those isolated from beef (14%) or pork (45%). Resistance to aminoglycosides was also noted at varying degrees. The most common resistance genes found in E. faecalis were aminoglycosides (aac, aphA3, aadE, sat4, aadA), macrolides (ermB, ermA), tetracyclines (tetM, tetL, tetO), streptogramin (vatE), bacitracin (bcrR) and lincosamide (linB). Virulence genes expressing aggregation substances (agg) and cytolysin (cylA, cylB, cylL, cylM) were found more frequently in poultry E. faecalis and were unconditionally associated with tetM, linB and bcrR resistance genes. Other virulence genes coding for adhesion (ace, efaAfs), gelatinase (gelE) were also found in the majority of E. faecalis. Significant statistical associations were found between resistance and virulence genotypes, suggesting their possible physical link on a common genetic element. This study underscores the importance of E. faecalis as a reservoir of resistance and virulence genes and their potential transfer to humans through consumption of contaminated undercooked meat. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Enterococci are known to be intrinsically resistant to several antimicrobials and have the ability to acquire antibiotic resistance via genetic mobile elements such as plasmids, transposons, or through chromosomal exchange or mutations (Hegstad et al., 2010). This ability of enterococci poses a major challenge for effective antimicrobial therapy in humans. Enterococci have been recognized as reservoir of antimicrobial resistance genes having the potential to transfer these genes to other bacteria in the food environment (Aarestrup et al., 2000; Hummel et al., 2007; Jackson et al., 2007). ⁎ Corresponding author at: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1 W1. Tel.: + 1 403 782 8106; fax: + 1 403 782 6120. E-mail address: [email protected] (M. Aslam).

The food chain has been recognized as a source of antimicrobial resistant enterococci. Studies have assessed antimicrobial resistance (AMR) in enterococci (Aslam et al., 2010; Rizzotti et al., 2005; Šustáková et al., 2004) out of concern that such resistant bacteria can transfer to humans via contaminated meat. Although genetic mechanisms of resistance in enterococci have been suggested (Coque, 2008), little information is available about the prevalence of resistance and the genes responsible for resistance in enterococci particularly Enterococcus faecalis isolated from retail meats (McGowan-Spicer et al., 2008). A number of virulence factors responsible for infections in humans have been described for Enterococcus spp. (Hancock and Gilmore, 2006). These virulence factors include aggregation and adhesions substances, cytolysin, gelatinase, extracellular surface proteins and pheromones (Ogier and Serror, 2008). A few studies have reported the presence of virulence factors in enterococci recovered mostly from

0168-1605/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2012.03.026

M. Aslam et al. / International Journal of Food Microbiology 156 (2012) 222–230

dairy foods and animals (Diarra et al., 2010; Mannu et al., 2003; Valenzuela et al., 2009). Information about the resistance and virulence genes found in animal derived-enterococci is helpful to understand and control AMR but it may not represent potential risks associated with their presence on retail meat. Various environmental and processing factors (low temperature, disinfectant use, and handling by workers) may affect the dynamics of resistant enterococci on retail meat. The harboring of virulence and resistance genes by E. faecalis may pose a human health risks if these bacteria are ingested through undercooked meat (Eaton and Gasson, 2001). To date no information is available about the diversity and distribution of resistance and virulence genes in enterococci especially in E. faecalis isolated from retail meats. Genotyping can show higher diversity than phenotypes and consequently allows more accurate comparisons between resistant bacterial populations. To better understand the epidemiology of antimicrobial resistance in enterococci, statistical correlation between AMR phenotypes, resistance and virulence genotypes with relation to meat types need to be explored. The statistical associations that may exist between resistance and virulence genotypes could suggest physical linkages of genes on same genetic elements (Diarra et al., 2010). Previously a number of studies have established such correlation for E. coli isolated from animals and meat (Aslam et al., 2009; Boerlin et al., 2005). Therefore the objectives of this study were to estimate frequency of resistance in Enterococcus spp. especially E. faecalis isolated from retail meats, to analyze resistance and virulence gene contents and to describe unconditional statistical associations between AMR phenotypes, resistance and virulence genotypes. 2. Materials and methods 2.1. Sampling procedure The sampling strategy established by Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) was used (CIPARS, 2007). This sampling plan involved continuous weekly sampling over a period of one year, from 19 census divisions in Alberta representing 40 sampling days (May 2007–April 2008). During each visit, four stores were randomly selected from a census division which included three chain stores and one independent/ butcher store. From each store, one retail-level sample each of fresh ground beef (regular, medium, or lean), skin-on chicken (leg, wing, breast or thigh), turkey (drumstick, wing or ground turkey), and pork (loins or chops) were purchased. A total of 16–18 samples were collected during each visit comprising four samples of each meat type per store. A total of 564 samples including raw chicken (n = 206), turkey (n = 91) and beef (n = 134) and pork (n = 133) were purchased from retail stores across Alberta. Although turkey samples were not available from some retail stores, a sufficient number were purchased to provide informative data. Samples were collected aseptically in plastic bags and transported on ice within a cooler to the laboratory for primary isolation of enterococci within 36 h.

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Lab Blender). A 50 mL of aliquot of the BPW from each sample was aseptically transferred to 50 mL of double strength Enterococcosel broth (Becton Dickinson). The broth was incubated at 35 °C for 24 h and examined for growth and blackening. If growth and blackening was observed, one loopful was streaked on an Enterococcosel agar (Becton Dickinson) plate for colony isolation and incubated aerobically at 35 °C for 48 h. The Enterococcosel plates were examined and colonies surrounded by blacken agar were purified and characterized. The identity of typical colonies was confirmed by testing for catalase and tetrazolium reduction. Tetrazolium reduction was determined by streaking the isolate onto Slanetz and Bartley agar (Oxoid, Basingstoke, England) and incubating at 35 °C for 48 h. Growth in nutrient broth containing 6.5% NaCl, and at pH 9.0 with incubation at 35 °C for 72 h was also observed. Positive colonies were further characterized based on hemolysis on sheep blood agar and fermentation of L-arabinose, mannitol and α-methyl-D-glucoside. Enterococci were identified to the genus level by PCR using a primer set targeting the tuf gene as described previously (Jackson et al., 2004). Three isolates from each positive sample were frozen in 1 mL sheep blood for further analysis. One isolate was randomly selected from each positive sample for species identification, AMR testing, and determination of resistance and virulence genes. 2.3. Antimicrobial susceptibility testing The antimicrobial susceptibility of enterococci was tested with a panel of 16 antimicrobials (antimicrobial abbreviations and breakpoints are shown in parentheses): ciprofloxacin (CIP; ≥4 μg/mL), daptomycin (DAP; ≥4 μg/mL), linezolid (LZD; ≥8 μg/mL), quinupristin/ dalfopristin (QDA; ≥4 μg/mL) vancomycin (VAN; ≥32 μg/mL) tigecycline (TGC; ≥1 μg/mL), erythromycin (ERY; ≥8 μg/mL), gentamicin (GEN; >500 μg/mL), kanamycin (KAN; ≥1024 μg/mL), lincomycin (LIN; ≥8 μg/mL), penicillin (PEN; ≥16 μg/mL), streptomycin (STR; >1000 μg/mL), tylosin (TYL; ≥32 μg/mL), nitrofurantoin (NIT; ≥128 μg/mL), chloramphenicol (CHL; ≥32 μg/mL) and tetracycline (TET; ≥16 μg/mL). These antimicrobials have been grouped into three categories based on their importance in human medicine (Veterinary Drugs Directorate, 2005). The category I antimicrobials (very high importance in human medicine) are CIP, DAP, LZD, QDA, TGC, and VAN; category II antimicrobials (medium importance) are ERY, GEN, KAN, LIN, PEN, STR, and TYL, and category III antimicrobials (low importance) are CHL, NIT and TET. The minimum inhibitory concentration (MIC) values were determined using an automated broth microdilution method (Sensititre®; Trek Diagnostic Systems Inc., Westlake, OH) with CMV2AGPF plate. The results were interpreted according to the guidelines set by the Clinical and Laboratory Standards Institute (CLSI, 2010) and where no CLSI interpretive criteria were available; the breakpoints described by CIPARS (2007) were used. Staphylococcus aureus ATCC 29213, E. faecalis ATCC 51299 and E. faecalis ATCC 29212 obtained from American Type Culture Collection were used as quality control organisms.

2.2. Microbiological procedures

2.4. Microarray assay for detection of species, resistance and virulence genes

One whole portion of pork, chicken or turkey (e.g. pork chop or loin, chicken leg or breast, turkey drum stick or wing) or 25 g of ground beef or turkey was placed in the stomacher bag and a total volume of 225 mL of Buffered Peptone Water (BPW; Becton Dickinson and Co. Mississauga, ON) was added. Stomacher bags containing portions of pork chop/loin or poultry leg/ breast were shaken for 15 s, allowed to stand for 15 min followed by shaking for another 15 s. The BPW containing ground beef or turkey was stomached for 30 s using a stomacher (Seward® Stomacher® 400 C

Bacterial DNA was isolated as described previously (Diarra et al., 2010). A custom microarray that provided information on the taxonomy, virulence and antibiotic resistance gene content of individual enterococcal isolates was used. The Enteroarray carrying 70 taxonomic probes (50-mer oligonucleotides), 17 virulence gene probes (70-mer oligonucleotides) and 171 antibiotic resistance gene probes (70-mer oligonucleotides) for a total of 258 probes was constructed and validated in previous studies (Diarra et al., 2010; Champagne et al., 2011).

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Taxonomic probes were designed against four genes (ddl, pheS, atpA and recA) to identify eighteen recognized Enterococcus species: E. asini, E. avium, E. casseliflavus, E. cecorum, E. columbae, E. dispar, E. durans, E. faecalis, E.faecium, E. gallinarum, E. hermanniensis, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E. saccharolyticus and E. sulfureus. The universal eubacterial probe EUB338 was used as a positive control. Description of the antibiotic resistance genes, hybridization, washing, scanning, image processing, scoring, and data analysis were done as previously described (Hamelin et al., 2007; Garneau et al., 2010; Champagne et al., 2011). 2.5. Statistical analysis Isolates were classified as either susceptible or resistant. Isolates of intermediate resistance were considered as susceptible for this study. A resistant phenotype was described as an isolate with MIC value greater than the breakpoint value of a tested antimicrobial(s). Descriptive analyses and unconditional univariate logistic regression were used to determine prevalence and examine associations between AMR phenotypes and genotypes, virulence genotypes and meat types using commercially available software (SPSS 17.0 for Windows; SPSS Inc., Chicago, IL). Association test of Cochran–Mantel– Haenszel and logistic analysis (proportional odds model) was used to determine a correlation between virulence and resistance genes by using the FREQ procedure of SAS Institute (2000). To examine unconditional associations between resistance and virulence genes, outcome and predictor variables included genes with prevalence over 5%. Associations that were statistically significant were reported as odd ratios (OR) with 95% confidence intervals (CI). An OR of >1 indicated a positive association between the outcome and predictor variable, while an OR of b1 indicated a negative association between the outcome and predictor variable. The P-value of 0.05 was used to declare significance. 3. Results

and 8% of pork; however, the prevalence was low in chicken (2% of isolates) and was not found in turkey. Enterococcus faecium was also not found in turkey meat and its prevalence was low in other three meat types. 3.2. Prevalence of AMR in Enterococcus spp. Only 3.4% of enterococci were susceptible to all tested antimicrobials. Resistance to CIP, DAP, LZD, and VAN antimicrobials, very important in human medicine, was not found in any enterococci (Table 1). Resistance to TGC, also a clinically important drug, was found in 5% of turkey-derived E. faecalis and only in 3% of E. faecalis from pork and chicken. About 4% of E. hirae from beef were resistant to TGC. Since E. faecalis is intrinsically resistant to LIN and QDA therefore resistance data for these two antimicrobials was not reported. Resistance to QDA was found in 78% of chicken and 50% of pork-derived E. faecium. Resistance to ERY was found in 47% of chicken-derived and 28% of turkey-derived E. faecalis. Only 2% E. faecalis from beef and 8% from pork showed ERY resistance. Resistance to KAN and TYL, drugs important in human medicine, was also higher in E. faecalis isolated from chicken (KAN 20%; TYL 49%) and turkey (KAN 15%; TYL 29%). Only 11% chicken-derived E. faecium showed resistance to KAN. Multidrug resistance (≥3 antimicrobials) was found in about 91% of E. faecalis from each of chicken and turkey meats; however, 14% of beef-derived and 45% pork-derived E. faecalis were multidrug resistant. 3.3. Unconditional statistical associations between AMR phenotypes Table 2 shows unconditional statistical associations observed between resistance phenotypes of highest prevalence (ERY, KAN, LIN, STR, QDA, TET, TYL). E. faecalis isolates with resistance to TET were 9.7 times more likely to also show resistance to TYL (P b 0.001) than isolates not showing resistance to TET. Other significant associations were noted for AMR phenotypes with resistances for ERY, KAN, LIN, and STR.

3.1. Prevalence of Enterococcus spp. 3.4. Distribution of resistance genes in Enterococcus spp. The most common Enterococcus spp. identified by the Enteroarray was E. faecalis which was found in >94% of chicken and turkey samples and in about 73% of beef and 86% of pork samples (data not shown). Enterococcus hirae was found in about 21% of beef samples

A total of 171 resistance genes and their variants were analyzed using the Enteroarray with only 12 genes being found having prevalence of ≥5% with 41 genes being detected at a prevalence of

Table 1 The prevalence (%) of antimicrobial resistance in E faecalis, E. faecium and E. hirae isolates from retail meats. Antimicrobial

CIP DAP LZD QDA TGC VAN ERY GEN KAN LIN PEN STR TYL CHL NIT TET

Turkey (n = 91)

Pork (n = 107)

E. Faecalis

Beef (n = 129) E. faecium

E. Hirae

E. faecalis

Chicken (n = 205) E. faecium

E. Hirae

E. faecalis

E. faecalis

E. faecium

E. Hirae

0 0 0 83 2 0 2 0 1 96 0 3 3 0 0 14

0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0

0 0 0 11 4 0 11 0 0 89 0 0 11 0 4 37

0 0 0 98 3 0 47 2 20 99 0 37 49 0 0 88

0 0 0 78 0 0 22 0 11 100 22 11 22 0 0 89

0 0 0 100 0 0 33 0 33 100 0 100 33 0 0 100

0 0 0 97 5 0 28 10 15 97 0 11 29 0 0 90

0 0 0 82 3 0 8 1 2 89 0 1 9 2 1 43

0 0 0 50 0 0 0 0 0 100 0 50 0 0 0 50

0 0 0 50 0 0 0 0 0 88 0 0 0 0 0 25

Abbreviations are listed in alphabetical order. CIP, ciprofloxacin; CHL, chloramphenicol; DAP, daptomycin; ERY, erythromycin; GEN, gentamicin; KAN, kanamycin; LIN, lincomycin; LZD, linezolid; NIT, nitrofurantoin; PEN, penicillin; QDA, quinupristin/dalfopristin; STR, streptomycin; TET, tetracycline TGC, tigecycline; TYL, tylosin; VAN, vancomycin. Number of E. faecalis isolates from beef, chicken, turkey and pork were 94, 192, 87 and 92, respectively. Number of E. faecium isolates from beef, chicken and pork were 3, 9 and 2, respectively. Number of E. hirae isolates from beef, chicken and pork were 27, 3 and 8, respectively.

M. Aslam et al. / International Journal of Food Microbiology 156 (2012) 222–230 Table 2 Unconditional statistical associationsa observed between AMR phenotypes found in E. faecalis recovered from retail meats. Antimicrobials ERY

KAN

LIN STR QDA TET

KAN STR QDA TET STR TET TYL QDA TET TET TYL TET TYL TYL

Odds ratio

95% CI

P-value

22.2 29.0 6.21 10.0 25.0 20.3 20.4 82.5 9.3 8.2 20.9 7.8 6.7 9.7

10.8–45.7 16.0–52.7 2.5–15.7 5.2–19.1 12.9–48.2 4.9–84.4 10.0–41.9 19.0–358.7 3.1–27.5 3.9–17.3 2.9–152.0 4.5–13.5 2.7–16.7 5.2–18.2

b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001

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(Fig. 1-A). It was found in 89% of chicken, 84% of turkey, 27% of beef and 48% of pork E. faecalis. About 11% and 78% of E. faecium isolated from chicken were positive for sat4 and vatE genes, respectively (Fig. 1-B). These genes were not found in E. faecium isolated from beef or pork. As expected, the msrC gene encoding some of the macrolide resistance was found in all E. faecium isolates from beef (3 isolates; 100%) and chicken (9 isolates; 100%) but only in one of two pork isolates (50%). Multiple resistance genes (>3) were more frequently isolated from poultry-derived E. faecalis (69% of chicken, 50% of turkey isolates; Fig. 2). About 15% of chicken-derived E. faecalis carried >10 resistance genes. On the other hand 19% of beef-derived and 14% of pork-derived isolates carried multiple (>3) resistance genes. 3.5. Unconditional statistical associations between resistance genes and Enterococcus spp. and meat types

a Only statistically significant associations are reported (P-value of b 0.05) and were corrected for the effect of meat types. Associations are not reported for E. faecium and E. hirae due to small number of isolates. QDA, quinupristin/dalfopristin; KAN, kanamycin; LIN, lincomycin; STR, streptomycin; TYL, tylosin; TET, tetracycline.

b5%. About 120 resistance genes were not detected in any isolate. Overall about 22% of enterococci did not carry any tested resistance gene and the majority of those isolates (55%) were from beef. The tetM was the most common resistance gene found in E. faecalis

Unconditional associations observed between resistance genes are shown in Table 3. Isolates carrying aac(6) gene were more likely to be positive for ermA, ermB, tetL tetM, sat4, aphA3 and linB genes than isolates not positive for this gene. Isolates positive for the sat4 gene were more likely to be positive for the tetL, bcrR and aminoglycoside genes (aphA3, aadE) than isolates not carrying this gene. Table 4 shows unconditional statistical associations observed between resistance genes of highest prevalence (acc(6), ermB, linB, sat4, tetL, tetM, bcrR, ermA, aadE, aphA3) and three Enterococcus spp. of

A 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Aminoglycosides

Tetracycline

bcrR

sat4

linB

ermB

ermA

tetO

tetM

tetL

aadE

aadA

aphA3

aac/aphD

aac

Beef Chicken Turkey Pork

MLSB

B 100%

Beef Chicken Pork

90% 80% 70% 60% 50% 40% 30% 20% 10%

Aminoglycosides

Tetracycline

bcrR

msrC

SatGvatE

sat4

linB

ermB

ermA

tetM

tetL

aphA3

aadA

aac

0%

MLSB

Fig. 1. Distribution of resistance genes in Enterococcus faecalis (A) and E. faecium (B) isolated from retail meats. Vertical bar represents percentage of isolates; horizontal bar represents resistance genes.

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M. Aslam et al. / International Journal of Food Microbiology 156 (2012) 222–230

45%

Beef

40%

Chicken

35%

Turkey

30%

Pork

25% 20% 15% 10% 5% 0% 1

2

3

4

5

6

7

8

9

10

11

Number of resistance genes Fig. 2. Distribution of multiple resistance genes in E. faecalis isolated from retail meats.

highest prevalence (E. faecalis, E. faecium and E. hirae). It should be noted here that the prevalence of E. faecium and E. hirae was low at 2.6% and 7.1% respectively. The odds of finding aac(6) and linB genes in E. faecium and E. faecalis were significantly higher than in E. hirae.

Table 3 Unconditional statistical associationsa between resistance genes found in E. faecalis recovered from retail meats. Outcome resistance gene

Predictor gene

aac(6)

ermB sat tetL tetM bcrR ermA aadE aphA3 linB sat4 tetL tetM bcrR ermA aadE aphA3 tetL bcrR ermA tetL bcrR aadE aphA3 tetM bcrR ermA aadE aphA3 bcrR ermA aadE aphA3 ermA aadE aphA3 aadE aphA3 aphA3

ermB

linB

sat4

tetL

tetM

bcrR

ermA aadE a

Odds ratio 16.7 159.6 10.0 7.9 6.3 19.2 229.3 110.1 4.1 175.9 16.6 7.2 7.5 11,413.8 53.4 42.3 3.3 8.8 3.9 27.2 3.5 565.2 1494.6 7.4 3.7 16.8 25.6 28.7 16.6 7.5 41.2 34.6 7.5 4.6 3.7 222.3 96.6 696.4

95% CI

P-value

10.0–27.8 37.8–673.1 5.9–16.9 4.0–15.6 3.7–10.6 11.3–32.5 54.4–966.0 33.3–364.1 2.2–7.7 24.0–1289.2 10.1–27.2 4.2–12.3 4.7–11.8 1322.5–98,502.9 18.9–150.9 14.9–120.1 1.7–6.3 3.4–22.8 2.1–7.5 8.4–88.8 1.8–6.8 155.5–2054.7 346.3–6451.0 4.7–11.5 2.6–5.3 10.3–27.4 9.1–71.7 8.8–93.5 10.3–26.7 4.4–13.0 5.7–299.7 4.7–252.4 4.8–11.8 2.4–8.8 1.9–7.2 30.4–1624.4 23.2–402.8 187.9–2581.2

b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 0.0004 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 b0.0009 0.0002 0.0004 b0.0009 b0.0009 0.0001 b0.0009 b0.0009 b0.0009

Only statistically significant associations are reported (P-value of b 0.05) and were corrected for the effect of meat types. Associations were not calculated for E. faecium due to small number of isolates.

Multiple statistical associations were found between resistance genes detected in enterococci and the meat types (beef-derived enterococci were used as the reference category). Isolates positive for any of the resistance genes used in this analysis were significantly more likely to be chicken-derived E. faecalis than beef-derived (Table 4). There were no resistance genes that were more likely to be significantly associated with pork-derived E. faecalis than those from beef. 3.6. Distribution of virulence genes and unconditional statistical association with resistance genes A higher percentage of E. faecalis isolated from all four meat types were positive for virulence genes (Fig. 3). The majority of E. faecalis isolated from all four meat types carried pheromone genes. The cytolysin expressing genes (cylA, cylB, cylL, cylM) were more frequently found in E. faecalis from turkey and chicken meats. Similarly the gene expressing aggregation substances (agg) was present more frequently (about 50% of isolates) in poultry E. faecalis. All E. faecium isolates from beef, chicken and pork carried the efmAfm virulence gene only; no E. faecium was isolated from turkey. The majority of E. faecalis isolates from all four meat types carried ≥9 virulence genes whereas the majority of E. faecium and E. hirae isolates carried only a single virulence gene (data not shown). One E. hirae (12%) isolated from pork carried eight virulence genes. Multiple statistical associations were observed between resistance and virulence genotypes of E. faecalis (Table 5). The genes coding resistances for aminoglycosides (aphA3, aadE, sat4), tetracycline (tetL, tetM), erythromycin (ermA, ermB), lincomycin (linB), and bacitracin (bcrR) were significantly associated with the majority of virulence genes. For example E. faecalis isolates carrying genes for cytolysin (cylA, cylB, cylL, cylM) and aggregation substance (agg) were more likely to be positive for linB (lincomycin), tetM (tetracycline) and bcrR (bacitracin) genes than isolates not carrying these genes (Table 5). 4. Discussion E. faecalis was the most common species isolated from all meat types followed by E. hirae and E. faecium. E. hirae was more common in beef samples than chicken or pork and was not found in turkey meat. Our study was consistent with a 2009 CIPARS study suggesting that E. faecalis was commonly found in retail meat samples collected from other provinces. However, NARMS (2008) data suggested that E. faecium was more commonly isolated from retail meats. Another study from the USA has also suggested that E. faecium was the most common Enterococcus spp. recovered from retail meats (Hayes et al.,

M. Aslam et al. / International Journal of Food Microbiology 156 (2012) 222–230 Table 4 Unconditional statistical associationa observed between resistance genes and Enterococcus spp. and meat types. Resistance gene

Enterococcus spp.

aac(6)

Overall E. faecium E. hirae Overall E. faecium E. hirae Overall E. faecium E. hirae Overall E. faecium E. hirae Overall E. faecium E. hirae

linB

tetL

tetM

bcrR

aac(6)

ermB

linB

sat4

tetL

tetM

bcrR

ermA

aadE

aphA3

Meat type Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey Overall Chicken Pork Turkey

Odds ratio

95% CI

9.2 0.2

2.8–30.1 0.05–.9

33.8 0.8

10.0–113.9 0.2–3.3

0.7 0.2

0.2–2.1 0.1–0.5

0.9 0.2

0.3–2.7 0.1–0.4

2.1 0.05

0.7–6.8 0.01–0.2

9.3 0.9 1.8

4.5–19.3 0.3–2.6 0.7–4.6

8.8 1.3 3.4

4.7–16.7 0.6–3.0 1.6–7.1

21.9 6.3 12.3

3.0–163.0 0.7–54.6 1.5–100.5

9.9 1.2 2.4

3.0–32.7 0.2–6.1 0.6–2.3

8.2 1.2 4.9

4.8–13.9 0.6–2.3 2.7–9.1

20.3 2.0 12.6

11.4–36.0 1.2–3.5 6.5–24.5

37.0 1.0 14.8

19.6–69.9 0.5–2.1 7.5–29.2

7.0 1.1 2.8

3.9–12.6 0.5–2.3 1.4–5.7

7.6 0.5 1.4

2.3–15.7 0.09–2.5 0.4–5.1

6.0 0.5 1.4

2.3–15.7 0.09–2.5 0.4–5.1

P-value b 0.001 b 0.001 0.031 b 0.001 b 0.001 0.703 0.002 0.506 0.001 b 0.001 0.806 b 0.001 b 0.001 0.212 b 0.001

b 0.001 b 0.001 0.895 0.199 b 0.001 b 0.001 0.477 0.001 0.002 0.003 0.096 0.019 b 0.001 b 0.001 0.817 0.230 b 0.001 b 0.001 0.595 b 0.001 b 0.001 b 0.001 0.011 b 0.001 b 0.001 b 0.001 0.983 b 0.001 b 0.001 b 0.001 0.876 0.003 b 0.001 b 0.001 0.376 0.572 b 0.001 b 0.001 0.376 0.572

a Enterococcus faecalis was used as a reference category for unconditional association between Enterococcus spp. Beef was used as a reference category for unconditional associations between meat types.

2003). It is not clear why the prevalence of Enterococcus spp. in Canada is different from the United States; nevertheless studies have found a higher prevalence of E. faecalis in chicken production environments and in the processed meat products (Šustáková et al., 2004; Hayes et al., 2004). In the present study all enterococci were susceptible to CIP, VAN, LZD and DAP, antimicrobials of great importance in human medicine. Fifteen E. faecalis isolates were resistant to the tigecycline, an antibiotic recommended for use in humans in the United States (Grossi, 2009; Gales et al., 2008). Tigecycline is a glycylcycline

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antibiotic with effectiveness against multidrug resistant Enterococcus spp. Consequently, the presence of such isolates in retail meat represents potential food safety and public health risks. Although a few E. faecalis isolated from all four meats showed resistance to CHL, NIT, and PEN; resistance to ERY, GEN, KAN and STR was found in more chicken and turkey isolates than those from beef or pork. Resistance to multiple (≥3) antimicrobials was also more common in isolates from chicken and turkey. The higher resistance found in E. faecalis from poultry may reflect higher antimicrobial usage during poultry production. Previous studies have also suggested a higher prevalence of enterococci in poultry meats showing resistance to multiple antimicrobials (Hayes et al., 2003; CIPARS, 2009; NARMS, 2008). Furthermore the inadequate hygienic procedures applied at the commercial poultry processing plants may not eliminate resistant enterococci from the retail meat. Further studies are needed to determine the possible routes of chicken meat contamination with resistant enterococci continuum from poultry production through slaughter and processing. Because of their relative abundance and their resistance to environmental factors, enterococci have been proposed as indicator bacteria for antimicrobial resistance, as well as indicators for the hygienic quality of food and water (Pierson et al., 2007). However, factors affecting the numbers of enterococci in the intestinal tract and feces are not well established. Commensal Enterococcus spp. in livestock and poultry could contaminate the food chain during processing (Aslam et al., 2010; Diarra et al., 2007). Workers carrying enterococci in their gastrointestinal tract and on their hands could also be the important in the dissemination of these bacteria if hygienic precautions are not taken. E. faecium is an important organism as its frequent occurrence in nosocomial infections presents a challenge in clinical settings. In addition E. faecium is becoming increasingly resistant to clinically important antimicrobials such as QDA and VAN. In the present study, although a small number of enterococci were E. faecium, resistance to clinically important antimicrobials was found. The most common resistance was to QDA, TET and LIN in chicken- and pork-derived E. faecium, whereas all E. faecium from beef were resistant to LIN only. Resistance to other clinically important antimicrobials VAN and CIP was not found in E. faecium isolates. These results are in agreement with AMR data from other provinces (CIPARS, 2007, 2009) and studies from the United States (NARMS, 2008). A higher prevalence of vancomycin resistant enterococci was reported in the animal production environment and on raw meats in Europe (Del Grosso et al., 2000; Borgen et al., 2001). Avoparcin, a glycopeptide was approved for use in animal production in Europe until 1995 and that correlated with a higher prevalence of vancomycin resistant enterococci in meat or animals. However, following a ban on its use in animal production, there has been a significant decrease in the prevalence of vancomycin resistant E. faecium in chicken (Hawkey, 2008). A recent report from Denmark did not find vancomycin resistance in chicken, beef or pork (DANMAP, 2009). In our study neither vancomycin resistance nor genes coding for this resistance were found. Studies from the United States and Canada reported similar findings (Hayes et al., 2003; NARMS, 2008; CIPARS, 2009). Avoparcin was never approved for use in animal production in the United States or Canada which may explain the absence of vancomycin resistance in our E. faecium isolates. Quinupristin-dalfopristin (streptogramin) is bacteriostatic against E. faecium but ineffective (due to intrinsic resistance) against E. faecalis. In the United States, this compound is approved for the treatment of some serious vancomycin resistant enterococci infections (Johnson and Livermore, 1999). In the present study, QDA resistant E. faecium were detected specifically in chicken meat. The relationship of this resistance and the use of virginiamycin (another streptogramin) in poultry production need to be established.

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Fig. 3. Distribution of virulence genes in Enterococcus faecalis isolated from retail meats. Vertical bar represents percentage of isolates; horizontal bar represents virulence genes.

Although numerous mechanisms of antibiotic resistance have been described in enterococci from variety of sources, little is known about the resistance genotype distribution of these bacteria in retail meats. In our study aadE, aphA3, sat4, ermB and tetL genes found in E. faecalis isolates were significantly associated, suggesting their presence on same genetic element. The aadE-aphA3-sat4 gene cluster located on transposon Tn5405 has been found in E. faecium (Werner et al., 2001). In our study a significant correlation between tet genes and the glycopeptide bacitracin (bcrR), macrolide (ermA) and aminoglycoside (aadE and aphA3) resistance genes was observed. It is possible that these genes are part of larger composite genetic element however; further research is needed to investigate this possibility. Tetracycline resistance was observed in E. faecalis isolated from all meat types and possibly mediated by the tetL and tetM genes because significant statistical association was found between tetracycline resistance and tetM gene (data not shown). A possible genetic link

between tetracycline resistance genes and other genes such as ermB has been also suggested (Cauwerts et al., 2007). The use of bacitracin or tetracycline as a growth promoter and/or therapeutic agent may therefore co-select for resistance to macrolide and aminoglycoside (Matos, 2009), which may be important as alternative therapy for enterococcal infections in humans. This study for the first time showed that clinically important resistance genes were more likely to be found in enterococci from retail poultry meats than retail pork or beef. This suggests that poultry meat especially retail chicken can play a role in the transfer of resistance genes to consumers through handling of meat or after ingestion of improperly cooked meat contaminated with enterococci. Further studies are needed to assess the frequency of consumer's exposure to enterococci carrying resistance genes. Multiple virulence factors found in enterococci can play a role in the pathogenesis of human disease. In this study the majority of

Table 5 Unconditional statistical associationsa observed between resistance and virulence genes. Outcome gene

Predictor gene

Odd ratios

95% CI

P-value

Outcome gene

Predictor gene

Odd ratios

95% CI

P-value

ace agrBfs efaAfs gelE agg cylA cylB cylM cylL agg cylA cylB cylL cylM efmAfm

tetM tetM tetM tetM tetM tetM tetM tetM tetM linB linB linB linB linB linB

2.5 4.6 3.8 4.4 7.9 9.4 11.5 12.4 10.4 3.4 4.6 3.8 4.1 4.3 71.4

1.5–4.1 2.9–7.4 2.2–6.8 2.8–7.0 4.6–13.5 4.8–18.5 5.7–23.4 5.9–26.1 5.1–21.1 1.8–6.5 2.4–8.7 2.0–7.3 2.2–7.7 2.2–8.0 15.0–339.2

0.0002 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001

ace agrBfs agg cylA cylB cylL cylM efaAfs efmAfm gelE

bcrR bcrR bcrR bcrR bcrR bcrR bcrR bcrR bcrR bcrR

3.9 5.9 13.3 24.4 23.3 24.7 27.5 4.8 5.0 5.2

2.2–7.0 3.4–10.0 8.1–21.9 11.6–51.3 11.5–47.2 11.7–52.1 12.5–60.7 2.5–9.3 1.1–23.3 3.1–8.9

b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 b.0001 0.021 b.0001

a Only statistical association observed between tetM, linB and bcrR resistance genes and five virulence factors (cytolysin, cylA, cylB, cylL, cylM; aggregation substance, agg; gelatinase, gelE; adhesion, ace) are reported. Statistical associations were also observed between resistance genes aphA3, sat4, aac(6), aadE, ermA, ermB, and tetL and above virulence factors expressing genes.

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E. faecalis isolates from all meats were positive for genes (ace, efaAfs) expressing adhesion factors and genes expressing pheromones (cpd1, cob, cCF10, cAM, cad1). But genes coding for cytolysin (cylA, cylB, cylL, cylM) and aggregating substance (agg) were more prevalent in poultry E. faecalis. The concurrent expression of cytolysin and aggregation virulence factors results in an increased pathogenicity of E. faecalis isolates (Gilmore et al., 2002) suggesting that poultryderived isolates may have more virulence potential than beef or pork E. faecalis. The majority of virulence genes found in E. faecalis were associated with the presence of clinically important resistance genes coding for aminoglycosides, lincosamides and macrolides antimicrobials. A potential linkage of virulence and resistance genes on the same genetic element raises concerns because of the possibility of coselection of the virulence along with resistance genes under selective pressure after the use of antimicrobials in animal production. In conclusion, E. faecalis was the most common Enterococcus spp. found in all four meat types. Except for tigecycline, resistance to very important clinical drugs was not found in enterococci isolated from retail meats. Resistance was more prevalent in poultry-derived enterococci especially from chicken. Statistical association observed between resistance phenotypes suggests that use of one antimicrobial may result in the appearance of resistance to unrelated classes of antimicrobials. The occurrence of linked resistance genes suggests their presence on common genetic elements and raises concerns about horizontal gene transfer to other bacteria sharing the same ecological niche. It appears that poultry-derived isolates may have virulence potential due to the expression of both cytolysin and aggregation factors. The majority of E. faecalis isolates carried multiple virulence genes that were found to be associated with resistance genes. Like the observed resistance gene linkage, the implied co-location on the same genetic element of virulence and AMR genes raises concerns about their potential for co-transfer to other enterococci. Although a few studies have reported the presence of resistance and virulence genes in enterococci to our knowledge, this is the first report assessing enterococcal AMR and virulence genotypes and their relevance to retail meats. Acknowledgments This study was supported by the Alberta Livestock and Meat Agency and Matching Investment Initiative funds from Agriculture and Agri-Food Canada. The technical support of Cara Service from Lacombe Research Centre, Lacombe and Heidi Rempel from Pacific Agri-Food Research Centre, Agassiz is greatly acknowledged. The authors would like to extend their thanks to Cheryl Turner, Barbara Dakin, Catherine Taylor, Jovana Kovacevic, Kyla Kennedy, and Deana Rolheiser from the Agri-Food Laboratories Branch, Alberta Agriculture and Rural Development for their technical support for initial bacterial isolation and confirmation. The authors greatly acknowledge the clerical support of Loree Verquin from Lacombe Research Centre. References Aarestrup, F.M., Agerso, Y., Gerner-Smidt, P., Madsen, M., Jensen, L.B., 2000. Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers and pigs in Denmark. Diagnostic Microbiology and Infectious Disease 37, 127–137. Aslam, M., Diarra, M.S., Service C, Rempel, H., 2009. Antimicrobial resistance genes in Escherichia coli isolates recovered from a commercial beef processing plant. Journal of Food Protection 72, 1089–1093. Aslam, M., Diarra, M.S., Service, C., Rempel, H., 2010. Characterization of antimicrobial resistance in Enterococcus spp. recovered from a commercial beef processing plant. Foodborne Pathogens and Disease 7, 235–241. Boerlin, P., Travis, R., Gyles, C.L., Reid-Smith, R., Janecko, N., Lim, H., Nicholson, V., McEwen, S.C., Friendship, R., Archambault, M., 2005. Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario. Applied and Environmental Microbiology 71, 6753–6761.

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