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Characteristic and antimicrobial resistance in Escherichia coli from retail meats purchased in the Czech Republic
Food Control 47 (2015) 401e406
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Characteristic and antimicrobial resistance in Escherichia coli from retail meats purchased in the Czech Republic a, b, *, Ivana Kola b, Katerina Bogdanovicova a, Rena ta Karpískova a, b Alena Sko ckova ckova a b
Department of Milk Hygiene and Technology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic Veterinary Research Institute, Brno, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2014 Received in revised form 14 July 2014 Accepted 19 July 2014 Available online 28 July 2014
The aim of this study was to characterize and compare Escherichia coli found in retail meats (pork, poultry, beef, venison), focusing on the detection of resistant genes that could be spread through the food chain and also for the presence of selected virulence genes. The resistance to antimicrobial agents was determined by disk diffusion method and E-test. Polymerase chain reaction was used for the detection of selected genes encoding for virulence factors (eaeA, hly, stx1, stx2) and genes encoding resistance to tetracycline, b-lactams and quinolones. Compared to beef isolates, the isolates from poultry and pork displayed higher resistant rates and also possessed more resistance genes. All together, 25.9% of the isolates were positive for the presence of the gene blaTEM, and the presence of any tet gene was detected in 23.1% of the isolates. The production of extended-spectrum b-lactamases was confirmed in one isolate from poultry as well as the presence of a plasmid-mediated gene qnrA attributed to the quinolone and fluoroquinolone resistance. Based on these results, it can be surmised that poultry is the most riskassociated meat source in terms of antibiotic resistance. The stx2e gene was detected in one isolate from pork samples, but this subtype is commonly associated with edema disease in pigs and is seldom found in humans. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Poultry meat Beef Pork Escherichia coli Resistance genes
1. Introduction Meat represents a substantial portion of the diet of most people worldwide. The world average annual consumption of pork is approximately 15 kg per person followed by poultry (12.6 kg) and beef (9.6 kg). The annual consumption of meat in the Europe is even higher (pork 34.2 kg, poultry 21.9 kg and beef 16.1 kg) (FAO, 2014). Because of the high consumption level, the safety and quality of meat in retail markets is an important public health issue. Escherichia coli are widespread gut commensal microorganisms in humans and warm-blooded animals, and are also an important zoonotic agent, which can be found in both animal and human infections (Duffy, Lynch, & Cagney, 2008). In the microbiological analysis of water and foodstuffs, E. coli is used to assess their hygienic quality, and its presence can also indicate the presence of enteric pathogens (Altalhi & Hassan, 2009). Furthermore, the level of antibiotic resistance in E. coli is considered to be a good indicator
* Corresponding author. Department of Milk Hygiene and Technology, University ho 1-3, 612 42 Brno, Czech of Veterinary and Pharmaceutical Sciences Brno, Palacke Republic. Tel.: þ420 541 562 722. ). E-mail address: [email protected] (A. Sko ckova http://dx.doi.org/10.1016/j.foodcont.2014.07.034 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
of the selection pressure exerted by the use of antibiotics (Lei et al., 2010) and of the resistance problems to be expected in infectious ndez, Cancelo, Díaz-Vega, Capita, & Alonsodiseases (Alvarez-Fern a Calleja, 2013). Meat and meat products can be easily contaminated by E. coli during animal evisceration after slaughter, through contact with tainted water or during meat handling (Alvarez-Fern andez et al., 2013). Because of the intensive use of antimicrobial agents in animal production, bacteria originating from food animals frequently carry a resistance to a range of antimicrobial agents, including those commonly used in humans (Hammerum & Heuer, 2009). The transfer of resistant bacteria between animals and humans through food products has been documented and could pose a threat to public health (Thorsteinsdottir, Haraldsson, Fridriksdottir, Kristinsson, & Gunnarsson, 2010). Moreover, antimicrobialresistant bacteria may represent a reservoir of resistance genes transferable to pathogenic or commensal bacteria in the human ndez et al., 2013), and therefore digestive tract (Alvarez-Fern a compromise the effective treatment of bacterial infections. In 2006 the European Union banned all feed antibiotics in animal husbandry (Regulation (EC) No, 1831/2003). However, producers' expectations regarding performance enhancement and
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illness prevention have led to an increased use of therapeutically valuable agents (Martins da Costa, Oliveira, Ramos, & Bernardo, 2011). During the last decade, a number of events have stressed the need for an increased awareness of the public health aspects related to antimicrobial resistance in animal husbandry. For example, the emergence of b-lactamases responsible for the resistance to b-lactam antibiotics, including cephalosporins, shows that risks to human health include the possibility of the horizontal transfer of resistance genes (Kaesbohrer et al., 2012). Another antibiotic resistance mechanism that has recently been on the increase in enterobacteria, is a resistance to fluoroquinolons et al., 2011). determined by plasmid-mediated qnr genes (Dolejska Both of these antimicrobials, third and fourth-generation cephalosporins and fluoroquinolons, are on the W.H.O. list of criticallyimportant antimicrobials for human medicine (FAO/WHO/OIE, 2008). This study should provide important information about the risk of consumer exposure to resistant E. coli, to help evaluate and compare the safety of different retail meats. Samples of venison were used as a control group because we assume there is no antimicrobial selection pressure in game meat. Because the food supply is now due to the international trade global, with the local microflora from the country of origin being spread to other countries, the results of this study could be important, not only for the Czech Republic, but for other countries as well. The objective of this study was to determine the prevalence of antimicrobial-resistant E. coli in meat (pork, poultry, beef, venison) within the retail market of the Czech Republic and to characterize obtained isolates focusing on the detection of extended-spectrum b-lactamases production and the presence of tet and plasmidmediated qnr genes. The pathogenic potential of E. coli isolates from retail meat based on their virulence gene profiles was also determined. 2. Material and methods 2.1. Sample collection A total of 322 refrigerated, individually packed fresh meat samples were purchased from various supermarkets in the Czech Republic during the years of 2012 and 2013. Together samples from eight cities and twenty supermarkets were investigated. The samples collected during one sampling were always from different processing plants and the time period between samplings was a minimum one month. A majority of the samples were of Czech origin (n ¼ 296) but imported samples from other European countries (n ¼ 26) were also covered in the study. The meat samples differed in animal origin (pork n ¼ 110, poultry n ¼ 80, beef n ¼ 82, and venison ¼ 50). The samples of poultry included samples of chickens, hens and turkey, both with and without the skin. All samples were individually wrapped in PE bags to avoid cross-contamination, refrigerated, and immediately after being collected, they were transported to the laboratory for examination. 2.2. Isolation and identification of E. coli In the laboratory, swabs were taken from the surface of the meat using sterile sponges (3M™ Sponge-stick, USA) and were placed in plastic bags with 30 ml BPW (Buffered Peptone Water, Oxoid, UK), homogenized on a Stomacher device, and incubated at 37 C overnight. When samples of whole chickens were investigated, 25 g of meat and skin from the neck was homogenized with 225 ml BPW and also incubated at 37 C overnight. Homogenates were
then substantially streaked on TBX agar (Tryptone Bile X-glucuronide medium, Oxoid, UK) and again incubated at 44 C overnight. From each positive sample, three suspected E. coli isolates were involved in the study for confirmation and determination of resistance to antimicrobials, but only isolates with different resistance phenotype from one sample were included in the study and further characterized. The confirmation of the suspected isolates from TBX agar was determined from the detection of oxidase (OXItest, Pliva-Lachema, CZ) and the production of indole (COLItest, Pliva-Lachema, CZ). 2.3. Antimicrobial susceptibility testing The susceptibility to antibiotics commonly used in clinical treatments was tested by disk diffusion method according to the CLSI protocol (2012) on Mueller-Hinton agar (Oxoid Ltd, UK) using the following antimicrobials and concentrations: ampicillin e AMP (10 mg), amoxicillin/clavulanic acid e AMC (30 mg), cefotaxime e CTX (30 mg), chloramphenicol e CMP (30 mg), streptomycin e STR (10 mg), kanamycin e KAN (30 mg), gentamicin e GEN (10 mg), sulfamethoxazol/trimethoprim e SXT (25 mg), trimethoprim e TMP (5 mg), tetracycline e TET (30 mg), nalidixic acid e NAL (30 mg), ciprofloxacin e CIP (5 mg), and colistin e COL (10 mg). Antibiotic disks were obtained from Oxoid Ltd (UK). The E. coli strains were evaluated based on the size of the zones of inhibition and classified as susceptible, intermediate resistant, or resistant according to the CLSI criteria for Enterobacteriaceae (2012). The breakpoints used can be seen in Table 2 and E. coli strain ATCC 25922 was used as a control. Strains showing resistance to one or more antimicrobial agents were considered resistant, and strains resistant to three or more groups of antimicrobials were considered multi-resistant. Obtained data were statistically evaluated using the Fisher's exact test. In resistant E. coli isolates where the zone of inhibition was larger than the minimum size (>6 mm) as well as in intermediate resistant isolates, the minimum inhibitory concentration (MIC) of antimicrobial substances was determined by the E-test (Oxoid Ltd, UK). In E. coli isolates where the bla gene was detected as well as in the cefotaxime-resistant colonies, a double disk synergy test , & Urba skova , 2007) was performed to (Hrab ak, Vanis, Bergerova determine the production of ESBL. Antibiotic disks with amoxicillin/clavulanic acid e AMC (30 mg), aztreonam e AZT (30 mg), ceftazidime e CAZ (30 mg), cefpodoxime e CPD (10 mg), cefpodoxime/clavulanic acid e CD (10 mg), cefotaxime e CTX (30 mg), cefepime e FEP (30 mg) were used and the synergistic effect was then observed. 2.4. Detection of selected virulence and resistance genes Polymerase chain reaction (PCR) was used for the detection of genes encoding selected virulence factors e eaeA, hly, stx1, and stx2, for resistance to tetracycline e tet(A), tet(B), tet(C), and tet(G), blactams e blaTEM, blaSHV, and blaCTx-M, and quinolones e qnrA, qnrB, qnrS. Bacterial DNA was isolated from an overnight culture on blood agar (BioRad, France) by lysis of the bacterial cell suspension at 95.5 C for 10 min, with the addition of 20% Chelex 100 (BioRad, France), followed by centrifugation at 13,000 g for three minutes. The supernatant was used as a DNA template. For the detection of the tet and bla genes, PCR according to Ng, Martin, Alfa, and Mulvey ~ as, Zarazaga, S (2001), Brin anez, Ruiz-Larrea, and Torres (2002) and Lewis, Herrera, Wickes, Patterson, and Jorgensen (2007) were performed, respectively. For the detection of qnr genes, primers reported by Cattoir, Poirel, Rotimi, Soussy, and Nordmann (2007) and Gay et al. (2006) were used. Isolates positive for the presence
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Table 1 List of primers for the detection of virulence and resistance genes used in the study. Primer
Primer sequence (50 -30 )
Amplified gene
tetA/F tetA/R tetB/F tetB/R tetC/F tetC/R tetG/F tetG/R TEM-F TEM-R SHV-F SHV-R PANCTX-M.F PANCTX-M.R CTXM1-F3 CTXM1-R2 TOHO1-2F TOHO1-1R CTXM825F CTXM825R CTXM914F CTXM914R QnrAm-F QnrAm-R QnrBm-F QnrBm-R QnrS-1 QnrS-2 EHEC hlyF EHEC hlyR stx1 F stx1 R stx2 F stx2 R eaeA F eaeA R
GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG TTG GTT AGG GGC AAG TTT TG GTA ATG GGC CAA TAA CAC CG CTT GAG AGC CTT CAA CCC AG ATG GTC GTC ATC TAC CTG CC CAG CTT TCG GAT TCT TAC GG GAT TGG TGA GGC TCG TTA GC TTC TTG AAG ACG AAA GGG C ACG CTC AGT GGA ACG AAA AC CAC TCA AGG ATG TAT TGT G TTA GCG TTG CCA GTG CTC G TTT GCG ATG TGC AGT ACC AGT AA CGA TAT CGT TGG TGG TGC CAT A GAC GAT GTC ACT GGC TGA GC AGC CG C CGA CGC TAA TAC A GCG ACC TGG TTA ACT ACA ATC C CGG TAG TAT TGC CCT TAA GCC CGC TTT GCC ATG TGC AGC ACC GCT CAG TAC GAT CGA GCC GCT GGA GAA AAG CAG CGG AG GTA AGC TGA CGC AAC GTC TG AGA GGA TTT CTC ACG CCA GG TGC CAG GCA CAG ATC TTG AC GGM ATH GAA ATT CGC CAC TG TTT GCY GYY CGC CAG TCG AA ACG ACA TTC GTC AAC TGC AA TAA ATT GGC ACC CTG TAG GC ACG ATG TGG TTT ATT CTG GA CTT CAC GTG ACC ATA CAT AT ACA CTG GAT GAT CTC AGT GG CTG AAT CCC CCT CCA TTA TG CCA TGA CAA CGG ACA GCA GTT CCT GTC AAC TGA GCA GCA CTT TG GTG GCG AAT ACT GGC GAG ACT CCC CAT TCT TTT TCA CCG TCG
tet(A)
210
tet(B)
659
tet(C)
418
tet(G)
844
blaTEM
1150
blaSHV
885
blaCTx-M
554
Lewis et al. (2007)
CTX-M group I
499
Pitout et al. (2004)
CTX-M group II
351
CTX-M group III
307
CTX-M group IV
474
qnrA
580
qnrB
264
qnrS
417
Gay et al. (2006)
hly
165
Fagan et al. (1999)
stx1
614
stx2
779
eaeA
890
of the blaCTx-M gene were further typed using PCR according to Pitout, Hossain, and Hanson (2004). The detection of virulence genes was performed using multiplex PCR according to Fagan, Hornitzky, Bettelheim, and Djordjevic (1999). All primers used in the study are listed in Table 1. PCR products were analyzed by gel electrophoresis in 1.5% agarose (Serva, Germany), followed by visualization on a transilluminator after being stained with ethidium bromide.
Amplicon size (bp)
Reference Ng et al. (2001)
~ as et al. (2002) Brin
Cattoir et al. (2007)
2.5. Serotyping and subtyping of stx2 gene The isolate positive to the stx2 gene was serotyped using a U, Alexa, & type microplate agglutination assay (Salajka, Salajkova Hornich, 1992). Agglutination was performed with a set of 70 types of O-antisera, including the most common O-serogroups. Subtyping of stx2 gene was performed by the method described , and Rychlík (2000). by Alexa, Hamrík, Salajka, Stoura cova
Table 2 Prevalence of resistant E. coli (%) from retail pork, poultry, beef and venison. Antimicrobials
AMP AMC CTX CMP STR KAN GEN SXT TMP TET NAL CIP COL Resistance to min. 1 AML Multi-resistance
b-lactams Chloramphenicol Aminoglycosides
Antimetabolites Tetracyclines Quinolones, Fluoroquinolones Polymyxins
Breakpoints (CLSI, 2012)
Percentage of resistance
R/I/S (mm)
Pork (n ¼ 32)
Poultry (n ¼ 29)
Beef (n ¼ 30)
Venison (n ¼ 13)
13/14e16/17 13/14e17/18 22/23e25/26 12/13e17/18 11/12e14/15 13/14e17/18 12/13e14/15 10/11e15/16 10/11e15/16 11/12e14/15 13/14e18/19 15/16e20/21 8/9e10/11
46.9 9.3 3.1 3.1 25.0 3.1 6.3 21.9 25.0 43.8 9.4 6.3 0.0 62.5 31.3
55.2 13.8 3.5 13.8 24.1 6.9 10.3 24.1 31.0 34.5 55.2 37.9 3.5 82.8 51.7
10.0 0.0 0.0 0.0 6.6 3.3 0.0 3.3 3.3 6.6 0.0 0.0 0.0 10.0 6.6
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Notes: n ¼ number of investigated isolates; R ¼ resistant; I ¼ intermediate resistant; S ¼ susceptible; AML ¼ antimicrobial agent; AMP (ampicillin), AMC (amoxicillin/ clavulanic acid), CTX (Cefotaxim), CMP (chloramphenicol), STR (streptomycin), KAN (kanamycin), GEN (gentamycin), SXT (sulfamethoxazole/trimethoprim), TMP (trimethoprim), TET (tetracycline), NA (nalidixic acid), CIP (ciprofloxacin), COL (colistin).
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3. Results and discussion In each of the positive samples only isolates with the same phenotype of resistance were detected, therefore just one isolate per sample was included in the study. A total of 104 E. coli isolates (pork n ¼ 32, poultry n ¼ 29, beef n ¼ 30, venison n ¼ 13) were obtained from retail raw meat samples, with 47 (45.2%) of these isolates being resistant to one or more groups of antimicrobial agents. Strains were highly variable with regard to resistance pattern and differences were observed between resistant rates in isolates form pork, poultry, beef and venison. The amount of isolates showing resistance to at least one antimicrobial tested was the highest in isolates from poultry (82.8%) and the lowest in isolates from venison (0.0%). Statistically significant differences (P < 0.05) in the percentage of isolates resistant to at least one antimicrobial agent were observed between isolates from poultry meat and beef, poultry meat and venison, pork and beef, and pork and venison. Difference in the percentage of isolates resistant to at least one antimicrobial agent between poultry meat and pork, and beef and venison was not significant. The percentage of isolates resistant to each antimicrobial agent is reported in Table 2. The number of antimicrobials tested an isolate showed resistance ranged from zero to a maximum of 10 in one isolate from the poultry meat and resistance to b-lactam antibiotics was detected the most often. All together, 25.9% of isolates were positive for the presence of the gene blaTEM, while the presence of any tet gene was detected in 23.1% of isolates. The gene blaCTx-M, as well as the gene qnrA, was detected in one isolate (1.0%). 3.1. Pork More than one half of the isolates (62.5%) from the pork samples were resistant to at least one antimicrobial agent, while ten (31.3%) were multi-resistant isolates (Table 3). This is significantly higher
Table 3 Phenotypic and genotypic characteristic of multi-resistant E. coli from retail meat. Source
Phenotype of resistance
No. of isolates
Resistance genes
Pork
AMP-AMC-STR-SXT-TMP-TET AMP-AMC-GEN-TET-NAL-CIP AMP-AMC-CTX-STR-GEN-TET STR-TET-NAL AMP-STR-SXT-TMP-TET AMP-SXT-TMP-TET
1 1 1 1 3 2
AMP-CMP-STR-KAN-SXT-TMPTET-NAL-CIP
1
blaTEM, blaTEM, blaTEM, tet(A) blaTEM, blaTEM, blaTEM, blaTEM,
AMP-AMC-CMP-KAN-TMP-TETNAL-CIP AMP-AMC-TET-NAL AMP-AMC-CMP-STR-GEN-SXTTMP-TET-NAL-CIP AMP-STR-TMP-NAL-CIP AMP-STR-NAL-CIP AMP-CTX-SXT-TMP TET-NAL-CIP AMP-SXT-TMP-TET-NAL STR-SXT-TMP-TET-NAL-COL AMP-TET-NAL-CIP AMP-STR-KAN-SXT-TMP-NAL-CIP AMP-CMP-TET STR-GEN-SXT-TMP-NAL-CIP AMP-STR-GEN-SXT-TMP-NAL-CIP AMP-CMP-NAL-CIP
1
blaTEM, tet(B)
1 1
blaTEM, tet(B) blaTEM, tet(A)
1 1 1 1 1 1 1 1 1 1 1 1
blaTEM blaTEM blaCTx-M tet(B) blaTEM, tet(A) e blaTEM, tet(A) blaTEM blaTEM, tet(A) e blaTEM e
AMP-STR-KAN-SXT-TMP-TET AMP-STR-TET
1 1
blaTEM, tet(B) blaTEM, tet(B)
Poultry
Beef
tet(B) tet(B) tet(B) tet(A) tet(B) tet(A) tet(A)
amount of multi-resistant isolates compared to the isolates from the beef and venison (P < 0.05). Resistance patterns were diverse, and although similar to the isolates from the poultry and beef samples, the predominant resistance in the pork samples was resistance to ampicillin and tetracycline (Table 2). Similarly to other € lzel, Ka €mpf, studies (Kaesbohrer et al., 2012; Schwaiger, Huther, Ho & Bauer, 2012; Thorsteinsdottir et al., 2010), frequent resistance to streptomycin, sulfamethoxazol/trimethoprim and trimethoprim was also detected in isolates from the pork samples. Several resistance genes were detected in isolates from the pork samples with blaTEM detected the most often (40.6%). Another bla gene responsible for the resistance to b-lactam antibiotics was not detected and no ESBL-producing E. coli was identified using the double disk synergy test. Three different tetracycline-resistant determinants were found in isolates from pork. Genes tet(A) and tet(B) were often detected in multi-resistant isolates (Table 2), whereas tet(C) gene was identified in two isolates that were monoresistant to tetracycline. Although there were isolates resistant to quinolons and fluoroquinolons, no qnr gene was detected in isolates from the pork. More details dealing with this problematic are explained in the section 3.2. One isolate from pork of Czech origin tested positive for the presence of the stx2 gene. This isolate did not belong to any of the 70 serogroups tested (including serogroups O26, O103, O111, O118, O121, O145, O157) and was sensitive to all antimicrobials. The gene stx2 encodes the production of Shiga toxin 2 and is typical of the group of Shiga toxigenic E. coli (STEC). Subtyping of stx2 gene revealed a subtype stx2e, which is commonly associated with edema disease in pigs, but is seldom found in human STEC infections and has not been associated with diarrhea or severe illnesses (Beutin et al., 2008). 3.2. Poultry A total of 24 (82.8%) isolates from the poultry samples were resistant to one or more groups of antimicrobials and fifteen (51.7%) were multi-resistant which is significantly higher amount of multiresistant isolates compared to the isolates from beef and venison (P < 0.05). Resistance patterns were highly diverse as can be seen in Table 3, however most of the resistant strains displayed resistance to ampicillin and nalidixic acid (Table 2). Similar to other studies (de Jong et al., 2009; Schwaiger et al., 2012; Thorsteinsdottir et al., 2010) isolates from the poultry samples displayed the highest resistant rates in all of the antimicrobials tested, except for streptomycin and tetracycline, and resistant rates for nalidixic acid and ciprofloxacin were significantly higher (P < 0.05) compared to the isolates from pork, beef and venison. The high resistance levels for nalidixic acid and ciprofloxacin are alarming. Quinolons, especially fluoroquinolons are critically important for treating serious infections from E. coli in both humans and animals. Therefore, continued surveillance is required to detect emerging fluoroquinolone-resistant phenotypes (Alvarez Fernandez et al., 2013). Frequent resistance to fluoroquinolons in poultry was also reported in Germany (Kaesbohrer et al., 2012), ndez et al., 2013) and Iceland (Thorsteinsdottir Spain (Alvarez-Fern a et al., 2010). The high prevalence of E. coli resistance to these antimicrobials in poultry samples may reflect a higher use of quinolons and fluoroquinolons in poultry than in pigs or cattle in the Czech Republic as stated in the report published by Institute for State Control of Veterinary Biologicals and Medicines (Hera, , Dorn, & Pokludov Koutecka a, 2010). Bacterial resistance to quinolons and fluoroquinolons is conventionally attributed to chromosomally encoded mechanisms that allow the alteration of quinolone targets (quinolone resistance determining region e QRDR) (Jones-Dias et al., 2013). In our study,
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we focused on the detection of plasmid-mediated quinolone resistance genes (qnrA, qnrB, qnrS) that are horizontally transferable. As qnr genes often confer low-level resistance to both quinolons and fluoroquinolons (Yue et al., 2008), all E. coli isolates from the study were used for amplification, and only one isolate from the poultry samples was positive for the qnrA gene. This isolate was identified as being sensitive to both nalidixic acid and ciprofloxacin using the disk diffusion method and MIC were of 4 mg ml1 and 0.012 mg ml1, respectively. The low number of detected qnr genes in our study, despite the high resistance rates for quinolons and fluoroquinolons, suggests that other resistant determinants were responsible for these resistances. As QRDR mutations are most likely the main quinolone resistance mechanism in animal isolates (Jones-Dias et al., 2013), we assume that this mechanism was responsible for quinolone and fluoroquinolone resistance in our study as well. The most prevalent resistance gene in isolates from the poultry samples, as well as in the isolates from beef and pork, was the gene blaTEM (41.4%), encoding the production of b-lactamase enzymes that hydrolyze the b-lactam ring, and inactivate the b-lactams (Ojer-Usoz et al., 2013; Smet et al., 2008). Production of extendedspectrum b-lactamases (ESBL) was proved in one isolate from poultry samples of Czech origin where the gene blaCTx-M was detected. This isolate with resistance phenotype AMP-CTX-SXTTMP was identified as ESBL-producing not only using PCR but also by using the double disk synergy test and further typing proved an affiliation to group I. This group includes CTX-M-1, -3, -10 to -12, -15, -22, -23, -28, -29, and -30 (Pitout et al., 2004). The minimum inhibitory concentration of this isolate to cefotaxime was 32 mg ml1. ESBL are capable of hydrolyzing third-generation cephalosporins and monobactams, and are plasmid-mediated b-lactamases that are easily transferable between different bacteria (Kanamori et al., 2011), therefore the low level of prevalence in the meat from the Czech retail outlet is a satisfactory result. Two different tetracycline-resistant determinants were found in isolates from poultry samples with the gene tet(A) being detected more frequently (20.7%) compared to the gene tet(B) (6.9%). Frequent detection of tet genes in our study corresponds to the high prevalence of tetracycline-resistant isolates. The main mechanisms responsible for the resistance of bacteria to tetracycline include an active efflux system, ribosomal protection and enzymatic inactivation (Koo & Woo, 2011). The resistance genes tet(A), tet(B) and tet(C) detected in our study encode the energy-dependent efflux system, which is the most common in gram-negative bacteria. No virulence gene was detected in isolates from the poultry samples. 3.3. Beef In the retail beef samples the low prevalence of resistant E. coli was observed and statistically significant differences (P < 0.05) in the percentage of resistant isolates to at least one antimicrobial agent compared to the isolates from poultry meat and pork were found. Only three (10.0%) isolates from the beef samples were resistant to at least one of the antimicrobial agents, and two of them were multi-resistant isolates. Lower levels of resistance to all antimicrobials in isolates of E. coli from cattle compared to the resistance level in E. coli from poultry and pigs were also published by other authors (EFSA, 2011; de Jong et al., 2009; Kaesbohrer et al., 2012), but the presence of resistant isolates in cattle is usually higher compared to the isolates from the beef samples investigated in our study. All resistant strains isolated from the beef displayed resistance to ampicillin, while both multi-resistant strains tested positive for
405
resistant genes blaTEM and tet(B). The mono-resistant strain possessed no bla gene, although it was resistant to ampicillin using the disk diffusion method. The zone of inhibition was close to the breakpoint and using the E-test, this strain was determined as intermediate (MIC of 16 mg ml1) according to the CLSI (2012). The presence of either the ESBL-producing isolate or the gene qnr, or even the presence of the virulence gene was not detected in isolates from the beef samples. 3.4. Venison The venison samples were used as the control group where we assume the low antibiotic selective pressure is only caused by the residues of antimicrobial substances in the environment, if any. All isolates obtained from the venison were susceptible to all antimicrobial agents tested in the study and no virulence gene was detected. 4. Conclusion In conclusion, compared to venison and beef isolates, E. coli isolates from poultry and pork displayed higher resistance rates, while possessing more resistance genes. The production of extended-spectrum b-lactamases was confirmed in one isolate from the poultry samples as well as the presence of plasmidmediated gene qnrA. Based on these results, it can be concluded that poultry is the most risk-associated meat source in terms of antibiotic resistance. Overall, the frequent presence of multiresistant isolates in retail meat is particularly important, allowing raw meat to be a significant reservoir of resistance genes including those that can be transferred to sensitive strains and then subsequently spread through the food chain. Acknowledgments The results of the project LO1218 were obtained with a financial support from MEYS of the CR under the NPU I program. References Alexa, P., Hamrík, J., Salajka, E., Stoura cov a, K., & Rychlík, I. (2000). Differentiation of verotoxigenic strains of Escherichia coli isolated from piglets and calves in the rní Medicína, 45, 39e43. Czech Republic. Veterina Altalhi, A. D., & Hassan, S. A. (2009). Bacterial quality of raw milk investigated by Escherichia coli and isolates analysis for specific virulence-gene markers. Food Control, 20, 913e917. ndez, E., Cancelo, A., Díaz-Vega, C., Capita, R., & Alonso-Calleja, C. Alvarez-Fern a (2013). Antimicrobial resistance in E. coli isolates from conventionally and organically reared poultry: a comparison of agar disc diffusion and Sensi Test Gram-negative methods. Food Control, 30, 227e234. Beutin, L., Krüger, U., Krause, G., Miko, A., Martin, A., & Strauch, E. (2008). Evaluation of major types of Shiga toxin 2e-producing Escherichia coli bacteria present in food, pigs and the environment as potential pathogens for humans. Applied and Environmental Microbiology, 74, 4806e4816. ~ as, L., Zarazaga, M., Sa nez, Y., Ruiz-Larrea, F., & Torres, C. (2002). b-lactamases in Brin ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrobial Agents and Chemotherapy, 46, 3156e3160. Cattoir, V., Poirel, L., Rotimi, V., Soussy, C. J., & Nordmann, P. (2007). Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBLproducing enterobacterial isolates. Journal of Antimicrobial Chemotherapy, 60, 394e397. Clinical and Laboratory Standards Institute (CLSI). (2012). Performance standards for antimicrobial susceptibility teststing. CLSI Document M100eS22. USA, PA: Clinical and Laboratory Standards Institute. , M., Jurcickova, Z., Literak, I., Pokludova, L., Bures, J., Hera, A., et al. (2011). Dolejska IncN plasmids carrying bla CTX-M-1 in Escherichia coli isolates on a dairy farm. Veterinary Microbiology, 149, 513e516. Duffy, G., Lynch, O. A., & Cagney, C. (2008). Tracking emerging zoonotic pathogens from farm to fork. Meat Science, 78, 34e42. European Food Safety Authority (EFSA). (2011). Panel on Biological Hazards (BIOHAZ); Scientifis opinion on the public health risks of bacterial strains producing
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extended-spectrum beta-lactamases and/or AmpC beta-lactamases in foodproducing animals. EFSA Journal, 9, 2322. European Union. (2003). Regulation no. 1831/2003 of the European parliament and of the council of 22 September 2003 on additives for use in animal nutrition. Official Journal of the European Union L, 268, 29e43. Fagan, P. K., Hornitzky, M. A., Bettelheim, K. A., & Djordjevic, S. P. (1999). Detection of shiga-like toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin (EHEC hlyA) genes in animal feces by multiplex PCR. Applied and Environmental Microbiology, 65, 868e872. FAO. (2014). Faostat: Food balance sheets. Available at http://faostat.fao.org/site/610/ DesktopDefault.aspx?PageID¼610#ancor. FAO/WHO/OIE. (2008). Joint FAO/OIE/WHO expert meeting on critically important antimicrobials. Report of meeting held in FAO, Rome, Italy, November 2007, Geneva, Switzerland. Available at http://www.who.int/foodborne_disease/ resources/Report_CIA_Meeting.pdf. Gay, K., Robicsek, A., Strahilevitz, J., Park, C. H., Jacoby, G., Barrett, T. J., et al. (2006). Plasmid-mediated quinolone resistance in non-Typhi serotypes of Salmonella enterica. Clinical Infectious Diseases, 43, 297e304. Hammerum, A. M., & Heuer, O. E. (2009). Human health hazards from antimicrobial-resistant Escherichia coli of animal origin. Clinical Infectious Diseases, 48, 916e921. Hera, A., Kouteck a, L., Dorn, D., & Pokludov a, L. (2010). Spotr eba antibiotik a anti v letech 2003e2010 [Consumption of anrní medicín parazitik ve veterina e v CR tibiotics and antiparasitics in veterinary medicine in the Czech Republic in years 2003e2010]. Available at http://www.uskvbl.cz/cs/informace/tiskove-centrum/ tiskprohl. k, J., Vanis, V., Bergerova , T., & Urba skova , P. (2007). Detection of extended Hraba spectrum beta lactamases (ESBL) and AmpC type beta lactamases in entervy Centra Epidemiologie a Mikrobiologie (SZÚ, Praha), 16, 31e36. obacteria. Zpra Jones-Dias, D., Manageiro, V., Francisco, A. P., Martins, A. P., Domingues, G., Louro, D., et al. (2013). Assessing the molecular basis of transferable quinolone resistance in Escherichia coli and Salmonella spp. from food-producing animals and food products. Veterinary Microbiology, 167, 523e531. de Jong, A., Bywater, R., Butty, P., Deroover, E., Godinho, K., Klein, U., et al. (2009). A pan-European survey of antimicrobial susceptibility towards human-use antimicrobial drugs among zoonotic and commensal enteric bacteria isolated from healthy food-producing animals. Journal of Antimicrobial Chemotherapy, 63, 733e744. Kaesbohrer, A., Schroeter, A., Tenhagen, B. A., Alt, K., Guerra, B., & Appel, B. (2012). Emerging antimicrobial resistance in commensal Escherichia coli with public health relevance. Zoonoses Public Health, 59, 158e165. Kanamori, H., Navarro, R. B., Yano, H., Sombrero, L. T., Capeding, M. R., Lupisan, S. P., et al. (2011). Molecular characteristics of extended-spectrum b-lactamases in
clinical isolates of Enterobacteriaceae from the Philippines. Acta Tropica, 120, 140e145. Koo, H., & Woo, G. (2011). Distribution and transferability of tetracycline resistance determinants in Escherichia coli isolated from meat and meat products. International Journal of Food Microbiology, 145, 407e413. Lei, T., Tian, W., He, L., Huang, X. H., Sun, Y. X., Deng, Y. T., et al. (2010). Antimicrobial resistance in Escherichia coli isolates from food animals, animal food products and companion animals in China. Veterinary Microbiology, 146, 85e89. Lewis, J. S., II, Herrera, M., Wickes, B., Patterson, J. E., & Jorgensen, J. H. (2007). First report of the emergence of CTX-M-type extended-spectrum b-lactamases (ESBLs) as the predominant ESBL isolated in a U.S. health care system. Antimicrobial Agents and Chemotherapy, 51, 4015e4021. Martins da Costa, P., Oliveira, M., Ramos, B., & Bernardo, F. (2011). The impact of antimicrobial use in broiler chickens on growth performance and on the occurrence of antimicrobial-resistant Escherichia coli. Livestock Science, 136, 262e269. Ng, L. K., Martin, I., Alfa, M., & Mulvey, M. (2001). Multiplex PCR for the detection of tetracycline resistant genes. Molecular and Cellular Probes, 15, 209e215. lez, D., Vitas, A. I., Leiva, J., García-Jalo n, I., Febles-Casquero, A., Ojer-Usoz, E., Gonza et al. (2013). Prevalence of extended-spectrum b-lactamase-producing Enterobacteriaceae in meat products sold in Navarra, Spain. Meat Science, 93, 316e321. Pitout, J. D., Hossain, A., & Hanson, N. D. (2004). Phenotypic and molecular detection of CTX-M-beta-lactamases produced by Escherichia coli and Klebsiella spp. Journal of Clinical Microbiology, 42, 5715e5721. Salajka, E., Salajkov a, Z., Alexa, P., & Hornich, M. (1992). Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic Escherichia coli strains isolated from postweaning diarrhoea in pigs. Veterinary Microbiology, 32, 136e175. € lzel, Ch, K€ Schwaiger, K., Huther, S., Ho ampf, P., & Bauer, J. (2012). Prevalence of antibiotic-resistant enterobacteriaceae isolated from chicken and pork meat purchased at the slaughterhouse and at retail in Bavaria, Germany. International Journal of Food Microbiology, 154, 206e211. Smet, A., Martel, A., Persoons, D., Dewulf, J., Heyndrickx, M., Catry, B., et al. (2008). Diversity of extended-spectrum beta-lactamases and class C beta-lactamases among cloacal Escherichia coli isolates in Belgian broiler farms. Antimicrobial Agents and Chemotherapy, 52, 1238e1243. Thorsteinsdottir, T. R., Haraldsson, G., Fridriksdottir, V., Kristinsson, K. G., & Gunnarsson, E. (2010). Prevalence and genetic relatedness of antimicrobialresistant Escherichia coli isolated from animals, foods and humans in Iceland. Zoonoses Public Health, 57, 189e196. Yue, L., Jiang, H. X., Liao, X. P., Liu, J. H., Li, S. J., Chen, X. Y., et al. (2008). Prevalence of plasmid-mediated quinolone resistance qnr genes in poultry and swine clinical isolates of Escherichia coli. Veterinary Microbiology, 132, 414e420.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Characteristic and antimicrobial resistance in Escherichia coli from retail meats purchased in the Czech Republic a, b, *, Ivana Kola b, Katerina Bogdanovicova a, Rena ta Karpískova a, b Alena Sko ckova ckova a b
Department of Milk Hygiene and Technology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic Veterinary Research Institute, Brno, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2014 Received in revised form 14 July 2014 Accepted 19 July 2014 Available online 28 July 2014
The aim of this study was to characterize and compare Escherichia coli found in retail meats (pork, poultry, beef, venison), focusing on the detection of resistant genes that could be spread through the food chain and also for the presence of selected virulence genes. The resistance to antimicrobial agents was determined by disk diffusion method and E-test. Polymerase chain reaction was used for the detection of selected genes encoding for virulence factors (eaeA, hly, stx1, stx2) and genes encoding resistance to tetracycline, b-lactams and quinolones. Compared to beef isolates, the isolates from poultry and pork displayed higher resistant rates and also possessed more resistance genes. All together, 25.9% of the isolates were positive for the presence of the gene blaTEM, and the presence of any tet gene was detected in 23.1% of the isolates. The production of extended-spectrum b-lactamases was confirmed in one isolate from poultry as well as the presence of a plasmid-mediated gene qnrA attributed to the quinolone and fluoroquinolone resistance. Based on these results, it can be surmised that poultry is the most riskassociated meat source in terms of antibiotic resistance. The stx2e gene was detected in one isolate from pork samples, but this subtype is commonly associated with edema disease in pigs and is seldom found in humans. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Poultry meat Beef Pork Escherichia coli Resistance genes
1. Introduction Meat represents a substantial portion of the diet of most people worldwide. The world average annual consumption of pork is approximately 15 kg per person followed by poultry (12.6 kg) and beef (9.6 kg). The annual consumption of meat in the Europe is even higher (pork 34.2 kg, poultry 21.9 kg and beef 16.1 kg) (FAO, 2014). Because of the high consumption level, the safety and quality of meat in retail markets is an important public health issue. Escherichia coli are widespread gut commensal microorganisms in humans and warm-blooded animals, and are also an important zoonotic agent, which can be found in both animal and human infections (Duffy, Lynch, & Cagney, 2008). In the microbiological analysis of water and foodstuffs, E. coli is used to assess their hygienic quality, and its presence can also indicate the presence of enteric pathogens (Altalhi & Hassan, 2009). Furthermore, the level of antibiotic resistance in E. coli is considered to be a good indicator
* Corresponding author. Department of Milk Hygiene and Technology, University ho 1-3, 612 42 Brno, Czech of Veterinary and Pharmaceutical Sciences Brno, Palacke Republic. Tel.: þ420 541 562 722. ). E-mail address: [email protected] (A. Sko ckova http://dx.doi.org/10.1016/j.foodcont.2014.07.034 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
of the selection pressure exerted by the use of antibiotics (Lei et al., 2010) and of the resistance problems to be expected in infectious ndez, Cancelo, Díaz-Vega, Capita, & Alonsodiseases (Alvarez-Fern a Calleja, 2013). Meat and meat products can be easily contaminated by E. coli during animal evisceration after slaughter, through contact with tainted water or during meat handling (Alvarez-Fern andez et al., 2013). Because of the intensive use of antimicrobial agents in animal production, bacteria originating from food animals frequently carry a resistance to a range of antimicrobial agents, including those commonly used in humans (Hammerum & Heuer, 2009). The transfer of resistant bacteria between animals and humans through food products has been documented and could pose a threat to public health (Thorsteinsdottir, Haraldsson, Fridriksdottir, Kristinsson, & Gunnarsson, 2010). Moreover, antimicrobialresistant bacteria may represent a reservoir of resistance genes transferable to pathogenic or commensal bacteria in the human ndez et al., 2013), and therefore digestive tract (Alvarez-Fern a compromise the effective treatment of bacterial infections. In 2006 the European Union banned all feed antibiotics in animal husbandry (Regulation (EC) No, 1831/2003). However, producers' expectations regarding performance enhancement and
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illness prevention have led to an increased use of therapeutically valuable agents (Martins da Costa, Oliveira, Ramos, & Bernardo, 2011). During the last decade, a number of events have stressed the need for an increased awareness of the public health aspects related to antimicrobial resistance in animal husbandry. For example, the emergence of b-lactamases responsible for the resistance to b-lactam antibiotics, including cephalosporins, shows that risks to human health include the possibility of the horizontal transfer of resistance genes (Kaesbohrer et al., 2012). Another antibiotic resistance mechanism that has recently been on the increase in enterobacteria, is a resistance to fluoroquinolons et al., 2011). determined by plasmid-mediated qnr genes (Dolejska Both of these antimicrobials, third and fourth-generation cephalosporins and fluoroquinolons, are on the W.H.O. list of criticallyimportant antimicrobials for human medicine (FAO/WHO/OIE, 2008). This study should provide important information about the risk of consumer exposure to resistant E. coli, to help evaluate and compare the safety of different retail meats. Samples of venison were used as a control group because we assume there is no antimicrobial selection pressure in game meat. Because the food supply is now due to the international trade global, with the local microflora from the country of origin being spread to other countries, the results of this study could be important, not only for the Czech Republic, but for other countries as well. The objective of this study was to determine the prevalence of antimicrobial-resistant E. coli in meat (pork, poultry, beef, venison) within the retail market of the Czech Republic and to characterize obtained isolates focusing on the detection of extended-spectrum b-lactamases production and the presence of tet and plasmidmediated qnr genes. The pathogenic potential of E. coli isolates from retail meat based on their virulence gene profiles was also determined. 2. Material and methods 2.1. Sample collection A total of 322 refrigerated, individually packed fresh meat samples were purchased from various supermarkets in the Czech Republic during the years of 2012 and 2013. Together samples from eight cities and twenty supermarkets were investigated. The samples collected during one sampling were always from different processing plants and the time period between samplings was a minimum one month. A majority of the samples were of Czech origin (n ¼ 296) but imported samples from other European countries (n ¼ 26) were also covered in the study. The meat samples differed in animal origin (pork n ¼ 110, poultry n ¼ 80, beef n ¼ 82, and venison ¼ 50). The samples of poultry included samples of chickens, hens and turkey, both with and without the skin. All samples were individually wrapped in PE bags to avoid cross-contamination, refrigerated, and immediately after being collected, they were transported to the laboratory for examination. 2.2. Isolation and identification of E. coli In the laboratory, swabs were taken from the surface of the meat using sterile sponges (3M™ Sponge-stick, USA) and were placed in plastic bags with 30 ml BPW (Buffered Peptone Water, Oxoid, UK), homogenized on a Stomacher device, and incubated at 37 C overnight. When samples of whole chickens were investigated, 25 g of meat and skin from the neck was homogenized with 225 ml BPW and also incubated at 37 C overnight. Homogenates were
then substantially streaked on TBX agar (Tryptone Bile X-glucuronide medium, Oxoid, UK) and again incubated at 44 C overnight. From each positive sample, three suspected E. coli isolates were involved in the study for confirmation and determination of resistance to antimicrobials, but only isolates with different resistance phenotype from one sample were included in the study and further characterized. The confirmation of the suspected isolates from TBX agar was determined from the detection of oxidase (OXItest, Pliva-Lachema, CZ) and the production of indole (COLItest, Pliva-Lachema, CZ). 2.3. Antimicrobial susceptibility testing The susceptibility to antibiotics commonly used in clinical treatments was tested by disk diffusion method according to the CLSI protocol (2012) on Mueller-Hinton agar (Oxoid Ltd, UK) using the following antimicrobials and concentrations: ampicillin e AMP (10 mg), amoxicillin/clavulanic acid e AMC (30 mg), cefotaxime e CTX (30 mg), chloramphenicol e CMP (30 mg), streptomycin e STR (10 mg), kanamycin e KAN (30 mg), gentamicin e GEN (10 mg), sulfamethoxazol/trimethoprim e SXT (25 mg), trimethoprim e TMP (5 mg), tetracycline e TET (30 mg), nalidixic acid e NAL (30 mg), ciprofloxacin e CIP (5 mg), and colistin e COL (10 mg). Antibiotic disks were obtained from Oxoid Ltd (UK). The E. coli strains were evaluated based on the size of the zones of inhibition and classified as susceptible, intermediate resistant, or resistant according to the CLSI criteria for Enterobacteriaceae (2012). The breakpoints used can be seen in Table 2 and E. coli strain ATCC 25922 was used as a control. Strains showing resistance to one or more antimicrobial agents were considered resistant, and strains resistant to three or more groups of antimicrobials were considered multi-resistant. Obtained data were statistically evaluated using the Fisher's exact test. In resistant E. coli isolates where the zone of inhibition was larger than the minimum size (>6 mm) as well as in intermediate resistant isolates, the minimum inhibitory concentration (MIC) of antimicrobial substances was determined by the E-test (Oxoid Ltd, UK). In E. coli isolates where the bla gene was detected as well as in the cefotaxime-resistant colonies, a double disk synergy test , & Urba skova , 2007) was performed to (Hrab ak, Vanis, Bergerova determine the production of ESBL. Antibiotic disks with amoxicillin/clavulanic acid e AMC (30 mg), aztreonam e AZT (30 mg), ceftazidime e CAZ (30 mg), cefpodoxime e CPD (10 mg), cefpodoxime/clavulanic acid e CD (10 mg), cefotaxime e CTX (30 mg), cefepime e FEP (30 mg) were used and the synergistic effect was then observed. 2.4. Detection of selected virulence and resistance genes Polymerase chain reaction (PCR) was used for the detection of genes encoding selected virulence factors e eaeA, hly, stx1, and stx2, for resistance to tetracycline e tet(A), tet(B), tet(C), and tet(G), blactams e blaTEM, blaSHV, and blaCTx-M, and quinolones e qnrA, qnrB, qnrS. Bacterial DNA was isolated from an overnight culture on blood agar (BioRad, France) by lysis of the bacterial cell suspension at 95.5 C for 10 min, with the addition of 20% Chelex 100 (BioRad, France), followed by centrifugation at 13,000 g for three minutes. The supernatant was used as a DNA template. For the detection of the tet and bla genes, PCR according to Ng, Martin, Alfa, and Mulvey ~ as, Zarazaga, S (2001), Brin anez, Ruiz-Larrea, and Torres (2002) and Lewis, Herrera, Wickes, Patterson, and Jorgensen (2007) were performed, respectively. For the detection of qnr genes, primers reported by Cattoir, Poirel, Rotimi, Soussy, and Nordmann (2007) and Gay et al. (2006) were used. Isolates positive for the presence
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403
Table 1 List of primers for the detection of virulence and resistance genes used in the study. Primer
Primer sequence (50 -30 )
Amplified gene
tetA/F tetA/R tetB/F tetB/R tetC/F tetC/R tetG/F tetG/R TEM-F TEM-R SHV-F SHV-R PANCTX-M.F PANCTX-M.R CTXM1-F3 CTXM1-R2 TOHO1-2F TOHO1-1R CTXM825F CTXM825R CTXM914F CTXM914R QnrAm-F QnrAm-R QnrBm-F QnrBm-R QnrS-1 QnrS-2 EHEC hlyF EHEC hlyR stx1 F stx1 R stx2 F stx2 R eaeA F eaeA R
GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG TTG GTT AGG GGC AAG TTT TG GTA ATG GGC CAA TAA CAC CG CTT GAG AGC CTT CAA CCC AG ATG GTC GTC ATC TAC CTG CC CAG CTT TCG GAT TCT TAC GG GAT TGG TGA GGC TCG TTA GC TTC TTG AAG ACG AAA GGG C ACG CTC AGT GGA ACG AAA AC CAC TCA AGG ATG TAT TGT G TTA GCG TTG CCA GTG CTC G TTT GCG ATG TGC AGT ACC AGT AA CGA TAT CGT TGG TGG TGC CAT A GAC GAT GTC ACT GGC TGA GC AGC CG C CGA CGC TAA TAC A GCG ACC TGG TTA ACT ACA ATC C CGG TAG TAT TGC CCT TAA GCC CGC TTT GCC ATG TGC AGC ACC GCT CAG TAC GAT CGA GCC GCT GGA GAA AAG CAG CGG AG GTA AGC TGA CGC AAC GTC TG AGA GGA TTT CTC ACG CCA GG TGC CAG GCA CAG ATC TTG AC GGM ATH GAA ATT CGC CAC TG TTT GCY GYY CGC CAG TCG AA ACG ACA TTC GTC AAC TGC AA TAA ATT GGC ACC CTG TAG GC ACG ATG TGG TTT ATT CTG GA CTT CAC GTG ACC ATA CAT AT ACA CTG GAT GAT CTC AGT GG CTG AAT CCC CCT CCA TTA TG CCA TGA CAA CGG ACA GCA GTT CCT GTC AAC TGA GCA GCA CTT TG GTG GCG AAT ACT GGC GAG ACT CCC CAT TCT TTT TCA CCG TCG
tet(A)
210
tet(B)
659
tet(C)
418
tet(G)
844
blaTEM
1150
blaSHV
885
blaCTx-M
554
Lewis et al. (2007)
CTX-M group I
499
Pitout et al. (2004)
CTX-M group II
351
CTX-M group III
307
CTX-M group IV
474
qnrA
580
qnrB
264
qnrS
417
Gay et al. (2006)
hly
165
Fagan et al. (1999)
stx1
614
stx2
779
eaeA
890
of the blaCTx-M gene were further typed using PCR according to Pitout, Hossain, and Hanson (2004). The detection of virulence genes was performed using multiplex PCR according to Fagan, Hornitzky, Bettelheim, and Djordjevic (1999). All primers used in the study are listed in Table 1. PCR products were analyzed by gel electrophoresis in 1.5% agarose (Serva, Germany), followed by visualization on a transilluminator after being stained with ethidium bromide.
Amplicon size (bp)
Reference Ng et al. (2001)
~ as et al. (2002) Brin
Cattoir et al. (2007)
2.5. Serotyping and subtyping of stx2 gene The isolate positive to the stx2 gene was serotyped using a U, Alexa, & type microplate agglutination assay (Salajka, Salajkova Hornich, 1992). Agglutination was performed with a set of 70 types of O-antisera, including the most common O-serogroups. Subtyping of stx2 gene was performed by the method described , and Rychlík (2000). by Alexa, Hamrík, Salajka, Stoura cova
Table 2 Prevalence of resistant E. coli (%) from retail pork, poultry, beef and venison. Antimicrobials
AMP AMC CTX CMP STR KAN GEN SXT TMP TET NAL CIP COL Resistance to min. 1 AML Multi-resistance
b-lactams Chloramphenicol Aminoglycosides
Antimetabolites Tetracyclines Quinolones, Fluoroquinolones Polymyxins
Breakpoints (CLSI, 2012)
Percentage of resistance
R/I/S (mm)
Pork (n ¼ 32)
Poultry (n ¼ 29)
Beef (n ¼ 30)
Venison (n ¼ 13)
13/14e16/17 13/14e17/18 22/23e25/26 12/13e17/18 11/12e14/15 13/14e17/18 12/13e14/15 10/11e15/16 10/11e15/16 11/12e14/15 13/14e18/19 15/16e20/21 8/9e10/11
46.9 9.3 3.1 3.1 25.0 3.1 6.3 21.9 25.0 43.8 9.4 6.3 0.0 62.5 31.3
55.2 13.8 3.5 13.8 24.1 6.9 10.3 24.1 31.0 34.5 55.2 37.9 3.5 82.8 51.7
10.0 0.0 0.0 0.0 6.6 3.3 0.0 3.3 3.3 6.6 0.0 0.0 0.0 10.0 6.6
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Notes: n ¼ number of investigated isolates; R ¼ resistant; I ¼ intermediate resistant; S ¼ susceptible; AML ¼ antimicrobial agent; AMP (ampicillin), AMC (amoxicillin/ clavulanic acid), CTX (Cefotaxim), CMP (chloramphenicol), STR (streptomycin), KAN (kanamycin), GEN (gentamycin), SXT (sulfamethoxazole/trimethoprim), TMP (trimethoprim), TET (tetracycline), NA (nalidixic acid), CIP (ciprofloxacin), COL (colistin).
et al. / Food Control 47 (2015) 401e406 A. Skockova
404
3. Results and discussion In each of the positive samples only isolates with the same phenotype of resistance were detected, therefore just one isolate per sample was included in the study. A total of 104 E. coli isolates (pork n ¼ 32, poultry n ¼ 29, beef n ¼ 30, venison n ¼ 13) were obtained from retail raw meat samples, with 47 (45.2%) of these isolates being resistant to one or more groups of antimicrobial agents. Strains were highly variable with regard to resistance pattern and differences were observed between resistant rates in isolates form pork, poultry, beef and venison. The amount of isolates showing resistance to at least one antimicrobial tested was the highest in isolates from poultry (82.8%) and the lowest in isolates from venison (0.0%). Statistically significant differences (P < 0.05) in the percentage of isolates resistant to at least one antimicrobial agent were observed between isolates from poultry meat and beef, poultry meat and venison, pork and beef, and pork and venison. Difference in the percentage of isolates resistant to at least one antimicrobial agent between poultry meat and pork, and beef and venison was not significant. The percentage of isolates resistant to each antimicrobial agent is reported in Table 2. The number of antimicrobials tested an isolate showed resistance ranged from zero to a maximum of 10 in one isolate from the poultry meat and resistance to b-lactam antibiotics was detected the most often. All together, 25.9% of isolates were positive for the presence of the gene blaTEM, while the presence of any tet gene was detected in 23.1% of isolates. The gene blaCTx-M, as well as the gene qnrA, was detected in one isolate (1.0%). 3.1. Pork More than one half of the isolates (62.5%) from the pork samples were resistant to at least one antimicrobial agent, while ten (31.3%) were multi-resistant isolates (Table 3). This is significantly higher
Table 3 Phenotypic and genotypic characteristic of multi-resistant E. coli from retail meat. Source
Phenotype of resistance
No. of isolates
Resistance genes
Pork
AMP-AMC-STR-SXT-TMP-TET AMP-AMC-GEN-TET-NAL-CIP AMP-AMC-CTX-STR-GEN-TET STR-TET-NAL AMP-STR-SXT-TMP-TET AMP-SXT-TMP-TET
1 1 1 1 3 2
AMP-CMP-STR-KAN-SXT-TMPTET-NAL-CIP
1
blaTEM, blaTEM, blaTEM, tet(A) blaTEM, blaTEM, blaTEM, blaTEM,
AMP-AMC-CMP-KAN-TMP-TETNAL-CIP AMP-AMC-TET-NAL AMP-AMC-CMP-STR-GEN-SXTTMP-TET-NAL-CIP AMP-STR-TMP-NAL-CIP AMP-STR-NAL-CIP AMP-CTX-SXT-TMP TET-NAL-CIP AMP-SXT-TMP-TET-NAL STR-SXT-TMP-TET-NAL-COL AMP-TET-NAL-CIP AMP-STR-KAN-SXT-TMP-NAL-CIP AMP-CMP-TET STR-GEN-SXT-TMP-NAL-CIP AMP-STR-GEN-SXT-TMP-NAL-CIP AMP-CMP-NAL-CIP
1
blaTEM, tet(B)
1 1
blaTEM, tet(B) blaTEM, tet(A)
1 1 1 1 1 1 1 1 1 1 1 1
blaTEM blaTEM blaCTx-M tet(B) blaTEM, tet(A) e blaTEM, tet(A) blaTEM blaTEM, tet(A) e blaTEM e
AMP-STR-KAN-SXT-TMP-TET AMP-STR-TET
1 1
blaTEM, tet(B) blaTEM, tet(B)
Poultry
Beef
tet(B) tet(B) tet(B) tet(A) tet(B) tet(A) tet(A)
amount of multi-resistant isolates compared to the isolates from the beef and venison (P < 0.05). Resistance patterns were diverse, and although similar to the isolates from the poultry and beef samples, the predominant resistance in the pork samples was resistance to ampicillin and tetracycline (Table 2). Similarly to other € lzel, Ka €mpf, studies (Kaesbohrer et al., 2012; Schwaiger, Huther, Ho & Bauer, 2012; Thorsteinsdottir et al., 2010), frequent resistance to streptomycin, sulfamethoxazol/trimethoprim and trimethoprim was also detected in isolates from the pork samples. Several resistance genes were detected in isolates from the pork samples with blaTEM detected the most often (40.6%). Another bla gene responsible for the resistance to b-lactam antibiotics was not detected and no ESBL-producing E. coli was identified using the double disk synergy test. Three different tetracycline-resistant determinants were found in isolates from pork. Genes tet(A) and tet(B) were often detected in multi-resistant isolates (Table 2), whereas tet(C) gene was identified in two isolates that were monoresistant to tetracycline. Although there were isolates resistant to quinolons and fluoroquinolons, no qnr gene was detected in isolates from the pork. More details dealing with this problematic are explained in the section 3.2. One isolate from pork of Czech origin tested positive for the presence of the stx2 gene. This isolate did not belong to any of the 70 serogroups tested (including serogroups O26, O103, O111, O118, O121, O145, O157) and was sensitive to all antimicrobials. The gene stx2 encodes the production of Shiga toxin 2 and is typical of the group of Shiga toxigenic E. coli (STEC). Subtyping of stx2 gene revealed a subtype stx2e, which is commonly associated with edema disease in pigs, but is seldom found in human STEC infections and has not been associated with diarrhea or severe illnesses (Beutin et al., 2008). 3.2. Poultry A total of 24 (82.8%) isolates from the poultry samples were resistant to one or more groups of antimicrobials and fifteen (51.7%) were multi-resistant which is significantly higher amount of multiresistant isolates compared to the isolates from beef and venison (P < 0.05). Resistance patterns were highly diverse as can be seen in Table 3, however most of the resistant strains displayed resistance to ampicillin and nalidixic acid (Table 2). Similar to other studies (de Jong et al., 2009; Schwaiger et al., 2012; Thorsteinsdottir et al., 2010) isolates from the poultry samples displayed the highest resistant rates in all of the antimicrobials tested, except for streptomycin and tetracycline, and resistant rates for nalidixic acid and ciprofloxacin were significantly higher (P < 0.05) compared to the isolates from pork, beef and venison. The high resistance levels for nalidixic acid and ciprofloxacin are alarming. Quinolons, especially fluoroquinolons are critically important for treating serious infections from E. coli in both humans and animals. Therefore, continued surveillance is required to detect emerging fluoroquinolone-resistant phenotypes (Alvarez Fernandez et al., 2013). Frequent resistance to fluoroquinolons in poultry was also reported in Germany (Kaesbohrer et al., 2012), ndez et al., 2013) and Iceland (Thorsteinsdottir Spain (Alvarez-Fern a et al., 2010). The high prevalence of E. coli resistance to these antimicrobials in poultry samples may reflect a higher use of quinolons and fluoroquinolons in poultry than in pigs or cattle in the Czech Republic as stated in the report published by Institute for State Control of Veterinary Biologicals and Medicines (Hera, , Dorn, & Pokludov Koutecka a, 2010). Bacterial resistance to quinolons and fluoroquinolons is conventionally attributed to chromosomally encoded mechanisms that allow the alteration of quinolone targets (quinolone resistance determining region e QRDR) (Jones-Dias et al., 2013). In our study,
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we focused on the detection of plasmid-mediated quinolone resistance genes (qnrA, qnrB, qnrS) that are horizontally transferable. As qnr genes often confer low-level resistance to both quinolons and fluoroquinolons (Yue et al., 2008), all E. coli isolates from the study were used for amplification, and only one isolate from the poultry samples was positive for the qnrA gene. This isolate was identified as being sensitive to both nalidixic acid and ciprofloxacin using the disk diffusion method and MIC were of 4 mg ml1 and 0.012 mg ml1, respectively. The low number of detected qnr genes in our study, despite the high resistance rates for quinolons and fluoroquinolons, suggests that other resistant determinants were responsible for these resistances. As QRDR mutations are most likely the main quinolone resistance mechanism in animal isolates (Jones-Dias et al., 2013), we assume that this mechanism was responsible for quinolone and fluoroquinolone resistance in our study as well. The most prevalent resistance gene in isolates from the poultry samples, as well as in the isolates from beef and pork, was the gene blaTEM (41.4%), encoding the production of b-lactamase enzymes that hydrolyze the b-lactam ring, and inactivate the b-lactams (Ojer-Usoz et al., 2013; Smet et al., 2008). Production of extendedspectrum b-lactamases (ESBL) was proved in one isolate from poultry samples of Czech origin where the gene blaCTx-M was detected. This isolate with resistance phenotype AMP-CTX-SXTTMP was identified as ESBL-producing not only using PCR but also by using the double disk synergy test and further typing proved an affiliation to group I. This group includes CTX-M-1, -3, -10 to -12, -15, -22, -23, -28, -29, and -30 (Pitout et al., 2004). The minimum inhibitory concentration of this isolate to cefotaxime was 32 mg ml1. ESBL are capable of hydrolyzing third-generation cephalosporins and monobactams, and are plasmid-mediated b-lactamases that are easily transferable between different bacteria (Kanamori et al., 2011), therefore the low level of prevalence in the meat from the Czech retail outlet is a satisfactory result. Two different tetracycline-resistant determinants were found in isolates from poultry samples with the gene tet(A) being detected more frequently (20.7%) compared to the gene tet(B) (6.9%). Frequent detection of tet genes in our study corresponds to the high prevalence of tetracycline-resistant isolates. The main mechanisms responsible for the resistance of bacteria to tetracycline include an active efflux system, ribosomal protection and enzymatic inactivation (Koo & Woo, 2011). The resistance genes tet(A), tet(B) and tet(C) detected in our study encode the energy-dependent efflux system, which is the most common in gram-negative bacteria. No virulence gene was detected in isolates from the poultry samples. 3.3. Beef In the retail beef samples the low prevalence of resistant E. coli was observed and statistically significant differences (P < 0.05) in the percentage of resistant isolates to at least one antimicrobial agent compared to the isolates from poultry meat and pork were found. Only three (10.0%) isolates from the beef samples were resistant to at least one of the antimicrobial agents, and two of them were multi-resistant isolates. Lower levels of resistance to all antimicrobials in isolates of E. coli from cattle compared to the resistance level in E. coli from poultry and pigs were also published by other authors (EFSA, 2011; de Jong et al., 2009; Kaesbohrer et al., 2012), but the presence of resistant isolates in cattle is usually higher compared to the isolates from the beef samples investigated in our study. All resistant strains isolated from the beef displayed resistance to ampicillin, while both multi-resistant strains tested positive for
405
resistant genes blaTEM and tet(B). The mono-resistant strain possessed no bla gene, although it was resistant to ampicillin using the disk diffusion method. The zone of inhibition was close to the breakpoint and using the E-test, this strain was determined as intermediate (MIC of 16 mg ml1) according to the CLSI (2012). The presence of either the ESBL-producing isolate or the gene qnr, or even the presence of the virulence gene was not detected in isolates from the beef samples. 3.4. Venison The venison samples were used as the control group where we assume the low antibiotic selective pressure is only caused by the residues of antimicrobial substances in the environment, if any. All isolates obtained from the venison were susceptible to all antimicrobial agents tested in the study and no virulence gene was detected. 4. Conclusion In conclusion, compared to venison and beef isolates, E. coli isolates from poultry and pork displayed higher resistance rates, while possessing more resistance genes. The production of extended-spectrum b-lactamases was confirmed in one isolate from the poultry samples as well as the presence of plasmidmediated gene qnrA. Based on these results, it can be concluded that poultry is the most risk-associated meat source in terms of antibiotic resistance. Overall, the frequent presence of multiresistant isolates in retail meat is particularly important, allowing raw meat to be a significant reservoir of resistance genes including those that can be transferred to sensitive strains and then subsequently spread through the food chain. Acknowledgments The results of the project LO1218 were obtained with a financial support from MEYS of the CR under the NPU I program. References Alexa, P., Hamrík, J., Salajka, E., Stoura cov a, K., & Rychlík, I. (2000). Differentiation of verotoxigenic strains of Escherichia coli isolated from piglets and calves in the rní Medicína, 45, 39e43. Czech Republic. Veterina Altalhi, A. D., & Hassan, S. A. (2009). Bacterial quality of raw milk investigated by Escherichia coli and isolates analysis for specific virulence-gene markers. Food Control, 20, 913e917. ndez, E., Cancelo, A., Díaz-Vega, C., Capita, R., & Alonso-Calleja, C. Alvarez-Fern a (2013). Antimicrobial resistance in E. coli isolates from conventionally and organically reared poultry: a comparison of agar disc diffusion and Sensi Test Gram-negative methods. Food Control, 30, 227e234. Beutin, L., Krüger, U., Krause, G., Miko, A., Martin, A., & Strauch, E. (2008). Evaluation of major types of Shiga toxin 2e-producing Escherichia coli bacteria present in food, pigs and the environment as potential pathogens for humans. Applied and Environmental Microbiology, 74, 4806e4816. ~ as, L., Zarazaga, M., Sa nez, Y., Ruiz-Larrea, F., & Torres, C. (2002). b-lactamases in Brin ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrobial Agents and Chemotherapy, 46, 3156e3160. Cattoir, V., Poirel, L., Rotimi, V., Soussy, C. J., & Nordmann, P. (2007). Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBLproducing enterobacterial isolates. Journal of Antimicrobial Chemotherapy, 60, 394e397. Clinical and Laboratory Standards Institute (CLSI). (2012). Performance standards for antimicrobial susceptibility teststing. CLSI Document M100eS22. USA, PA: Clinical and Laboratory Standards Institute. , M., Jurcickova, Z., Literak, I., Pokludova, L., Bures, J., Hera, A., et al. (2011). Dolejska IncN plasmids carrying bla CTX-M-1 in Escherichia coli isolates on a dairy farm. Veterinary Microbiology, 149, 513e516. Duffy, G., Lynch, O. A., & Cagney, C. (2008). Tracking emerging zoonotic pathogens from farm to fork. Meat Science, 78, 34e42. European Food Safety Authority (EFSA). (2011). Panel on Biological Hazards (BIOHAZ); Scientifis opinion on the public health risks of bacterial strains producing
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