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Direct detection of antibiotic resistance genes inspecimens of chicken and pork meat

Cristiana Garofalo a, Carla Vignaroli b,⁎, Giada Zandri b, Lucia Aquilanti a, Donatella Bordoni a,Andrea Osimani a, Francesca Clementi a, Francesca Biavasco b

a Department of Food Sciences, Università Politecnica delle Marche, via Ranieri, Montedago, 60131 Ancona, Italyb Institute of Microbiology and Biomedical Sciences, Università Politecnica delle Marche, via Ranieri, Montedago, 60131 Ancona, Italy

Received 17 July 2005; received in revised form 19 May 2006; accepted 7 July 2006

Abstract

Antibiotic resistance (AR) in bacteria, a major threat to human health, has emerged in the last few decades as a consequence of the selectivepressure exerted by the widespread use of antibiotics in medicine, agriculture and veterinary practice and as growth promoters in animalhusbandry.

The frequency of 11 genes [tet(M), tet(O), tet(K), erm(A), erm(B), erm(C), vanA, vanB, aac (6′)-Ie aph (2″)-Ia, mecA, blaZ] encodingresistance to some antibiotics widely used in clinical practice was analysed in raw pork and chicken meat and in fermented sausages as well as infaecal samples from the relevant farm animals using a molecular approach based on PCR amplification of bacterial DNA directly extracted fromspecimens.

Some of the 11 AR genes were highly prevalent, the largest number being detected in chicken meat and pig faeces. The genes found mostfrequently in meat were tet(K) and erm(B); vanB and mecAwere the least represented. All 11 determinants were detected in faecal samples exceptmecA, which was found only in chicken faeces. erm(B) and erm(C) were detected in all faecal samples. The frequency of AR genes was notappreciably different in meat compared to faecal specimens of the relevant animal except for vanB, which was more prevalent in faeces.

Our findings suggest that AR genes are highly prevalent in food-associated bacteria and that AR contamination is likely related to breedingrather than processing techniques.

Finally, the cultivation-independent molecular method used in this work to determine the prevalence of AR genes in foods proved to be a rapidand reliable alternative to traditional tools.© 2006 Elsevier B.V. All rights reserved.

Keywords: Antibiotic resistance genes; Direct DNA recovery; Nested PCR; Meat samples

1. Introduction

The widespread use of antibiotics in human and veterinarymedicine and in livestock breeding is a cause of growing concern(Teuber, 1999; Schwarz et al., 2001; Normark and Normark,2002; Turnidge, 2004). Antimicrobial agents have been used infeeds as growth promoters in Europe (Wegener et al., 1999; Dritzet al., 2002) and North America (National Research Council,1999) for nearly half a century, contributing to the increasingemergence of resistant bacteria in humans (Witte, 1997; van den

Bogaard and Stobberingh, 2000; Gorbach, 2001; Phillips et al.,2004; Lu et al., 2004).

In Sweden, antimicrobial agents have been banned from useas growth promoters since 1986 (Wierup, 2001). Voluntarywithdrawal of all antimicrobial growth promoters from Danishbroiler production since February 1998 resulted in a significantdecline in resistance to avilamycin, erythromycin, vancomycinand virginiamycin (Emborg et al., 2003). In 1997 avoparcin,tylosin, spiramycin, bacitracin and virginiamycin were bannedfrom use in animal feeds in the European Union (World HealthOrganization, 2001) due to their structural relatedness to anti-microbial agents used in human medicine (Phillips et al., 2004).Discontinuation of these practices has resulted in decreased

International Journal of Food Microbiology 113 (2007) 75–83www.elsevier.com/locate/ijfoodmicro

⁎ Corresponding author. Tel.: +39 071 2204643; fax: +39 071 2204693.E-mail address: c.vignaroli@univpm.it (C. Vignaroli).

0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2006.07.015

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antibiotic resistance in animals, food products and humans insome European countries (Pantosti et al., 1999; Klare et al.,1999; Aarestrup et al., 2001; Phillips et al., 2004), though not incomplete eradication (Bager et al., 1999; Aarestrup et al., 2001;Johnsen et al., 2005).

The presence of antibiotic resistance (AR) genes in animalsand food raises even greater concern, since AR is carried bymobile genetic elements like plasmids, transposons and chro-mosomal cassettes (Salyers et al., 1995; Rowe-Magnus et al.,2001), which can promote intra- and inter-specific, and eveninter-generic transfer (Davies, 1994; Biavasco et al., 1996;

Gevers et al., 2003b). Bacterial ecosystems exist at differentlevels (environment, human and animal intestine, foods) wheresimple or complex routes of AR gene transfer among bacterialpopulations can occur (Teale, 2002). The transfer of resistancegenes to pathogenic or opportunistic bacteria poses a seriousthreat, since infections caused by these micro-organisms cannotbe treated with common antibiotics (Gorbach, 2001; Normarkand Normark, 2002; Phillips et al., 2004).

For several decades, studies of the selection and dissemina-tion of antibiotic resistances have mainly focused on clinicallyrelevant bacterial species. More recently, the hypothesis hasbeen advanced that saprophytic bacteria may act as reservoirs ofAR genes found in human pathogens (van den Bogaard andStobberingh, 2000; Gorbach, 2001). Such reservoirs can bepresent in the intestines of farm animals exposed to antibioticsand may thus contaminate raw meat even when hygienicstandards and regulations are complied with (Taylor, 1999;Gorbach, 2001; Sørensen et al., 2001; Sundsfjord et al., 2001).Non-pathogenic antibiotic-resistant bacteria like lactobacilli andenterococci are increasingly being isolated from poultry, swine,calf (Tannock et al., 1994; Lin et al., 1996; Giraffa, 2002; vanden Bogaard et al., 2002; Gevers et al., 2003a; Chingwaru et al.,2003; Harada et al., 2004) and from healthy human faeces(Aarestrup et al., 2000a; van den Bogaard and Stobberingh,2000). Bacteria involved in food fermentation and those used asstarter cultures may also constitute AR reservoirs (Giraffa,2002; Danielsen and Wind, 2003; Franz et al., 2003). Raw meatand fermented foods are therefore potential vehicles for the

Table 1Bacterial control strains used in this study

Bacterial strain AR gene Reference or source

S. pyogenes 7020 tet(M) Inst. colla

E. faecalis JH2-2TET tet(O) Giovanetti et al. (2003)E. faecium ET18 tet(K) Inst. colla

S. haemolyticus 955 erm(A) Inst. colla

E. faecium PE1 erm(B) Inst. colla

Staphylococcus spp. SE12 erm(C) Inst. colla

E. faecium BM4147 vanA Leclercq et al. (1988)E. faecalis ATCC 51299 vanB ATCCb

E. faecium M48 aac (6′)-Ieaph (2″)-Ia

Inst. colla

S. aureus 27R mecA Frank et al. (1995)S. aureus ATCC29213 blaZ ATCCb

a Inst. coll, collection of the Microbiology Institute, Università Politecnicadelle Marche, Ancona (Italy).b ATCC, American Type Culture Collection.

Table 2PCR primers used in this study

Target gene Primer sequence (5′→3′) Positiona Ta (°C) Product size (bp) Reference/accession no.b

tet(M) 1-GAACTCGAACAAGAGGAAAGC 595–615 62 740 Olsvik et al. (1995)2-ATGGAAGCCCAGAAAGG T 1334–13161a-ACCCGTATACTATTTCATGCACTc 325–347 48 1115 X929472a-CCTTCCATAACCGCATTTTGc 1439–1420

tet(O) 1-AACTTAGGCATTCTGGCTCAC 195–215 62 519 Olsvik et al. (1995)2-TCCCACTGTTCCATATGCTCA 709–689

tet(K) 1-TCGATAGGAACAGCAGTA 187–204 55 169 Ng et al. (2001)2-CAGCAGATCCTACTCCTT 355–338

erm(A) 1-TCTAAAAAGCATGTAAAAGAA 43–63 50 645 Sutcliffe et al. (1996)2-CTTCGATAGTTTATTAATATTAGT 687–6641a-CAGGAAAAGGACATTTTACCAAc 116–137 50 572 Sutcliffe et al. (1996)/X032162-CTTCGATAGTTTATTAATATTAGT 687–664

erm(B) 1-GAAAAGGTACTCAACCAAATA 43–63 54 639 Sutcliffe et al. (1996)2-AGTAACGGTACTTAAATTGTTTAC 681–658

erm(C) 1-TCAAAACATAATATAGATAAA 212–232 50 642 Sutcliffe et al. (1996)2-GCTAATATTGTTTAAATCGTCAAT 1125–1102

vanA 1-GGGAAAACGACAATTGC 176–192 58 732 Dutka-Malen et al. (1995)2-GTACAATGCGGCCGTTA 907–891

vanB 1-ATGGGAAGCCGACAGTC 174–190 58 635 Dutka-Malen et al. (1995)2-GATTTCGTTCCTCGACC 808–792

mecA 1-GGGATCATAGCGTCATTATTC 380–400 58 1429 Predari et al. (1991) and Murakami et al. (1991)2-AGTTCTGCAGTACCGGATTTGC 1808–1787

blaZ 1-ACTTCAACACCTGCTGCTTTC 652–672 58 240 Martineau et al. (2000)/M602532-TAGGTTCAGATTGGCCCTTAGc 889–869

aac (6′)-Ie 1-GAGCAATAAGGGCATACCAAAAATC 731–755 58 505 Kao et al. (2000)aph (2″)-Ia 2-CCGTGCATTTGTCTTAAAAAACTGG 1235–1211a The position given is from the first base of the coding sequence.b Reference or GenBank accession number of sequence takes into account the primer design.c Primer designed for this study.

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spread of antibiotic-resistant bacteria and/or antibiotic resis-tances along the food chain to the consumer (Sørensen et al.,2001; Hayes et al., 2003, Franz et al., 2003) raising majorconcerns with regard to food safety (Taylor, 1999; Aarestrupet al., 2000a; Donabedian et al., 2003; Franz et al., 2003).

In the present work, pork and chicken specimens collected inplants producing raw meat products and/or fermented sausageswere analysed for the presence of 11 genes encoding clinicallyrelevant AR by employing a method of detection based on PCRassays using DNA directly extracted from meat samples.Chicken and pig faeces collected in the same plants were alsoanalysed to assess the involvement of farm animal faecalcarriage in the occurrence of AR genes in foods.

2. Materials and methods

2.1. Bacterial control strains

The 11 antibiotic-resistant strains (each carrying one of theAR genes of interest) used as positive controls in PCR reactionsare listed in Table 1, together with their source. Enterococcusfaecalis JH2-2 (Jacob and Hobbs, 1974) was used as a negativecontrol in all PCR reactions.

2.2. Sampling

Sampling was carried out throughout 2002 at plants makingraw meat products and/or fermented sausages in Marche

(central Italy). Chicken carcass and chicken-and-pork skewerspecimens were obtained from two meat plants; pig carcass,minced pork and fresh and fermented sausage samples camefrom two producers of fermented sausages. Faeces wereobtained from the slaughterhouses of the same four plants bydissection of pig and chicken entrails. Overall, there were 108specimens, 88 meat (28 chicken, 46 pork and 14 skewer) and 20faecal (13 broiler and 7 pig) samples.

2.3. DNA extraction and quantification

For meat specimens, 10 g of each sample was homogenisedtwice in 10 ml of TE (10 mM Tris–HCl pH 7.8 and 1 mMEDTA) in a Stomacher apparatus (400 Circulator, InternationalPBI, Milan, Italy) at 260 rpm for 1 min. Bacterial DNA wasextracted from 1 ml of each homogenate essentially followingthe protocol described by Drake et al. (1996). After the secondcentrifugation the protocol was modified by treating pellets withlysozyme, RNase DNase-free and proteinase K in order toobtain complete lysis of bacterial cells, and then by adding880 mM Na perchlorate before the phenol–chloroform extrac-tion procedure. Skewer homogenates contained both chickenand pork.

For faecal specimens, we used the QIAamp DNA stool minikit (Qiagen S.p.A., Milan, Italy) following the manufacturer'srecommendations with minor modifications to enhance DNAyield. One gram of each sample was diluted 10-fold in0.9% NaCl solution and homogenised in the Stomacher

Table 3PCR nested primers used in this study

Target gene Primer sequence (5′→3′) Positiona Ta (°C) Product size (bp) Reference/accession no.b

tet(M) 3-CTTAGGAAAATGGGGATTCCc 346–365 48 1009 X929474-GCGGTGATACAGATAAACCc 1354–13361-GAACTCGAACAAGAGGAAAGC 595–615 62 512 Olsvik et al. (1995)/X929474a-GATGCCCTTTTGGAAATCTCc 1106–1087

tet(O) 3-TACCAGTGGTGCAATTGCAGAc 57–277 58 419 Y077804-TTATATGGGGATGCTGCCCAAc 675–655

tet(K) 3-GAACAGCAGTATATGGAAc 194–211 50 118 S674494-AAAAAGTGATTGTGACCAc 311–294

erm(A) 1a-CAGGAAAAGGACATTTTACCAAc 116–137 58 572 Sutcliffe et al. (1996)/X032162-CTTCGATAGTTTATTAATATTAGT 687–6643-CTATAGAAATTGATGGAGGCTTAc 170–192 58 518 Sutcliffe et al. (1996)/X032162-CTTCGATAGTTTATTAATATTAGT 687–664

erm(B) 3-CAATTCCCTAACAAACAGAGGc 262–282 60 420 Sutcliffe et al. (1996)/U863752-AGTAACGGTACTTAAATTGTTTAC 681–658

erm(C) 3-GTAATTTCGTAACTGCCATTc 596–615 52 502 M179904-GCATGTTTTAAGGAATTGTTc 1097–1078

vanA 3-GTAGGCTGCGATATTCAAAGC 358–378 60 231 Bell et al. (1998)4-CGATTCAATTGCGTAGTCCAAT 588–567

vanB 3-GGTGCGATACAGGGTCTGTTc 313–332 58 479 U004564-GGAATGTCTGCTGGAACGATc 791–772

mecA 3-AAAATCGATGGTAAAGGTTGGC 1276–1297 55 533 Murakami et al. (1991)2-AGTTCTGCAGTACCGGATTTGC 1808–1787

blaZ 1-ACTTCAACACCTGCTGCTTTC 652–672 58 173 Martineau et al. (2000)4-TGACCACTTTTATCAGCAACC 824–804

aac (6′)-Ie 1-GAGCAATAAGGGCATACCAAAAATC 731–755 62 220 Kao et al. (2000)/M13771aph (2″)-Ia 4-GCCACACTATCATAACCACTc 950–931a The position given is from the first base of the coding sequence.b Reference or GenBank accession number of sequence takes into account the primer design.c Primer designed for this study.

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apparatus at 200 rpm for 30 s. The filtrate was centrifuged at5000 rpm for 15 min, the pellet was suspended in ASL buffer(included in the kit) and collected by centrifugation. After lysisof bacterial cells, PCR inhibitor compounds of faecal origin wereremoved and DNAwas purified according to the manufacturer'sinstructions.

DNA was quantified using the DyNA Quant 200 kit(Amersham Biosciences GE Healthcare, Milan, Italy) and theDyNA Quant 200 Fluorometer fluorescence reader (AmershamBiosciences) according to Teare et al. (1997).

To establish the successful recovery of bacterial DNA and theabsence of PCR inhibitor substances, a preliminary PCR assaytargeting the conserved regions of 16S rRNA was carried outusing the Y1–Y2 primer pair (Young et al., 1991). Briefly, theamplification reaction was performed in a total volume of 50 μlcontaining 20 ng DNA and the following mix components: 1×reaction buffer (DiaTech S.r.l., Jesi, Italy), 0.6 U Taq polymerase(DiaTech S.r.l.), 0.2 mM dNTPs, and 1.5 mM MgCl2. Initialdenaturation was performed at 94 °C for 3 min, followed by30 cycles of: 94 °C for 45 s, 55 °C for 45 s and 72 °C for 60 s, andfinal extension at 72 °C for 7 min. The 16S rDNA ampliconswere analysed on 1.5% agarose (Bio-Rad Laboratories,Richmond, CA) gel, using a 100-bp ladder (M-medical, Milan,Italy) as a molecular weight standard.

2.4. PCR and nested PCR for amplification of AR genes

Five μl of the DNA extract, corresponding to about 10–20 ngof bacterial DNA, or 5 μl of the PCR product was used for PCRand nested PCR assays in a total volume of 50 μl containing: 1×buffer [0.5 M KCl and 0.1 M Tris–HCl (pH 8.3)], 50 pmol ofeach primer, 2.5 or 3.0 mM MgCl2, (3.0 mM MgCl2 for theamplification of erm genes in both assays and of aac (6′)-Ie aph(2″)-Ia, abbreviated as aac-aph, in nested PCR), 0.1 or 0.25 mM

dNTPs (depending on MgCl2 concentration) and 1.5 U hot-startTaq DNA polymerase (AmpliTaq Gold, Applied Biosystem,Foster City, CA). PCR assays (35 amplification cycles) includedheating at 95 °C for 10 min and final extension at 72 °C for7 min. Thermal cycling for the different genes was optimised asfollows. PCR for tet(M), tet(K) and tet(O): 2 min at 94 °C, 1 minat 48–62 °C, 2 min at 72 °C; for erm(A), erm(B), erm(C): 1 minat 94 °C, 1 min at 50–54 °C, 1 min at 72 °C; for vanA, vanB,blaZ, mecA, aac-aph: 30 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C.Nested PCR for tet(M), tet(K), aac-aph, blaZ, mecA, erm(B)and erm(C): 30 s at 94 °C, 30 s at 50–62 °C, 30 s at 72 °C; forerm(A): 1 min at 94 °C, 1 min at 58 °C, 1 min at 72 °C; for vanA:50 s at 94 °C, 45 s at 60 °C, 50 s at 72 °C; for tet(O) and vanB:30 s at 94 °C, 30 s at 58 °C, 45 s at 72 °C. Positive (Table 1) andnegative (E. faecalis JH2-2) controls were used in each PCRprocedure together with two reagent blanks (PCRmixture addedwith water or TE buffer employed in the DNA extraction step).

Primer pair sequences and position, target genes, annealingtemperature and amplicon size are listed in Tables 2 and 3 forPCR and nested PCR assays, respectively. The primers designed

Table 4Chicken and pork samples giving positive results after PCR and nested PCR assays

Sample category Assay No. of positive samples

tet(M) tet(O) tet(K) erm(A) erm(B) erm(C) vanA vanB mecA blaZ aac-aph

Poultry meat (28) PCRa 11 11 15 19 14 11 20 0 0 20 12n-PCRb 17 15 23 23 21 24 28 5 2 23 16

Pork carcass portions (12) PCRa 2 0 8 0 12 5 4 0 0 8 4n-PCRb 12 8 12 7 12 10 6 0 0 10 7

Pork minced meat (12) PCRa 1 0 10 0 10 6 1 0 0 10 4n-PCRb 12 4 12 5 12 9 7 1 0 12 7

Fresh sausages (13) PCRa 0 0 12 2 12 6 5 0 0 11 6n-PCRb 13 9 13 7 13 10 7 0 0 13 9

Fermented sausages (9) PCRa 1 0 9 0 9 1 3 0 1 9 3n-PCRb 9 3 9 4 9 7 6 0 1 9 8

Skewers (14) PCRa 1 0 7 1 14 7 1 0 0 8 2n-PCRb 14 8 12 4 14 12 7 0 1 11 9

Total meat (88) 77 47 81 50 81 72 61 6 4 78 56Chicken faeces (13) PCRa 4 4 2 7 13 9 7 5 0 10 3

n-PCRb 9 5 6 12 13 13 11 7 1 12 6Pig faeces (7) PCRa 7 7 2 7 7 5 2 6 0 4 5

n-PCRb 7 7 6 7 7 7 7 6 0 7 7Total faeces (20) 16 12 12 19 20 20 18 13 1 19 13a PCR: positive after PCR.b n-PCR: positive after PCR and nested PCR.

Fig. 1. Comparison of the frequencies of the different AR genes in meat andfaeces. meat samples;▪ faecal samples.

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for this study were based on the sequences published in GenBank(accession numbers in Tables 2 and 3) using the Primer3 software(Whitehead Institute for Biomedical Research, Cambridge, MA).

All assays were carried out in a Gene Amp 9700thermocycler (Applied Biosystem). Amplicons were analysedon 1–2% agarose gel (Bio-Rad) using a 50-bp, a 100-bp, or a 1-kb ladder (all from M-medical), depending on amplicon size, asa molecular weight standard. Gels were run at 3 V/cm (constantvoltage) in 0.5× TBE (45 mM Tris, 45 mM boric acid, 1 mMEDTA) and stained with 0.5 μg/ml ethidium bromide. Digitalimages were viewed and captured using the Image Master VDS

(Amersham Biosciences) equipped with the Multi-Analystsystem (Bio-Rad).

2.5. Detection limits of the PCR assays

The limits of detection of PCR and nested PCR protocolswere tested on meat samples for all the AR genes studied,whereas for mecA, vanA and tet(M) they were also assessed onfaecal samples. To determine the protocols' detection limit, foreach gene a negative sample was randomly selected for spiking.For those genes for which all meat samples gave a positive

Fig. 2. AR gene frequency in meat and faecal samples. meat samples; ▪ faecal samples.

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result, a raw meat specimen aseptically collected from pigmuscle was used for spiking. In both cases, 25-g aliquots of thesample were suspended in 225 ml peptone-water (Oxoid Ltd.,Basingstoke, England) and homogenised twice at 260 rpm for1 min. For each gene, 9 ml aliquots of the homogenates wereautoclaved and separately inoculated with 1 ml of 10-fold serialdilutions (from 10−1 to 10−8) obtained from overnight culturescontaining about 109 CFU ml−1 of antibiotic-resistant strainsused as positive controls (Table 1). Spiked samples wereimmediately subjected to DNA extraction as described above,and 5 μl of the resulting suspension was used as a template inPCR assays. Viable cells actually inoculated into eachhomogenate were counted on Brain Heart Infusion Agar(BHIA, Oxoid) plates. The detection limit of each PCR andnested PCR assay was defined as the lowest spike (CFU/ml ofsample) yielding a positive result.

2.6. Statistical analysis

The frequencies of the different AR genes were calculated asthe ratio of positive samples to total number of samples. Thefrequencies of the different AR genes within meat and faecessamples, and of each AR gene in faeces to meat were com-pared using Fisher's test. Statistical analysis was performed withthe S-PLUS 6 statistical program (S-PLUS 6.1 for Windows,Professional Edition, Release 1). Pb0.05 was regarded asstatistically significant.

3. Results

3.1. AR gene frequency in meat and faecal samples

Eighty-eight samples of chicken and pork meat and 20 faecalsamples from the same animals were screened by PCR andnested PCR for the presence of genes encoding resistance tosome antibiotics widely used in clinical practice, i.e. tetracy-cline, erythromycin, vancomycin, penicillin, methicillin andgentamicin. The AR genes tet(M), tet(O), tet(K), erm(A), erm(B), erm(C), vanA, vanB, blaZ and aac-aph were selectedbecause they are found both in food of animal origin and inhuman specimens (Sundsfjord et al., 2001; Aarestrup et al.,2000a,b). mecAwas also included to evaluate a role for food inthe spread of human MRSA infection. Results are reported inTable 4.

As expected, nested PCR yielded a larger number of positivesamples. This increment was much greater in meat specimens.The largest increment was observed for tet(M), which wasdetected in all pork and skewer samples, followed by tet(O) anderm(A). Of the latter, tet(O) was never detected using PCR,whereas erm(A) was detected in fresh sausage and in chicken-and-pork skewers. mecA and vanB showed a very low preva-lence even after the nested PCR assays.

In meat, tet(K) and erm(B) were the predominant genes(frequency, 0.92) and vanB and mecA were the least abundant(0.07 and 0.045, respectively) (Fig. 1). In particular, vanAwas theprevalent gene in chicken meat (Pb0.05) compared to tet(O),tet(M), erm(B), vanB, aac-aph and mecA, as was recovered

from all samples, followed by erm(C), blaZ, erm(A), tet(K), anderm(B) (Fig. 2A). As regards pork, tet(M), tet(K) and erm(B)were detected in all samples followed by blaZ. Statistical analysisyielded significant results (Pb0.05) for tet(M), tet(K) and erm(B)against erm(A), vanB, andmecA in all types of pork meat, and forblaZ against erm(A), vanB, and mecA in minced meat and insausages (Fig. 2B–E). VanB was detected only in minced pork(Fig. 2C) and mecA only in fermented sausages (Fig. 2E).

In skewers, erm(B) and tet(M) were the prevalent genes inall samples (Pb0.05 against tet(O), erm(A), vanA, vanB, aac-aph and mecA), followed by tet(K) and erm(C), and finallyblaZ (Fig. 2F).

All 11 determinants were present in faeces except mecA,which was found only in chicken faeces (frequencyb0.1). erm(B) and erm(C) were found in all samples, followed by erm(A),blaZ, and vanA at frequencies ranging from 0.84 to 0.92 inchicken faeces (Fig. 2G) and at a frequency of 1.0 in pig faeces(Fig. 2H). The remaining AR determinants exhibited differentfrequencies that depended on animal origin. Overall, AR geneswere more prevalent in pig (Fig. 2H), all pig faeces samplesbeing positive for tet(M), tet(O), erm(A) and erm(B) even afterthe first PCR assay (Table 4). In chicken faeces (Fig. 2G) theerm genes and blaZ were the most frequent (Pb0.05) comparedto mecA, aac-aph, tet(O) and tet(K), while the prevalence ofvanA was significant (Pb0.01) only against mecA.

To assess the transmission of AR genes from animals tomeat, we compared their frequency in faecal to meat samplesand found that tet(K) was significantly more frequent (Pb0.05)in meat and erm(A), erm(C) and vanB in faeces (Fig. 1).

3.2. Detection limits of the PCR assays

Analysis of the limits of detection of the PCR protocols forthe 11 AR genes (Table 5) showed that in meat samples theyranged from 102 [erm(C)] to 108 CFU/ml [tet(K), erm(A) and

Table 5Detection limits of PCR and nested PCR protocols targeting the different ARgenes

AR genes PCR (CFU/ml)a Nested PCR (CFU/ml)a

Meat samplestet(M) 105 1tet(O) 107 104

tet(K) 108 10erm(A) 108 103

erm(B) 103 10erm(C) 102 100

aac (6′)-Ie aph (2″)-Ia 108 102

blaZ 103 100

mecA 105 102

vanA 106 100

vanB 105 10

Faecal samplestet(M) 107 102

vanA 107 102

mecA 107 102

a Lowest spike of bacterial control strain detectable by PCR or nested PCRprotocols.

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aac-aph]. When the tet(M), vanA and mecA protocols weretested on faecal samples, all showed a detection limit of107 CFU/ml.

The nested PCR protocols yielded visible amplicons for bothmeat and faecal samples when homogenates were spiked with100 to 104 CFU/ml of the bacterial control strains. Detectionlimits ranged from 100 to 102 CFU/ml for all AR genes but erm(A) (103 CFU/ml) and tet(O) (104 CFU/ml). On the whole, PCRwith nested primers allowed improvement on the results of PCRby 102 [erm(C)] to 107 [tet(K)] times.

4. Discussion

The widespread and indiscriminate use of antibiotics inhuman and veterinary medicine and in livestock breeding hasled to a spread of AR among both pathogenic and commensalmicro-organisms (Witte, 1997; Phillips et al., 2004). In par-ticular, use of antibiotics as livestock growth promoters appearsto have created large reservoirs of AR genes with the potentialto be transferred both horizontally and vertically. The actualsignificance of these findings for public health is however stillunclear (Witte, 2000; Franz et al., 2003; Phillips et al., 2004;Turnidge, 2004). The same antibiotic-resistant bacterial cloneshave been described in food and human specimens, eitherclinical and from the community (Bates et al., 1994; Sundsfjordet al., 2001; Gelsomino et al., 2002; Franz et al., 2003), and thesame AR genes have been isolated from human and food strains(Schwarz et al., 2001; Sundsfjord et al., 2001; Donabedianet al., 2003).

This study was undertaken to establish the presence of ARgenes in pork and chicken products, which are staples in humandiet and could constitute a significant route for the spread ofresistances to clinically important antibiotics. The highprevalence in both meat and faeces of most of the AR genesinvestigated in this study deepens our concern.

Resistance genes to macrolides and tetracyclines were themost prevalent in our specimens. Among the former, the mostfrequent was erm(B). Its detection even before nested PCRsuggests the existence of several copies of this gene, in line withits location on transposable elements (Clewell et al., 1995) aswell as with the frequent presence of erythromycin-resistantbacteria in animals and meat (Aarestrup et al., 2000a; van denBogaard et al., 2002). This finding may also be related to the lowdetection limit of our PCR protocol. With regard to tetracyclineresistance, tet(M) and tet(K) were the most prevalent genes inpork meat, with tet(M) being detected in all samples includingskewers.

These findings suggest a prevalence of erm(B)- and tet(M)-carrying micro-organisms in both pigs and pork processinglines, and confirm the diffusion of tetracycline resistance (Ng etal., 2001; Villedieu et al., 2003) among Gram-negative (van denBogaard and Stobberingh, 2000) as well as Gram-positivegenera (Chopra and Roberts, 2001; Gevers et al., 2003a).Localization of tet(M) and erm(B) on conjugative transposonsof the Tn916–Tn1545 family (Clewell et al., 1995) and of tet(K) on small plasmids (Oppegaard et al., 2001) in closeassociation with insertion sequences (Yazdankhah et al.,

2000) could contribute to the high prevalence of these genesin our samples.

The high frequency of vanA in chicken samples (both faecesand meat) could be due to the persistence of vanA plasmids infarms and factories, in line with previous data (Aarestrup et al.,2001; Johnsen et al., 2005), whereas the very low prevalence ofvanB in all meat products correlates with the low rate ofisolation of vanB enterococci from both food and clinicalspecimens in Europe (Robredo et al., 2000; Del Grosso et al.,2000; Goossens et al., 2003).

The lack of appreciable differences in the frequency ofseveral AR genes between faeces and meat suggests that theflow of AR genes from animals to meat does not take place inthe factory (through surface contamination, processing orhandling) but is more likely related to breeding techniques.Accordingly, in chicken faeces and meat the prevalent ARgenes were erm(A), erm(B), erm(C), vanA and blaZ, whilemecA was the least frequent. In pig faeces and pork theprevalent genes were tet(M) and erm(B), whereas mecA wasdetected exclusively in fermented sausages. A different dis-tribution between faecal and meat specimens was observed inchicken for vanB, which was much more frequent in faeces, andfor tet(K), which was detected more often in meat. Even thoughthese differences were not significant, they are nonethelessimportant and are probably to be ascribed to the small studysample. When all faecal specimens were compared with allmeat products independently of their origin, a significant dif-ference emerged not only for tet(K) and vanB but also forerm(A) and erm(C), the last three being more frequent infaeces. The prevalence of vanB in faeces could be attributed toDNA from intestinal micro-organisms that did not survive theprocessing phase. Similarly, the decrease in the number ofsamples positive for tet(O), erm(C) and vanA from pig faecescompared to pork meat suggests their carriage by AR intestinalmicro-organisms progressively overcome by food-associatedbacteria, including those used as starters, which survive morereadily in meat.

The absence of mecA in pig faeces and pork products butfermented sausages suggests contamination by mecA staphylo-cocci of human origin during meat processing that may beascribed to the fact that methicillin is exclusively used in humanmedicine and to the high salt tolerance of staphylococci.

Although use of tetracycline, virginiamycin and avoparcin asgrowth promoters in feeds was banned five years ago, resis-tances to antibiotics of the same classes (tetracyclines,macrolides and glycopeptides) are still highly prevalent. Ourfindings suggest the persistence of resistant clones, probablydue to a selective pressure towards multi-resistant geneticelements, related to supplementation of animal feeds withlicensed antimicrobials, metals (Hasman and Aarestrup, 2005)or disinfectants (Schweizer, 2001; Aiello and Larson, 2003). Aphysical linkage between AR genes and genes conferring otherselective advantages reported for glycopeptides (Johnsen et al.,2005) may also explain these findings.

Use of nested PCR improved detection of AR genes by up to107 times with respect to PCR and afforded reliable compar-ison of frequencies thanks especially to more homogeneous

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detection limits, which ranged between 100 and 102 CFU/ml formost genes.

Although PCR and nested PCR assays do not allow todiscriminate between AR genes from viable and non-viablemicro-organisms and free DNA, use of a molecular methodproved to be a rapid and reliable alternative to traditional toolsto determine the prevalence of antimicrobial resistance genes infoods. One major advantage is that it allows to overcome theproblems associated with isolation and cultivation of bacteriafrom meat. The molecular approach allows indirect, but simpleand reliable screening of food samples for the presence of ARfood-associated bacteria. A combination of cultivation-inde-pendent and isolation approaches is warranted when moreaccurate evaluation of AR diffusion from animals to man via thefood chain is required.

Acknowledgements

This work was supported by “Fondazione Cassa diRisparmio di Verona, Vicenza, Belluno e Ancona”.

We are grateful to Dr. Sandra Torriani of the Scientific andTechnological Department, University of Verona (Italy), forproviding some references strains and to Dr. Luigi Ferrante forassistance with statistical analysis.

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