Antibiotic resistance genes and identification of staphylococci collected from the production chain of swine meat commodities

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1 Food Microbiology 25 (2008) Short communication FOOD MICROBIOLOGY Antibiotic resistance genes and identification of staphylococci collected from the production chain of swine meat commodities Desj Simeoni a, Lucia Rizzotti a, Piersandro Cocconcelli b,c, Simona Gazzola b,c, Franco Dellaglio a, Sandra Torriani a, a Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, Verona, Italy b Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, Piacenza, Italy c Centro Ricerche Biotecnologiche, Università Cattolica del Sacro Cuore, Via Milano 26, Cremona, Italy Received 29 November 2006; received in revised form 20 March 2007; accepted 6 September 2007 Available online 15 September Abstract Staphylococci harbouring antibiotic resistance (AR) genes may represent a hazard for human health and, as other resistant foodrelated bacteria, they contribute to the spread of AR. In this study, we isolated resistant staphylococci from an entire swine production chain and investigated the occurrence of 11 genes [aac(6 0 )Ie-aph(2 00 )Ia, blaz, meca, vana, vanb, erma, ermb, ermc, tet(m), tet(o) and tet(k)] encoding resistance to some antibiotics largely used in clinical practice. The 66 resistant staphylococcal isolates were identified as Staphylococcus epidermidis (27 isolates), Staphylococcus aureus (12), Staphylococcus xylosus (12), Staphylococcus simulans (5), Staphylococcus pasteuri (4), Staphylococcus carnosus (3), Staphylococcus lentus (2) and Staphylococcus sciuri (1). Specific-PCR detection of AR genes showed the prevalence of the tet(k) gene in most of the isolates (89.4%), followed by tet(m) and ermc (about 75%); meca was detected in more than half of S. aureus and S. epidermidis isolates. The genes vana and vanb were not retrieved. It was found that a high proportion of coagulase-positive and -negative isolates are multidrug-resistant and some of them carry up to six AR genes. Our findings show that the swine production chain is a source of antibiotic-resistant staphylococci suggesting the importance of resistance surveillance in the food production environment. r 2007 Elsevier Ltd. All rights reserved. Keywords: Antibiotic resistance genes; PCR detection; Food chain; Staphylococcus; Multidrug resistance 1. Introduction Staphylococci are ubiquitous Gram-positive bacteria that represent part of the normal bacterial microflora of the skin and mucosal surfaces of humans and animals. Some staphylococcal species, such as Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus saprophyticus, are well known for their implications in human health diseases (Yugueros et al., 2000). On the other hand, some species of coagulase-negative staphylococci (CNS) are considered technologically important in the manufacturing processes of various meat-derived products, especially dry fermented sausages, where they are used as Corresponding author. Tel.: ; fax: address: sandra.torriani@univr.it (S. Torriani). starters to ensure the quality and safety of the final product (Aymerich et al., 2003). In the last decades, the spread of antibiotic resistance (AR) in bacteria, including staphylococci, is increasing and may represent a hazard for human health. Among antibioticresistant staphylococci, multidrug-resistant S. aureus strains are of great public concern since resistances make more difficult the treatment of infections. Moreover, a number of CNS, such as several S. epidermidis strains, are important hospital-acquired infection agents and the 80 90% of these isolates are methicillin-resistant (de Mattos et al., 2003). Transfer of antibiotic-resistant bacteria to humans (or their AR genes to pathogens) via the food chain has already been reported (Angulo et al., 2004; Phillips et al., 2004). Some studies have evidenced the spreading of resistant bacteria, such as staphylococci, enterococci and lactic acid bacteria, during meat processing (Gevers et al., /$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi: /j.fm

2 D. Simeoni et al. / Food Microbiology 25 (2008) ; Huys et al., 2005; Rizzotti et al., 2005) and also the transfer of AR determinants in natural microenvironments between bacteria of different origins (Cocconcelli et al., 2003). Thus, food-related bacteria, including the components of starters, have the potential to serve as a reservoir of AR genes with the hazard of transferring these determinants to other commensal or pathogenic species. Up to now, many researches have focused on the spread of resistant S. aureus in clinical environments (Marchese et al., 2000; Roberts et al., 2000; da Silva Coimbra et al., 2003), whereas a restricted number of investigations, or Antimicrobial Resistance Research Programs (e.g. DAN- MAP), have regarded the presence in food of antibioticresistant S. aureus (Huys et al., 2005) and resistant CNS (Perreten et al., 1998). The aim of this study was to evaluate the occurrence of staphylococci resistant to various antimicrobial agents in the entire production chain of swine meat commodities, from animal farming to final foods. Resistant staphylococcal isolates were collected and examined for the presence of 11 AR genes by means of specific PCRs; species identification and genetic correlation among the isolates were determined, as well. 2. Materials and methods 2.1. Isolation of antibiotic-resistant staphylococci Staphylococci were isolated from samples obtained from various steps of the production chain of two factories producing swine meat commodities located in the North of Italy. The analysed samples included: eight faecal specimens collected from individual pigs; eight dry feedstuffs; 14 raw and processed pork meat products (raw unprocessed pork products, carcasses, raw minced pork, fresh sausages) after processing in the slaughterhouses, and six dry fermented sausages ripened at the same factories. Each sample was suspended in sterile 0.9% NaCl, then an aliquot of this suspension was added to Trypticase Soy Broth (Oxoid Italia, Milan, Italy) plus 4% NaCl and incubated overnight at 37 1C. Aliquots of this culture were streaked on selective plates containing Mannitol Salt Agar (MSA, Oxoid) supplemented with one of the following antibiotics: erythromycin (final concentration 16 mgml 1 ), gentamycin (32 mgml 1 ), oxacillin (4 mgml 1 ), penicillin G (0.5 mgml 1 ), tetracycline (32 mgml 1 ), vancomycin (4 mgml 1 ), according to the breakpoint values defined by NCCLS (2004). After incubation (37 1C; 24 h), representative colonies were picked and subcultured in tubes containing Brain Heart Infusion (BHI; Oxoid) added with the antimicrobial agent at the same concentration used for isolation. The antibiotic-resistant isolates were purified and maintained at 80 1C in 25% (v/v) glycerol Bacterial strains and DNA extraction The staphylococcal isolates were deposited in the laboratory culture collection. S. aureus subsp. aureus DSM T, S. epidermidis DSM T, Staphylococcus carnosus subsp. carnosus DSM T and Staphylococcus xylosus DSM T were used as reference strains. Total genomic DNA was extracted and purified from 2-ml overnight cultures as described by Marmur (1961) Identification and typing of staphylococci Selected presumptive staphylococci were identified by internal transcribed spacer-pcr (ITS-PCR) as described by Jensen et al. (1993) and following the amplification conditions of Mendoza et al. (1998). The amplicons were resolved by agarose (3%, w/v) gel and each profile was visually compared with those obtained from the staphylococcal reference strains. The identification of isolates was then confirmed either by species-specific PCRs for S. aureus, S. epidermidis, S. carnosus and S. xylosus (Martineau et al., 1996, 1998; Aymerich et al., 2003) or by 16S rrna gene partial sequencing. A portion of the 16S rrna gene was amplified by PCR as previously described (Rizzotti et al., 2005). The PCR products were purified by using the Wizard SV Gel and PCR Clean-Up system according to the package insert (Promega Corporation, Madison, WI) and sequenced at Biomolecular Research (BMR) Centre, University of Padova, Italy. The BlastN program software was used for nucleotide sequence analysis. The RAPD typing method was carried out with the primer D8635 on DNA extracted from the isolates as described by Akopyanz et al. (1992). The patterns were analysed using Gel Compar 4.0 software (Applied Math, Kortrijk, Belgium) PCR detection of antimicrobial resistance genes The presence of genes involved in resistance to aminoglycosides [aac(6 0 )Ie-aph(2 00 )Ia gene], beta-lactams (blaz, meca), glycopeptides (vana, vanb), macrolide-lincosamidestreptogramins (erma, ermb, ermc) and tetracyclines [tet(m), tet(o), tet(k)] was determined in the isolates by PCR amplification using the specific primers and the conditions reported by Rizzotti et al. (2005) Determination of phenotypic antimicrobial resistance The resistances of staphylococci to specific antibiotics were checked streaking each isolate on MSA plates added with the antimicrobial agents at the concentrations reported above. Minimum inhibitory concentrations (MICs) were also determined for some antimicrobial agents by the agar dilution method (NCCLS, 2004) on Isosensitest agar plates (Oxoid) added with 10% BHI.

3 198 ARTICLE IN PRESS D. Simeoni et al. / Food Microbiology 25 (2008) Results and discussion 3.1. Isolation and identification of resistant staphylococci Growth of antibiotic-resistant staphylococci was observed for 22 out of 36 food and food-related matrices and a total of 66 resistant staphylococcal isolates were considered for a deeper investigation on their taxonomic position and AR pattern. The combination of different genetic approaches was applied to accurately identify the isolates. A preliminary identification at the species level was obtained with the ITS-PCR assay. However, very similar 16S 23S rdna ITS patterns can be displayed by diverse staphylococcal species (Blaiotta et al., 2003). Consequently, species-specific PCRs and/or 16S rrna partial sequencing were applied to confirm the identification. The occurrence and distribution of Staphylococcus species among the resistant isolates are shown in Table 1. S. epidermidis was the predominant species and was isolated from all kinds of analysed matrices except dry fermented sausages. All the S. aureus isolates were found only in fresh sausages, these meat products are generally at low risk, with respect to consumer health, if properly cooked. On the other hand, S. xylosus was the unique species that was isolated from dry fermented sausages, probably because of its better adaptation to the environmental conditions prevailing in these commodities Prevalence of AR genes among resistant staphylococcal isolates Each staphylococcal isolate was tested for the presence of the 11 AR genes. The PCR amplification results are summarised in Table 2. All except for six staphylococcal isolates held more than one AR gene and a high proportion of these staphylococci (72.7%) carried almost four different AR genes. In particular, three S. epidermidis isolates carried six AR genes that confer resistance to five different antibiotics. Table 1 Identification and origin of the antibiotic-resistant staphylococcal isolates Species No. of isolates (%) No. of isolates from isolation matrix Faeces Feedstuff Raw meat Processed meat Dry fermented sausage S. epidermidis 27 (40.9) S. aureus 12 (18.2) 12 S. xylosus 12 (18.2) S. simulans 5 (7.6) 5 S. pasteuri 4 (6.1) 4 S. carnosus 3 (4.6) 3 S. lentus 2 (3.0) 2 S. sciuri 1 (1.5) 1 Total : Not detected. Table 2 Incidence of AR genes in the antibiotic-resistant staphylococcal isolates Species No. of isolates No. of positive isolates a tet(m) tet(o) tet(k) erma ermb ermc aac6- aph2 b blaz meca S. epidermidis S. aureus S. xylosus S. simulans S. pasteuri S. carnosus S. lentus S. sciuri AR incidence (%) : Not detected. a vana, vanb genes were not detected in any of the isolates. b Shortening of aac(6 0 )Ie-aph(2 00 )Ia.

4 D. Simeoni et al. / Food Microbiology 25 (2008) These results confirm the large spread of multidrugresistant bacteria, including staphylococci, which can be isolated from different environments, such as clinical, animal and food samples (Santos Sanches et al., 2000; Khan et al., 2005). The fact that 72.7% of our isolates carried two tetracycline resistance determinants reveals a great diffusion of this type of resistance. The carriage of multiple tet genes was commonly found in individual Gram-positive bacteria (Schwarz et al., 1998; Huys et al., 2005; Rizzotti et al., 2005). The high incidence of tet(k) and tet(m) genes in the isolated staphylococci, 89.4% and 74.2%, respectively, can be explained by their usual genetic locations. In fact, the presence of tet(k) gene on small multicopy plasmids and tet(m) on conjugative transposons (Tn916 Tn1545 family) contributes to the spread of these determinants (Chopra and Roberts, 2001). Furthermore, we found tet(o) only in one S. epidermidis strain; even Schwarz et al. (1998) observed tet(o) only in few staphylococcal isolates and Schmitz et al. (2001) did not retrieved this gene in tetracycline-resistant S. aureus. A large number (75.8%) of the resistant staphylococci isolated here harboured ermc gene. This gene is frequently located on small multicopy plasmids (Khan et al., 2002), which are present in many different staphylococcal species. The ermb gene is often carried by conjugative transposons (Khan et al., 2002) and this could explain the diffusion of ermb (56.1%) in the staphylococci isolated in this study. Conversely, the erma gene was detected in a lower number of isolates. The resistance to aminoglycosides [aac(6 0 )Ie-aph(2 00 )Ia gene] was found only in seven isolates belonging to the S. epidermidis species, mainly derived from animal faeces; indeed this gene is usually more diffused in staphylococci of human origin (Werckenthin et al., 2001). The blaz gene for beta-lactam resistance was detected in 63.6% of the staphylococci, including all S. aureus isolates and 77.8% of the S. epidermidis isolates, indicating that this determinant is widely spread among the CNS, too. The resistance to methicillin (oxacillin), encoded by meca gene, was detected in 36.4% of the analysed staphylococci, comprising 58.3% of the S. aureus isolates and 40.5% of the isolated CNS (44.4% of S. epidermidis strains). The meca determinant may cause severe problems in humans for the treatment of methicillin-resistant S. aureus (MRSA) (Lee, 2003) and, in recent years, for S. epidermidis infections, too (Kitao, 2003). In this study, the vana and vanb genes for vancomycin resistance were not detected. Such antibiotic was the only uniformly effective antimicrobial treatment for staphylococcal infections but several cases of vancomycin-resistant staphylococci, including S. epidermidis with a low-level (8 16 mgml 1 ) resistance, have been published (Krcmery and Sefton, 2000). In our study, a S. epidermidis strain showed a vancomycin MIC of 6 mgml 1. Probably, the same mechanism of resistance retrieved in vancomycinintermediate S. aureus was involved in the reduced susceptibility of this strain (Hanaki et al., 1998). A study of the French National Monitoring Program (RESABO) showed that CNS were less susceptible to antimicrobial agents than coagulase-positive staphylococci (Werckenthin et al., 2001). Conversely, our results showed a comparable pattern of AR genes between CNS and S. aureus isolates. Since the plates used for isolation contained only one antimicrobial agent, the resistance pattern of each staphylococcal isolate was established for the other antibiotics. A close correspondence between resistance and PCR results was found for erythromycin, gentamycin, penicillin G and tetracycline. Instead, this correlation was not observed for several isolates that carry meca gene in the presence of oxacillin as previously described by Martineau et al. (2000) that reported the occurrence of S. aureus strains with the meca gene but susceptible to oxacillin Strain typing To determine molecular epidemiological relatedness among staphylococcal isolates belonging to the same species, RAPD patterns were generated. The profiles obtained from the S. aureus isolates are shown in Fig. 1. Numerical analysis of the RAPD profiles revealed a large genetic polymorphism among the staphylococcal isolates, especially among S. aureus and S. epidermidis, even if high similarity percentages were given by some of them isolated from the same matrix (data not shown). These data were combined with the results of the AR gene screening showing the absence of clonal strains. As a consequence no relevant contamination events have Fig. 1. RAPD profiles obtained with the primer D8635 from S. aureus isolates (lanes 1 12) and reference strain (lane 13). Lanes M: DNA molecular size marker (1 kb plus DNA ladder, Invitrogen, Italy).

5 200 ARTICLE IN PRESS D. Simeoni et al. / Food Microbiology 25 (2008) occurred between different steps of the production chain under consideration, even if a higher number of strains should be analysed to confirm this. In conclusion, this investigation provided evidences of the wide occurrence of AR genes in resistant staphylococci derived from various steps of the production chain of swine meat commodities. It emerged that both coagulase-positive and -negative resistant staphylococci carry a number of AR genes demonstrating the spread of multidrug resistance in bacteria of food origin too, and the importance of resistance surveillance in the food production environment. Acknowledgement This study was supported by the Fondazione Cassa di Risparmio di Verona, Vicenza, Belluno e Ancona. References Akopyanz, N., Bukanov, N.O., Westblom, T.U., Kresovich, S., Berg, D.E., DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. 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