Antibiotic resistance of coagulase-negative staphylococci associated with food and used in starter cultures

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Abstract

The resistance of 330 coagulase-negative staphylococci (CNS) associated with food or used in starter cultures and belonging to the species Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus piscifermentans, Staphylococcus equorum, Staphylococcus succinus and Staphylococcus xylosus, against 21 antibiotics was determined using the disk diffusion method. The incidence and number of resistances was found to be species and source of isolation dependent. Most strains of S. equorum (63%), S. succinus (90%) and S. xylosus (95%) exhibited resistances against up to seven antibiotics, whereas only few strains of S. carnosus (12%) and S. piscifermentans (27%) were antibiotic resistant. Resistances to lincomycin, penicillin, fusidic acid, oxacillin, ampicillin and tetracycline were predominant. Among strains of S. xylosus, the incidence of resistance ranged from 22% for tetracycline up to 69% for penicillin. Concerning the source of isolation, resistances were often determined in strains of S. equorum, S. succinus and S. xylosus isolated from cheese (87%) and sausage (83%), and strains of S. xylosus obtained from meat starter cultures (93%). Remarkably, all CNS were sensitive to the clinically important antibiotics chloramphenicol, clindamycin, cotrimoxazol, gentamicin, kanamycin, linezolid, neomycin, streptomycin, synercid and vancomycin. The phenotypic resistances to ß-lactam antibiotics, lincomycin and tetracycline were verified by PCR amplification and could be traced back to the genes blaZ, lnuA and tetK, respectively. This study permitted a comprehensive insight into the incidence of antibiotic resistances in food-associated CNS.

Introduction

To date, the genus Staphylococcus contains 41 validly described species (DSMZ, 2008) that are traditionally grouped into coagulase-positive (CPS) and coagulase-negative staphylococci (CNS). Main habitats are skin, skin glands and mucous membranes of humans and animals, and only a few species are of special importance in foods and for human health. The CPS Staphylococcus aureus causes food intoxications and is involved in severe human infections, whereas the CNS Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus saprophyticus are opportunistic pathogens (Götz et al., 2006, Le Loir et al., 2003). Among CNS, the species S. carnosus (ssp. carnosus and utilis), S. condimenti, S. equorum (ssp. equorum and linens), S. piscifermentans, S. succinus (ssp. casei and succinus) and S. xylosus are either associated with foods or play a major role in the food processing industry. Strains of S. carnosus and S. xylosus are traditionally used in starter cultures for meat fermentations (Hammes and Hertel, 1998, Ordóñez et al., 1999). The other species are often isolated from fermented foods and therefore may have the potential for future application in starter or protective cultures (Bockelmann, 2002, Place et al., 2002, Place et al., 2003, Probst et al., 1998, Schlafmann et al., 2002, Tanasupawat et al., 1992).

Due to the intensive use of antibiotics in public health and animal husbandry, antibiotic resistance in pathogens including S. aureus has been an increasing medical problem during the last decades (Mazel and Davies, 1999). Research in recent years showed that resistances to antibiotics, including resistance to some antibiotics of therapeutic importance, also occur in strains of the important starter organism S. carnosus and S. xylosus (Gardini et al., 2003, Holley and Blaszyk, 1998, Kastner et al., 2006, Martín et al., 2006, Mauriello et al., 2000, Teuber et al., 1996), and in a few cases the underlying genes could be detected (Kastner et al., 2006, Perreten et al., 1997). Starter organisms are purposely added in high numbers (107–108CFU/g) to produce fermented foods. In addition, the CNS belonging to the microbiota of spontaneously fermented foods were shown to occur in numbers of 106 to 107CFU/g (Bockelmann and Hoppe-Seyler, 2001, Ercolini et al., 2003, Mauriello et al., 2004, Parente et al., 2001, Rantsiou et al., 2005, Rodriguez et al., 1996, Rossi et al., 2001, Teuber et al., 1996). Thus, enormous amounts of living bacteria are incorporated into the human body. Antibiotic resistances determinants contained in starter organisms or naturally occurring CNS may thus be transferred to commensals or pathogenic bacteria (Teuber et al., 1999). In addition, horizontal transfer of resistance gene was shown to occur in food (Teuber et al., 1999) and may be facilitated by the food matrix (Hertel et al., 1995). Therefore, the question arises regarding the contribution of food as a reservoir for the spread of antibiotic resistance (Franz et al., 2005, Teuber, 1999, Werner et al., 1997, Witte, 1999).

Recently, the European Food Safety Authority (EFSA) has undertaken the task to establish a concept for the safety assessment of microorganisms used in food and feed production. The proposed “qualified presumption of safety” (QPS) system (EFSA, 2004) is similar in concept and purpose to the GRAS system (Burdock and Carbin, 2004) in the USA. It is based on four pillars dealing with establishing identity, body of knowledge, possible pathogenicity and end use. With regard to possible pathogenicity, it is interesting to note that clinical isolates of S. carnosus, S. equorum, S. succinus and S. xylosus have recently been described (Couto et al., 2001, Dominguez et al., 2002, Novakova et al., 2006, Petinaki et al., 2001). This raises the question to the presence of virulence factors, or other potential disease-causing threats. In addition, horizontal transfer of antibiotic resistance determinants is considered an important safety issue. Although food isolates of S. xylosus and S. equorum have been described to produce enterotoxins (Bautista et al., 1988, Rodriguez et al., 1996, Vernozy-Rozand et al., 1996) and studies about the incidence of antibiotic resistances have been published, there is still a need to obtain sufficient knowledge about safety issues of food-associated CNS.

In this study the incidence of resistance against 21 antibiotics among 330 strains of food-associated CNS was investigated using the disk diffusion test according to the NCCLS guidelines. Phenotypic resistances were confirmed using PCR detection systems for the known antibiotic resistance genes in staphylococci.

Section snippets

Bacterial strains and growth conditions

In this study 330 CNS and the type strains of Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus equorum, Staphylococcus succinus, Staphylococcus piscifermentans and Staphylococcus xylosus were used. The CNS were isolated from various foods, commercial starter cultures and from patients in clinics. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 35218 served as reference strains for disk diffusion testing (NCCLS, 2003). S. xylosus VF5 (containing tetK gene, Perreten

Incidence of phenotypic antibiotic resistances and multiple resistances

Three-hundred-thirty food-associated CNS isolates were investigated for their susceptibility to antibiotics. The species affiliation of strains obtained from different working groups in Europe was verified by partial rRNA sequence analysis resulting in 106, 2, 11, 64, 10 and 137 strains of S. carnosus, S. condimenti, S. piscifermentans, S. equorum, S. succinus and S. xylosus, respectively. Twenty-one antibiotics were chosen according to the recommendations of NCCLS (2003) for testing clinical

Discussion

In this study, we showed that among food-associated staphylococci the incidence of resistant strains and the number of antibiotic resistances within strains are notably higher for S. xylosus, S. equorum and S. succinus (in the following named the S. xylosus-group) when compared to S. carnosus, S. condimenti and S. piscifermentans (in the following named the S. carnosus-group). This finding is in agreement with previous studies showing that strains of S. carnosus exhibit markedly less antibiotic

Acknowledgments

We thank M. Kranz for the excellent technical assistance during sequencing, Annika Schmidt for assistance in performance of disk diffusion testing and Labor Enders & Partner (Stuttgart, Germany) for advice and support. We are indebted to Charles M.A.P. Franz for critical reading of the manuscript. This study was supported by the Landesstiftung Baden-Württemberg (Project No. BA 150134).

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