Staphylococci isolated from ready-to-eat meat – Identification, antibiotic resistance and toxin gene profile

doi:10.1016/j.ijfoodmicro.2016.09.001

International Journal of Food Microbiology

Volume 238, 5 December 2016, Pages 113-120

https://doi.org/10.1016/j.ijfoodmicro.2016.09.001 Get rights and content

Highlights

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Staphylococci isolated from ready-to-eat meat were analyzed.
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All isolates were identified as coagulase-negative staphylococci.
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S. equorum and S. vitulinus were the most prevalent species.
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68% of isolates exhibited antibiotic resistance.
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80% of isolates harbored SAgs genes.

Abstract

The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance.

Introduction

The genus Staphylococcus is divided into 52 species and 28 subspecies that are grouped into coagulase-positive (CPS) and coagulase-negative staphylococci (CNS) (http://www.bacterio.net). Most staphylococcal species are harmless and reside normally on the skin and mucous membranes of humans and other organisms (Chajęcka-Wierzchowska et al., 2014, Dubois et al., 2010, Guran and Kahya, 2015, Podkowik et al., 2012, Podkowik et al., 2016). There are also well known staphylococcal species responsible for a wide variety of diseases of animals and humans (van Duijkeren et al., 2008).

The pathogenic capacity of staphylococci is attributed to a combination of invasive properties, production of extracellular factors and antibiotic resistance. Staphylococcal toxins include toxic shock syndrome toxin 1 (TSST-1), exfoliative toxins (ETA to ETD), staphylococcal enterotoxins (SEs; SEA to SEE, SEG to SEI, SER) with demonstrated emetic activity, and staphylococcal enterotoxin-like (SEl) proteins. All the toxins listed above possess superantigenic activity and were designated as staphylococcal superantigens (SAgs) (Bukowski et al., 2010, Fowoyo and Ogunbanwo, 2016, Stich et al., 2010, Vasconcelos et al., 2011). Staphylococcal food poisoning is an intoxication that results from the consumption of foods containing sufficient amounts of one (or more) of the above-mentioned toxins (Dinges et al., 2000, Le Loir et al., 2003). Enterotoxigenic CNS strains, including organisms responsible for outbreaks of food poisoning have also been described by several authors (Batista et al., 2013, Podkowik et al., 2016, Vasconcelos et al., 2011, Zell et al., 2008). Additionally, it was reported that CNS may be a possible reservoir of enterotoxin genes typically identified in S. aureus (Ławrynowicz-Paciorek et al., 2007, Vasconcelos and da Cunha, 2010).

In addition to toxin production, an important factor determining the pathogenicity of staphylococci is associated with their antibiotic resistance. In recent years, a systematic growth in the number of antibiotic-resistant staphylococcal strains in the human environment has been observed. Most research concerning antibiotic resistance of staphylococci isolated from food focuses on S. aureus, whereas less attention is paid to other species (Gao et al., 2012). Antibiotic-resistant strains other than S. aureus were also found in food (Gardini et al., 2003, Guran and Kahya, 2015, Martin et al., 2006, Podkowik et al., 2012) and genes encoding microbial resistance to tetracycline, erythromycin and β-lactams have been detected in CNS isolated from starter cultures, probiotic bacteria, fermented food and meat (Chajęcka-Wierzchowska et al., 2014, Guran and Kahya, 2015, Simeoni et al., 2008). Also in this case, different species of staphylococci have been suggested as a reservoir of antibiotic resistance genes (Chajęcka-Wierzchowska et al., 2014, Kloos and Bannerman, 1994, Neu, 1992) which can be transferred to S. aureus, making it resistant to multiple agents (Al-Masaudi et al., 1991).

Humans are the most important source of staphylococci, especially S. aureus and S. epidermidis but also S. hominis, S. haemolyticus, S. saprophyticus, S. capitis, S. warneri, S. simulans and S. cohnii. Because human skin staphylococci are not indigenous microbiota of raw foods, contamination is mainly associated with improper handling of cooked or processed foods. Food handlers may contaminate raw materials, equipment and finished products via manual contact or through respiratory secretions (Kadariya et al., 2014, Katsaras et al., 1985). Air, dust and food contact surfaces can also serve as vehicles in the transfer of staphylococci to foodstuffs (Bhatia and Zahoor, 2007).

Food-borne diseases are of major concern worldwide (Kadariya et al., 2014). Among the predominant bacteria involved in food-borne diseases, staphylococci (especially S. aureus) are a principal cause of gastroenteritis resulting from the consumption of contaminated food (Le Loir et al., 2003). In this case, meat and meat products have been one of the most frequently reported types of food involved in such outbreaks (Hennekinne et al., 2012, Van Loo et al., 2007). Currently, a great popularity of ready-to-eat meat products that are obtained and cut into appropriate portions or slices at the store upon request can be observed. Such products can be purchased in small quantities and eaten relatively quickly, which may reduce the risk of pathogenic microorganisms growing in the food. On the other hand, such food products are touched repeatedly by shop staff and also come into frequent contact with various equipment and store surfaces, which may be conducive to their contamination.

Therefore, the aim of this study was to analyze the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store upon request, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated.

Section snippets

Bacterial isolates

Bacteria were isolated from 45 ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami, pork luncheon meat, sliced upon request in 5 randomly selected butcher shops in Szczecin, Poland. A 10 g portion of each food sample was homogenized in 90 ml buffered peptone water (1%, w/v Graso, Poland), incubated overnight at 37 °C and 0.1 ml was plated on Mannitol Salt Agar medium (MSA, Graso, Poland), which allowed to culture all currently described species of staphylococci.

Results

The PCR used in this study enabled amplification of all the genes investigated including gap, mecA, ses, sels, eta, etd, and tst-1 in DNA from control S. aureus strains (Fig. 1). The PCR products from the positive controls were equal to those previously described by Holtfreter et al. (2007), Oliveira and de Lencastre (2002) and Yugueros et al. (2000). For no-template and negative controls, no amplicon was generated and nonspecific reactions were not observed.

Discussion

In view of the large number of cases in which staphylococci are implicated in outbreaks of food-related infections and food poisoning (Batista et al., 2013, Chajęcka-Wierzchowska et al., 2015, Fowoyo and Ogunbanwo, 2016, Podkowik et al., 2016, Vasconcelos et al., 2011, Zell et al., 2008), the present article provides the analysis of antibiotic resistance of a variety of staphylococci isolated from ready-to-eat food sliced at the store upon the customer's request and shows the possible risk of

Conclusions

In conclusion, the present study further extends previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance, serving as a reservoir of antibiotic resistance and SAgs genes. The high prevalence of CNS carrying staphylococcal enterotoxin genes also highlights the potential for food poisoning caused by these species.

Acknowledgments

The authors would like to thank the Dean of the Faculty of Biotechnology and Animal Husbandry (Grant No. 517-01-027-3323/17) for providing financial support to this project (young researcher grant for K. Fijałkowski). We would also like to thank Professor Bröker from the University of Greifswald in Germany for providing the reference staphylococcal strains and Ms. Bogna Rusak from the West Pomeranian University of Technology, Szczecin, Poland for her contributions during isolation and

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Staphylococci isolated from ready-to-eat meat – Identification, antibiotic resistance and toxin gene profile

Highlights

Abstract

Introduction

Section snippets

Bacterial isolates

Results

Discussion

Conclusions

Acknowledgments

Int. J. Food Microbiol.

J. Food Prot.

Food Microbiol.

Vet. J.

J. Food Prot.

Syst. Appl. Microbiol.

Lancet

Int. J. Food Microbiol.

Int. J. Food Microbiol.

Pediatr. Clin. N. Am.

Vet. Microbiol.

Int. J. Food Microbiol.

Int. J. Food Microbiol.

Int. J. Food Microbiol.

Food Microbiol.

Vet. Microbiol.

Int. J. Food Microbiol.

FEMS Microbiol. Lett.

Antimicrobial resistance and gene transfer in Staphylococcus aureus

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Exfoliative toxins of Staphylococcus aureus

Toxins

Occurrence and antibiotic resistance of enterococci in ready-to-eat food of animal origin

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J. Clin. Microbiol.

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Microbiol. Immunol.

Exotoxins of Staphylococcus aureus

Clin. Microbiol. Rev.

Identification of a variety of Staphylococcus species by matrix-assisted laser desorption ionization-time of flight mass spectrometry

J. Clin. Microbiol.