Abstract
Sixty-seven staphylococcal isolates belonging to 12 species were obtained from 70 ready-to-eat food products. Staphylococcus aureus (n=25), and Staphylococcus epidermidis (n=13) were dominant. Susceptibility to penicillin, oxacillin, tetracycline, clindamycin, gentamicin, erythromycin, ciprofloxacin, and vancomycin was determined. All investigated S. aureus isolates were resistant to at least one antibiotic, and fifteen isolates were resistant to four and more antibiotics. Thirty-eight coagulase-negative staphylococci (CNS) isolates were resistant to at least one antibiotic, and seventeen to four and more antibiotics. Fifteen CNS isolates were mecA positive, and grew in the presence of 6 μg/mL oxacillin. All S. aureus isolates were mecA-negative. Arginine catabolic mobile element (ACME) was found in seven S. epidermidis isolates. Five S. epidermidis isolates harbored ica operon, ACME and were able to form biofilm. Three of them also possessed IS256 element and were mecA-positive. The expression of icaA gene was comparable in five ica-positive S. epidermidis isolates. One of six mecA positive S. epidermidis isolates was classified as sequence type (ST)155, one as ST110, and two as ST88. Two methicillin-resistant Staphylococcus epidermis (MRSE) belonged to new STs, that is, ST362, and ST363. Enterotoxin genes were found in 92% of S. aureus isolates. No enterotoxin gene was detected in analyzed CNS population. We show that ready-to-eat products are an important source of antibiotic-resistant CNS and potentially virulent strains of S. epidermidis, including genotypes undistinguishable from hospital-adapted clones.
Introduction
S
Materials and Methods
Bacterial strains
Seventy samples of ready-to-eat porcine, bovine, and chicken meat products were taken during a 4-month period from five supermarkets in Wrocław (Poland). Staphylococci were cultured on Giolitti-Cantoni broth and Baird-Parker agar. One CNS and/or one S. aureus isolate per food sample were characterized. The S. aureus and CNS isolates were identified by using catalase and coagulase tests. The CNS strains were identified by API STAPH 32 (bioMerieux, Warsaw, Poland) and 16S rDNA sequence analysis (htpp://rdna4.ridom.de/static/primer.html). Reference MRSA strain MICRO-PL 031515 and MSSA strain MICRO-PL 030950 were provided by Professor Waleria Hryniewicz of the National Institute of Medicines, Warsaw, Poland.
Preparation of bacterial DNA
DNA was prepared from 2 mL of bacterial suspension from an overnight culture in brain-heart infusion as described in Bania et al. (2006).
Antimicrobial resistance of staphylococci
Staphylococcal isolates were plated on Oxa-screen agar containing 6 μg/mL oxacillin and 4% NaCl (bioMerieux). The results were interpreted according to the EUCAST document E. Def. 3.1. mecA gene was detected according to Milheiriço et al. (2007).
Susceptibility to penicillin (10 units/disc), tetracycline (30 μg/disc), clindamycin (2 μg/disc), gentamicin (10 μg/disc), erythromycin (15 μg/disc), ciprofloxacin (5 μg/disc), and vancomycin (30 μg/disc) (Oxoid Ltd., Basingstoke, United Kingdom) was tested by the disk-diffusion method and interpreted according to the CLSI document M100-S20 (CLSI, 2010).
Detection of virulence-associated genes in staphylococci and biofilm formation of S. epidermidis
The detection of enterotoxin genes was as previously described (Bania et al., 2006). The presence of ACME, IS256, and ica cluster was confirmed according to Diep et al. (2008), Kozitskaya et al. (2004), and Cho et al. (2002), respectively. The expression of ica operon was determined by real-time polymerase chain reaction on iQ5 system (BioRad, Warsaw, Poland) as described by Conlon et al. (2002). The ability of S. epidermidis to form biofilm was tested as described by Jones et al. (1992).
Genotyping of S. epidermidis
Sequence type (ST) of mecA-positive S. epidermidis was determined according to Thomas et al. (2007). Assignment of the ST was performed by using the mlst.net platform.
Results and Discussion
Contamination of food by antibiotic-resistant staphylococci could be a prerequisite for further colonization of human nose or skin. Therefore, food has been considered a vehicle for its transmission.
Sixty-seven staphylococcal isolates belonging to 12 species were obtained from 70 ready-to-eat products. S. aureus (n=25) and S. epidermidis (n=13) constituted dominant species. The remaining species were Staphylococcus pasteuri (n=7), Staphylococcus hemolyticus (n=4), Staphylococcus carnosus (n=2), Staphylococcus saprophyticus (n=5), Staphylococcus sciuri (n=3), Staphylococcus chromogenes (n=2), Staphylococcus capitis (n=3), Staphylococcus xylosus (n=1), Staphylococcus equorum (n=1), and Staphylococcus lugdunensis (n=1). S. epidermidis and other CNS species including S. haemolyticus and S. capitis were found to be a common cause of device-associated infections in neonatal, oncology, and intensive care units (Kloos and Bannerman, 1994; Kozitskaya et al., 2004).
Antibiotic resistance is considered an important pathogenic trait of staphylococci (Jones et al., 1992). All S. aureus isolates investigated here were resistant to at least 1 antibiotic, and 15 isolates (60%) were resistant to 4 and more antibiotics. Thirty-eight CNS isolates (90%) were resistant to at least 1 antibiotic, and 17 (40%) to 4 and more antibiotics. None of the S. aureus isolates characterized in this work was mecA-positive, thus confirming that MRSA rarely occurs in foodstuffs. In turn, 15 (36%) of food-derived CNS harbored mecA gene. All S. sciuri, S. chromogenes, S. lugdunensis, two S. haemolyticus, and one S. pasteuri isolate harboring mecA gene grew in the presence of 6 μg/mL oxacillin. Six S. epidermidis isolates (46%) were classified as methicillin-resistant Staphylococcus epidermis (MRSE). Since ready-to-eat (RTE) food does not need thermal processing before consumption, it could serve as a vector for the spread of antibiotic-resistant microorganisms. Twenty-four S. aureus and 37 CNS isolates were resistant to penicillin. Eleven S. aureus and 18 CNS isolates were resistant to tetracycline. Erythromycin resistance was found in 14 S. aureus and 26 CNS isolates. Eight S. aureus and five CNS isolates were resistant to gentamicin. Eighteen S. aureus and eight CNS isolates were resistant to ciprofloxacin. Resistance to clindamycin was found in 12 S. aureus, and 12 CNS isolates. All staphylococci were susceptible to vancomycin.
The presence of enterotoxin-like genes was reported in CNS, but no enterotoxin gene could be detected in the population analyzed here. Among S. aureus, toxin genes were detected in 23 (92%) of the isolates. Twenty of them harbored the egc1, sec, and sell; four of these also possessed tst; and another one, selk. The remaining three harbored only selp gene.
S. epidermidis virulence factors are associated with the pathogenicity of hospital strains (Kloos and Bannerman, 1994). ACME was found in seven (54%) S. epidermidis isolates, from which four (67%) were mecA-positive. Four ACME-positive isolates harbored arc and opp-3 genes; two, arc gene; and one possessed only opp-3 gene. Five ACME-positive isolates harbored the ica operon, and formed biofilm, and three of these possessed IS256 (Table 1). The expression of icaA RNA was comparable in five analyzed isolates.
ACME, arginine catabolic mobile element; ST, sequence type.
ST was determined in the six MRSE. One of them was classified as ST155, one as ST110, and two as ST88. ST88, ST155, and ST110 had been already isolated from human patients (Miragaia et al., 2009). Two new allelic profiles were determined, that is, ST362 and ST 363. We provided the first data on genotypes of S. epidermidis from food.
Our results demonstrate that ready-to-eat food can be considered an important source of antibiotic-resistant staphylococci and potentially virulent strains of S. epidermidis. Some of the food-derived MRSE represent genotypes undistinguishable from hospital-adapted clones.
Footnotes
Acknowledgment
This work was supported by Wrocław University of Environmental and Life Sciences (project no. 404/GW/2010).
Disclosure Statement
No competing financial interests exist.