2541 Journal of Food Protection, Vol. 70, No. 11, 2007, Pages 2541–2548 Copyright ᮊ, International Association for Food Protection Comparison of Antibiogram, Staphylococcal Enterotoxin Productivity, and Coagulase Genotypes among Staphylococcus aureus Isolated from Animal and Vegetable Sources in Korea JIN SAN MOON,1 AE RI LEE,2 SEUNG HYEUP JAW,3 HYUN MI KANG,1 YI SEOK JOO,1 YONG HO PARK,4 MAL NAM KIM,2 AND HYE CHEONG KOO4* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/11/2541/1681190/0362-028x-70_11_2541.pdf by guest on 29 September 2021 1Department of Bacteriology, National Veterinary Research and Quarantine Service, Anyang, Gyeonggi-do, Republic of Korea; 2Department of Biology, Sangmyung University, Seoul, Republic of Korea; 3Department of Biotechnology, Pulmuone, Seoul, Republic of Korea; and 4KRF Zoonotic Disease Priority Research Institute, Department of Microbiology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul, Republic of Korea MS 07-207: Received 18 April 2007/Accepted 5 July 2007 ABSTRACT Staphylococcal food poisoning is caused by enterotoxin-producing Staphylococcus aureus. We investigated the prevalence of such organisms in samples of bovine mastitic milk (n ϭ 714), raw meat (n ϭ 139), and vegetables (n ϭ 616). We determined the degrees of relatedness of isolates as indicated by antibiogram, staphylococcal enterotoxin (SE) productivity, and coagulase gene restriction fragment length polymorphism analysis. We examined 297 S. aureus isolates and found SE production in 57 (31.8%), 4 (7.8%), and 49 (73.1%) isolates from raw milk, raw meat, and vegetables, respectively. A high proportion of the isolates obtained from milk produced more than two types of toxins (mainly SEA, SEB, and/or SEC), whereas isolates from raw meat and vegetables primarily produced SEA alone. Most isolates were sensitive to cephalothin (97.6%), gentamicin (80.8%), erythromycin (79.5%), and tetracycline (72.7%), but were resistant to penicillin (90.2%) and ampicillin (88.9%). The proportion of antibiotic-resistant isolates differed according the source of the bacteria; the milk and vegetable isolates were more resistant to penicillin and ampicillin than were the meat isolates (P Ͻ 0.05), whereas tetracycline resistance was limited to the milk and vegetables isolates. The coagulase genotypes (I to XII) varied with the source of the organism, and only a few genotypes prevailed in each source: II (42.4%) and IV (24%) types in isolates from milk, IX (35.3%) and XI (45%) from raw meat, and III (40.3%) and XII (32.8%) from vegetables. These findings suggest that remarkable differences exist in antibiogram, SE productivity, and coagulase genotypes, resulting in limited clonal transmission of S. aureus into various food sources. As enterotoxin production only occurs when S. aureus grows to high numbers, staphylococcal food poisoning can be prevented by proper refrigeration. Staphylococcus aureus is recognized worldwide as a duced food poisoning. The SEs produced by S. aureus or- common cause of infection in humans and animals. S. au- ganisms are thought to be the primary virulence factors reus produces a spectrum of exotoxins and other determi- associated with this illness (5). Because of their importance nants of virulence that contribute to its pathogenicity. in terms of public health and food safety, an efficient meth- Staphylococcal enterotoxins (SEs) are recognized as being od of screening livestock and vegetables for enterotoxins is the most important virulence factors involved in cases of required. food poisoning in humans (6). In Korea, food poisoning A total of 18 kinds of SE genes (sea to see, seg to ser, outbreaks occurred primarily by consumption of meat and seu) have been found (39), and SEA, SEB, SEC, and (27.9%), shellfish and its processed products (26%), and SED are strongly correlated with invasive S. aureus infec- ready-to eat meals (24%), such as kimbap and packed lunch tions in humans (6). Unlike well-known SE types (sea to boxes, according to the food poisoning statistic data re- see), the newly described SE genes (seg to seu) lack some ported by the Korean Food and Drug Administration be- traits causing emesis in primates or have not been investi- tween 2000 and 2002 (22). In fact, 9.62% of food poisoning gated yet, and these are named staphylococcal enterotoxin– outbreaks in Korea have been caused by S. aureus, the third like proteins. In Korea, SEA, temperature-stable SEB, and most common pathogen being outnumbered only by Vibrio SEH have been the most frequently recovered SEs from parahaemolyticus and Salmonella species (32). cases of food poisoning, and seh was always detected with Milk products (12), raw meat (including uncooked sea or seg in 59.6% (198 of 332) of all S. aureus intoxi- smoked ham) (3), and vegetarian food products (13) are cations in Korea (8, 15). reported to be associated with S. aureus enterotoxin-in- A number of molecular epidemiological studies have been conducted on the enterotoxin-producing S. aureus iso- * Author for correspondence. Tel: ϩ82-2-880-1288; Fax: ϩ82-2-871- lated from bovine mastitic milk and other foods, as well as 7524; E-mail: [email protected]. from humans, in order to detect reservoirs and sources of 2542 MOON ET AL. J. Food Prot., Vol. 70, No. 11 infection, and to monitor the spread of S. aureus within, TABLE 1. Overview of Staphylococcus aureus strains isolated in among, and beyond specific animal populations (6). PCR- this study based coagulase genotyping by restriction fragment length S. aureus–positive polymorphism (RFLP) analysis of the 3Ј end of the gene Samples samplesa encoding staphylococcal coagulase has been suggested as a simple and effective method for typing S. aureus isolates Type No. No. % for use in epidemiological studies (1, 18). In previous stud- Livestock Milk, rawb 714 179 25 ies, we used PCR-RFLP to carry out the coagulase geno- products Beef, raw 46 16 35 typing of enterotoxin-producing S. aureus organisms iso- Pork, raw 46 17 37 lated from bovine mastitic milk in Korea (33). But the pub- Chicken, raw 47 18 38 lished data on the use of this typing method to distinguish Subtotal 853 230 27 among S. aureus strains isolated from different types of Vegetables Lettuce 84 16 19 livestock and vegetables in Korea are scarce. Bean sprouts 60 13 22 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/11/2541/1681190/0362-028x-70_11_2541.pdf by guest on 29 September 2021 The overall frequency of nosocomial infections caused Green bean sprouts 60 9 15 by methicillin-resistant S. aureus (MRSA) has increased Spinach 48 6 13 and is currently estimated to exceed 64 to 72% (430 of 682 Sprouted vegetables 42 6 14 and 18,750 of 26,042) in Korea (26, 32). Although several Young radish 24 3 13 studies on the isolation of MRSA from raw chicken meat Chinese cabbage 48 2 4 or bovine mastitic milk (29, 30) have been reported with a Others 250 12 5 low frequency of MRSA in livestock, information regard- Subtotal 616 67 11 ing the isolation and characteristics of MRSA from animals Total 1,469 297 20 and vegetable sources is limited. Therefore, the aim of this a study was to investigate the prevalence of enterotoxin-pro- Only one S. aureus strain could be isolated from each S. aureus– ducing S. aureus organisms in bovine mastitic milk, raw positive sample. b Bovine mastitic milk. meat (pork, beef, and chicken), and vegetables in Korea and to determine their phenotypic and genetic relatedness by antibiogram, SE productivity, and coagulase genotype grew on this agar with the formation of characteristic zones and patterns. rings due to lipolysis, proteolysis, and tellurite reduction, were selected to obtain pure colonies to be grown on brain heart infu- MATERIALS AND METHODS sion agar (Oxoid). The isolates were initially identified by the method described by Roberson et al. (37), which included gram Sampling of bovine mastitic milk, raw meats, and vege- staining, a coagulase test, a DNase test, a Voges-Proskauer test, tables, and bacterial identification. The milk was obtained from and a mannitol fermentation test. The isolates were finally con- 714 dairy cows with mastitis that were being raised on 52 farms firmed using a Vitek gram-positive identification card (bio- located across the South Korea (Table 1). The milk samples, Me´rieux, Marcy l’Etoile, France). The isolates were kept refrig- which were collected between January 2003 and December 2004 erated at Ϫ70ЊC until further use. according to the protocol described by the National Mastitis Coun- Detection of enterotoxin production in S. aureus. SE pro- cil (16), contained more than 200,000 somatic cells per ml, which duction was detected using a SET-RPLA (staphylococcal entero- was measured by a Milkoscan 4000 (Foss Electric Co., Hillerød, toxin A, B, C, and D detection by reverse passive latex aggluti- Denmark). A 10-␮l aliquot was taken from each sample and nation) kit according to the manufacturer’s instructions (Oxoid). spread over blood agar plates (Bacto-Agar; Difco, Becton Dick- inson, Sparks, Md.) containing 5% washed sheep erythrocytes and Antibiotic susceptibility test. The MICs of methicillin and incubated at 37ЊC for 24 h. Colonies suspected of being staphy- vancomycin resistance and the resistance to other antibiotics using lococci were subcultured on blood agar plates. a disk diffusion test on Mueller-Hinton agar (Difco, Becton Dick- A total of 139 samples of raw meat (46 beef, 46 pork, and inson) were determined as recommended by the Clinical and Lab- 47 chicken samples) were collected at random from supermarkets oratory Standards Institute (CLSI) (10). Commercially available or conventional markets in large cities in Korea, including Seoul, disks (BBL, Becton Dickinson, Sparks, Md.) were used as rec- Busan, Daejon and Gwangju, between May and August 2004.