Detection of enterotoxin genes of Staphylococcus sp isolated from nasal cavities and hands of food handlers

Brazilian Journal of Microbiology (2010) 41: 59-65 ISSN 1517-8382 DETECTION OF ENTEROTOXIN GENES OF STAPHYLOCOCCUS SP ISOLATED FROM NASAL CAVITIES AND HANDS OF FOOD HANDLERS Rall, V.L.M.1*; Sforcin, J.M.1; Augustini, V.C.M.1; Watanabe, M.T.1; Fernandes Jr., A.1; Rall, R.2; Silva, M.G.3; Araújo Jr., J.P.1 1 Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brasil; 2 Faculdade de Tecnologia, Botucatu, SP, Brasil; 3 Departamento de Patologia, Universidade Estadual Paulista, Botucatu, SP, Brasil. Submitted: May 08, 2008; Returned to authors for corrections: April 20, 2009; Approved: September 28, 2009. ABSTRACT Food handlers, an important factor in food quality, may contain bacteria that are able to cause foodborne disease. The present study aimed to research coagulase-negative (CNS) and -positive staphylococci (CPS) in 82 food handlers, analyzing nasal and hand swabs, with identification of 62 CNS (75.6%) and 20 CPS strains (24.4%). Staphylococcal enterotoxins genes were investigated by PCR. In 20 CPS strains, 19 were positive for one or more genes. The percentage of CNS presenting genes for enterotoxins was high (46.8%). Despite of the staphylococcal species, the most common gene was sea (35.4%), followed by seh and sej (29.2%). The detection of new staphylococcal enterotoxins (SEs) genes showed a higher pathogenic potential in this genus. The presence of these gene points out the importance of CNS not only as contaminant bacteria but also as a pathogen. Key words: Staphylococcal enterotoxins, coagulase-negative staphylococci, S. aureus, food handler. INTRODUCTION manipulation of food by carriers of this microorganism (7). The nasal mucosa has been described as the most important source Food handlers contribute to food safety, being potential of propagation, being colonized in the first days of life Among sources of bacteria that causes foodborne diseases due to the staphylococci, S. aureus is considered the most important to introduction of pathogens during its processing, distribution man and can be found in the nasal mucosa in 20 to 50% of and manipulation (1). adults in cutaneous pleats, armpits, as well as in inguinal and Staphylococcus aureus, produces enterotoxins and is considered one of the greatest causes of intoxication although perineal areas (33). The staphylococcal enterotoxins are considered its found persistently or temporarily in human nasal superantigens, characterized by simultaneous connections to microbiota, without causing any symptoms. The presence of the major complex of histocompatibility class II in an antigen these bacteria in food occurs frequently due to inappropriate presenting cell and T cell receptors, without the presence of *Corresponding Author. Mailing address: Department of Microbiology and Immunology, Institute of Biosciences, Sao Paulo State University - UNESP. Post Office Box 510. 18618-000, Botucatu, Sao Paulo, Brazil.; Tel. (+5514) 3811-6240 Fax (+5514) 3815-3744.; Email: 59 Rall, V.L.M. et al. specific antigens, resulting in systemic effects such as high fever, vomiting, diarrhea, as well as hepatic and renal dysfunctions (6). Staphylococcal isolation and identification (13) After incubation, five black colonies from each plate (presence and absence of halo) were identified. The screening Classic antigenic SE have been identified as SEA, SEB, tests used were Gram staining and production of catalase and SEC1, SEC2, SEC3, SED and SEE (4). Ren et al. (26) have coagulase. The coagulase-positive strains were submitted to the sequenced the gene of toxin H. In 1998, Munson et al. kit "Staphytect Test Dry Spot" (Oxoid). The two positive identified and characterized seg and sei genes and Zhang et al. clumping species were submitted to the VP test (S. aureus- (34) found the gene sej in the same plasmid that encoded sed. positive and S intermedius-negative). Recently, several other toxins have been described and their The coagulase-negative staphylococcal isolates were genes have been sequenced, known as SEK, SEL, SEM, SEN, identified using API Staphy and according to Kloos and SEO, SEP, SEQ, SER and SEU (8,11, 14, 21,22,23). Besides Bannerman (10), with ornithine decarboxylase presence, β SEH, SEI and SEG, which present emetic activity (17,30), the hemolysis production, urea degradation, novobiocin resistance involvement of other SEs in foodborne outbreaks is not clear and anaerobic thioglycolate growth. Sensibility tests for yet. Other coagulase-positive staphylococci (CPS) species, bacitracin (0.04U) and furazolidone (100 µg) were performed such as S. hycus, S. intermedius and several coagulase-negative before API, in order to separate CNS from Kocuria, according staphylococci (CNS), have also been involved in cases of to Bannerman & Peacock (2). outbreaks (32). The present work aimed to investigate the presence of coagulase-positive and -negative staphylococci in nasal mucosa and hands of food handlers at industrial kitchens in the city of DNA isolation For DNA isolation, a GFX commercial kit (GE Healthcare) was used, according to supplier instructions. Botucatu and to detect the genes responsible for SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ production. Detection of genes encoding staphylococcal enterotoxins Primers used in the detection of SE genes are listed in MATERIAL AND METHODS Table1. PCR amplifications were composed of 2.5 µL PCR Buffer Material for analysis 10x (Invitrogen), 1.0 µM of MgCl2 (Invitrogen), 200 µM dNTP Botucatu is a small town and there are only 3 industrial (Invitrogen), 1 U of Taq DNA Polymerase (Invitrogen), 10 kitchens with a significant number of workers. Apparently, picomols of each primer, 3 µL of the DNA sample, and sterile they are clean but didn’t follow GHPs or HACCP ultrapure water in order to reach 25 µl (qsp) (Milli-Q Plus, implemented. Millipore). Samples were collected from the hands (interdigital PCR protocol was performed in PTC-100 (MJ region, indexfingers, thumbs and palms of both right and left Research, Inc., USES) using the following amplification hands) and anterior nares of 82 food handlers, distributed cycles: initial denaturation for 5 minutes at 94ºC and 30 cycles among 3 kitchens in the city, with a moist swab (saline), during at 94ºC for 2 minutes for denaturation and 72ºC for 1 minute meal preparation. One swab was used in each region. for extension. The various temperatures used in the annealing The swabs were streaked on Baird-Parker plates (Difco) step are shown in Table 1. Final extension was performed at immediately after collection. As soon as possible (until 1 hour), 72ºC for 5 minutes. The PCR-amplified samples were analyzed these plates were incubated at 35ºC for 48 hours in our by electrophoresis for 30 minutes at 125V (Electrophoresis laboratory. Power Supply Model EPD 600 - Amersham-Pharmacia 60 Enterotoxi (...truncated)