Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding

Revista da Sociedade Brasileira de Medicina Tropical 45(5):579-585, Sep-Oct, 2012 Major Article Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding Tiane Martin de Moura[1], [2], Fabrício Souza Campos[3], Pedro Alves d’Azevedo[4], Sueli Teresinha Van Der Sand[1], [2], Ana Cláudia Franco[1], [3], Jeverson Frazzon[5] and Ana Paula Guedes Frazzon[1], [2] [1]. Programa de Pós-Graduação em Microbiologia Agrícola e do Ambiente, Departamento de Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. [2]. Laboratório de Bacteriologia, Departamento de Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. [3]. Laboratório de Virologia, Departamento de Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. [4]. Laboratório de Cocos Gram-Positivos, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS. [5]. Instituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. ABSTRACT Introduction: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. Methods: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. Results: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). Conclusions: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans. Keywords: Staphylococcal enterotoxin. Coagulase. Antimicrobial-resistance. INTRODUCTION Black pudding or blood sausage is a type of sausage, made from the blood, fat, and skin of cattle or pig, stuffed into natural or synthetic casing, and tied manually. This kind of sausage is very popular in south Brazil, Argentina, and Uruguay. Animal products are susceptible to microorganism contamination, and bacteria present in food could cause foodborne disease or be a possible route for the transfer of antimicrobial resistance to humans1,2. Members of the genus Staphylococcus are gram-positive cocci, and are natural inhabitants of the skin and mucous membranes of humans and animals. Currently, according to literature3, this genus comprises 45 species, which are divided into 2 groups: coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS), based on the ability to coagulate rabbit plasma. On the one hand, some CoNS species are components of the natural microbiota of food, and play an important role in the manufacturing processes of diverse meat-derived products; in particular, in dry fermented sausages, they act as starters to ensure the quality and safety of the final products4. On the other hand, CoPS and CoNS species, such as Staphylococcus aureus, Staphylococcus epidermidis, and S. saprophyticus, are well Address to: Dra. Tiane Martin de Moura. PPGMAA/Deptº Microbiologia/UFRGS. Av. Sarmento Leite 500/sala 209, 90050-170 Porto Alegre, RS, Brasil. Phone: 55 51 3308-3935; Fax 55 51 3308-4111 e-mail: Received in 12/09/2011 Accepted in 13/03/2012 www.scielo.br/rsbmt known for their implications in human health and disease. S. aureus is considered to be one of the most common pathogens responsible for the outbreaks in 1994 and 1998 in São Paulo (Brazil); in general, 51.5% of the outbreaks were caused by S. aureus5. In addition, the incidence of nosocomial infections caused by CoNS has increased in the last few years. In Brazil6 and the United States of America (USA), CoNS are the most common cause of nosocomial infections in the intensive care nursery. They are responsible for blood stream infections in neonates, also causing infections of the urinary tract, wounds, bloodstream, and the endocardium in immunocompetent individuals, where S. saprophyticus is the most prevalent species7. A prospective study was conducted from June 2001 to May 2002 in a hospital burn unit, with 252 patients; 49 (19.4%) of these developed clinically and microbiologically proven sepsis and the most prevalent bacteria were S. aureus and CoNS8. Perhaps the most notable virulence factors associated with staphylococci are the heat-stable enterotoxins (SEs) produced by certain strains. These toxins are a leading cause of gastroenteritis, including vomiting, abdominal cramping, diarrhea, and malaise, in 3–10 h following the consumption of preformed toxin by susceptible individuals9. The SEs are classified into 5 classical serological types: SEA, SEB, SEC1,2,3, SED, and SEE, but recently other enterotoxins were described in the literature, including SEG, SEH, SEI, SER, SES, SET and the enterotoxin-like proteins SElJ, SElK, SElL, SElM, SElN, SElO, SE1P, SE1Q, and SElU10. Among the CoPS, S. aureus is frequently responsible for outbreaks of food poisoning, due to its ability to express 7 different toxins. However, other CoPS, such as S. intermedius and Staphylococcus hyicus 579 Moura TM et al - Enterotoxin genes and antibiotic resistance in staphylococci from food can also express enterotoxins11. During the period from 1999 to 2009, 6,349 outbreaks of foodborne diseases were reported in Brazil, and 20.5% of these cases were caused by Staphylococcus spp. Furthermore, enterotoxigenic CoNS have also been isolated from the hands of food handlers and food, demonstrating the importance of CoNS in public health12,13. The use of antimicrobial agents in animal husbandry, as a growth promoter, has a selective effect in the emergence and maintenance of resistant bacteria in animals, a (...truncated)