Biotyping and enterotoxigenicity of coagulase-positive and coagulase-negative staphylococci isolated from different raw meat

Document Type : Full paper (Original article)


1 Department of Food Hygiene, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

2 Graduated from Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

3 Department of Medical Microbiology, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran


Background: Staphylococcal enterotoxins (SEs) are produced in foods under favorable conditions and considered a potential biological threat. Aims: The study was performed to detect enterotoxigenic genes of A and B in coagulase-positive (CPS) and coagulase-negative staphylococci (CNS) to evaluate biotypes and antibiotic resistance of isolated Staphylococcus aureus from different meat. Methods: A total of 160 meat swab samples were collected from lamb, water buffalo, cattle, and chicken carcasses. Presumptive colonies on Baird Parker agar were subjected to biochemical identification, including Gram staining, catalase, oxidase, and coagulase activity. Relevant colonies separately were subjected to the polymerase chain reaction (PCR) assay for identification of Staphylococcus genus, enterotoxigenic genes (sea and seb) and the thermonuclease gene (nuc) specific for the S. aureus. The antibiotic susceptibility test was also carried out using five antibiotics. Results: Totally, 150 Staphylococcus spp. were isolated from the samples among which 135 (90%) isolates harbored sea gene, meanwhile, none of the isolates contained seb gene. Twenty-five S. aureus confirmed by PCR from which 15 isolates (60%) belonged to host specific (HS), 7 isolates (28%) belonged to non-host specific (NHS) biotypes, while 3 isolates (12%) were non-typable. Overall, 68%, 56%, 16%, 12%, and 8% of isolates were resistant to penicillin, trimethoprim, gentamicin, oxacillin, and erythromycin, respectively. Conclusion: The meats provided from this area were contaminated with enterotoxigenic and antibiotic-resistance staphylococci, which can threaten the health of the consumers. The study showed that not only CPS contain enterotoxin A gene, but also CNS isolates possess this gene, especially in buffalo meat, and thus they are recognized as potential hazards in different meats.


Main Subjects

Adwan, K (2014). Fast DNA isolation and PCR protocols for detection of methicillin-resistant staphylococci. Folia Microbiol., 59: 5-8.
Al-Ashmawy, MA; Sallam, KI; Abd-Elghany, SM; Elhadidy, M and Tamura, T (2016). Prevalence, molecular characterization, and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus isolated from milk and dairy products. Foodborne Path. Dis., 13: 156-162.
Al-Tarazi, YH; Albetar, MA and Alaboudi, AR (2009). Biotyping and enterotoxigenicity of staphylococci isolated from fresh and frozen meat marketed in Jordan. Food Res. Int., 42: 374-379.
Angulo, FJ; Nargund, VN and Chiller, TC (2004). Evidence of an association between use of anti-microbial agents in food animals and anti-microbial resistance among bacteria isolated from humans and the human health consequences of such resistance. J. Vet. Med., Series B. 51: 374-379.
Ataee, RA; Karami, A; Izadi, M; Aghania, A and Ataee, MH (2011). Molecular screening of staphylococcal enterotoxin B gene in clinical isolates. Cell J. (Yakhteh). 13: 187-192.
Atanassova, V; Meindl, A and Ring, C (2001). Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham a comparison of classical culturing detection and RFLP-PCR. Int. J. Food Microbiol., 68: 105-113.
Aycicek, H; Cakiroglu, S and Stevenson, TH (2005). Incidence of Staphylococcus aureus in ready-to-eat meals from military cafeterias in Ankara, Turkey. Food Cont. 16: 531-534.
Baniardalan, S; Mohammadzadeh, A; Pajohi-Alamoti, M; Mahmoodi, P and Sadeghinasab, A (2017). Detection of toxic shock toxin (tst) gene in Staphylococcus aureus isolated from bovine milk samples. Bulgarian J. Vet. Med., 20: 236-243.
Bellio, A; Chiesa, F; Gallina, S; Bianchi, DM; Macori, G; Bossi, D and Decastelli, L (2019). Insight into the distribution of staphylococci and their enterotoxins in cheeses under natural conditions. Front Microbiol., 9: 3233.
Brakstad, OG; Aasbakk, K and Maeland, JA (1992). Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol., 30: 1654-1660.
CLSI (Clinical and Laboratory Standards Institute) (2006). Methods for antimicrobial dilution and disk susceptibility testing of in-frequently isolated or fastidious bacteria. Approved Guideline (M45-A). Clinical and Laboratory Standards Institute, USA.
Cunha, MDLRD; Rugolo, LMSDS and Lopes, CADM (2006). Study of virulence factors in coagulase-negative staphylococci isolated from newborns. Mem. Inst. Oswaldo Cruz. 101: 661-668.
Dastmalchi Saei, H; Ahmadi, M; Mardani, K and Batavani, RA (2009). Molecular typing of Staphylococcus aureus isolated from bovine mastitis based on polymorphism of the coagulase gene in the north west of Iran. Vet. Microbiol., 137: 202-206.
Dehkordi, FS; Gandomi, H; Basti, AA; Misaghi, A and Rahimi, E (2017). Phenotypic and genotypic characterization of antibiotic resistance of methicillin-resistant Staphylococcus aureus isolated from hospital food. Antimicrob. Res. Infect. Control. 6: 104-115.
Den Heijer, CD; Van Bijnen, EM; Paget, WJ; Pringle, M; Goossens, H; Bruggeman, CA; Schellevis, FG; Stobberingh, EE and APRES Study Team (2013). Prevalence and resistance of commensal Staphylococcus aureus, including meticillin-resistant S. aureus, in nine European countries: a cross-sectional study.  Lancet Infect. Dis., 13: 409-415.
Devriese, LA (1984). A simplified system for biotyping Staphylococcus aureus strains isolated from different animal species. J. Appl. Bacteriol., 56: 215-220.
Fitzgerald, JR; Reid, SD; Ruotsalainen, E; Tripp, TJ; Liu, M; Cole, R; Kuusela, P; Schlievert, PM; Jarvinen, A and Musser, JM (2003). Genome diversification in Staphylococcus aureus: molecular evolution of a highly variable chromosomal region encoding the staphylococcal exotoxin-like family of proteins. Infect. Immun., 71: 2827-2838.
Foster, TJ (2017). Antibiotic resistance in Staphylococcus aureus. Current status and future prospects. FEMS Microbiol. Reviews. 41: 430-449.
Havaei, SA; Assadbeigi, B; Esfahani, BN; Hoseini, NS; Rezaei, N and Havaei, SR (2015). Detection of mecA and enterotoxin genes in Staphylococcus aureus isolates associated with bovine mastitis and characterization of Staphylococcal cassette chromosome mec (SCCmec) in MRSA strains. Iranian J. Microbiol., 7: 161-167.
Hennekinne, JA; De Buyser, ML and Dragacci, S (2012). Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation.FEMS Mic. Reviews. 36: 815-836.
Huber, H; Ziegler, D; Pflüger, V; Vogel, G; Zweifel, C and Stephan, R (2011). Prevalence and characteristics of methicillin-resistant coagulase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons. BMC Vet. Res., 7: 1-7.
Isigidi, BK; Mathieu, AM; Devriese, LA; Godard, C and Hoof, JV (1992). Enterotoxin production in different Staphylococcus aureus biotypes isolated from food and meat plants. J. Appl. Bacteriol., 72: 16-20.
Jay, JM; Loessner, MJ and Golden, DA (2005). Staphylococcal gastroenteritis. Modern Food Microbiol., PP: 545-566.
Johnson, WM; Tyler, SD; Ewan, EP; Ashton, FE; Pollard, DR and Rozee, KR (1991). Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. J. Clin. Microbiol., 29: 426-430.
Kadariya, J; Smith, TC and Thapaliya, D (2014). Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. BioMed. Res. Int., 2014: 1-9.
Kim, NH; Yun, AR and Rhee, MS (2011). Prevalence and classification of toxigenic Staphylococcus aureus isolated from refrigerated ready-to-eat foods (sushi, kimbab and California rolls) in Korea. J. Appl. Microbiol., 111: 1456-1464.
Kitai, S; Shimizu, A; Kawano, J; Sato, E; Nakano, C; Kitagawa, H; Fujio, K; Matsumura, K; Yasuda, R and Inamoto, T (2005). Prevalence and characterization of Staphylococcus aureus and enterotoxigenic Staphylococcus aureus in retail raw chicken meat throughout Japan. J. Vet. Med. Sci., 67: 269-274.
Kloos, WE and Bannerman, TL (1994). Update on clinical significance of coagulase-negative staphylococci. Clin. Microbiol. Rev., 7: 117-140.
Kukhtyn, MD; Horyuk, YV; Horyuk, VV; Yaroshenko, TY; Vichko, OI and Pokotylo, OS (2017). Biotype characterization of Staphylococcus aureus isolated from milk and dairy products of private production in the western regions of Ukraine. Regulatory Mech. Bio., 8: 384-388.
Mahyudin, NA; Sahil, SM; Radu, S; Mahmud, NK and Rashid, A (2019). Multiple drug resistance among Staphylococcus aureus strains isolated from cutting boards of commercial food premises: A threat to food and public health safety. J. Bchem. Mic. Btech., 7: 48-51.
Manjarrez López, AM; Díaz Zarco, S; Salazar García, F; Valladares Carranza, B; Gutiérrez Castillo, ADC; Barbabosa Plliego, A; Alavera Rojas, M; Alonso Fresan, MU and Velázquez Ordoñez, V (2012). Staphylococcus aureus biotypes in cows presenting subclinical mastitis from family dairy herds in the Central–Eastern State of Mexico. Rev. Mex. CiencPecu. 3: 265-274.
Murray, P (2003). Manual of clinical microbiology. 8th Edn., Washington, D.C., ASM Press. PP: 726-742.
Neskovic, A (2008). Characterization of coagulase positive Staphylococci from pig carcasses from Swedish slaughterhouses. Independent thesis advanced level, Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. P: 22.
Normanno, G; La Salandra, G; Dambrosio, A; Quaglia, NC; Corrente, M; Parisi, A; Santagada, G; Firinu, A; Crisetti, E and Celano, GV (2007). Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. Int. J. Food Microbiol., 115: 290-296.
Nurjadi, D; Olalekan, AO; Layer, F; Shittu, AO; Alabi, A; Ghebremedhin, B; Schaumburg, F; Hofmann-Eifler, J; Van Genderen, PJ; Caumes, E and Fleck, R (2014). Emergence of trimethoprim resistance gene dfrG in Staphylococcus aureus causing human infection and colonization in sub-saharan Africa and its import to Europe. J. Antimic. Chemother., 69: 2361-2368.
Nurjadi, D; Schäfer, J; Friedrich-Jänicke, B; Mueller, A; Neumayr, A; Calvo-Cano, A; Goorhuis, A; Molhoek, N; Lagler, H; Kantele, A and Van Genderen, PJ (2015). Predominance of dfrG as determinant of trimethoprim resistance in imported Staphylococcus aureus. Clin. Microbiol. Infec., 21: 1095.e5-1095.e9.
Pereira, V; Lopes, C; Castro, A; Silva, J; Gibbs, P and Teixeira, P (2009). Charaecterization for entrotoxin production virulance factors, and antibiotic susceptibility of Staphylococcus aureus is odates from various food in Portugal. Food Microbiol., 26: 278-282.
Piette, A and Verschraegen, G (2009). Role of coagulase-negative staphylococci in human disease. Vet. Microbiol., 134: 45-54.
Podkowik, M; Park, JY; Seo, KS; Bystroń, J and Bania, J (2013). Enterotoxigenic potential of coagulase-negative staphylococci. Int. J. Food Microbiol., 163: 34-40.
Rall, VLM; Sforcin, JM; de Deus, MFR; de Sousa, DC; Camargo, CH; Godinho, NC; Galindo, LA; Soares, TCS and Araujo Jr, JP (2010). Polymerase chain reaction detection of enterotoxins genes in coagulase-negative staphylococci isolated from Brazilian Minas cheese. Fb. Path. Dis., 7: 1121-1123.
Sarrafzadeh Zargar, MH; Hosseini Doust, R and Mohebati Mobarez, A (2014). Staphylococcus aureus enterotoxin, A gene isolated from raw red meat and poultry in Tehran, Iran. Int. J. Enteric Pathog., 2: 1-6.
Soltan Dallal, MMS; Salehipour, Z; Eshraghi, S; Mehrabadi, JF and Bakhtiari, R (2010). Occurrence and molecular characterization of Staphylococcus aureus strains isolated from meat and dairy products by PCR-RFLP. Annals Microbiol., 60: 189-196.
Turchi, B; Bertelloni, F; Marzoli, F; Cerri, D; Tola, S; Azara, E; Longheu, CM; Tassi, R; Schiavo, M; Cilia, G and Fratini, F (2020). Coagulase negative staphylococci from ovine milk: Genotypic and phenotypic characterization of susceptibility to antibiotics, disinfectants and biofilm production. Small Rumi. Res., 183: 106030.
Vasconcelos, NG; Pereira, VC; Júnior, JA and da Cunha, MDL (2011). Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil. J. Appl. Microbiol., 111: 749-762.
Veras, JF; do Carmo, LS; Tong, LC; Shupp, JW; Cummings, C; dos Santos, DA; Cerqueira, MMOP; Cantini, A; Nicoli, JR and Jett, M (2008). A study of the enterotoxigenicity of coagulase-negative and coagulase-positive staphylococcal isolates from food poisoning outbreaks in Minas Gerais, Brazil. Int. J. Infect. Dis., 12: 410-415.
Wang, H; Wang, H; Bai, Y; Xu, X and Zhou, G (2018). Pathogenicity and antibiotic resistance of coagulase-negative staphylococci isolated from retailing chicken meat. LWT. 90: 152-156.
Younis, GA; Elkenany, RM; Radwan, MM and Allah, MMA (2017). Antimicrobial resistant profiles of methicillin-resistant Staphylococcus aureus (MRSA) recovered from broiler chicken populations in Egypt. Indian J. Poult. Sci., 52: 332-337.