Antimicrobial Resistance and Detection of the mec A Gene Besides Enterotoxin-Encoding Genes Among Coagulase-Negative Staphylococci Isolated from Clam Meat of Anomalocardia brasiliana
Restricted accessResearch articleFirst published online December, 2013
Antimicrobial Resistance and Detection of the mec A Gene Besides Enterotoxin-Encoding Genes Among Coagulase-Negative Staphylococci Isolated from Clam Meat of Anomalocardia brasiliana
The marine clam Anomalocardia brasiliana is a candidate as a sentinel animal to monitor the contamination levels of coliforms in shellfish-harvesting areas of Brazil's northeastern region. The aim of the present study was to search enterotoxin-encoding genes plus the mecA gene among coagulase-negative staphylococci (CNS) isolates from shellfish meats of A. brasiliana. The specimen clam (n=48; 40 clams per sample) was collected during low tide in the bay area of Mangue Seco from April through June 2009, and random samples of chilled and frozen shelled clam meat (n=33; 250 g per sample) were obtained from retail shops from January through March 2012. Seventy-nine CNS isolates were identified, including Staphylococcus xylosus, S. cohnii spp. urealyticus, S. sciuri, and S. lentus. A high percentage of isolates resistant to erythromycin (58.5%), penicillin (51.2%), and tetracycline (43.9%), and the fluoroquinolones levofloxacin (39%) and ciprofloxacin (34.1%) were recorded from those environmental samples. Isolates from retail shops were particularly resistant to oxacillin (55.3%) and penicillin (36.8%). All CNS resistant to oxacillin and/or cefoxitin were positive for the presence of the mecA gene, but phenotypically susceptible to vancomycin. Also, the enterotoxin-encoding genes seg and seh were detected through multiplex–polymerase chain reaction in 77.7% and 88.8% of the isolates from environmental samples, versus 90.5% and 100% of the isolates from retail shops, respectively. The data reveal the risk to public health due to consuming raw or undercooked shellfish containing enterotoxigenic plus methicillin-resistant CNS.
Get full access to this article
View all access options for this article.
References
1.
AlmeidaC, SoaresF. Microbiological monitoring of bivalves from the Ria Formosa Lagoon (south coast of Portugal): A 20 years of sanitary survey. Mar Pollut Bull, 2012; 64:252–262.
2.
AltschulSF, MaddenTL, SchafferAA, ZhangJ, ZhangZ, MillerW, LipmanDJ. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res, 1997; 25:3389–3402.
BautistaL, GayaP, MedinaM, NuñezM. A quantitative study of enterotoxin production by sheep milk staphylococci. Appl Environ Microbiol, 1988; 54:566–569.
5.
BeckerK, RothR, PetersG. Rapid and specific detection of toxigenic Staphylococcus aureus: Use of two multiplex PCR enzyme immunoassay for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J Clin Microbiol, 1998; 36:2548–2553.
BloemendaalALA, BrouwerEC, FluitAC. Methicillin resistance transfer from Staphylocccus epidermidis to methicillin-susceptible Staphylococcus aureus in a patient during antibiotic therapy. PLoS ONE, 2010; 5:e11841.
8.
Brazil, Ministry of Agriculture. Instrução normativa n° 62, de 26 de agosto de. Oficializa os Métodos Analíticos Oficiais para Análises Microbiológicas para Controle de Produtos de Origem Animal e Água. Diário Oficial da União. Brasília, DF, Brasil: Seção 1, September18, 2003; 14.
9.
[CLSI] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Second Informational SupplementCLSI Document M100-S22. Wayne, PA: CLSI, 2012.
10.
CLSI. Methods for Dilution Susceptibility. Tests for Bacteria That Grow Aerobically. Approved Standard, 6thCLSI Document M7-A6. Wayne, PA: CLSI, 2003.
11.
CoutoI, SanchesIS, Sá-LeãoR, De LencastreH. Molecular characterization of Staphylococcus sciuri strains isolated from humans. J Clin Microbiol, 2000; 38:1136–1143.
12.
FreitasMFL, LuzIS, Silveira-FilhoVM, JúniorJWP, StamfordTLM, MotaRA, SenaMJ, AlmeidaAMP, BalbinoVQ, Leal-BalbinoTC. Staphylococcal toxin genes in strains isolated from cows with subclinical mastitis. Pesqui Vet Bras, 2008; 28:617–621.
13.
FrigattoEA, MachadoAM, PignatariAC, GalesAC. Is the cefoxitin disk test reliable enough to detect oxacillin resistance in coagulase-negative staphylococci?J Clin Microbiol, 2005; 43:2028–2029.
HammadAM, WatanabeW, FujiiT, ShimamotoT. Occurrence and characteristics of methicillin-resistant and -susceptible Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci from Japanese retail ready-to-eat raw fish. Int J Food Microbiol, 2012; 156:286–289.
16.
HanssenAM, KjeldsenG, SollidJUE. Local variants of staphylococcal cassette chromosome mec in sporadic methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci: Evidence of horizontal gene transfer?Antimicrob Agents Chemother, 2004; 48:285–296.
17.
HuDL, MainaEK, OmoeK, InoueF, YasujimaM, NakaneA. Superantigenic toxin genes coexist with specific staphylococcal cassette chromosome mec genes in methicillin-resistant Staphylococcus aureus. Tohoku J Exp Med, 2011; 225:161–169.
18.
HungKH, YanJJ, LuYC, ChenHM, WuJJ. Evaluation of discrepancies between oxacillin and cefoxitin susceptibility in coagulase-negative staphylococci. Eur J Clin Microbiol Infect Dis, 2011; 30:785–788.
19.
IkedaT, TamateN, YamaguchiK, MakinoS. Mass outbreak of food poisoning disease caused by small amounts of staphylococcal enterotoxins A and H. Appl Environ Microbiol, 2005; 71:2793–2795.
20.
ItoT, KatayamaY, HiramatsuK. Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob Agents Chemother, 1999; 43:1449–1458.
21.
JainA, AgarwalJ, BansalS. Prevalence of methicillin-resistant, coagulase-negative staphylococci in neonatal intensive care units: Findings from a tertiary care hospital in India. J Med Microbiol, 2004; 53:941–944.
22.
JorgensenHJ, MathisenT, LovsethA, OmoeK, QvaleKS, LoncarevicS. An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk. FEMS Microbiol Lett, 2005; 252:267–272.
23.
KondoY, ItoT, MaXX, WatanabeS, KreiswirthBN, EtienneJ, HiramatsuK. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: Rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob Agents Chemother, 2007; 51:264–274.
24.
Le LoirY, BaronF, GautierM. Staphylococcus aureus and food poisoning. Genet Mol Res, 2003; 2:63–76.
25.
MartinezDI, OliveiraAJFC. Faecal bacteria in Perna perna (linnaeus, 1758) (Mollusca: Bivalvia) for biomonitoring coastal waters and seafood quality. Braz J Oceanogr, 2010; 58:29–35.
26.
MouraTM, CamposFS, d'AzevedoPA, Van Der SandST, FrancoAC, FrazzonJ, FrazzonAP. Prevalence of enterotoxin–encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding. Rev Soc Bras Med Trop, 2012; 45:579–585.
27.
OliveiraJ, CunhaA, CastilhoF, RomaldeJL, PereiraMJ. Microbial contamination and purification of bivalve shellfish: Crucial aspects in monitoring and future perspectives—A mini-review. Food Control, 2011; 22:805–816.
28.
OmoeK, MachikoI, ShimodaY, HuDL, UedaS, ShinagawaK. Detection of seg, seh and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh or sei genes. J Clin Microbiol, 2002; 40:857–862.
29.
PereiraML, Do CarmoLS, SantosEJ, PereiraJL, BergdollMS. Enterotoxin H in Staphylococcal food poisoning. J Food Prot, 1996; 59:559–561.
30.
PodkowikM, ParkJY, SeoKS, BystronJ, BaniaJ. Enterotoxigenic potential of coagulase-negative staphylococci. Int J Food Microbiol, 2013; 163:34–40.
31.
PotasmanI, PazA, OdehM. Infectious outbreaks associated with bivalve shellfish consumption: A worldwide perspective. Clin Infect Dis, 2002; 35:921–928.
32.
RallVL, SforcinJM, de DeusMF, de SousaDC, CamargoCH, GodinhoNC, GalindoLA, SoaresTC, AraújoJP.Jr. Polymerase chain reaction detection of enterotoxins genes in coagulase-negative staphylococci isolated from Brazilian Minas cheese. Foodborne Pathog Dis, 2010; 7:1121–1123.
RodríguezM, NúñezF, CórdobaJJ, BermúdezE, AsensioMA. Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential. Appl Environ Microbiol, 1996; 62:1897–1902.
35.
RosecJP, GigaudO. Staphylococcal enterotoxin genes of classical and new types detected by PCR in France. Int J Food Microbiol, 2002; 77:61–70.
36.
RossneyAS, ShoreAC, MorganPM, FitzgibbonMM, O'ConnelB, ColemanDC. The emergence and importation of diverse genotypes of methicillin-resistant Staphylococcus aureus (MRSA) harboring the panton-valentine leukocidin gene (pvl) reveal that pvl is a poor marker for community-acquired MRSA strains in Ireland. J Clin Microbiol, 2007; 45:2554–2563.
37.
TamuraK, PetersonD, PetersonN, StecherG, NeiM, KumarS. MEGA 5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol, 2011; 28:2731–2739.
38.
TokueY, ShojiS, SatohK, WatanabeA, MotomiyaM. Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother, 1992; 36:6–9.
39.
UdoEE, Al-BustanMA, JacobLE, ChughTD. Enterotoxin production by coagulase-negative staphylococci in restaurant workers from Kuwait City may be a potential cause of food poisoning. J Med Microbiol, 1999; 48:819–823.
40.
ValleJ, Gomez-LuciaE, PirizS, GoyacheJ, OrdenJA, VadilloS. Enterotoxin production by staphylococci isolated from healthy goats. Appl Environ Microbiol, 1990; 56:1323–1326.
41.
VernozyRC, MazuyC, PrevostG, LapeyreC, BesM, BrunY, FleuretteJ. Enterotoxin production by coagulase-negative staphylococci isolated from goats milk and cheese. Int J Food Microbiol, 1996; 30:271–280.
42.
WoRMS, World Register of Marine Species. http://www.marinespecies.org/. 2012 December 3.
43.
WuS, PiscitelliC, de LencastreH, TomaszA. Tracking the evolutionary origin of the methicillin resistance gene: Cloning and sequencing of a homologue of mecA from a methicillin susceptible strain of Staphylococcus sciuri. Microb Drug Resist, 1996; 2:435–441.
