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New Staphylococcus aureus genotyping method based on exotoxin (set) genes.

ABSTRACT: A variety of methods for genotyping Staphylococcus aureus isolates exists: the two most widely used methods are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Here, we describe a sequence-based genotyping method based on genes encoding S. aureus superantigen-like proteins, which belong to a family of exotoxins called staphylococcal exotoxins. The sequences of PCR-amplified internal fragments of three different set genes (set2, set5, and set7) of 61 well-characterized clinical methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates and reference strains were compared. Phylogenetic analysis was performed based on single-nucleotide polymorphisms (SNP). The SNP dendrograms of the set gene sequences differentiated the 61 isolates into 22 distinct subgroups, designated exotoxin sequence types (ETST), while the standard seven-gene MLST profiles differentiated the same 61 isolates into 19 subgroups. Of the 19 different MLST subgroups, 16 corresponded to 16 distinct ETST groups. However, three MLST subgroups, ST1, ST30, and ST36, were each further separated into more than one ETST subgroup. The exotoxin-based genotyping method was able to discriminate MRSA and MSSA isolates according to their specific epidemiological characteristics. This SNP analysis of the three set genes is thus equally or more discriminatory than the seven-gene MLST method, providing a good alternative typing tool for a laboratory that has sequencing capability.

SUBMITTER: Aguiar-Alves F

PROVIDER: S-EPMC1594596 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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New Staphylococcus aureus genotyping method based on exotoxin (set) genes.

Aguiar-Alves Fábio F Medeiros Fred F Fernandes Octavio O Gudziki Pereira Rossiane Maria RM Perdreau-Remington Françoise F Riley Lee W LW

Journal of clinical microbiology 20060801 8

A variety of methods for genotyping Staphylococcus aureus isolates exists: the two most widely used methods are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Here, we describe a sequence-based genotyping method based on genes encoding S. aureus superantigen-like proteins, which belong to a family of exotoxins called staphylococcal exotoxins. The sequences of PCR-amplified internal fragments of three different set genes (set2, set5, and set7) of 61 well-characteri ...[more]

PMID: 16891484

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Project description:Staphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus in foods. In the present study, 64 S. aureus isolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains by spa typing, multilocus sequence typing (MLST), exotoxin gene content, and antimicrobial resistance. The strains belonged to 10 clonal complexes (CCs): CC5 (29.0%), CC30 (25.8%), CC45 (16.1%), CC8, CC15 (two strains each), CC1, CC22, CC25, CC59, and CC121 (one strain each). They contained hemolysin genes (90.3%); lukED (77.4%); exfoliatin genes eta, etd (6.5% each), and etb (3.2%); tst (25.8%); and the following enterotoxin or enterotoxin-like genes or clusters: sea (38.7%), seb (12.9%), sec (16.1%), sed-selj with or without ser (22.9%), selk-selq (6.5%), seh, sell, selp (9.7% each), egc1 (32.3%), and egc2 (48.4%). The number of se and sel genes ranged from zero to 12. All isolates carrying tst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spa type t002, sequence type 5 [ST5]; spa type t2173, ST5) were methicillin resistant and harbored staphylococcal cassette chromosome mec (SCCmec) IVd. Six (19.4%) were mupirocin resistant, and one (spa type t120, ST15) from a food handler carried mupA (MIC, 1,250 μg/ml). Resistance to ampicillin (blaZ) (61.3%), erythromycin (ermA-ermC or ermC) (25.8%), clindamycin (msrA-msrB or msrB) (16.1%), tetracycline (tetK) (3.2%), and amikacin-gentamicin-kanamycin-tobramycin (aphA with aacA plus aphD or aadD) (6.5%) was also observed. The presence of S. aureus strains with an important repertoire of virulence and resistance determinants in the food chain represents a potential health hazard for consumers and merits further observation.

Project description:BACKGROUND: A limitation of both culture-based and molecular methods of screening for staphylococcal infection is that current tests determine only the presence or absence of colonization with no information on the colonizing strain type. A technique that couples polymerase chain reaction to mass spectrometry (PCR/ESI-MS) has recently been developed and an assay validated to identify and genotype S. aureus and coagulase-negative staphylococci (CoNS). METHODS: This study was conducted to determine the rates, risk factors, and molecular genotypes of colonizing Staphylococcus aureus in adult patients presenting to an inner-city academic emergency department. Participants completed a structured questionnaire to assess hospital and community risks for infection with methicillin-resistant S. aureus (MRSA). Nasal swabs were analyzed by PCR/ESI-MS to identify and genotype S. aureus and CoNS. RESULTS: Of 200 patients evaluated, 59 were colonized with S. aureus; 27 of these were methicillin-resistant strains. Twenty-four of the 59?S. aureus carriers were co-colonized with a CoNS and 140 of the 200 patients were colonized exclusively with CoNS. The molecular genotypes of the 59?S. aureus strains were diverse; 21 unique molecular genotypes belonging to seven major clonal complexes were identified. Eighty-five of 200 patients carried strains with high-level mupirocin resistance. Of these eighty-five participants, 4 were colonized exclusively with S. aureus, 16 were co-colonized with S. aureus and CoNS, and 65 were colonized exclusively with CoNS. CONCLUSION: The prevalence of S. aureus and methicillin-resistant S. aureus colonization in a random sample of patients seeking care in Emergency Department was 29.5% and 13.5%, respectively. A substantial fraction of the S. aureus-colonized patients were co-colonized with CoNS and high-level mupirocin-resistant CoNS. Determining the molecular genotype of S. aureus during intake screening may prove valuable in the future if certain molecular genotypes become associated with increased infection risk.

Dataset Information

New Staphylococcus aureus genotyping method based on exotoxin (set) genes.

Publications

New Staphylococcus aureus genotyping method based on exotoxin (set) genes.

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