Dataset Information

Double-locus sequence typing using clfB and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus.

ABSTRACT: Sequence-based epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) has recently been promoted because it results in unambiguous data sets that can be organized in local and global databases. The replacement of previous typing methods, such as the highly discriminatory pulsed-field gel electrophoresis (PFGE), has been attempted with various markers and typing schemes, including spa typing and multilocus sequence typing. However, despite a number of advantages, none of these methods showed convincing evidence for performance in epidemiological typing comparable to that of PFGE. By using three sets of 48 MRSA strains comprising isolates that were (i) genetically highly diverse, (ii) genetically related, and (iii) obtained from long-term carriers, we analyzed the performance of the four highly polymorphic S. aureus markers: clfA, clfB, fnbA, and spa. Typeability, discriminatory power, in vivo stability, and evolution of these markers were compared to those of PFGE. Clearly, none of the markers alone could match the discriminatory power of PFGE (63 genotypes; index of discrimination of 0.96). Instead, this could be achieved by combining markers in pairs. We showed that by using only 3' partial sequences of approximately 500 bp, the majority of each marker's discriminatory power was displayed, and using the partial sequences, the best performance was obtained with the combination of clfB and spa (57 genotypes; index of discrimination of 0.94). Genetic changes were not observed for any of the sequence markers over a period of 3 years and in the case of partial sequences for a period of more than 4 years. This is in contrast to PFGE where changes occurred after several months. The genetic differences found between isolate pairs of long-term carriers and among highly related isolates indicated clonal evolution. A typing scheme based on 500-bp 3' partial sequences of clfB and spa is proposed.

SUBMITTER: Kuhn G

PROVIDER: S-EPMC1828982 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Publications

Double-locus sequence typing using clfB and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus.

Kuhn G G Francioli P P Blanc D S DS

Journal of clinical microbiology 20061108 1

Sequence-based epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) has recently been promoted because it results in unambiguous data sets that can be organized in local and global databases. The replacement of previous typing methods, such as the highly discriminatory pulsed-field gel electrophoresis (PFGE), has been attempted with various markers and typing schemes, including spa typing and multilocus sequence typing. However, despite a number of advantages, none of the ...[more]

PMID: 17093014

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Project description:Sequencing of the spa gene of methicillin-resistant Staphylococcus aureus (MRSA) is used for assigning spa types to e.g., detect transmission and control outbreaks. Traditionally, spa typing is performed by Sanger sequencing but has in recent years been replaced by whole-genome sequencing (WGS) in some laboratories. Spa typing by WGS involves de novo assembly of millions of short sequencing reads into larger contiguous sequences, from which the spa type is then determined. The choice of assembly program therefore potentially impacts the spa typing result. In this study, WGS of 1,754 MRSA isolates was followed by de novo assembly using the assembly programs SPAdes (with two different sets of parameters) and SKESA. The spa types were assigned and compared to the spa types obtained by Sanger sequencing, regarding the latter as the correct spa types. SPAdes with the two different settings resulted in assembly of the correct spa type for 84.8% and 97.6% of the isolates, respectively, while SKESA assembled the correct spa type in 98.6% of cases. The misassembled spa types were generally two spa repeats shorter than the correct spa type and mainly included spa types with repetition of the same repeats. WGS-based spa typing is thus very accurate compared to Sanger sequencing, when the best assembly program for this purpose is used. IMPORTANCE spa typing of methicillin-resistant Staphylococcus aureus (MRSA) is widely used by clinicians, infection control workers, and researchers both in local outbreak investigations and as an easy way to communicate and compare MRSA types between laboratories and countries. Traditionally, spa types are determined by Sanger sequencing, but in recent years a whole-genome sequencing (WGS)-based approach has become increasingly used. In this study, we compared spa typing by WGS using different methods for assembling the genome from short sequencing reads and compared to Sanger sequencing as the gold standard. We find substantial differences in correct assembly of spa types between the assembly methods. Our findings are therefore important for the quality of WGS based spa typing data being exchanged by clinical microbiology laboratories.

Dataset Information

Double-locus sequence typing using clfB and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus.

Publications

Double-locus sequence typing using clfB and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus.

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