title: Mapping the genetic diversity within major clonal complexes of meticillin-resistant Staphylococcus aureus utilizing genome-wide fluorescent amplified fragment length polymorphism markers.
abstract: The genetic diversity between major meticillin-resistant Staphylococcus aureus (MRSA) lineages was probed using fluorescent amplified fragment length polymorphism (FAFLP) as a random genome sampling tool. Genomic DNA was digested with endonucleases BglII and Csp6I and a subset of the restricted fragments were amplified using the primer pair BglII+A and Csp6I+0. Sixty-seven FAFLP profiles consisting of 46-68 amplified fragments ranging in size from 50 to 600 bp were exhibited amongst the 71 isolates analysed. Cluster analysis of FAFLP data revealed concordance with spa typing and MLST clonal complexes (CC), with isolates of each CC grouping in the same FAFLP cluster. Furthermore, FAFLP could differentiate subtypes within the homogeneous CC22 isolates and also between MLST sequence types 8 and 239. The discriminatory power of FAFLP was 0.998 compared to values of 0.975 and 0.909 for spa typing and MLST, respectively. Thus, FAFLP analysis proved to be a rapid, reproducible and high-resolution tool that displayed the microheterogeneity within MRSA lineages. Using FAFLP data, lineage-specific fragments were identified and sequenced; these encoded toxins, antibiotic resistance determinants and bacteriophage resistance factors. Lineage-specific sequence variations were observed, which may provide insights into the evolution and fitness of successful lineages. This will also aid in the development of rapid and high-throughput diagnostic PCR-based assays for the identification of MRSA lineages in resource-poor settings.
authors.affiliation: Microbiology Services Division Colindale, Health Protection Agency - Department for Bioanalysis and Horizon Technologies, London NW9 5EQ, UK.
authors.forename: Darshana
authors.initials: D
authors.lastname: Desai
authors.affiliation: Microbiology Services Division, Cambridge Laboratory, Health Protection Agency, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge CB2 0QW, UK.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ellington
authors.affiliation: Microbiology Services Division Colindale, Health Protection Agency - Department for Bioanalysis and Horizon Technologies, London NW9 5EQ, UK.
authors.forename: Catherine
authors.initials: C
authors.lastname: Arnold
authors.affiliation: Microbiology Services Division Colindale, Health Protection Agency - Department for Bioanalysis and Horizon Technologies, London NW9 5EQ, UK.
authors.forename: Meeta
authors.initials: M
authors.lastname: Desai
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D015373:Bacterial Typing Techniques; D004269:DNA, Bacterial / Q000032:analysis*; D014644:Genetic Variation; D016680:Genome, Bacterial; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics*; D058885:Multilocus Sequence Typing
chemical_list: D004269:DNA, Bacterial