Progress toward rapid and accurate Staphylococcus aureus strain typing.

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), continues to be one of the most important bacterial pathogens in humans and is responsible for a constantly increasing number of nosocomial and community-acquired infections. Bacterial strain typing, or subspecies identification, has become an important clinical tool for investigating suspected outbreaks and evaluating nosocomial transmission. Adequate and precise typing of S. aureus isolates that permits the monitoring of local outbreaks and widerscale dissemination of specific dangerous clones is of great concern (1–3 ). Similar to typing in other microbial organisms, S. aureus typing has been greatly influenced and improved by molecular techniques. Differences in the various molecular methods used to define different genomic characteristics greatly affect their essential parameters, including discriminatory power and test turnaround time (1–3 ). The most important methods currently used for MRSA typing are pulsedfield gel electrophoresis (PFGE) (4 –7 ), multilocus sequence typing (MLST) (8, 9 ), spa (staphylococcal surface protein A) typing (1, 6, 10, 11 ), multilocus variable tandem repeat analysis (MLVA) (12, 13 ), and repeat-element PCR (rep-PCR) typing (7, 14, 15 ). These methods have been used to investigate the evolution of the MRSA clones that have emerged since the 1960s and to study their worldwide dissemination. Tremendous progress in molecular-typing methods has been made, with optimization and standardization of sequence-based technologies offering broad applicability and high throughput; however, no single S. aureus typing method has yet provided rapid and reliable information that is sufficient to permit prompt action to control infections. Because S. aureus has a highly clonal population structure, many different chromosomal markers indexing polymorphisms in coding or noncoding regions will provide largely congruent classifications of isolates into clonal lineages. PFGE is considered the gold standard in typing for a variety of bacteria, including S. aureus (4 –7 ). This method is technically demanding, however, with a low throughput and difficulties in achieving interlaboratory comparability and standardization. PFGE may be too discriminatory for long-term epidemiologic investigations, and it fails to account for genetic changes within a single, globally distributed clonal lineage of MRSA (6, 9 ). With the indexing of allelic polymorphisms in 7 neutral housekeeping genes (9 ), MLST has been considered the reference method for defining the core genetic population structure of S. aureus (8 ). As a DNA sequencing– based method, MLST allows full reproducibility of results between laboratories, but it has lower resolution and remains too labor-intensive and costly for use as a primary typing tool. spa typing, which evaluates sequence polymorphism in the variable X region of the spa gene, which encodes staphylococcal surface protein A, has become popular for MRSA typing (6, 10, 11 ). As a sequencing-based method for a single locus, spa typing possesses moderate discriminatory power, between PFGE and MLST; however, repeat-based spa typing is subject to misclassification bias due both to horizontal DNA transfer and to recombination and homoplasy (16 ). Typing techniques that are rapid, easy, and relatively inexpensive are based on PCR amplification methods, which include rep-PCR typing and MLVA. rep-PCR typing, which is based on multicopy elements in the staphylococcal genome, has been shown to be variably discriminatory but poorly reproducible among laboratories when in-house protocols are used (14 ). A kit-based rep-PCR assay combined with a commercial fingerprint-analysis platform (DiversiLab typing system; bioMérieux) showed excellent reproducibility and moderate discriminatory power as a screening tool to rule out potential MRSA outbreaks in hospitals (7, 15 ). MLVA schemes share the rep-PCR advantages of rapid turnaround times and high throughput, but their discriminatory power depends on the number and types of loci for variable number of tandem DNA repeats/staphylococcal interspersed repeat units that are analyzed. As with rep-PCR, no stan1 Departments of Pathology and Medicine, Vanderbilt University School of Medicine, Nashville, TN. * Address correspondence to the author at: Molecular Infectious Disease Laboratory, Vanderbilt University Hospital, 4605 TVC, Nashville, TN 37232-5310. Fax 615-343-8420; e-mail [email protected]. Received August 25, 2009; accepted September 17, 2009. Previously published online at DOI: 10.1373/clinchem.2009.135707 2 Nonstandard abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; PFGE, pulsed-field gel electrophoresis; MLST, multilocus sequence typing; MLVA, multilocus variable tandem repeat analysis; rep-PCR, repeat-element PCR; DLA, direct linear analysis. Clinical Chemistry 55:12 2074–2076 (2009) Editorials