BackgroundMolecular typing of methicillin-resistant Staphylococcus aureus (MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.Methodology/Principal FindingsA new MLVA scheme for S. aureus was validated using 1681 S. aureus isolates collected from Dutch patients and 100 isolates from pigs. MLVA using 8 tandem repeat loci was performed in 2 multiplex PCRs and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. The assessed number of repeats was used to create MLVA profiles consisting of strings of 8 integers that were used for categorical clustering. MLVA yielded 511 types that clustered into 11 distinct MLVA complexes which appeared to coincide with MLST clonal complexes. MLVA was at least as discriminatory as PFGE and twice as discriminatory as spa-sequence typing. There was considerable congruence between MLVA, spa-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness.ConclusionsThe MLVA described in this study is a high throughput, relatively low cost genotyping method for S. aureus that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology.

Shortly after the introduction of methicillin in 1959, methicillin-resistant Staphylococcus aureus (MRSA) emerged as an important human pathogen (1). Currently, MRSA is held responsible for numerous hospital-acquired infections worldwide, such as skin infections and toxic shock syndrome (2). MRSA also emerged as a community-acquired pathogen and in recent years has increasingly been isolated from livestock (3, 4). Livestock-associated MRSA (LA-MRSA) can be separated from other MRSA strains, as all strains belong to one multilocus sequence type (MLST) clonal complex. Since the first detection of LA-MRSA in 2003, it has been found in many countries worldwide (5-7). In the Netherlands, LA-MRSA has become quite prominent, making up approximately 40% of all MRSA strains isolated from humans that were sent to the National Institute for Public Health and the Environment (RIVM) for molecular typing as part of the Dutch national MRSA surveillance.From 2002 to 2007, all Dutch MRSA isolates were typed with pulsed-field gel electrophoresis (PFGE) using SmaI, but because of its labor-intensive character and the subjectivity involved in interpretation, this method was replaced by spa typing in 2007 (8, 9). In addition, multilocus variable-number tandem-repeat analysis (MLVA) was introduced for S. aureus in 2008 (10-12), and since then all isolates have been characterized by MLVA and spa typing.Although spa and MLVA typing are very well suited for characterizing most MRSA isolates, they provide very low discriminatory power for isolates belonging to MLVA complex 398 (MC398) (10, 13). The limited differentiation of MC398 isolates, representing LA-MRSA, has impeded investigations on transmission events and possible outbreaks caused by LA-MRSA.Transmission of LA-MRSA has been described in multiple reports, suggesting that human-to-human transmission of LA-MRSA is less likely to occur than that of other MRSA lineages (14,15). However, these reports describe the transmission of the MC398 (or CC398) clade based on epidemiology and limited molecular characterization of isolates, making it difficult to interpret if actual transmission events with LA-MRSA did occur.Recently, a new high-resolution typing technique for LA-MRSA was introduced named whole-genome mapping (13). Using this method, epidemiologically unrelated LA-MRSA isolates that were previously indistinguishable by spa and MLVA typing can now be differentiated. Furthermore, the method is able to identify transmission events between livestock veterinarians and their household, showing its potential as a typing tool for LA-MRSA.In the study presented here, we further investigated the potential of whole-genome mapping to identify possible transmission of

Since 2007, livestock-associated meticillin-resistant Staphylococcus aureus (LA-MRSA) has become the predominant MRSA clade isolated from humans in the Netherlands. To assess possible temporal changes, we molecularly characterised over 9,000 LA-MRSA isolates submitted from 2003 to 2014 to the Dutch MRSA surveillance. After an initial rapid increase with a peak in 2009 (n = 1,368), the total number of submitted LA-MRSA isolates has been slowly decreasing to 968 in 2014 and over 80% of LA-MRSA belonged to one of three predominant MLVA/spa-types. Next generation sequencing (n=118) showed that MT569/t034 isolates were genetically more diverse than MT398/ t011 and MT572/t108. Concurrent with the decrease in LA-MRSA, fewer people reported having contact with livestock and this was most prominent for people carrying MT569/t034 LA-MRSA. The proportion of LA-MRSA isolated from infection-related materials increased from 6% in 2009, to 13% in 2014 and most of these isolates originated from patients older than 50 years of age. Remarkably, 83% of these patients reported not having contact with livestock. The results reveal an ongoing change in the genotypic and epidemiological characteristics of Dutch LA-MRSA isolated from humans with the emergence of a LA-MRSA subclade independent of livestock exposure, suggesting LA-MRSA starts to resemble non-LA-MRSA in terms of transmissibility and pathogenicity.

In 2007 in the Netherlands, 30% of all human isolates of methicillin-resistant Staphylococcus aureus (MRSA) sent to the National Institute for Public Health and the Environment could not be typed by pulsed-field gel electrophoresis (non-typable (NT)-MRSA). Molecular characterisation of the NT-MRSA isolates revealed 27 different spa types and two distinct SCCmec types, type IV and V. All NT-MRSA isolates were closely related based on spa and multi-locus sequence typing and belonged to the ST398 lineage. The rapid increase of NT-MRSA (ST398) isolates over the last years shows the importance of this relatively new clonal lineage.

MLVA is superior to spa typing and will suffice to characterize MRSA isolates for surveillance.