Changing characteristics of livestock-associated meticillin-resistant Staphylococcus aureus isolated from humans - emergence of a subclade transmitted without livestock exposure, the Netherlands, 2003 to 2014.

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.

Multiple-locus variable number tandem repeat analysis is superior to spa typing and sufficient to characterize MRSA for surveillance purposes.

AIM: Assess the best approach to type methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcal protein A (spa) typing, multiple-locus variable number tandem repeat analysis (MLVA) or both. MATERIALS & METHODS: Discriminatory power of spa typing and MLVA was determined using 20,771 MRSA isolates. RESULTS: There were twice as many MLVA types (MTs) as spa types present in the collection. Among the top 70% of the isolates, 37 spa types and 139 MTs were found. MLVA diversity among the top-10 spa types was high (diversity index 0.96), while spa diversity among the top-10 MTs was much lower (diversity index 0.83). The probability that two MRSA isolates with the same spa type also had the same MT was low (Wallace's coefficient 0.27). By contrast, most MRSA isolates yielding the same MT also had the same spa type (Wallace's coefficient 0.90). CONCLUSION: MLVA is superior to spa typing and will suffice to characterize MRSA isolates for surveillance.

High Resolution Typing by Whole Genome Mapping Enables Discrimination of LA-MRSA (CC398) Strains and Identification of Transmission Events.

After its emergence in 2003, a livestock-associated (LA-)MRSA clade (CC398) has caused an impressive increase in the number of isolates submitted for the Dutch national MRSA surveillance and now comprises 40% of all isolates. The currently used molecular typing techniques have limited discriminatory power for this MRSA clade, which hampers studies on the origin and transmission routes. Recently, a new molecular analysis technique named whole genome mapping was introduced. This method creates high-resolution, ordered whole genome restriction maps that may have potential for strain typing. In this study, we assessed and validated the capability of whole genome mapping to differentiate LA-MRSA isolates. Multiple validation experiments showed that whole genome mapping produced highly reproducible results. Assessment of the technique on two well-documented MRSA outbreaks showed that whole genome mapping was able to confirm one outbreak, but revealed major differences between the maps of a second, indicating that not all isolates belonged to this outbreak. Whole genome mapping of LA-MRSA isolates that were epidemiologically unlinked provided a much higher discriminatory power than spa-typing or MLVA. In contrast, maps created from LA-MRSA isolates obtained during a proven LA-MRSA outbreak were nearly indistinguishable showing that transmission of LA-MRSA can be detected by whole genome mapping. Finally, whole genome maps of LA-MRSA isolates originating from two unrelated veterinarians and their household members showed that veterinarians may carry and transmit different LA-MRSA strains at the same time. No such conclusions could be drawn based spa-typing and MLVA. Although PFGE seems to be suitable for molecular typing of LA-MRSA, WGM provides a much higher discriminatory power. Furthermore, whole genome mapping can provide a comparison with other maps within 2 days after the bacterial culture is received, making it suitable to investigate transmission events and outbreaks caused by LA-MRSA.

Multiple-locus variable number tandem repeat analysis of Staphylococcus aureus: comparison with pulsed-field gel electrophoresis and spa-typing.

BACKGROUND: Molecular 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 FINDINGS: A 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. CONCLUSIONS: The 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.