Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen able to colonize humans, companion animals and livestock. MRSA in livestock was first reported in 1972 from cases of bovine mastitis, in Belgium where MRSA was found to originate from humans (Devriese et al., 1972). Since then MRSA in animals has been reported infrequently, mostly in companion animals, again originating from humans (Crombé et al., 2013). In 2005 (Voss et al., 2005) identified a new lineage of MRSA, sequence type (ST) 398 grouping within clonal complex (CC) 398 (http://saureus.mlst.net) associated with livestock and able to colonize humans. Following this, livestock associated (LA) MRSA; namely CC398 has been reported globally, in horses, cattle and poultry (Butaye et al., 2016).

In Europe, LA-MRSA has been widely reported from various countries in pigs, poultry and cattle (Butaye et al., 2016), with infrequent reports from the UK. The number of cases of LA-MRSA in farm animals in the UK is gradually increasing with recent reports from, horses, cattle, poultry, pigs, and pork meat products (Loeffler et al., 2009; Paterson et al., 2014). This increase in the number of cases suggests the gradual emergence and change in epidemiology of LA-MRSA among UK livestock. LA-MRSA has been shown to transfer from animals and humans by direct exposure to livestock (Voss et al., 2005). Colonization of LA-MRSA in humans is rare and tends to be asymptomatic and usually transient (Pletinckx et al., 2013). However, should an infection in an immunocompromised individual arise it may be difficult to treat with routine antibiotics, such as penicillin. It is therefore it is important to establish the virulence and antimicrobial potential of these pathogens currently emerging in the UK to ensure antibiotcs are still effective, if treatment is required.

Molecular genotyping methods have long been used to understand the genetic background of S. aureus strains, these have included; DNA microarray for detection of resistance and virulence genes; spa typing which looks at variable repeats within the S. aureus protein A (spa); multilocus sequence typing (MLST) of housekeeping genes and classification of the mobile genetic element Staphylococcal cassette chromosome (SCCmec), the defining feature of MRSA (Stefani et al., 2012). Several types of SCCmec have been described by the International Working Group on the Classification of Staphylococcal cassette Chromosome (2009), the various SCCmec types can be defined according to the combination of their ccr and mec complexes.

Although, the principles behind the typing methods described above remain useful for characterization and differentiation of MRSA isolates; employing these methods can be laborious and time consuming. Advances in comparative genomics using whole genome sequencing (WGS), allows for a more rapid method for identifying genetic determinants and for studying phylogenetic relationships between groups of isolates based on the core genome rather than a finite panel of selected genes.

In this study we compared whole genomes sequences (WGS) of UK LA-MRSA isolates collected between 2013 and 2015, to LA-MRSA animal isolates from European and non-European countries. All isolates looked at in this study belonged to the CC398 lineage. UK LA-MRSA livestock isolates selected for WGS were identified from a panel of more than 1000 S. aureus strains recovered from pig caeca collected from abattoir and from samples submitted for routine diagnostic testing (Loeffler et al., 2009; Anonymous, 2016) during this period. WGS of methicillin-susceptible S. aureus (MSSA) CC398 isolates from animals were also included as comparators to the MRSA isolates as it has been speculated that MRSA CC398 evolved from MSSA CC398 following acquisition of the SCCmec element (Vandendriessche et al., 2014).

The aims of the study were to establish the phylogenetic relationship between LA-MRSA CC398 recovered in the UK and those circulating in other countries, and to identify similarities/differences in the carriage of: antimicrobial resistance, heavy metal resistance, disinfectant resistance and virulence genes, to provide insight into the emergence, evolution and possible adaptation of LA-MRSA in livestock in the UK.

The antimicrobial resistance and virulence potential of LA-MRSA has been well documented for mainland Europe and other countries globally (Guardabassi et al., 2007; Price et al., 2012). However, there is currently little data published on the pathogenicity, antimicrobial resistance and phylogeny of UK LA-MRSA CC398 directly isolated from animal sources (Ward et al., 2014). It is therefore important to establish how UK LA-MRSA characteristics compare with those circulating in Europe and elsewhere, especially as LA-MRSA isolates have been found to carry genes that confer resistance to antimicrobials used to treat serious veterinary and human infections.

Also, WGS and downstream applications are increasingly being applied to pathotype microorganisms, to provide a view on the epidemiology, phylogeny and microevolution of bacterial pathogens; including MRSA (Fitzgerald, 2012; Price et al., 2012; Tong et al., 2015). Advances in comparative genomics, using WGS, allows for a more rapid and accurate method to determine phylogenetic relationships between groups of isolates compared to more traditional typing and screening methods such as MLST and DNA microarray. For phylogeny, WGS analysis considers nsSNPs of a much larger number of core/common genes (usually several hundred) present in a set of isolates when predicting phylogeny in comparison to typing schemes such as MLST, which only looks at seven house-keeping genes (http://pubmlst.org/saureus/). For detection of virulence and AMR genes, WGS has the potential to probe for the presence of an infinite number of genes, and the ability to identify emerging variants whilst DNA microarrays can only probe a limited number of virulence and AMR determinants (Piccinini et al., 2012). Therefore, in this study, WGS was used to determine the phylogeny and molecular characteristics of 12 LA-MRSA CC398s isolated from animals in the UK after screening more than a thousand S. aureus, between 2013 and 2015. As LA-MRSA CC398 is a zoonotic pathogen that has been reported infrequently in the UK, but is more prevalent elsewhere in Europe, WGS data from CC398 isolates from European (including UK) and non-European countries were compared.

Molecular characteristics that were explored in isolates using WGS included AMR, HMR, DR, and virulence. The use of WGS to infer AMR in MRSA is becoming more common place and has been argued to be at least as reliable as phenotypic testing (Aanensen et al., 2016). The results from this study show most LA-MRSA isolates, including several from the UK, harbored multiple AMR genes with the number ranging between 3 and 12, per isolate. In some instances multiple genes encoding resistance to the same antimicrobial class (e.g., macrolides) were present, which was not unexpected. For the subset of isolates with phenotypic data available, there was good correlation (>96%) between the geno- and pheno-types of most antimicrobials compared, except phenicols, which was nevertheless >85% and the aminoglycosides (75%). This suggests there may be genes/chromosomal mutations responsible for the latter phenotypes that were not included in our pipeline, which requires further investigation. The presence of multiple AMR genes in our LA-MRSA isolates raises concern as they add potentially transferable resistances to the bacterial gene pool present in humans and animals that can be passed to other bacteria including other strains of S. aureus.

It has been considered that CC398 originated in humans, probably as a MSSA and transferred to animals where it evolved by losing some of the virulence genes associated with human adaptation allowing successful colonization of a broader host range, and acquiring the mecA gene (Price et al., 2012). This was followed by acquisition of the SCCmec element (Vandendriessche et al., 2014), conferring resistance to β-lactams and also to tetracycline. Tetracycline resistance is attributed to its historic use in veterinary medicine as a growth promoter added to animal feed, a practice now being banned across the EU. Another resistance attributed to its use in animal feed is resistance to zinc (Cavaco et al., 2011). From this study the mecA gene was present in all isolates excluding the MSSA strains. Genes encoding tetracycline resistance were ubiquitous; all isolates, except one harbored tetM, which is well established in the CC398 livestock clade (Stegger et al., 2013). One MSSA strain conferred tetracycline resistance through tetK and tetL.

Presence of zinc and cadmium resistance genes was variable in the panel of isolates included in this study. The czrC gene confers resistance to both zinc and cadmium, and was present in all the UK CC398s relating to food animals but not in the 3 UK isolates from horses. Although the sample size was limited to 89 isolates, due to lack of WGS data from LA-MRSA CC398 being widely available, our results showed a strong correlation between the SCCmecV (associated with ccrC complex) and carriage of czrC, supporting a report by Cavaco et al. (2010) who concluded that a variant of SCCmecV carried czrC (designated subtype Vc). Their study also showed 100% correlation between the presence of czrC and phenotypic resistance to zinc (Cavaco et al., 2010), therefore it is likely that isolates in our panel harboring czrC would show resistance to zinc phenotypically. The majority of ccrC isolates from Europe (~86%) showed correlation between presence of ccrC and czrC, this was greater than in the non-European isolates (~60%). In contrast, only 6% of isolates harboring ccrA2B2 carried the czrC gene. The low incidence czrC genes in ccrA2B2 isolates may impact on the prevalence of this lineage in the future, if environmental selection pressure from heavy metals such as zinc increases or ccrA2B2 isolates containing czrC may become more widespread. Zinc is approved for inclusion in animal feed in some European countries and the selective effects of such inclusion on the occurrence of MRSA have been reported (Moodley et al., 2011) and debated (Burch, 2014), Our results suggest a possible impact on the epidemiology and incidence of different LA-MRSA subtypes, which require further scrutiny. In contrast, only 23% of isolates carried the cadD genes conferring resistance to cadmium only, which has been detected as a contaminant in animal feed (Farmer and Farmer, 2000). Genes encoding resistance to quaternary ammonium compounds which are used widely as disinfectants on farms and the food industry were present in just 9% of isolates.

The WGS data were also used to infer phylogeny, which clustered the European isolates within two main lineages, EU t011 and t034 that separated according to spa type. EU t011 included the majority of European spa-type t011 isolates present in our panel which were from a variety of animal host species i.e., host generalist, including turkeys, pigs, rats and horses. These isolates formed a distinct lineage but there was clear separation of isolates harboring ccrA2B2, part of the SCCmec IV cassette, from isolates harboring ccrC, part of the SCCmec V cassette. All seven UK spa-type t011 isolates from pig, turkey and horse were within this host generalist lineage. The second European lineage (EU t034) predominantly included spa-type t034 isolates from pigs; which was distinct from the non-European t034 isolates that included isolates from pigs but also from turkeys, which indicates radiation and adaptation of spa type t034 in different host species. The five remaining UK LA-MRSA CC398 isolates clustered within the EU t034 branch, and included several isolates from diseased pigs and one from a cow with mastitis. In fact out of the six UK MRSA CC398 isolates that were recovered from diseased animals, MRSA was thought to be responsible for clinical disease only in APHA02. APHA02 was recovered from swabs taken from skin lesion of diseased piglets, where subsequent culture yielded profuse growth of haemolytic S. aureus colonies. Downstream testing by PCR of suspect colonies showed presence of mecA (Hall et al., 2015). The occurrence of UK MRSA isolates of both spa-type t011 and t034 in different sub-lineages indicates separate incursions of LA-MRSA CC398 into the UK, rather than expansion of a single MRSA clone into different animal hosts.

It is worth noting that several MSSA isolates clustered within the EU t034 sub-lineage, this could indicate instability and loss of the SCCmec V in these isolates or point to multiple independent acquisition events.

It has been proposed that CC398 strains have lost genes associated with virulence in humans as a result of anthroponotic transmission, which has resulted in low level epidemicity in humans (Fitzgerald, 2012). None of the isolates explored in this study carried any human associated virulence genes, in concordance with a previous study (Price et al., 2012) and the above suggestions. However, SAAV_2008 and SAAV_2009 virulence genes associated with the avian adapted φAVβ prophage that have been reported by several authors (Price et al., 2012; Argudin et al., 2013; Abdelbary et al., 2014), were identified in nine MSSA isolates from the US from turkeys and pigs. The avian prophage genes were not detected in any of the UK isolates, including the turkey MRSA isolate (APHA01). Presence of the φAVβ prophage in MSSA but not MRSA in this and other studies (Argudin et al., 2013; Abdelbary et al., 2014) may suggest an association between the φAVβ prophage and MSSA isolates, which requires further investigation.

In conclusion, our data has shown a European origin for the 12 UK CC398 MRSAs from livestock, as demonstrated by the phylogenetic reconstruction performed using WGS data. There are indications from this reconstruction that there have been multiple independent incursions into the UK rather than clonal expansion following a single introduction. To our knowledge this is the first such study which has been performed using WGS data from UK CC398 LA-MRSA livestock isolates. The collection of strains assembled may have some bias as it only includes isolates available to us and WGS from CC398 LA-MRSA of livestock origin available from public archives. In future we hope to repeat the analysis with a larger collection of CC398 isolates from the UK, as well as globally to determine how representative these isolates may be of those that are found in the UK. The number and types of AMR genes present in the UK isolates were similar to other CC398 LA-MRSA of European origin. The virulence genes included in our pipeline were not detected in any of the UK isolates, despite one isolate being associated with disease (APHA02). It is possible that this LA-MRSA isolate harbors virulence determinants that were not included in our pipeline or that other factors predisposed the piglets to an opportunistic staphylococcal infection. Lastly, genes encoding resistance to heavy metals such as zinc and cadmium were present in ~86% of European isolates including the UK CC938 livestock isolates; the degree to which these genes contribute to survival, dissemination and selection of LA-MRSA in livestock is worthy of further study.

We believe this is an important study because it not only indicates the emergence and possible origins of LA-MRSA in UK livestock but its potential to transmit and evolve through different animal host species. It is only by identifying the clonal origin of UK LA-MRSA isolates alongside continual monitoring of genetic traits that appropriate biosecurity and control measures, as well as methods for restricting transmission, can be considered in future.

Laboratory work – MS, MAA, BP, MAb, JR, and RE. Data analysis and Bioinformatics – JN, AK, MD, BP, and MFA. Manuscript preparation and review – MA, AK, CT, MAA, MS, JN, MD, PB, AL, BP, and RE.

This work was funded by the Veterinary Medicines Directorate through grants VM0506 and RDVM0518 to CT and MFA. MAA was supported by a research grant from the Fundación Alfonso Martín Escudero.