Staphylococcus aureus genomics and the impact of horizontal gene transfer

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Contents lists available at ScienceDirect

International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm

Mini Review

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Staphylococcus aureus genomics and the impact of horizontal gene transfer

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Jodi A. Lindsay ∗

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Q2 Division of Infection and Immunity, St George’s, University of London, Cranmer Terrace, London SW17 0RE, United Kingdom

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a r t i c l e

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a b s t r a c t

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Article history: Available online xxx

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Keywords: Mobile genetic elements Transduction Restriction modification Selection Fitness Evolution

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Introduction

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Whole genome sequencing and microarrays have revealed the population structure of Staphylococcus aureus, and identified epidemiological shifts, transmission routes, and adaptation of major clones. S. aureus genomes are highly diverse. This is partly due to a population structure of conserved lineages, each with unique combinations of genes encoding surface proteins, regulators, immune evasion and virulence pathways. Even more variable are the mobile genetic elements (MGE), which encode key proteins for antibiotic resistance, virulence and host-adaptation. MGEs can transfer at high frequency between isolates of the same lineage by horizontal gene transfer (HGT). There is increasing evidence that HGT is key to bacterial adaptation and success. Recent studies have shed light on new mechanisms of DNA transfer such as transformation, the identification of receptors for transduction, on integration of DNA pathways, mechanisms blocking transfer including CRISPR and new restriction systems, strategies for evasion of restriction barriers, as well as factors influencing MGE selection and stability. These studies have also lead to new tools enabling construction of genetically modified clinical S. aureus isolates. This review will focus on HGT mechanisms and their importance in shaping the evolution of new clones adapted to antibiotic resistance, healthcare, communities and livestock. © 2013 Published by Elsevier GmbH.

S. aureus is a common cause of human and animal infection, causing a wide range of superficial through to invasive and toxinmediated diseases. The normal habitat is the nares and mucous membranes, and 30% of humans are persistently colonized. Infection is generally with the host’s own colonizing strain and is as a result of a breach of the protective skin layer, and a failure of the immune system – predominantly the innate system and neutrophils – to clear the organism (Lindsay, 2013). The first S. aureus genomes were sequenced in 2001 and revealed the core metabolic, regulatory and virulent capacity of the organism, although the function of many genes is still unknown (Kuroda et al., 2001). Subsequent genomes differed substantially from the original sequences, and paved the way for population biology studies. Comparisons of whole genomes revealed S. aureus populations are clonal, with ten dominant lineages colonizing and infecting humans, and additional lineages found in animals (Lindsay et al., 2006; Sung et al., 2008). The lineages differ in hundreds of genes that are present/absent, or have variant regions within genes, and include surface proteins, gene regulators

∗ Tel.: +44 0208 725 0445. E-mail address: [email protected]

and immune evasion pathways. In addition, there are SNP differences in housekeeping and other stable genes. Isolates belonging to the same lineage are remarkably conserved, even when separated by time and space. For example, the original MW2 isolate of community-associated methicillin-resistant S. aureus (MRSA) of the lineage clonal complex (CC)1 from the USA and a methicillinsusceptible osteomyelitis CC1 isolate from the UK had only 285 differing SNPs within coding regions in the lineage specific genome (Holden et al., 2004). In contrast, the MGEs of these two strains differ markedly. MGEs account for 15–20% of the genome of S. aureus isolates, and include bacteriophage, S. aureus pathogenicity islands, plasmids, transposons and staphylococcal cassette chromosomes (SCC) (Lindsay, 2010). MGEs vary substantially more than the core genomes of lineages. Snapshots of the genomes of S. aureus populations by whole genome sequencing reveals closely clustered organisms with remarkably different MGE profiles, indicating frequent transfer and loss of whole elements. This is important because the identification of genes encoded on MGEs is revealing important resistance, and host-adaptation mechanisms. They can also encode toxins such as Panton-Valentine leukocidin, the most important toxins for food poisoning such as entertoxins A, B and C, and toxic shock syndrome toxin. As we understand populations and their distribution better, it is becoming clearer that MGEs are key to the evolution of new clones that adapt to new niches and cause novel clinical and economic problems (Lindsay, 2010).

1438-4221/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.ijmm.2013.11.010

Please cite this article in press as: Lindsay, J.A., Staphylococcus aureus genomics and the impact of horizontal gene transfer. Int. J. Med. Microbiol. (2013), http://dx.doi.org/10.1016/j.ijmm.2013.11.010

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Examples of these new clones include healthcare-associated MRSA (HA-MRSA) that are resistant to all types of ␤-lactam antibiotics as well as the fluoroquinolones. These isolates emerged in the 1980s, and are fitter than the early types of MRSA seen in hospitals (Knight et al., 2012). They did not replace sensitive S. aureus but caused an additional disease burden. High levels of MRSA in a hospital prevent the use of ␤-lactams as prophylactic or empirical therapy, meaning inferior antibiotics are relied upon for both preventing and treating many infections. Community-associated MRSA (CA-MRSA) cause severe skin and soft tissue infections in healthy patients in the community. These infections do not respond to ␤-lactams and often require hospitalization and systemic antibiotics, overloading hospital services. Livestock associated MRSA (LA-MRSA) heavily colonize pig and veal calves on farms, leading to colonization of farm workers and vets who are then susceptible to infection. In each of these cases, evolution and selection of these clones is strongly associated with resistance, immune evasion and virulence factors encoded on MGEs (Lindsay, 2010). However, we do not see S. aureus isolates with all MGEs, and we do not even see S. aureus with all of the most important resistance or virulence or host adaption pathways. Therefore, there is much scope for new S. aureus emerging that could cause much greater problems for healthcare, agriculture and potentially other reservoirs by broadening host range, virulence factor and toxin carriage and resistance profile. Our greatest concerns might be fully drug resistant clones in healthcare, healthcare clones that cause severe infection in healthy patients, community clones that spread more easily, or livestock clones that frequently infect animals or carry toxins for food poisoning. The reasons why such clones have not emerged is not clear. However, we are discovering more about MGEs, the genes they carry, how they transfer, barriers to their transfer and factors that influence stability. This greater understanding helps us to predict which strains may evolve in the future and the environmental conditions that may enhance their emergence and selection.

Horizontal gene transfer mechanisms The physical process of transferring DNA from one bacterial cell to another can be accomplished by generalized transduction, conjugation or transformation. Generalized transduction is likely to be the major method for transfer of DNA between S. aureus cells as phage required for transfer are prevalent in S. aureus populations and the process is very efficient in the laboratory.

Generalized transduction Transduction simply describes the transfer of DNA from one cell to another via bacteriophage (phage), and typically refers to the transfer of the phage’s own DNA from one cell to another and its integration into the bacterial chromosome at a specific site. An integrated bacteriophage (prophage) is generally stable in the chromosome and is passed to daughter cells during bacterial replication. Prophage can be induced under stress, leading to excision of the prophage DNA, replication of phage DNA, synthesis of new phage proteins including heads and tails, and packaging of the phage DNA into virulent phage particles. Lysis of the bacterial cell releases infectious phage particles which bind specifically to other S. aureus cells. The receptor for phage binding has recently been discovered to be glycosylated wall teichoic acid (Xia et al., 2011; Winstel et al., 2013). Phage particles inject their DNA into the recipient cell where it either integrates into the chromosome as a prophage (lysogenic pathway), or goes on to generate more infectious phage particles and kill the recipient host (lytic pathway).

These lysogenic, double-stranded DNA phage are common in S. aureus, with most naturally occurring isolates carrying between one and four different prophage types (Lindsay et al., 2006). At least eight families have been described, each with a unique integrase gene and corresponding insertion sites in the bacterial chromosome (McCarthy et al., 2012b). The phage genomes are typically 45 kb in size, and some are known to encode bacterial virulence factors such as enterotoxin A or Panton-Valentine leukocidin, while others encode immune evasion strategies such as chemotaxis inhibitory protein or complement inhibition proteins, and these presumably provide a benefit to the host bacterium. Generalized transduction is a variation of the transduction process, where the newly forming phage particles package bacterial chromosomal or plasmid DNA instead of phage DNA. It is not known if this is accidental or programmed, and why only some bacteriophage do this but not others. The pseudo-phage particles are released during cell lysis, bind to S. aureus receptors on recipient cells, and inject the DNA into the new cell. This DNA is not phage, and therefore does not integrate as like the lysogenic phage, nor does it trigger the lytic lifecycle and kill the recipient cell. Some host DNA appears to be preferentially packaged by generalized transducing phage leading to very high transfer frequencies. S. aureus pathogenicity islands are 15 kb mobile genetic elements related to phage, but that do not have the genes necessary for making phage particles (Lindsay et al., 1998). They can be packaged into miniature phage particles where the necessary proteins are donated by phage (Ruzin et al., 2001). Particular SaPI are preferentially packaged by particular phage (Maiques et al., 2007). SaPIs can encode toxins such as toxic shock syndrome toxin and enterotoxins B and C as well as resistances. Plasmids are also thought to be packaged into phage particles at higher frequency that other DNA, and package as concatemers of sequential phage DNA that is then resolved in the recipient cells (Novick et al., 1986). It is likely that all types of DNA can be packaged by generalized transduction, with a maximum load of around 45 kb. Transduction does not require direct contact between the donor and recipient bacteria. Free phage particles can be released by growing cultures, and are relatively stable. Phage therapy studies have shown that they can also survive in mammalian hosts although some are sequestered by the reticuloendothelial system (Merril et al., 1996). S. aureus bacteriophage are particularly host specific, and this ensures that S. aureus MGE are rarely found in other species or genera. Winstel et al. (2013) have shown that altered glycosylation of the S. aureus bacteriophage receptor is sufficient to block phage binding, but also to prevent HGT from other S. aureus. The divergent S. aureus lineage ST395 has altered glycosylation of wall teichoic acid and this could explain why it does not carry typical S. aureus MGE. Continued evolutionary divergence may lead to new sub-species or species. Conjugation Conjugation involves the transfer of DNA from one cell to another directly through a connecting tube (pilus) or pore (Grohmann et al., 2003). In S. aureus pili are not seen, and it is presumed that pores are formed between neighbouring cells in close proximity. The tra genes encode the necessary proteins and show similarity to type IV secretion systems (Guglielmini et al., 2013) and are carried exclusively on mobile genetic elements. Conjugative plasmids are large, generally over 45 kb and therefore too large to transfer by transduction, and can carry an extensive and variable range of antibiotic and heavy metal resistance genes (McCarthy and Lindsay, 2012; Liu et al., 2013). A recent survey of published and sequenced plasmids in S. aureus revealed tra genes in 4 of 39 plasmid groups and 13 of 243 plasmids (McCarthy and Lindsay, 2012).

Please cite this article in press as: Lindsay, J.A., Staphylococcus aureus genomics and the impact of horizontal gene transfer. Int. J. Med. Microbiol. (2013), http://dx.doi.org/10.1016/j.ijmm.2013.11.010

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Conjugative plasmids also require a nicking enzyme for transfer (Climo et al., 1996; Edwards et al., 2013). Tra genes can also be found on conjugative transposons, the most common being the Tn916 family encoding tetM resistance which, unusually, can be found in multiple genera of Gram positive bacteria (Roberts and Mullany, 2011). S. aureus is also a known recipient of enterococcal tra positive plasmids (Clewell et al., 1985; Sung and Lindsay, 2007), including those that carry high level vancomycin resistance which is of great clinical importance (Weigel et al., 2003). A recent study has suggested that a conjugative plasmid in the recipient S. aureus enhances this process, possibly due to an extracellular product such as a pheromone (Zhu et al., 2013), although the vancomycin and methicillin resistant progeny have a growth defect (Lindsay, 2013). Overall, tra genes are rare, and while they are an important mechanism of transfer, they are not distributed throughout the population in the same manner as lysogenic bacteriophage.

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Transformation is the uptake of free DNA from the environment by a bacterium, usually via bacterially encoded competence proteins that form a pore-like structure in the bacterial cell membrane. This mechanism is extremely efficient in a range of bacteria, such as Streptococcus pneumonia where it was exploited in the earliest genetic studies to prove the role of DNA in conferring phenotype (Lorenz and Wackernagel, 1994). Early studies to identify if S. aureus could take up free DNA from the environment revealed very low transfer efficiency. This transfer was dependent on the presence of prophage or lytic phage, and subsequently it was shown that this transfer required phage proteins, specifically tail proteins (Thompson and Pattee, 1981; Jackson et al., 1983). These tail structures bound to the cell in the presence of DNA and calcium, suggesting DNA transfers into the cell via a similar mechanism to transduction. As this was not the typical transformation process dependent on bacterial competence proteins, it was referred to as DNA mediated transformation, or pseudotransformation. Large fragments of DNA could be transformed, sufficient to construct the early maps of the S. aureus chromosome (Stahl and Pattee, 1983). Since naturally occurring S. aureus are virtually all lysogenic, and cell cultures release lytic phage at free DNA at low frequency, this could account for low level DNA transfer in S. aureus populations. It has now been demonstrated that S. aureus can also undergo natural transformation, via bacterially encoded competence proteins (Morikawa et al., 2012). Expression of competence is controlled by the sigma H factor which is also required for stabilization of lysogenic phage. Surprisingly, the naturally occurring copy of the S. aureus sigH gene is unable to switch on competence until it duplicates itself and in the process changes its promoter region. This spontaneous chromosomal arrangement occurs at low frequency (<10−5 ), so that only a small fraction of the population express the required sigma factor. sigH expression also requires particular nutritional requirements, and in addition is under posttranscriptional control, possibly via a small RNA. When sigH is supplied on a plasmid and expressed at high level, transformation occurs at extremely low frequency (10−9 ). Although most of the experiments were performed in lysogenic bacteria, strain N315 cured of phage and carrying sigH on a plasmid was able to take up DNA, suggesting the process is indeed phage independent. By careful optimization of the system, an SCCmec element from N315 was able to be transformed into an N315 deficient recipient, suggesting this process could also be involved in very rare transfer events of large regions of DNA that do not fit into transducing phage heads. These transformation pathways may be responsible for the rare transfer of very large pieces of DNA that have moved into some isolates creating unique S. aureus hybrid strains. Examples include

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the ST239 lineage derived from a CC8 lineage background that replaced a 550 kb region (20% of the genome) with a SCCmec positive fragment from the CC30 lineage (Robinson and Enright, 2004). Similarly, the CC30 lineage has branched into several variants due to exchange of large regions to generate ST34 and ST42 variants. They may also be responsible for the transfer of the large SCCmec type I–III elements from coagulase negative staphylococci.

Replication or integration of transferred DNA Transferred DNA must be able to replicate itself or integrate into the chromosome or a resident plasmid, otherwise it will become diluted by the next round of bacterial growth and division. Plasmids can circularize and replicate themselves autonomously. Integration is often a very specific process with specific integration targets driven by enzymes encoded on mobile genetic elements (MGE). Integration and replication processes are efficient leading to high transfer rates. They are also tightly regulated, and competition between MGEs can result in failure to transfer DNA successfully. In contrast, chromosomal DNA integrates via homologous recombination dependent on the relatively inefficient RecA system. Bacteriophages and S. aureus pathogenicity islands (SaPIs) encode specific integrases that catalyze the integration of the whole element into a target site (Maiques et al., 2007). There are at least eight different types of integrase in S. aureus bacteriophages, and each one has a different target site (McCarthy et al., 2012b). Similarly there are at least five different SaPI integrases and each has a unique target site (Lindsay et al., 2008). The integrases are used as Q3 the basis of grouping different phage and SaPI into distinct families. Target sites are defined by att flanking regions, and the elements are integrated in a site specific way. Once integrated, the prophage in the lysogenic state down-regulates expression of the integrase and any excisionase function phage encoded regulatory proteins (Mir-Sanchis et al., 2012). These proteins may also account for phage immunity, switching off promotors of incoming phage and preventing successful integration. SCC elements are large pieces of DNA that insert at the orfX gene in S. aureus chromosome and often encode methicillin resistance genes. There are several types, partially characterized by the types of ccr gene they carry. ccr genes catalyze excision and integration of the element into the orfX location (Kaito et al., 2008; Ito et al., 2004). Transposons encode integrases that ensure insertion of the element into either the chromosome or a plasmid. Some transposon integrases are site specific such as Tn554 (Bastos and Murphy, 1988), but others insert randomly such as Tn551 (Pattee, 1981). Plasmids delivered by transduction as multimers must be resolved and re-circularize (Novick et al., 1986) while those delivered by conjugation as single stranded linear DNA must circularize and replicate the double strand (Climo et al., 1996). Plasmids replicate independently of the chromosome using their self-encoded rep genes. The expression level of the Rep protein is very tightly controlled by antisense RNAs, and the level of expression determines the copy number of the plasmid in the cell (Rasooly and Novick, 1993; Liu et al., 2013). Therefore, if a new plasmid enters a cell where a plasmid with the same Rep is controlling copy number, there will be competition between these two plasmids for the Rep protein and one is likely to be lost. This is the likely mechansim for plasmid incompatibility (Novick, 1987). In a recent study of 243 plasmids, 39 plasmid groups were identified, each with one or more of 21 different types of rep gene (McCarthy and Lindsay, 2012). Chromosomal DNA delivered to a new cell does not have a specific pathway for integration and risks loss unless it can be incorporated into the host chromosome. Homologous recombination is dependent on the RecA system for DNA repair (Bayles et al., 1994) and involves the new DNA meeting a choromosomally located

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related gene sequence, and RecA catalyzing a strand cross-over reaction likely in response to chromosomal DNA damage (Ayora et al., 2011). In S. aureus this system is relatively inefficient, as in the laboratory double cross-over mutants with a resistant cassette are extremely difficult to generate, whereas in other laboratory strains such as E. coli they can be isolated routinely.

Barriers to transfer Once DNA enters the bacterial cell, it is also subject to attack by bacterial mechanisms designed to protect the host from foreign DNA. Protection is necessary because lytic bacteriophage can dessimate a bacterial population.

Type I RM The S. aureus Type I RM system is found in nearly all isolates of S. aureus (Waldron and Lindsay, 2006). A Type I R-M system requires three genes, hsdR (restriction), hsdM (modification), and hsdS (specificity) (Murray, 2000) and all three are typically found together. The S. aureus Sau1 system is unusual, and consists of one copy of hsdR, and two different copies of hsdMS at distant locations (Waldron and Lindsay, 2006; Roberts et al., 2013). Each gene product forms a subunit that combines as a complex of M2 S or R2 M2 S. M2 S catalyzes the transfer of a methyl group to adenine residues within a specific target sequence and protects the DNA from restriction. The R2 M2 S complex is a restriction endonuclease that recognizes DNA hemi-methylated at the same target sequence, binds and slides along the DNA until it meets another complex sliding in the opposite direction and cleaves the DNA. The cleavage site is nonspecific and distant to the target site. The S subunit determines the specific sequence target to be methylated or restricted (Murray, 2000) The hsdS genes are highly conserved amongst isolates belonging to the same lineage or clonal complex (CC), but differ substantially between lineages (Waldron and Lindsay, 2006). Therefore, S. aureus lineages see the DNA from other lineages as ‘foreign’. Isolates with the same hsdS genes exchange DNA at high frequency, but transfer frequency is hundreds of times lower between isolates from different lineages with different hsdS variants (Waldron and Lindsay, 2006; Roberts et al., 2013). This likely contributes to the independent evolution of S. aureus lineages. Comparison of the mobile genetic elements carried in S. aureus populations of different lineages suggests frequent HGT, but restriction between lineages (McCarthy and Lindsay, 2012, 2013; McCarthy et al., 2012b). Thus plasmids and bacteriophage types can be associated with particularly lineages and this likely impacts on their ability to adapt to new environments, hosts and virulence. Identification of the target sites that the Sau1 system binds to is difficult because the DNA is not digested at the target site. Plasmid libraries are screened to narrow down and then confirm the specific sequence. Using this approach we have identified the targets sites of the first three S. aureus lineages, CC1, CC5 and CC8 (Roberts et al., 2013). The CC8 hsdS genes are identical to those found in ST239. Unusually, the CC8 lineage shares one hsdS sequence with CC1 isolates, and the other with CC5. The distribution of the target sites in S. aureus genomes and MGEs reveals that conjugative plasmids have specifically lost the targets sites for the unique CC5 system. Therefore, these plasmids are able to transfer without restriction barriers from CC8 isolates to CC5 isolates, but not vice versa (Roberts et al., 2013). Conjugative plasmids encoding antibiotic resistance genes are found in the USA in clinically significant CA-MRSA and HAMRSA CC8 as well as HA-MRSA CC5 lineages, suggesting these plasmids are indeed transferring between lineages and could be

accelerating the evolution of multi-drug resistant clones (Roberts et al., 2013). Construction of genetically modified S. aureus in the laboratory relies on RN4220 and a few other strains that will accept foreign DNA. RN4220 has a double mutation in both the type I hsdR gene and the type IV nuclease (see below) (Waldron and Lindsay, 2006; Corvaglia et al., 2010), explaining why it is an effective intermediate. This strain can accept DNA from E. coli, and crucially it can modify the DNA according to a CC8 lineage pattern, thus making it possible to transfer the DNA to a CC8 isolate with an intact CC8 RM system. RN4220 itself is not a good choice of strain for investigating virulence as it is phage negative, agr defective and has a multitude of SNPs (Nair et al., 2011). Now that the target sequences of major S. aureus lineages have been identified, it will be possible to construct shuttle plasmids missing these target sites for transfer of DNA into and between specific lineages without having to pass through RN4220 (Roberts et al., 2013). Isolates belonging to the bovine lineage ST151 typically have a mutation in both copies of the hsdS gene, so that the Sau1 system is non-functional (Sung et al., 2008). These isolates can take up foreign DNA at high frequency, although interestingly they are relatively susceptible to antibiotics and do not carry unusual MGE types. Perhaps the bovine niche in the UK where they were isolated has relatively low levels of antibiotic resistant S. aureus. This lineage appears to be the exception, and all other S. aureus appear to have functional type I RM systems, indicating they regularly see mobile DNA. Type II RM Type II RM describe the restriction enzymes regularly in use in the molecular biology laboratory because they target and digest specific DNA sequences. These enzymes typically bind to a specific 4–8 nt palindromic sequence and digest reproduceably at this site, often leaving sticky ends. The RM system consists of two enzymes, a modification and a matching restriction enzyme. The modification enzyme modifies host DNA, while the restriction enzyme binds to the same site on incoming DNA if it is unmodified and digests it. Early studies identified the Sau3AI RM in S. carnosus (Seeber et al., 1990). Functional Type II RM have been described in S. aureus, such as Sau42I (Dempsey et al., 2005). However, it is notable that Type II RM appear to be relatively rare in S. aureus and are typically carried on MGE. This implies that the introduction of the MGE causes modification of the host cell DNA, and prevents transfer of DNA into the cell from any donors unless they are similarly modified. Because the systems are rare, this may represent an evolutionary dead-end for the cell that now cannot acquire foreign DNA. A possible exception may be the MRSA CC398 lineage associated with livestock (LA-MRSA). These strains were initially identified because their DNA was uniquely resistant to digestion by the type II RM SmaI, suggesting the SmaI target site of CCCGGG is modified in these strains (Bens et al., 2006). Whole genome sequencing of a CC398 isolate (Schijffelen et al., 2010) reveals a putative Type II RM, provisionally titled SauSTORF2546, that may be responsible. It is carried on a putative core variable region that is conserved in many CC398 isolates and nearby to several transposases. Type IV RM Type IV systems are novel because they recognize and digest DNA that is modified (rather than unmodified) at a specific target sequence. The S. aureus type IV system (originally referred to as a type III system until its function was fully described) is widespread in S. aureus isolates, and is called SauUS1 (Corvaglia et al., 2010; Xu et al., 2011). In S. aureus, only the gene for the restriction enzyme is present, and therefore this system is not involved in modifying

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DNA in S. aureus. The restriction enzyme recognizes and digests introduced DNA when it has been donated from E. coli that modifies cysteines in the DNA via the dcm system (Xu et al., 2011). It seems unusual that all S. aureus should evolve to carry an enzyme for preventing DNA transfer from E. coli, as these two species are not thought to share DNA. There is no evidence that HGT between these species is shaping bacterial evolution. However, understanding this restriction barrier is very useful for understanding barriers to HGT and the construction of genetically modified S. aureus strains (Sung and Lindsay, 2007; Veiga and Pinho, 2009). Plasmids can be constructed in a dcm-E. coli background and easily transformed directly into S. aureus (Xu et al., 2011; Monk, 2012; Prax et al., 2013). This increases the frequency of DNA transfer into S. aureus clinical isolates without having to pass through a restriction-deficient strains such as RN4220 and ensures much simpler construction of genetically modified isolates.

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CRISPR loci are clustered, regularly interspaced, short palindromic repeats that function to inhibit transfer of specific MGEs that have matching sequences to the CRISPR spacers (Marraffini and Sontheimer, 2008). In staphylococci, they were first described in the S. epidermidis isolate RP62a, which contains a CRISPR locus that includes a spacer that is homologous to a region of the nickase gene encoded on staphylococcal conjugative plasmids and required for plasmid replication. The spacers are transcribed and processed into small RNAs that interfere with the nickase gene (Marraffini and Sontheimer, 2008). The exact mechanism of target interference due to the neighbouring CRISPR associated genes (cas) genes has not been described in staphylococci, but it is likely that binding to the plasmid DNA in the recipient cell targets it for digestion via CRISPR encoded nucleases (Makarova et al., 2011). No other CRISPR region has been identified in other sequenced S. epidermidis strains. It is also very rare in S. aureus genomes, and has been reported three times. The first was in the CC75 lineage of S. aureus from northern Australia which represent the most divergent branch of the S. aureus phylogenetic tree (Holt et al., 2011). An MRSA of the unusual ST779 sequence type from an Irish hospital revealed a CRISPR locus associated with the SCCmec, and 5/13 additional isolates from the same ST were also positive (Kinnevey et al., 2013). Of 16 Canadian isolates of CC398 MRSA, the lineage associated with livestock (LA-MRSA), six carried CRISPR loci in the SCCmec region (Golding et al., 2012). At present it is not known if the CRISPR systems in S. aureus are functional. CRISPR, like RM systems, can be regarded as bacterial immune systems protecting the cell from attack by invading DNA such as plasmids and phage (Makarova et al., 2011). In the case of CRISPRs, ‘vaccination’ is possible by incorporating fragments of the attacking DNA into the locus. However, since each introduced spacer targets a unique MGE, staphylococci would require very large variety of spacer regions to protect them from the very large number of known MGE types. This would limit its effectiveness as an immune system. Interestingly, recent studies have suggested that the presence of CRISPR systems in streptococci prevent HGT of virulence and resistance genes, and thus might be selected against in rapidly evolving bacterial pathogens (Bikard et al., 2012). Therefore, S. aureus probably does not rely on CRISPR for immunity due to the wide variety of MGEs moving between isolates, and because these MGEs encode useful functions that provide a selective advantage in its normal habitats.

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Once new DNA has transferred, evaded digestion and replicated, it can start to exert an influence on the host cell. Whether it

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survives is dependent on the fitness costs versus advantages of the new DNA to the cell in its environmental niche. Since there is so much variation in MGE in S. aureus populations, transfer and loss must both occur at substantial frequency. When an MGE becomes fixed in a population, it likely is exerting a selective advantage. Resistance genes provide a substantial selective advantage to their host when exposed to their corresponding antibiotic. S. aureus isolates have developed resistance to all known antibiotic classes, most often due to genes carried on MGE (Lindsay, 2013). However, the majority of isolates that colonize humans are only resistant to ␤-lactam antibiotics. Resistance is maintained in populations that are continuously exposed. One of these environments is healthcare where certain clones have become successful and spread in hospitals. They are typically resistant to both methicillin and the fluoroquinolones, and are known as healthcare-associated MRSA (HA-MRSA). Depending on the geography and hospital, the proportion of HA-MRSA causing S. aureus infections can range from a few percent to over 60% (Stefani et al., 2012). These clones can carry resistance to macrolides, aminoglycosides, fusidic acid, rifampicin, tetracyclines, mupirocin, linezolid, daptomycin and very rarely to glycopeptides (Lindsay, 2012). However, no strains have acquired resistance to all antibiotics (Lindsay, 2013). We have recently reported that HAMRSA clones appear to acquire and lose a range of resistances at high frequency, suggesting a shuffling of MGE in response to multiple and intermittent antibiotic exposures (Knight et al., 2012). This is likely to be pronounced in the skin and mucous membranes of colonized patients in hospitals receiving prophylactic, empirical and therapeutic treatments, including for non-staphylococcal infections. Our studies also suggested that HA-MRSA clones that shuffle resistances most efficiently have a selective advantage in hospitals (Knight et al., 2012). The carriage of methicillin resistance on the largest SCCmec elements is known to have a fitness cost (Ender et al., 2004). The fact that HA-MRSA clones carrying these elements survived and spread in hospitals shows the advantage of the resistance phenotype outweighed the cost. However, the newer and leaner SCCmec elements do not appear to have a fitness cost (Lee et al., 2007; Knight et al., 2013), and the most successful of the HA-MRSA clones – CC22 SCCmecIV – carries a lean element and is becoming the dominant clone in an increasing number of countries. This lineage may have an additional fitness advantage that is unrelated to the SCCmec (Knight et al., 2013). So far, a specific fitness cost associated with other MGE, including the large plasmids often found in HA-MRSA, has not been identified (Knight et al., 2013). Since they are lost frequently, it is possible that the relevant selective environment has not yet been identified. The community-associated MRSA (CA-MRSA) also carry the lean SCCmecIV element. These clones are typically resistant to ␤-lactams, but are often sensitive to other antibiotics. The epidemiological feature most commonly associated with CA-MRSA are bacteriophage encoding Panton-Valentine leukocidin. This toxin may play a role in virulence, but it is unclear why this factor may contribute to a selective advantage for colonization and spread in healthy populations, and especially children, athletes, the military, prisons and other communities in close physical contact. At present this is restricted to certain regions such as the USA and Asia (Lindsay, 2010). Livestock-associated MRSA (LA-MRSA) have spread rapidly in pig and veal calf farms in mainland Europe and the USA since their first description in 2005. These clones are SCCmecIII positive and resistant to tetracyclines, often with more than one resistance gene (McCarthy et al., 2012). ␤-Lactams and tetracyclines are widely used in intensive agriculture. At present, these clones do not cause infection in pigs but heavy colonization leads to colonization of farm workers and vets, and can lead to subsequent human

Please cite this article in press as: Lindsay, J.A., Staphylococcus aureus genomics and the impact of horizontal gene transfer. Int. J. Med. Microbiol. (2013), http://dx.doi.org/10.1016/j.ijmm.2013.11.010

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infection. The rapid selection and extensive spread in the farms suggest a substantial selective pressure for survival of these clones over other colonizing flora. Epidemiological studies also strongly implicate certain MGEs with host-niches. The clearest example is the phi3 bacteriophage family that often encode immune evasion pathways, such as staphylococcal complement inhibitory protein (scin), chemotaxis inhibitory protein (chips) and staphylokinase (sak). These proteins specifically target immune pathways of the human host. The phi3 phage are predominantly found in human isolates, and are much less prevalent in animal isolates of S. aureus (Sung et al., 2008). Other examples include ruminant specific coagulants encoded on SaPIs in ruminants (Guinane et al., 2010), and avian specific immune evasion pathways in bird isolates (Lowder et al., 2009) The sasX gene has recently been shown to contribute to the colonization and pathogenesis of MRSA ST239 isolates. It is encoded on an unusual large bacteriophage-like element, and is spreading to other clones, and appears to be selecting for waves of MRSA in Asia (Li et al., 2012). MGEs can carry genes that strongly alter their selection in unusual ways. Toxin-antitoxin systems encoded on MGE have been reported in S. aureus, and have a fitness cost if they are lost. During loss, the anti-toxin remaining in the cell has a shorter half-life than the toxin. The toxin is released from the anti-toxin and either kills the host or prevents cell growth (Fu et al., 2009; Sayed et al., 2012). Unexpectedly, some MGEs can encode inhibitors of spreading and virulence. A gene called fudoh is found in some SCCmecII elements and has these properties. Presumably, the element has only survived and been selected in S. aureus populations because the benefits of the SCCmec element were more beneficial than the cost to the cell.

Conclusions MGEs that transfer between S. aureus dramatically alter the ability of strains to evade antibiotics, colonize new hosts and adapt to new environments. The major transfer system in S. aureus is generalized transduction, although conjugation can occur, followed by transformation at much lower frequency. MGE transfer successfully because they replicate or integrate into the chromosome or plasmids efficiently. The major barrier to S. aureus gene transfer are the RM systems, particularly type I. CRISPR have been described but are rare. The type I RM system ensures the relatively independent evolution of S. aureus lineages and reduced transfer of DNA between lineages. Successful MGEs must provide a selective advantage to the host that outweighs their fitness cost or they will be lost. The major selective advantage is antibiotic resistance, and host immune evasion in the normal habitat of the skin and mucous membranes. There is evidence that acquisition and loss is frequent and bacteria with flexible genomes are selected for. In contrast, when an MGE becomes fixed in a population, it likely is exerting a selective advantage. These rules are crucial for our understanding of how S. aureus populations are being shaped by genetic and selective pressures. They allow us to understand how and why certain clones are selected in different environments and to predict risks for the evolution of increasingly virulent and resistant strains. The rules are also leading to a new generation of tools to genetically manipulate S. aureus and evade both type I and type IV restriction systems. S. aureus populations are continuing to evolve, partly in response to our use of antibiotics and healthcare and agricultural practices. New clones causing economic and social costs are likely to emerge in a variety of settings in the short-to-medium term.

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