BACKGROUND
methicillin-resistant staphylococcus aureus  are versatile and highly adaptive bacteria that are a major cause of hospital-associated  infections, and are emerging to be a common cause of community-associated  and livestock-associated  infections.
resistance to every antibiotic commonly prescribed is reported, and therefore the treatment and control of mrsa populations is difficult; this is of global concern.
resistance and virulence genes are often carried on mobile genetic elements , such as bacteriophage, plasmids and transposons  <cit> .
dissemination of these genes through s. aureus populations by horizontal gene transfer  will lead to strains that are both more resistant and more virulent  <cit> .
plasmids carry a diverse range of antimicrobial and biocide resistance genes and can carry toxin genes  <cit> .
resistances to antimicrobial agents carried by s. aureus plasmids include aminoglycosides, β-lactams and macrolides.
recently, the sequencing of s. aureus plasmids originating from different bacterial environments has revealed novel resistance genes, such as the apma and vgac genes encoding resistance to apramycin and streptogramin a, respectively  <cit> .
in addition, heavy metal resistance genes are often carried on plasmids  <cit> .
toxin genes carried on s. aureus plasmids include exotoxin b , a toxin that causes blistering of the skin, and the toxins enta, entg, entj and entp  <cit> .
the classification of plasmids has historically been determined by incompatibility groups based on the finding that two plasmids with the same replication  proteins cannot be stably maintained in the same cell  <cit> .
more recently this method has been developed based on the sequence of the rep genes  <cit> .
the sequence of a large number of plasmids isolated from s. aureus has now been released into the public domain; however there is currently no clear understanding of how virulence genes and resistance genes are linked to rep genes and plasmids.
such knowledge is fundamental in understanding the spread of resistance and virulence.
additional barriers to the spread of plasmids between bacteria are the restriction-modification  systems.
two systems have been described in s. aureus; the type iii r-m system protects bacteria against foreign dna originating from other bacterial species  <cit> , whilst the type i  r-m system protects bacteria against dna originating from isolates of different s. aureus lineages  <cit> .
the type i rm system consists of a restriction subunit  and a modification subunit  that can cleave and methylate dna, and a specificity subunit  that determines the specificity of the restriction and modification.
each lineage of s. aureus encodes unique sequence specificity hsds genes; and this means that dna originating from different lineages by hgt is detected as foreign dna and is digested, whilst dna originating from the same lineage is detected as self dna and remains undigested.
therefore, exchange of mges between lineages is infrequent  <cit> .
human s. aureus can be grouped into  <dig> major clonal complex  lineages and many minor lineages  <cit> .
each lineage has a unique but highly conserved combination of genes encoding surface and secreted proteins  <cit> .
however, there is much variation in the carriage of mges within a lineage suggesting that hgt is frequent within a s. aureus lineage  <cit> .
our specific aims of this study were  to extend the rep family classification to  <dig> sequenced s. aureus plasmids,  to characterise the distribution of rep genes amongst the sequenced plasmids,  to assess the distribution of  <dig> resistance and virulence genes between plasmids, and  to investigate the distribution of plasmids between 254 s. aureus isolates from  <dig> different lineages using microarray analysis.
the overall aim was to better understand the dissemination of plasmids, resistance and virulence genes in s. aureus populations.
we report  <dig> unique plasmid groups each with a unique combination of rep genes, and demonstrate that resistance and virulence genes are associated with plasmid groups and with lineage.
both of these findings suggest that genetic pressures are restraining the evolution of increasingly resistant and virulent s. aureus strains.
RESULTS
characterisation of rep families
a total of  <dig> rep families were assigned.
<dig> families  match those previously characterised by jensen et al.
<cit> .
<dig> rep families are newly characterised in this study.
<dig> orphan rep sequences were also identified; in plasmids pavx , pwbg <dig> , pwbg <dig> , pkks <dig> , prj <dig> , sap099b .
plasmid groups possess unique combinations of rep genes
a total of  <dig> plasmid groups of staphylococcus aureus  were assigned  based on the combination of rep genes each plasmid possessed.
each plasmid group had a unique combination of rep gene sequences.
<dig> of the  <dig> sequenced plasmids contain orphan rep sequences and were not assigned to a plasmid group.
<dig> plasmid groups carried  <dig> rep sequence,  <dig> plasmid groups carried  <dig> rep sequences and  <dig> plasmid groups carried  <dig> rep sequences.
the large number of plasmid groups with more than  <dig> rep gene indicates high levels of recombination between s. aureus plasmids.
we note that in the majority of cases there was no difference in the length of a rep gene that appeared on single rep plasmids or multi-rep plasmids.
the number of plasmids belonging to each plasmid group varied considerably .
the average length of plasmids belonging to plasmid groups varied .
nine plasmid groups have small genomes  and carried few genes.
<dig> plasmid groups have large genomes  and carried a diverse range of genes.
<dig> of these  <dig> large plasmid groups possessed more than  <dig> rep gene sequence.
many of these large plasmids carried rep genes found in small plasmids indicating recombination and integration of smaller plasmids.
13/ <dig> plasmids carried plasmid conjugation transfer  a-m genes.
all plasmids from groups pgsa <dig>  pgsa <dig> and pgsa <dig> possessed traa-m genes, whilst plasmids from group pgsa <dig> possess homologs of trae, trag and trai.
conjugation ability is therefore tightly linked with the replication machinery and rep sequences of rep <dig> and rep <dig>  respectively.
resistance genes and virulence genes are associated with plasmid groups
the distribution of antimicrobial resistance, biocide resistance and heavy metal resistance genes found on plasmids was investigated .
the same resistance gene profile was found amongst all members of  <dig> plasmid groups .
for example, small plasmids belonging to pgsa <dig> all carried the ermc gene, and differed only by snps and insertions and deletions suggesting they are clonal .
however, in  <dig> other small plasmid groups completely different resistance gene profiles existed.
for example, the  <dig> plasmids belonging to the pgsa <dig> plasmids carried either cat, tetk, str or vgaa.
in contrast, larger plasmids carried more resistance genes, and  <dig> plasmid groups had more than one resistance gene profile.
the majority of variation within these plasmid groups was due to the addition of resistance genes to a set of core conserved resistance genes or due to different combinations of the same resistances.
for example, pgsa <dig> plasmids carried blaz and caddx with or without aac/aph, aade, apha, bcra, ip <dig>  mphbm, qaca, sat and tcaa .
toxin genes were rare amongst the sequence plasmids.
etb was only found in petb.
the genes enta, entg and entj were tightly associated with pgsa <dig> .
these genes were also present in a single member of the pgsa <dig> group suggesting that these genes can move to other plasmids.
entp was associated with pgsa <dig> .
interestingly, these toxin genes were most frequently found on plasmids carrying more than  <dig> rep gene.
some resistance genes had strong associations with particular rep genes and plasmid groups.
the tetracycline resistance gene tetk was found in pgsa <dig> plasmids indicating that the gene is tightly linked with the rep <dig> gene .
the chloramphenicol resistance gene cat was found only in pgsa <dig>  pgsa <dig> and pgsa <dig> plasmids.
other resistance genes were not associated with particular rep genes or plasmid groups; arsc, blaz, caddx, qaca.
microarray analysis reveals that rep, resistance and virulence genes are associated with s. aureus lineage
microarray analysis showed that there was a differential distribution of 4/ <dig> rep genes represented on the microarray  .
rep <dig> genes were found in isolates belonging to s. aureus lineages cc <dig>  cc <dig>  cc <dig> and cc <dig> but were rare in other major lineages.
rep <dig> gene was commonly found in cc <dig> s. aureus, but was rare in other major lineages.
rep <dig> was found commonly in cc <dig> isolates.
rep <dig> was found s. aureus isolates belonging to lineages cc <dig>  cc <dig>  cc <dig>  cc <dig> and cc <dig>  but was rare in other lineages.
rep <dig> were rare in all the s. aureus isolates included in our analysis.
this analysis demonstrates an association of rep genes with s. aureus lineages.
this is likely to be driven by both clonal expansion and by more frequent hgt within lineages than between lineages.
we also assessed the distribution of other plasmid genes between s. aureus lineages.
the presence of plasmid conjugation transfer a-m genes was rare amongst the s. aureus isolates in our collection and was not associated with lineage .
interestingly, antimicrobial resistance genes and heavy metal resistance genes were associated to lineage.
arsc was common in mrsa cc <dig> and cc <dig> isolates, but rare amongst other lineages.
blaz was common in all human lineages of s. aureus but was rare in animal lineages of s. aureus.
cada presence was associated with mrsa cc <dig>  cc <dig> and cc <dig> lineages, whilst caddx was widely distributed and associated with  <dig> different lineages.
erma presence was associated with cc <dig> and cc <dig> lineages.
qaca was associated with cc <dig> lineage.
<dig> of  <dig>  resistance genes represented on the microarray are rare in the isolates we have analysed and were not distributed in a lineage dependent manner.
we note that some of these genes may be carried on other elements or on integrated plasmids and this cannot be determined by microarray alone, for example tetm can also be carried on transposons such as tn <dig>
discussion
in this study we extended a previously proposed plasmid classification system to characterise rep genes from  <dig> plasmids that appear in the public domain  <cit> .
we characterised  <dig> rep families, of which  <dig> are newly described in this study.
whilst performing this analysis we noted that many plasmids carried more than one rep gene, we therefore assigned plasmids into groups based on the combination of rep genes carried.
a total of  <dig> plasmid groups were assigned, and interestingly 20/ <dig> groups of sequenced plasmid carry more than one rep gene sequence.
this indicates that recombination between s. aureus plasmids has occurred frequently.
recombination between s. aureus plasmids has been described, but the mechanisms and the frequency of such recombination events is not clearly understood  <cit> .
recombination should be a mechanism that transfers virulence and resistance genes into new plasmid groups.
the highly mosaic structure of plasmids seen suggests frequent recombination, but if this was completely random then resistance and virulence genes would not be associated to particular plasmid groups.
surprisingly, this was not the case.
we found that some resistance and virulence genes were associated with plasmid groups; for example all pgsa <dig> carried the ermc gene.
this suggests there are tight associations between particular rep and resistance gene combinations.
resistance and virulence genes that had wider plasmid distributions were typically located on transposable elements that can “hop” between plasmids.
this included blaz located on tn <dig> and caddx on insertion sequence  elements  <cit> .
we also found evidence of movement of genes tightly linked to specific plasmids;  the virulence genes enta, entg and entj are tightly linked with pgsa <dig>  but were also found in a single plasmid that belongs to pgsa <dig>  and  the bacitracin resistance gene bcra that is tightly linked to the pgsa <dig> plasmids, was also found in 1/ <dig> pgsa <dig> plasmids.
this argues that recombination can disseminate resistance and virulence genes into new plasmids, though this is rare.
why is plasmid recombination not completely random?
recombination is likely to generate non-functional plasmids, or novel plasmids that cannot out-compete their parental plasmids.
because of the rm system it is possible that some plasmids do not come into contact because they are restricted to a small number of lineages.
some plasmids will be selected for because they provide a benefit to their hosts in specific environments.
in addition, plasmids may be incompatible and this means that certain plasmids may not survive well in the same cell.
indeed, this study also showed that the distribution of plasmids in s. aureus is lineage associated.
this could limit the opportunities for plasmids in different lineages to recombine.
there are two possible explanations for lineage associations of plasmids.
firstly, plasmids are distributed by clonal expansion and passed to daughter cells during replication.
we found evidence that this occurs frequently, such as the cc <dig> isolates included in our analysis which represent a single dominant clone of invasive mrsa from a hospital in london, u.k.  <cit> .
all isolates carried the same rep genes; this is evidence that clonal expansion can be a cause of plasmid distributions being lineage associated.
our conclusions are supported by the recent finding that usa <dig>  isolates carried highly conserved plasmids  <cit> .
the second explanation is that plasmids are transferred between isolates frequently, but are blocked by efficient rm barriers, reducing transfer between isolates of different lineages.
we found evidence that this occurs in s. aureus populations.
many plasmids were lineage associated but only found in some isolates, including those from different times and locations, indicating loss of plasmids as well as transfer.
the plasmids and resistances carried by our s. aureus isolates are reflective of the selective exposures existing in u.k. environments.
isolates originating from different countries may belong to different lineages and come into contact with the different exposures and carry different plasmids and resistances, or carry them at different frequencies  <cit> .
antibiotic usage and host specific plasmids are therefore also likely to have roles in controlling plasmid dissemination.
the sequenced s. aureus plasmids may not be representative of all plasmid diversity, as they originate from a small number of lineages from only a few countries.
it is generally accepted that plasmids that contain the same origin of replication are incompatible and cannot survive within the same cell  <cit> .
this study has identified a diverse range of rep genes and rep gene combinations.
biological tests are required to determine the incompatibility of plasmid groups, and to draw conclusions on the importance of this phenomenon in limiting plasmid recombination.
mges in other bacterial species may be additional sources of novel resistance and virulence genes that can move into s. aureus populations.
importantly, the vana gene in vancomycin-resistant s. aureus  isolates is carried on a transposon tn <dig> which is commonly found in vancomycin-resistant enterococci  <cit> .
in some vrsa isolates the entire enterococcal plasmid has been maintained, whilst in others tn <dig> has moved onto a staphylococcal plasmid.
both genetic events suggest that enterococcal plasmid have successfully transferred into s. aureus bacteria.
future studies are required that assess the mosaicism of staphylococcal and enterococcal plasmids in order to understand the frequency of recombination and gene exchange between such bacterial species.
hgt mechanisms spread resistance and virulence genes between bacteria and populations.
in s. aureus, two major hgt mechanisms have been described for plasmid movement  plasmid conjugation via the conjugation transfer  complex, and  bateriophage generalized transduction.
in addition, it is possible that smaller plasmids can hitchhike larger plasmids that carry the tra complex and be transferred from donor to recipient bacteria  <cit> .
we found that the tra genes were rare amongst the sequenced plasmids  and were rare amongst our collection of 254 s. aureus isolates.
bacteriophage generalized transduction can transfer dna fragments of less than 45kb.
we found that  <dig>  % of plasmids could theoretically be transferred by generalized transduction as they have genomes that are <45kb in length.
<dig> of the  <dig> plasmids >45kb in length carry the tra genes.
collectively, this data suggests that conjugative plasmids and plasmid conjugation are infrequent, and that bacteriophage transduction is likely to be the most frequent transfer mechanism of plasmids, particularly non-conjugative plasmids.
CONCLUSIONS
plasmids are a principal driver of the spread of virulence and resistance genes in s. aureus populations via hgt, which is blocked by lineage specific r-m systems.
this study has demonstrated that resistance and virulence genes are associated with plasmid groups, and that plasmids are associated with s. aureus lineage.
this is evidence that genetic pressures and rm barriers are limiting the evolution of more resistant and more virulent s. aureus strains.

 methicillin-resistant staphylococcus aureus are versatile and highly adaptive bacteria that are a major cause of hospital-associated infections, and are emerging to be a common cause of community-associated and livestock-associated infections. resistance to every antibiotic commonly prescribed is reported, and therefore the treatment and control of mrsa populations is difficult; this is of global concern. resistance and virulence genes are often carried on mobile genetic elements, such as bacteriophage, plasmids and transposons. dissemination of these genes through s. aureus populations by horizontal gene transfer will lead to strains that are both more resistant and more virulent. plasmids carry a diverse range of antimicrobial and biocide resistance genes and can carry toxin genes. resistances to antimicrobial agents carried by s. aureus plasmids include aminoglycosides, β-lactams and macrolides. recently, the sequencing of s. aureus plasmids originating from different bacterial environments has revealed novel resistance genes, such as the apma and vgac genes encoding resistance to apramycin and streptogramin a, respectively. in addition, heavy metal resistance genes are often carried on plasmids. toxin genes carried on s. aureus plasmids include exotoxin b, a toxin that causes blistering of the skin, and the toxins enta, entg, entj and entp. the classification of plasmids has historically been determined by incompatibility groups based on the finding that two plasmids with the same replication proteins cannot be stably maintained in the same cell. more recently this method has been developed based on the sequence of the rep genes. the sequence of a large number of plasmids isolated from s. aureus has now been released into the public domain; however there is currently no clear understanding of how virulence genes and resistance genes are linked to rep genes and plasmids. such knowledge is fundamental in understanding the spread of resistance and virulence. additional barriers to the spread of plasmids between bacteria are the restriction-modification systems. two systems have been described in s. aureus; the type iii r-m system protects bacteria against foreign dna originating from other bacterial species, whilst the type i r-m system protects bacteria against dna originating from isolates of different s. aureus lineages. the type i rm system consists of a restriction subunit and a modification subunit that can cleave and methylate dna, and a specificity subunit that determines the specificity of the restriction and modification. each lineage of s. aureus encodes unique sequence specificity hsds genes; and this means that dna originating from different lineages by hgt is detected as foreign dna and is digested, whilst dna originating from the same lineage is detected as self dna and remains undigested. therefore, exchange of mges between lineages is infrequent. human s. aureus can be grouped into major clonal complex lineages and many minor lineages. each lineage has a unique but highly conserved combination of genes encoding surface and secreted proteins. however, there is much variation in the carriage of mges within a lineage suggesting that hgt is frequent within a s. aureus lineage. our specific aims of this study were to extend the rep family classification to sequenced s. aureus plasmids, to characterise the distribution of rep genes amongst the sequenced plasmids, to assess the distribution of resistance and virulence genes between plasmids, and to investigate the distribution of plasmids between 254 s. aureus isolates from different lineages using microarray analysis. the overall aim was to better understand the dissemination of plasmids, resistance and virulence genes in s. aureus populations. we report unique plasmid groups each with a unique combination of rep genes, and demonstrate that resistance and virulence genes are associated with plasmid groups and with lineage. both of these findings suggest that genetic pressures are restraining the evolution of increasingly resistant and virulent s. aureus strains.  characterisation of rep families a total of rep families were assigned. families match those previously characterised by jensen et al.. rep families are newly characterised in this study. orphan rep sequences were also identified; in plasmids pavx, pwbg, pwbg, pkks, prj, sap099b. plasmid groups possess unique combinations of rep genes a total of plasmid groups of staphylococcus aureus were assigned based on the combination of rep genes each plasmid possessed. each plasmid group had a unique combination of rep gene sequences. of the sequenced plasmids contain orphan rep sequences and were not assigned to a plasmid group. plasmid groups carried rep sequence, plasmid groups carried rep sequences and plasmid groups carried rep sequences. the large number of plasmid groups with more than rep gene indicates high levels of recombination between s. aureus plasmids. we note that in the majority of cases there was no difference in the length of a rep gene that appeared on single rep plasmids or multi-rep plasmids. the number of plasmids belonging to each plasmid group varied considerably. the average length of plasmids belonging to plasmid groups varied. nine plasmid groups have small genomes and carried few genes. plasmid groups have large genomes and carried a diverse range of genes. of these large plasmid groups possessed more than rep gene sequence. many of these large plasmids carried rep genes found in small plasmids indicating recombination and integration of smaller plasmids. 13/ plasmids carried plasmid conjugation transfer a-m genes. all plasmids from groups pgsa pgsa and pgsa possessed traa-m genes, whilst plasmids from group pgsa possess homologs of trae, trag and trai. conjugation ability is therefore tightly linked with the replication machinery and rep sequences of rep and rep respectively. resistance genes and virulence genes are associated with plasmid groups the distribution of antimicrobial resistance, biocide resistance and heavy metal resistance genes found on plasmids was investigated. the same resistance gene profile was found amongst all members of plasmid groups. for example, small plasmids belonging to pgsa all carried the ermc gene, and differed only by snps and insertions and deletions suggesting they are clonal. however, in other small plasmid groups completely different resistance gene profiles existed. for example, the plasmids belonging to the pgsa plasmids carried either cat, tetk, str or vgaa. in contrast, larger plasmids carried more resistance genes, and plasmid groups had more than one resistance gene profile. the majority of variation within these plasmid groups was due to the addition of resistance genes to a set of core conserved resistance genes or due to different combinations of the same resistances. for example, pgsa plasmids carried blaz and caddx with or without aac/aph, aade, apha, bcra, ip mphbm, qaca, sat and tcaa. toxin genes were rare amongst the sequence plasmids. etb was only found in petb. the genes enta, entg and entj were tightly associated with pgsa. these genes were also present in a single member of the pgsa group suggesting that these genes can move to other plasmids. entp was associated with pgsa. interestingly, these toxin genes were most frequently found on plasmids carrying more than rep gene. some resistance genes had strong associations with particular rep genes and plasmid groups. the tetracycline resistance gene tetk was found in pgsa plasmids indicating that the gene is tightly linked with the rep gene. the chloramphenicol resistance gene cat was found only in pgsa pgsa and pgsa plasmids. other resistance genes were not associated with particular rep genes or plasmid groups; arsc, blaz, caddx, qaca. microarray analysis reveals that rep, resistance and virulence genes are associated with s. aureus lineage microarray analysis showed that there was a differential distribution of 4/ rep genes represented on the microarray. rep genes were found in isolates belonging to s. aureus lineages cc cc cc and cc but were rare in other major lineages. rep gene was commonly found in cc s. aureus, but was rare in other major lineages. rep was found commonly in cc isolates. rep was found s. aureus isolates belonging to lineages cc cc cc cc and cc but was rare in other lineages. rep were rare in all the s. aureus isolates included in our analysis. this analysis demonstrates an association of rep genes with s. aureus lineages. this is likely to be driven by both clonal expansion and by more frequent hgt within lineages than between lineages. we also assessed the distribution of other plasmid genes between s. aureus lineages. the presence of plasmid conjugation transfer a-m genes was rare amongst the s. aureus isolates in our collection and was not associated with lineage. interestingly, antimicrobial resistance genes and heavy metal resistance genes were associated to lineage. arsc was common in mrsa cc and cc isolates, but rare amongst other lineages. blaz was common in all human lineages of s. aureus but was rare in animal lineages of s. aureus. cada presence was associated with mrsa cc cc and cc lineages, whilst caddx was widely distributed and associated with different lineages. erma presence was associated with cc and cc lineages. qaca was associated with cc lineage. of resistance genes represented on the microarray are rare in the isolates we have analysed and were not distributed in a lineage dependent manner. we note that some of these genes may be carried on other elements or on integrated plasmids and this cannot be determined by microarray alone, for example tetm can also be carried on transposons such as tn discussion in this study we extended a previously proposed plasmid classification system to characterise rep genes from plasmids that appear in the public domain. we characterised rep families, of which are newly described in this study. whilst performing this analysis we noted that many plasmids carried more than one rep gene, we therefore assigned plasmids into groups based on the combination of rep genes carried. a total of plasmid groups were assigned, and interestingly 20/ groups of sequenced plasmid carry more than one rep gene sequence. this indicates that recombination between s. aureus plasmids has occurred frequently. recombination between s. aureus plasmids has been described, but the mechanisms and the frequency of such recombination events is not clearly understood. recombination should be a mechanism that transfers virulence and resistance genes into new plasmid groups. the highly mosaic structure of plasmids seen suggests frequent recombination, but if this was completely random then resistance and virulence genes would not be associated to particular plasmid groups. surprisingly, this was not the case. we found that some resistance and virulence genes were associated with plasmid groups; for example all pgsa carried the ermc gene. this suggests there are tight associations between particular rep and resistance gene combinations. resistance and virulence genes that had wider plasmid distributions were typically located on transposable elements that can “hop” between plasmids. this included blaz located on tn and caddx on insertion sequence elements. we also found evidence of movement of genes tightly linked to specific plasmids; the virulence genes enta, entg and entj are tightly linked with pgsa but were also found in a single plasmid that belongs to pgsa and the bacitracin resistance gene bcra that is tightly linked to the pgsa plasmids, was also found in 1/ pgsa plasmids. this argues that recombination can disseminate resistance and virulence genes into new plasmids, though this is rare. why is plasmid recombination not completely random? recombination is likely to generate non-functional plasmids, or novel plasmids that cannot out-compete their parental plasmids. because of the rm system it is possible that some plasmids do not come into contact because they are restricted to a small number of lineages. some plasmids will be selected for because they provide a benefit to their hosts in specific environments. in addition, plasmids may be incompatible and this means that certain plasmids may not survive well in the same cell. indeed, this study also showed that the distribution of plasmids in s. aureus is lineage associated. this could limit the opportunities for plasmids in different lineages to recombine. there are two possible explanations for lineage associations of plasmids. firstly, plasmids are distributed by clonal expansion and passed to daughter cells during replication. we found evidence that this occurs frequently, such as the cc isolates included in our analysis which represent a single dominant clone of invasive mrsa from a hospital in london, u.k.. all isolates carried the same rep genes; this is evidence that clonal expansion can be a cause of plasmid distributions being lineage associated. our conclusions are supported by the recent finding that usa isolates carried highly conserved plasmids. the second explanation is that plasmids are transferred between isolates frequently, but are blocked by efficient rm barriers, reducing transfer between isolates of different lineages. we found evidence that this occurs in s. aureus populations. many plasmids were lineage associated but only found in some isolates, including those from different times and locations, indicating loss of plasmids as well as transfer. the plasmids and resistances carried by our s. aureus isolates are reflective of the selective exposures existing in u.k. environments. isolates originating from different countries may belong to different lineages and come into contact with the different exposures and carry different plasmids and resistances, or carry them at different frequencies. antibiotic usage and host specific plasmids are therefore also likely to have roles in controlling plasmid dissemination. the sequenced s. aureus plasmids may not be representative of all plasmid diversity, as they originate from a small number of lineages from only a few countries. it is generally accepted that plasmids that contain the same origin of replication are incompatible and cannot survive within the same cell. this study has identified a diverse range of rep genes and rep gene combinations. biological tests are required to determine the incompatibility of plasmid groups, and to draw conclusions on the importance of this phenomenon in limiting plasmid recombination. mges in other bacterial species may be additional sources of novel resistance and virulence genes that can move into s. aureus populations. importantly, the vana gene in vancomycin-resistant s. aureus isolates is carried on a transposon tn which is commonly found in vancomycin-resistant enterococci. in some vrsa isolates the entire enterococcal plasmid has been maintained, whilst in others tn has moved onto a staphylococcal plasmid. both genetic events suggest that enterococcal plasmid have successfully transferred into s. aureus bacteria. future studies are required that assess the mosaicism of staphylococcal and enterococcal plasmids in order to understand the frequency of recombination and gene exchange between such bacterial species. hgt mechanisms spread resistance and virulence genes between bacteria and populations. in s. aureus, two major hgt mechanisms have been described for plasmid movement plasmid conjugation via the conjugation transfer complex, and bateriophage generalized transduction. in addition, it is possible that smaller plasmids can hitchhike larger plasmids that carry the tra complex and be transferred from donor to recipient bacteria. we found that the tra genes were rare amongst the sequenced plasmids and were rare amongst our collection of 254 s. aureus isolates. bacteriophage generalized transduction can transfer dna fragments of less than 45kb. we found that  % of plasmids could theoretically be transferred by generalized transduction as they have genomes that are <45kb in length. of the plasmids >45kb in length carry the tra genes. collectively, this data suggests that conjugative plasmids and plasmid conjugation are infrequent, and that bacteriophage transduction is likely to be the most frequent transfer mechanism of plasmids, particularly non-conjugative plasmids.  plasmids are a principal driver of the spread of virulence and resistance genes in s. aureus populations via hgt, which is blocked by lineage specific r-m systems. this study has demonstrated that resistance and virulence genes are associated with plasmid groups, and that plasmids are associated with s. aureus lineage. this is evidence that genetic pressures and rm barriers are limiting the evolution of more resistant and more virulent s. aureus strains. 
plasmids often carry resistance genes and virulence genes that can disseminate through s. aureus populations by horizontal gene transfer mechanisms. sequences of s. aureus plasmids in the public domain and data from multi-strain microarrays were analysed to investigate the distribution of resistance genes and virulence genes on s. aureus plasmids, and the distribution of plasmids between s. aureus lineages. sequenced plasmids were assigned to plasmid groups that each possessed a unique combination of rep genes. we show some resistance genes and virulence genes were associated with specific plasmid groups suggesting there are genetic pressures preventing recombination of these genes into novel plasmid groups. this study argues that genetic pressures are restraining the spread of resistance and virulence genes amongst s. aureus plasmids, and amongst s. aureus populations, delaying the emergence of fully virulent and resistant strains. whole genome microarray analysis revealed that plasmid rep, resistance and virulence genes were associated with s. aureus lineages, suggesting restriction-modification barriers to hgt of plasmids between strains exist. staphylococcus aureus is major human and animal pathogen.
 staphylococcus aureus is major human and animal pathogen. plasmids often carry resistance genes and virulence genes that can disseminate through s. aureus populations by horizontal gene transfer mechanisms. sequences of s. aureus plasmids in the public domain and data from multi-strain microarrays were analysed to investigate the distribution of resistance genes and virulence genes on s. aureus plasmids, and the distribution of plasmids between s. aureus lineages.  a total of plasmid rep gene families, of which were novel to this study, were characterised using a previously proposed classification system. sequenced plasmids were assigned to plasmid groups that each possessed a unique combination of rep genes. we show some resistance genes and virulence genes were associated with specific plasmid groups suggesting there are genetic pressures preventing recombination of these genes into novel plasmid groups. whole genome microarray analysis revealed that plasmid rep, resistance and virulence genes were associated with s. aureus lineages, suggesting restriction-modification barriers to hgt of plasmids between strains exist. conjugation transfer complex genes were rare.  this study argues that genetic pressures are restraining the spread of resistance and virulence genes amongst s. aureus plasmids, and amongst s. aureus populations, delaying the emergence of fully virulent and resistant strains. 