methicillin - resistant staphylococcus aureus ( mrsa ) is recognized as one of the most common pathogens of both nosocomial and community - acquired infections worldwide . as a feature distinct from methicillin - susceptible s.  aureus ( mssa )
, mrsa has a transmissible genome element , staphylococcal cassette chromosome mec ( sccmec ) , inserted in a specific site of the chromosome .
the sccmec in mrsa has been differentiated into at least 12 genetic types ( i  xii ) , , among which types i  iii have been traditionally associated with hospital - acquired mrsa ( ha - mrsa ) , while type iv and v have been commonly found in community - acquired mrsa ( ca - mrsa ) . however , in recent years , ca - mrsa with the dominant sccmec types ( iv and v ) has been brought to healthcare settings causing nosocomial infections , , , which makes distinction between ha- and ca - mrsa more difficult in terms of sccmec type .
the pathogenesis of many ca - mrsa strains have been attributed to the production of panton - valentine leukocidin ( pvl ) , a two - component toxin encoded by two genes , lukf - pv and luk - s - pv , which are carried on lysogenic bacteriophages , .
accordingly , pvl - positive s.  aureus is associated with severe symptoms in a wide spectrum of infections including skin and soft tissue infections and necrotizing pneumonia , .
prevalence of ca - mrsa harbouring pvl genes has been increasing recently in hospitalized patients as well as healthy individuals in the community , .
distribution and spread of mrsa clones on a global scale have been revealed by genetic classifications with multilocus sequence typing and sccmec typing , .
several ha - mrsa clones including st5-mrsa - sccmec ii ( st5-ii , ny / japan clone ) and st22-iv ( emrsa-15 ) are known as pandemic clones predominating in east asia / north america and europe , respectively . in contrast , various clones have been documented for ca - mrsa which are distributed locally or predominate in a region , often associated with international spread .
globally predominant ca - mrsa includes five clones , i.e. st1 ( usa400 clone ) , st8 ( usa300 clone ) , st30 ( south west pacific clone ) , st59 ( taiwan clone ) and st80 ( european clone ) , among which st8 and st30 are considered pandemic as a result of its distribution to every continent . in asia , two pandemic ha - mrsa clones with st5 and st239 are disseminating , whereas various ca - mrsa clones including those with st8 , st30 , st59 , st72 and st772 have been reported . in nepal , the prevalence of mrsa from clinical specimens in hospitals has been described to be 2669% in several studies via antimicrobial susceptibility testing , , , , , although the rate varies depending on the types of infections or specimens examined .
a recent study revealed a high prevalence of pvl genes in nosocomial isolates of mrsa and mssa ( 26% and 52% respectively ) .
however , in nepal , there have been no studies conducted on genotypes ( st and sccmec types ) of clinical mrsa isolates , particularly pvl - positive isolates .
we found high prevalence of pvl in mrsa and mssa , as well as the presence of pvl - positive st772 mrsa - v ( bengal bay clone ) .
a deletion variant of elastin binding protein gene was first identified in st22 s.  aureus isolates and its origin was analysed .
from august 2012 to october 2012 , about 200 s.  aureus isolates were collected from two general hospitals ( approximately 100 isolates each ) with more than 500 beds in kathmandu , nepal .
the main specimen of the isolates was pus ( n  =  84 ) , followed by urine ( n  =  12 ) , sputum and blood ( n  =  2 each ) . a single isolate from each individual patient was subjected to study .
bacterial isolation and species identification were performed by conventional microbiological methods , and the presence of nuclease gene was confirmed by multiplex pcr .
individual bacterial strains were stored in microbank ( pro - lab diagnostics , richmond hill , on , canada ) at  80c and recovered when they were analysed .
staphylococcal 16s rrna , nuc , meca , pvl gene ( luks - pv / lukf - pv ) and acme - arca ( arginine deiminase gene ) were detected for all isolates by multiplex pcr assay as described by zhang et  al . .
minimum inhibitory concentrations against 18 antimicrobial agents based on the broth microdilution test were measured by using dry plate  eiken dp32 ( eiken chemical , tokyo , japan ) .
breakpoints defined in the clinical laboratory standards institute guidelines were used to distinguish between resistant and susceptible strains for most of drugs examined .
staphylocoagulase genotype ( coa type ) of s.  aureus isolates was determined by multiplex pcr using previously published primers and conditions . for the strains for which the coa types were not determined for i  x by the multiplex pcr , partial coa sequences ( d1 , d2 and the central regions ) were determined as described previously ,   to assign the coa genotype by sequence identity via blast search ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . for selected isolates , sequence type ( st ) was determined according to the scheme of multilocus sequence typing , and agr group was classified as described previously .
presence of genes encoding enterotoxins and other toxins , adhesins , other proteins related to virulence and antimicrobial resistance genes was analysed by multiplex or uniplex pcr using primers described previously , .
the sequence of the gene encoding elastin binding protein ( ebps ) was determined by pcr and direct sequencing , as described previously .
multiple alignment of ebps sequences determined in the present study and those retrieved from the genbank database was performed by the multalin interface ( http://multalin.toulouse.inra.fr/multalin/ ) . the lalign program ( european bioinformatics institute ; http://www.ebi.ac.uk/tools/psa/lalign/nucleotide.html )
a phylogenetic tree of ebps was constructed by the neighbour - joining method by mega 5.01 software , statistically supported by bootstrapping with 1000 replicates .
full - length ebps sequences of strains np173 , np177 and np199 determined in the present study were deposited in the genbank database under accession numbers kt951674kt951676 respectively .
among the 100 s.  aureus clinical isolates examined , 32 isolates were mrsa which had sccmec type v ( n  =  20 , 62.5% ) or type iv ( n  =  2 , 6.3% ) ( table  1 ) , while sccmec type was not identified for ten isolates .
pvl genes were detected in 78% ( 25/32 ) of mrsa and 71% ( 48/68 ) of mssa isolates .
the most common coagulase genotypes of pvl - positive mssa and mrsa were iva and via , respectively .
genetic characteristics were analysed for 17 isolates as representatives of pvl - positive mrsa ( seven isolates ) , pvl - positive mssa ( six isolates ) , pvl - negative mrsa ( three isolates ) and a pvl - negative mssa isolate ( table  2 ) .
the pvl - positive mrsa isolates belonged to st1 , st22 , st88 or st772 .
the st772 was identified into mrsa with sccmec v , coa - via and agr - ii ( one pvl - negative and four pvl - positive isolates ) , as well as two pvl - positive mssa .
mrsa isolates with or without pvl were mostly resistant to ampicillin , gentamicin and levofloxacin , and had generally more drug resistance genes than pvl - positive mssa , although some meca - positive isolates ( mrsa ) were susceptible to oxacillin ( supplementary table  s1 ) .
although luke - lukd and haemolysin genes were detected in most isolates examined , st772 isolates ( mrsa and mssa ) and st22 mrsa had more enterotoxin genes than st1 and st88 mrsa and st30 mssa isolates ( table  2 ) .
in the present study , we first demonstrated the presence of st772-mrsa - v and st22-mrsa - iv in nepal . st772 and st22 have been reported as epidemic clones associated with infections in both community and healthcare settings in india , , , .
st772 mssa was originally reported in bangladesh ; thereafter , st772-mrsa - v was identified in india , followed by transmission to east / southeast asia , australia , new zealand , the middle east and europe .
this clone is colloquially referred to as the bengal bay clone , and it is mostly pvl positive and relatively multiresistant compared to other ca - mrsa .
in addition to the increasing prevalence of st772-mrsa - v in india , detection of this clone outside india has been related to travel history to or from india , , .
because of its adjacent location , it is conceivable that st772-mrsa - v in india might have been readily transmitted to nepal , or it may have been originally endemic in nepal as well as india .
although the elastin binding protein ( ebps ) gene ( ebps ) was detected in all the isolates examined by pcr with primers described previously , pcr products that were shorter than the expected size ( 652  bp ) were found in four st22 isolates ( data not shown ) , among which three isolates ( np173 , np177 and np199 ; pvl - positive mrsa , pvl - negative mrsa and pvl - positive mssa respectively ) were further analysed for their ebps gene sequences . these ebps genes were revealed to be a variant ( ebps - v ) with an internal deletion of 180  bp encoding a 60 aa sequence . by blast search
, sequences similar to ebps - v were identified in strain 71a_s11 ( st22 ) , 93b_s9 and y12 ( st121 ) and 6850 ( st50 ) .
alignment of the deduced amino acid sequences of the ebps - v identified in the present study with those of other representative s.  aureus strains is shown in fig .  1
. the deleted portion ( 60 aa ) of ebps - v in st22 nepalese strains , corresponding to aa 199258 of ebps from strain col , was identical to those found in strains 71a_s11 , 93b_s9 , y12 and 6850 ( genbank accession nos .
phylogenetic analysis of the ebps from various s.  aureus strains , including ebps - v , revealed the presence of three major lineages ( i , ii and iii ) ( fig .  2 ) .
lineage ii contained the st22 cluster , which consisted of ebps - v from nepalese strains and intact ebps in st22 strains as emrsa-15 .
nucleotide sequence identity of intact ebps within the same lineage was more than 98.8% , while it was 95.298% between different lineages ( supplementary table  s2 ) .
ebps - v of st22 nepalese strains showed > 99% identity with each other , but slightly lower identity was found in st121 strains ( 95.695.7% ) .
the variant of ebps with a 180  bp deletion was first reported for isolates from orthopaedic infections in italy , although their st was not identified .
thereafter we identified a similar ebps variant ( ebps - v ) in st121 mssa isolates in myanmar   as well as in st121 isolates in bangladesh   and japan .
the present study elucidated that ebps - v of st22 and st121 belong to different lineages , suggesting that the 180  bp deletion event in ebps might have occurred in st22 s.  aureus and st121 s.  aureus independently .
ebps , one of the adhesins that binds to host cellular matrix factors involved in biofilm formation , is produced by most mrsa examined so far , , .
ebps is a cell - surface molecule and mediates binding of bacterial cell to soluble elastin peptides and tropoelastin , , with its n - terminal region ( aa 1434 ) a ligand - binding domain exposed on the surface of the cell and two ( h1 and h3 ) among the three putative hydrophobic domains spanning the cell membrane .
although the functional and structural changes caused by the deletion in st22 isolates are unknown , the n - terminal region must be exposed on the surface of cell for the normal function of ebps - v ; accordingly , the h2 region is necessary to span the membrane instead of the deleted h1 region ( fig .  1 , supplementary fig . 
s1 ) . in that case , only the form of the cytoplasmic portion of ebps - v may be different from that of ebps , which may be possible to cause any functional difference .
it was notable in the present study that all the st22 s.  aureus , including mrsa and mssa , harboured ebps - v , while intact ebps was also found in other previously reported st22 strains including emrsa-15 .
in contrast , all the st121 s.  aureus analysed to date harboured ebps - v 
 , , . the st121 clone is known to be a common cause of skin and soft tissue infection disseminating globally , mostly meca negative but pvl positive , exhibiting higher virulence . although the association of ebps - v with the enhanced virulence of the st121 clone is not known , if the presence of ebps - v affects phenotypic trait of st22 s.  aureus ( e.g. virulence or fitness advantage to environment ) , it is possible that the proportions of st22 isolates with ebps and ebps - v may change over time epidemiologically .
further studies thus may be necessary to monitor the prevalence of st22 s.  aureus with ebps - v .
 genetic characteristics were analysed for recent clinical isolates of methicillin - resistant and -susceptible staphylococcus aureus ( mrsa and mssa respectively ) in kathmandu , nepal . 
 mrsa isolates harbouring panton - valentine leukocidin ( pvl ) genes were classified into st1 , st22 and st88 with sccmec - iv and st772 with sccmec - v ( bengal bay clone ) , while pvl - positive mssa into st22 , st30 and st772 . 
 st22 isolates ( pvl - positive mrsa and mssa , pvl - negative mrsa ) possessed a variant of elastin binding protein gene ( ebps ) with an internal deletion of 180  bp , which was similar to that reported for st121 s.  aureus previously outside nepal . 
 phylogenetic analysis indicated that the ebps variant in st22 might have occurred independently of st121 strains . 
 this is the first report of st772 pvl - positive mrsa in nepal and detection of the deletion variant of ebps in st22 s.  aureus . 