staphylococci constitute one of the major normal flora in skin , nasal cavities and mucosal membranes of humans . however , they are known as common causes of various infections in both healthcare and community settings . while approximately 30% of healthy individuals are colonized with staphylococcus aureus asymptomatically , this bacterium causes various infections , including skin and soft tissue infections ( ssti ) , bactaeremia and pneumonia . healthcare - associated ( ha ) methicillin - resistant staphylococcus aureus ( mrsa ) has been recognized as a primary cause of nosocomial infections that acquired multiple drug resistance , associated with its global spread since the 1960s . thereafter , community - acquired ( ca ) mrsa have also emerged as cause of infections in individuals who have no healthcare - associated risk , , posing a public health concern worldwide . coagulase - negative staphylococci ( cns ) , ubiquitously distributed to humans , have been also increasing as nosocomial pathogens mainly as a result of development of prosthetic devices and invasive medical technologies . representative species causing infections are s.  epidermidis and s.  haemolyticus , which often acquire drug resistance , including methicillin resistance via same genetic mechanism as that of mrsa . methicillin resistance of staphylococcus is characterized by the presence of a transmissible genome element , staphylococcal cassette chromosome mec ( sccmec ) , which is inserted in the chromosome of bacterial cell . sccmec in mrsa has been differentiated into at least 11 genetic types ( i  xi ) , , among which types i to iii are commonly found in ha - mrsa , while type iv and v were reported to be frequently in ca - mrsa . however , in the present circumstances , ca - mrsa with the dominant sccmec types have been brought to healthcare settings , , , which makes distinction between ha- and ca - mrsa more difficult in terms of sccmec type . the initially identified ca - mrsa strains were characterized by production of panton - valentine leukocidin ( pvl ) , a two - component leukolytic toxin , which is associated with severe symptoms in a wide spectrum of infections , , including ssti 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 , . in myanmar , s.  aureus has been reported to be the major pathogen in bloodstream infections and the third most common bacteria in blood cultures from febrile children , . however , to our knowledge , there is no epidemiologic study on staphylococci from healthcare settings in myanmar , and thus information is not available for drug resistance and genetic characteristics on recent clinical isolates of s.  aureus , including mrsa , and cns . although we previously reported genetic traits of mrsa and methicillin - susceptible s.  aureus ( mssa ) isolates from hospital , community and food poisoning cases in myanmar , the epidemiologic features was not determined because of the low numbers of isolates analysed . in the present study , drug resistance and genetic traits , including prevalence of meca , acme ( arginine catabolic mobile element ) and pvl genes , was analysed for clinical isolates of staphylococci in a tertiary - care hospital in myanmar . a total of 128 staphylococcus strains were collected from patients admitted to north okkalapa general hospital , yangon , myanmar , between january 2012 and august 2013 . the main specimen of the isolates was wound swab of surgical site infections ( 57% ) , followed by high vaginal swab ( 12% ) , blood ( 11% ) , pus ( 10% ) and other specimens ( sputum , urine , ear exudate ) ( 10% ) . a single isolate from an individual patient bacterial isolates grown on agar plates were examined by conventional microbiologic methods , and their species were determined by bbl crystal gram - positive i d kit ( becton dickinson microbiology systems , cockeysville , md , usa ) . individual bacterial strains were stored in microbank ( pro - lab diagnostics , richmond hill , on , canada ) at  80c and recovered when they were analysed . the presence of 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 . 
 . sccmec type and acme type were also determined by multiplex pcr using previously published primers and conditions , . for major staphylococcal species , 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 ) for gram - positive cocci . breakpoints defined in the clinical laboratory standards institute ( clsi ) guidelines were used to distinguish between resistant and susceptible strains for most of the drugs examined . the 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 , sequences of d1 , d2 and the central region of coa were determined as described previously ,   to assign the coa genotype by sequence homology . sequence identity to the known coa types was analysed by basic local alignment search tool ( blast ; http://blast.ncbi.nlm.nih.gov/blast.cgi ) . for selected isolates , sequence type ( st ) was determined according to the scheme of multilocus sequence typing ( mlst ) , and agr group classification and protein a gene ( spa ) typing were performed as described previously , . the presence of genes encoding enterotoxins and other toxins , adhesins , other proteins related to virulence and antimicrobial resistance genes were analysed by multiplex or uniplex pcr using primers described previously . partial sequence of the gene encoding elastin - binding protein ( ebps ) was determined by pcr and direct sequencing as described previously . multiple alignment of nucleotide and amino acid sequences determined was performed by the clustal w 2.1 program ( dna data bank of japan ( ddbj ) , http://clustalw.ddbj.nig.ac.jp/ ) . full - length staphylocoagulase gene ( coa ) sequence of strain mmr - v determined in the present study was deposited in the genbank database under accession number kt599478 . among the 128 staphylococcal isolates obtained in the study period , the dominant species identified were s.  aureus ( n  =  50 , 39% ) and s.  haemolyticus ( n  =  45 , 35% ) , followed by s.  epidermidis ( n  =  8 , 6% ) and s.  saprophyticus ( n  =  7 , 5% ) . s.  aureus and s.  haemolyticus were isolated from wound swabs at high rates ( 65% and 62% , respectively ) , while s.  haemolyticus was the main species among isolates from blood culture ( 43% , 6/14 ) . the majority of s.  haemolyticus ( 71% , 32/45 ) and s.  epidermidis ( 75% , 6/8 ) possessed meca , while the detection rate of mrsa was only 8% ( 4/50 ) , and all the four mrsa had type iv sccmec ( table  1 ) . although the sccmec of some s.  haemolyticus and s.  epidermidis isolates was assigned to types iv and v , most of the isolates ( 71% , 27/38 ) were untypable . acme - arca was detected in two and one isolates of s.  haemolyticus and s.  epidermidis , respectively , and their acme was classified into type ii . pvl genes were detected in 20  s.  aureus isolates ( 40% ) , among which only one isolate was mrsa . pvl - positive s.  aureus were mostly isolated from pus or wound swabs ( table  2 ) . while the four mrsa isolates showed resistance to oxacillin and ampicillin , they were mostly susceptible to all other antimicrobial agents , except for gentamicin and erythromycin ( table  3 ) . mssa isolates were susceptible to most antimicrobial agents while showing low resistance rates to ampicillin , erythromycin , gentamicin and sulfamethoxazole / trimethoprim . in contrast , meca - positive s.  haemolyticus showed high resistance rates to ampicillin , cephalosporins , erythromycin and levofloxacin . similar to mrsa , meca - positive s.  epidermidis were susceptible to most of the antimicrobial agents except for oxacillin . none of the staphylococcal isolates was resistant to vancomycin , linezolid and fosfomycin . among the 50  s.  aureus isolates , ten staphylocoagulase ( coa ) genotypes were identified by multiplex pcr or sequencing , and the most common type was va ( n  =  19 ) , followed by viia ( n  =  8) , via ( n  =  6 ) , and iiia and viib ( n  =  4 ) ( table  4 ) . full - length coa was determined for an mssa strain mmr - v of which the coa type was untypable by the pcr assay . sequence identity of coa - d1 region and -d2 plus central regions of mmr - v to the known 12 coa types were 64.770.3% and 69.789.2% , respectively ( supplementary table  s1 ) . according to the criteria to determine coa type ( subtype ) proposed by watanabe et  al . , i.e. > 90% identity of the d1 region ( coa type ) and > 90% identity of the d1 and central region ( coa subtype ) , the staphylocoagulase gene of mmr - v was considered not to be classified into the known 12 coa types . while mrsa belonged to three coa types ( iiia , ivb , viib ) , pvl - positive isolates were assigned to five coa types ( iiia , va , via , viia , xiiia ) , with va being dominant , followed by via . mlst was performed for 27 isolates , i.e. 20 pvl - positive and 7 pvl - negative s.  aureus isolates , resulting in identification of 11 sts ( table  2 , table  5 ) . st3206 ( cc1 ) of two pvl - positive mssa isolates and st3075 of a pvl - negative mssa isolate were newly identified in the present study . pvl - positive isolates were differentiated into six sts ( st88 , st121 , st1153 , st1155 , st1930 , st3206 ) , among which st121 was dominant ( nine isolates , 45% of pvl - positive s.  aureus ) and found in only mssa , and other sts were identified in one to three isolates . a pvl - positive mrsa , strain mmr-42a , belonged to st88 , coa type iiia , agr type iii , and spa type t729 . the other three mrsa isolates were classified into st6 and st59 ( table  5 ) . st88 was also identified in pvl - positive mssa from blood and wound , which exhibited different patterns of toxin / virulence factors and drug resistance from those of a pvl - positive st88 mrsa . st121 pvl - positive mssa isolates belonged to coa type va and agr type iv , and harboured five to six enterotoxin genes , the bone sialoprotein gene ( bbp ) , and a variant of the elastin binding protein gene ( ebps - v ) with an internal 180  bp deletion as described previously . the st59  s.  aureus , both mrsa ( two strains ) and mssa ( one strain ) without pvl had more resistance genes ( ermb , aac(6)-ie - aph(2)-ia ) than other mrsa and pvl - positive mssa , showing resistance to more antimicrobial agents . in the present study , prevalence and drug resistance of staphylococcal species and genetic traits of s.  aureus were elucidated for clinical isolates in a tertiary hospital in myanmar . distinctive features in this study were the high prevalence and antimicrobial resistance trend of s.  haemolyticus , the low rate of mrsa , and the high rate of pvl among mssa . among cns species , s.  haemolyticus has been described as occasionally the second most frequent clinical isolates after s.  epidermidis , causing primarily bloodstream infections associated with the use of central venous catheters . in the present study , with a lower number of blood isolates ( 11% ) , the frequency of s.  haemolyticus was comparable to s.  aureus and higher than that of s.  epidermidis , suggesting the significance of this species in skin infections as well . in agreement with the view of this species having a great capacity to develop resistance to multiple classes of antimicrobial agents , , a high meca - positive rate associated with high resistance rates to various antimicrobial agents of s.  haemolyticus although the meca - positive rate in s.  aureus was still low , a high rate of methicillin - resistant s.  haemolyticus as well as s.  epidermidis may alert us to the potential increase of drug - resistant isolates among staphylococcal species , including mrsa . in the present study , the detection rate of pvl genes among s.  aureus was notably high ( 40% ) , which may be related to a high proportion of wound swabs and pus ( 67% ) in the specimens examined . detection of pvl genes in six different sts among 20  s.  aureus isolates suggests dissemination of pvl phages to multiple clones , while only the dominant clone , st121 , appears to spread within hospitals . strain mmr-42a is the first pvl - positive mrsa isolated in myanmar , having sccmec - iv and genetic types st88/spa - t729/agr - iii / coa - iii . the st88 mrsa with sccmec iv or v has been reported in both community and hospital settings in africa ( mostly in east africa ; tanzania and madagascar ) , ,   and asia ( mostly in china ) , , , and less frequently in europe , , , exhibiting agr type iii and various spa types , with t186 being dominant . the spa type t729 detected in strain mmr-42a is genetically closely related to t186 and was described also for st88 mrsa isolates in africa , suggesting close relatedness to the previously described st88 clone . pvl is associated with a part of st88 mrsa as well as mssa . in bangladesh and china , neighbouring countries to myanmar , pvl - positive st88 mssa and/or mrsa was reported , , . detection of st88 mrsa in myanmar suggests the potential spread of this clone in asia , and there should be concern in healthcare settings resulting from the presence of pvl in this clone . st121 mssa , mostly belonging to agr - iv , are distributed worldwide ( mainly africa , asia and europe ) as a common cause of ssti , often associated with pvl , while mrsa with this st is rare , . in our previous study in myanmar on s. aureus isolates from wound / pus , food poisoning and healthy adults , pvl genes were detected in only st121 mssa strains with coa - va / agr - iv from wounds in hospitalized patients . in the present study , st121 was dominant among pvl - positive isolates and showed genetically identical traits to those of previous mssa strains in myanmar . s.  aureus in myanmar has been previously revealed to harbour the genes of bone sialoprotein and a variant of elastin binding protein with 180  bp deletion , which was also found in pvl - positive st121 mssa in the present study . therefore , we suggest that a single st121 pvl - positive s.  aureus clone has been persisting as a cause of ssti in myanmar . although virulence of st121 mssa might be increased with pvl and other toxins , this clone is generally susceptible to most antimicrobial agents . thus , active promotion of early detection and treatment is recommended for infections with this clone in healthcare settings . it is of note that a novel staphylocoagulase genotype , coa xiii , was assigned to a pvl - positive mssa strain ( mmr - v ) belonging to the rare st1153 , which was isolated from 55-year - old patient with wound infection . the d1 and d2 regions of staphylocoagulase which define the coa genotype ( subtype ) is considered to be responsible for antibody recognition as well as contact with prothrombin , . accordingly , genetic diversity of the d1/d2 regions is suggested to be caused by selection with antibody and/or prothrombin in the host . hence , increased virulence is concerned with the emergence of s.  aureus with the new coa type as a result of the absence of immune response against the novel antigen of the virulence factor . in the present study , st1930 mssa was resistant to erythromycin and levofloxacin , which is the distinctive feature of resistance among pvl - positive isolates . although the significance of st1930 mssa is not evident , s.  aureus with cc96 , to which st1930 belongs , is revealed to secrete variable to high levels of alpha toxin , suggesting relevance to the increased virulence . therefore , the prevalence of the novel pvl - positive mssa clones st1153 and st1930 should be carefully monitored in myanmar . despite low mrsa rates among s.  aureus in the present study , it was notable that st59 was identified in two isolates with sccmec - iv as well as an mssa isolate . these isolates were pvl negative , however , resistant to multiple antimicrobial agents harbouring resistance genes such as erm(b ) . st59 ( cc59 ) mrsa has been classified into some groups , , with pvl - positive strains with sccmec - v predominating in taiwan and other asian countries ( taiwan clone ) , sccmec - iv - harbouring pvl - positive strains known as usa1000 clone mostly restricted to the united states and pvl - negative ( or positive ) mrsa with sccmec - iv or v in australia . the st59 pvl - negative mrsa - iv , with the same genetic traits as the st59 isolates in the present study , was also detected at a high rate in the nasal cavities of children in taiwan . thus , we suggest that st59  s.  aureus may be distributed widely in asia as well as australia and may occasionally acquire sccmec and/or pvl phage , associated with their clonal spread . in myanmar , caution may be necessary for the st59 mrsa in hospitals regarding acquisition of pvl genes and more drug resistance . in summary , the present study elucidated drug resistance and genetic traits of clinical isolates of staphylococci in a tertiary - care hospital in myanmar . further studies are needed in this country to survey the prevalence of methicillin - resistant cns , mrsa and pvl - positive s.  aureus and their drug resistance for control of staphylococcal infections in healthcare settings .
prevalence , drug resistance and genetic characteristics were analysed for a total of 128 clinical isolates of staphylococci obtained from a tertiary hospital in myanmar . 
 the dominant species were s.  aureus ( 39% ) and s.  haemolyticus ( 35% ) , followed by s.  epidermidis ( 6% ) and s.  saprophyticus ( 5% ) . the majority of s.  haemolyticus isolates ( 71.1% ) harboured meca , showing high resistance rates to ampicillin , cephalosporins , erythromycin and levofloxacin , while methicillin - resistant s.  aureus ( mrsa ) was only 8% ( four isolates ) among s.  aureus with type iv sccmec . 
 panton - valentine leukocidin ( pvl ) genes were detected in 20 isolates of s.  aureus ( 40% ) , among which only one isolate was mrsa belonging to sequence type ( st ) 88/agr - iii / coa - iiia , and the other 19 methicillin - susceptible s.  aureus ( mssa ) isolates were classified into six sts ( st88 , st121 , st1153 , st1155 , st1930 , st3206 ) . 
 an st1153 mssa isolate with pvl was revealed to belong to a novel coa type , xiiia . 
 st121  s.  aureus was the most common in the pvl - positive mssa ( 47% , 9/19 ) , harbouring genes of bone sialoprotein and variant of elastin binding protein as a distinctive feature . 
 although pvl - positive mssa was susceptible to most of the antimicrobial agents examined , st1930 isolates were resistant to erythromycin and levofloxacin . 
 st59 pvl - negative mrsa and mssa had more resistance genes than other mrsa and pvl - positive mssa , showing resistance to more antimicrobial agents . 
 this study indicated higher prevalence of meca associated with multiple drug resistance in s.  haemolyticus than in s.  aureus , and dissemination of pvl genes to multiple clones of mssa , with st121 being dominant , among hospital isolates in myanmar .