methicillin resistant staphylococcus aureus ( mrsa ) was initially reported as a nosocomial pathogen responsible for adult infections .
ca - mrsa has been recognized as a pathogen in adults and children without traditional risk factors for mrsa acquisition .
children colonized with mrsa are potential reservoirs for the spread of mrsa in the community [ 2 , 3 ] .
furthermore , the infants and newborns with immunological immaturity , especially those born prematurely and those requiring specialized care , remained the major group susceptible to ca - mrsa infections .
most ca - mrsa strains were associated with skin and soft tissue infections ( ssti ) and necrotizing pneumonia .
the incidence of pediatric ssti has increased rapidly in the previous decade [ 57 ] .
notably , community isolates differ significantly from nosocomial strains by the antimicrobial pattern and virulence profile .
it is known that resistance to beta - lactams is mediated by the meca gene carried by a mobile genetic element called staphylococcal cassette chromosome mec ( sccmec ) .
ca - mrsa have been described as strains harboring the sccmec type iv , type v , or type vii [ 8 , 9 ] and remained susceptible to the majority of antimicrobial agents other than beta - lactams .
furthermore , these strains have been found to carry virulence genes encoding a leukocyte - killing toxin called the panton - valentine leukocidin ( pvl ) determinant . in this report , we characterize clinical mrsa strains isolated from children hospitalized in the pediatric department at the university hospital of monastir , tunisia .
we are interested to investigate the phenotypic and genotypic markers of these isolates including antimicrobial resistance , sccmec type , pvl genes , pulsed - field gel electrophoresis ( pfge ) patterns , and multilocus sequence typing ( mlst ) of seven unlinked housekeeping genes ( arcc , aroe , glpf , gmk , pta , tpi , and yqil ) .
eight mrsa strains were collected from clinical specimens of hospitalized children in the pediatric department at the university hospital of monastir , tunisia , during a three - month period ( from june to august 2013 ) .
the subjects were 6 boys and 2 girls , aged from 12 days to 18 months .
the isolates were associated with skin infections : cutaneous abscesses ( 7 cases ) and facial cellulites ( 1 case ) . 

s. aureus were identified according to standard bacteriological procedures : gram strain reactions , colony morphology , catalase , the ability to coagulate the rabbit plasma , and latex agglutination test ( bio - rad ) .
susceptibility to the following antibiotics penicillin g , cefoxitin , moxalactam , kanamycin , amikacin , tobramycin , gentamicin , erythromycin , lincomycin , tetracycline , pristinamycin , furans , ofloxacin , trimethoprim - sulfamethoxazole , rifampicin , fusidic acid , fosfomycin , mupirocin , high mupirocin , vancomycin , teicoplanin , and linezolid was determined according to the recommendations of the committee for antimicrobial testing of the french society of microbiology ( casfm ) ( http://www.sfm-microbiologie.org/ ) .
methicillin resistance was determined by the disk diffusion method testing oxacillin disk ( 30  g ) on mueller - hinton agar supplemented with 2% sodium chloride .
multiplex polymerase chain reaction ( pcr ) was applied to determine the sccmec types according to a previous method described by oliveira and de lencastre .
pvl genes ( luks - pv , lukf - pv ) were detected by pcr as previously described .
the isolates were genotyped by pulsed - field gel electrophoresis ( pfge ) using the restriction enzyme smai according to the method previously described .
a chromosomal dna digest of s. aureus strain nctc 8325 was used as the reference strain .
allelic profiles and sequence type ( st ) were designated using the mlst database ( http://www.mlst.net ) .
the genbank accession numbers of staphylococcal gene sequences , arcc , aroe , glpf , gmk , pta , tpi , and yqil , determined in this study were , respectively , jf495119 , jf495120 , jf495121 , jf495122 , jf495123 , jf495124 , and jf495125 .
in this study we investigate 8 mrsa strains isolated from patients hospitalized in the pediatric department .
all isolates were identified as mrsa strains by the determination of methicillin resistance using oxacillin disk diffusion method .
antimicrobial susceptibility showed that all isolates were susceptible to the majority of 22 antibiotics tested ( see section 2 ) with the exception of the beta - lactams ( oxacillin , penicillin g , and cefoxitin ) .
all strains were resistant to kanamycin and only two of them were resistant to erythromycin .
for all clinical isolates the detection of meca gene and the identification of sccmec type were performed by amplification from genomic dna , using multiplex pcr method according to oliveira and de lencastre method . for each isolate ,
2 amplified fragments were obtained : a 162  bp fragment and a 342  bp fragment .
these two pcr products correspond to the amplification of meca gene and specific sccmec type iv locus ( dcs ) , respectively .
amplification of pvl genes ( luks - pv and lukf - pv ) was performed also on the genomic dna extracted from all strains . for each strain ,
the amplicon obtained has 433  bp ; thus all mrsa strains harbor pvl genes .
these results revealed that our clinical isolates have the peculiarities of ca - mrsa : susceptibility to the majority of antimicrobial agents and carrying sccmec iv and pvl genes . to investigate the clonality of these ca - mrsa isolates ,
pfge pattern analysis demonstrated that they are distributed on three pulsotypes arbitrary designated a , b , and c ( figure 1 ) .
six isolates carried the pulsotype a and two isolates carried the pulsotypes b and c , respectively .
ca - mrsa strains were also characterized by multilocus sequencing of internal fragments of seven housekeeping genes ( arcc , aroe , glpf , gmk , pta , tpi , and yqil ) .
nucleotide sequence analysis revealed that all isolates possess the same unique sequence type designated st80 .
mrsa strain is known as a main cause of infections for children and young adults .
mrsa strains investigated in this study are isolated from patients aged from 12 days to 18 months .
six children have been admitted with mrsa skin infections ; hence , these data indicate that these infections were community - acquired but were not necessarily caused by community mrsa strains .
however , the real site of mrsa acquisition is not readily determined because the community mrsa may designate mrsa colonization or a strain responsible for community infection detected in the community but not necessary acquired in the community .
two other ones are an 18-month - old child admitted with immunodeficiency and a 12-day - old child admitted with fever .
as referred to the definition of community mrsa infection , these isolates may be transmitted to these two patients from community .
ca - mrsa strains have been reported as a cause of colonization and infection in neonatal intensive care units in many countries [ 16 , 17 ] .
furthermore , nasal carriage may be a possible explanation of the transmission of mrsa among these patients . the same observation was reported by frazee et al . who considered that ca - mrsa is a common pathogen in cutaneous abscesses due to nasal carriage preceding infections .
it is interesting to note that newborns and young children , due to their immature immune systems , are easily infected by mrsa .
several similar studies reported that the majority of patients with mrsa infections were young children [ 6 , 18 ] .
for all strains antimicrobial resistance showed that they were resistant to oxacillin and susceptible to all non - beta - lactams .
however the resistance to kanamycin and erythromycin has been also observed . molecular characterization of mrsa isolates by the identification of sccmec type and the detection of luks - pv and lukf - pv genes revealed that all strains harbored the sccmec type iv and pvl genes . according to these results , our strains have been classified as community - acquired strains .
described sccmec iv and pvl genes as markers for ca - mrsa [ 8 , 19 ] .
it has been reported that the cassette type iv and pvl genes have been found in some nosocomial mrsa strains [ 2022 ] .
pfge analysis showed that ca - mrsa strains belonged to the same clone according to criteria of tenover et al . .
 full analyses of molecular typing results suggest that isolates belong to the ca - mrsa st80 clone .
in fact , this clone is being increasingly reported in the community worldwide and mainly detected in europe .
this antibiotic resistance pattern seems to be different from european strains  st80 ,  which were resistant to tetracycline and fusidic acid [ 23 , 24 ] .
however , ca - mrsa has emerged within the hospital setting , posing a significant public health threat .
so , what is most worrying is that these strains affect frequently newborns and young children and eventually cause potentially serious infections .
in fact some mrsa epidemic clones have been reported to cause skins and soft tissue infections as well as severe diseases .
clone is recognized as a predominant clone in europe , the united states , and tunisia .
this clone could become a health problem worldwide particularly that ca - mrsa strains were associated mainly with the presence of pvl genes which have an important impact on virulence .
these observations urge emphasizing infection control measures to monitor the transmission of highly virulent ca - mrsa in our hospital .
 community associated methicillin resistant staphylococcus aureus ( ca - mrsa ) is an emerging pathogen increasingly reported to cause skin and soft tissue infections for children . 
 the emergence of highly virulencet ca - mrsa strains in the immunodeficiency of young children seemed to be the basic explanation of the increased incidence of ca - mrsa infections among this population . 
 the subjects of this study were 8 patients hospitalized in the pediatric department at the university hospital of monastir . 
 the patients were young children ( aged from 12 days to 18 months ) who were suffering from mrsa skin infections ; two of them had the infections within 72  h of their admission . 
 the isolates were classified as community isolates as they all carried the staphylococcal cassette chromosome mec ( sccmec ) iv and pvl genes . 
 epidemiological techniques , pulsed - field gel electrophoresis ( pfge ) and multilocus sequence typing ( mlst ) , were applied to investigate ca - mrsa strains . 
 analysis of molecular data revealed that mrsa strains were related according to pfge patterns and they belonged to a single clone st80 . 
 antimicrobial susceptibility tests showed that all strains were resistant to kanamycin and 2 strains were resistant to erythromycin . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion and Conclusion