streptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide , particularlyin lower income countries .
pneumococcal diseases are the leading source of vaccine preventable deaths , mostly due to community - acquired pneumonia ( cap ) , accounting for approximately 11% of all deaths in children under 5 years old ( 1 ) .
colonization of the nasopharynx is a necessary step along the path to pneumococcal disease ( pd ) ( 2 ) .
pneumococcal conjugate vaccines ( pcv ) reduce nasopharyngeal carriage of serotypes which included in the vaccine by conferring capsular - specific immunity .
experience from countries where conjugate vaccines have been introduced has shown rapid and sustained carriage reduction of vaccine serotypes ( vt ) following vaccination ( 3 ) .
the microorganism produces a plethora of virulence factors , including the polysaccharide capsule , several surface - located proteins , and the toxin pneumolysin ( 5 , 6 ) .
the polysaccharide capsule is highly efficient in protecting the bacteria from opsonophagocytosis ( 7 ) .
surface proteins of s. pneumoniae ( pneumococcus ) have been investigated for their role in pneumococcal pathogenicity and as candidate antigens for protein based vaccines ( 8) . among the surface - associated proteins ,
the pneumococcal surface protein a ( pspa ) andc ( pspc ) are the best characterised choline - binding proteins ( 6 , 9 ) .
pneumolysin is a cytoplasmic toxin released by autolysis of the cell and it is a very important virulence factor with multiple effects .
the major autolytic enzyme of the pneumococcus is lyta ( nacetylmuramoyl - l - alanine - amidase ) , which is responsible for the deoxycholate- and penicillin induced cell lysis in the stationary phase , having a great clinical importance ( 10 ) . the lyta - encoded major autolysin of s. pneumoniae is a member of a widely distributed group of cell wall - degrading enzymes located in the cell envelope and postulated to play roles in avariety of physiological functions ( 11 ) .
rrga is a virulence factor in a murine lung infection model and has a varied distribution among serotypes of s.pneumoniae ( 12 ) .
s. pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits , rrga , rrgb and rrgc ( 13 ) .
some of the pneumococcal virulence factors are potential targets for protein- based pneumococcal vaccine production .
thus , in this study the presence of three genes were detected among the isolates of s. pneumoniae .
a total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory .
these samples were collected from children attending day care centers in different geographical areas of mashhad , iran .
both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours .
the plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier ( 14 ) .
the optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin ( oxoeid ) in an atmosphere of 5% co2 .
the genomic dna of the bacterial isolates were extracted by dnaase tissue kit ( kiagen , tehran , iran ) .
the presence of rrga , pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays .
oligonucleotides which were used as primers to amplify particular sequences of s. pneumoniae amplification conditions for lyta and pspc genes were : 94 c for 2 min , 25cycles of 94 c for 10 s , 58 c for 15 s , and 72c for1 min , followed by a final extension at 72 c for 5 min .
amplification conditions for rrga gene were : 95c for 2 min , 25cycles of 95c for 30 s , 51c for 30 s , and 72c for 90 s , followed by a final extension step at 72c for 5 min .
one amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company ( south korea ) .
sequences were examined for identity with published sequence data from national center for biotechnology information ( ncbi ) .
a total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory .
these samples were collected from children attending day care centers in different geographical areas of mashhad , iran .
both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours .
the plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier ( 14 ) .
the optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin ( oxoeid ) in an atmosphere of 5% co2 .
the genomic dna of the bacterial isolates were extracted by dnaase tissue kit ( kiagen , tehran , iran ) .
the presence of rrga , pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays .
amplification conditions for lyta and pspc genes were : 94 c for 2 min , 25cycles of 94 c for 10 s , 58 c for 15 s , and 72c for1 min , followed by a final extension at 72 c for 5 min .
amplification conditions for rrga gene were : 95c for 2 min , 25cycles of 95c for 30 s , 51c for 30 s , and 72c for 90 s , followed by a final extension step at 72c for 5 min .
one amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company ( south korea ) .
sequences were examined for identity with published sequence data from national center for biotechnology information ( ncbi ) .
among 260 nasopharanx samples from healthy children under 6 years , 59 isolates were confirmed as s. pneumoniae .
distribution of the lyta , rrga and pspc genes among isolates of s.pneumoniae were determined .
rrga and pspc were also found in17 ( 28.81% ) , and 2 ( 3.38% ) isolates respectively .
five patterns of these genes were seen among s. pneumoniae isolates : lyta ( n=33 , 55.93% ) , rrga ( n=2 , isolates , 3.38% ) , pspc ( n=1 1.69% ) , lyta+rrga ( n=16 , 27.11% ) and lyta+ rrga+ pspc ( n=1 , 1.69% ) .
the amplicon represented expected sequences with more than 90% identity with published data from ncbi ( genbank : ef 560634.1 ) .
the amplicon represented expected sequences with more than 90% identity with published data from ncbi ( genbank : ef 560634.1 ) .
it remains unclear , however , why some children develop invasive disease , whereas in the majority of cases , colonization remains asymptomatic , a combination of bacterial virulence and host factors may be responsible ( 18 ) .
we studied the s. pneumoniae isolates giving special reference to identification and distribution of virulence markers such as autolysin among the isolates from nasopharynx .
this exercise may help in understanding the factors contributing to the pathogenicity of s. pneumoniae .
further , there are numerous reports giving us increasing evidences on the role of lyta in pneumococcal pathogenesis suggesting that this might be more appropriate as vaccine antigen against s. pneumoniae infections .
the cpsa was used as a novel genetic marker specific for identification of s. pneumoniae and to differentiate it from the closely viridans group streptococci as well as other pneumococcus - like streptococci such as s. pseudopneumoniae ( 19 ) .
many virulence genes contribute to the colonization of s. pneumoniae ; however , our study only demonstrates this for pspc , rrga and lyta genes .
thus , synergistic effect and correlation of these virulence factors is necessary for our future work .
our results showed that most of the isolates had lyta gene ( 84.74% ) which has an important role in colonization .
this observation is in concordance with previous report which was showed that all of the unencapsulated isolates of s. pneumoniae were negative for lyta and psaa by pcr .
detection of lyta and psaa in six encapsulated isolates for which a serotype could be determined was negative ( 9 ) .
false negative results were obtained by the pcr assays for these two genes may be due to mutations or sequence variation . on the other hand , lyta is essentially a rather conserved gene displaying limited genetic variation ( 11 ) . in the study of whatmore ,
33 out of 62 isolates of s. pneumoniae were selected to represent a diverse range in terms of serotype , clinical association , and time and place of isolation .
autolysin which was found in all strains , might appear to be a suitable target virulence , and apparently highly conserved , for inclusion in a potential vaccine ( 11 ) .
the pneumococcal protein lyt a is the major autolysin of s. pneumoniae , it has an important function in pathogenesis by releasing pneumolysin and plays a fundamental biological role in bacterial lysis after exposure to certain antibiotics ( 19 ) .
it has been reported that the lyta gene has higher specificity than the pspc for identification of s. pneumoniae ( 15 , 20 ) . in our study , only 2 out of 59 isolates of s. pneumoniae ( 3.38% ) had pspc gene .
given the high sequence diversity of pspc , it is unlikely that pspc alone can be a vaccine antigen to provide protection from across different pneumococcal strains ( 16 ) . 

it was showed that rrga is central in pilus - mediated adherence and disease , even in the absence of polymeric pilus production ( 13 ) .
however , it has been demonstrated that numerous protein virulence factors are involved in the pathogenesis of pneumococcal disease ( 21 ) .
hence , new vaccine designs are focused on the surface proteins ( e. g. , pspa and pspc ) , cytolysin , and pneumolysin ( 22 ) .
we concluded that the gene cpsa was specific and highly conserved among s. pneumoniae isolates which were colonized in nasopharynx . on the other hand
, we showed that lyta gene was the most frequent genes among the s. pneumoniae isolates , and combination of rrga , lyta was the most observed pattern.thus this should allow for appropriate screening of adhesin - based vaccines to prevent infections by streptococci .
 background and objectives : many surface proteins are implicated in nasopharyngeal colonization and pathogenesis of streptococcus pneumoniae . 
 some of these factors are candidate antigens for protein based vaccines . 
 new vaccine designs focus on the surface proteins ( e. g. , pspa and pspc ) and also cytolysin , and pneumolysin . in this study , 
 3 key virulence genes , lyta , pspc , and rrga , which encoded surface proteins , were detected among s. pneumoniae isolates.materials and methods : a total of 260 nasopharyngeal swabs were collected from healthy children under 6 years old attending day care centers in mashhad , iran . 
 isolates of s. pneumoniae were confirmed by optochin susceptibility and colony appearance and also by pcr for cpsa gene . 
 the presence of lyta , pspc , and rrga genes were also detected by pcr.results:a total of 59 isolates were confirmed as s. pneumoniae . among these isolates , 50 ( 84.74% ) , 19 ( 32.20% ) , and 2 ( 3.38% ) 
 were positive for lyta , rrga , and pspc genes respectively . 
 the presence of these genes among s.pneumoniae isolates were as follows : 1 ) rrga , lyta , pspc ( 1 isolate ) , 2 ) rrga , lyta(17isolates ) , 3 ) pspc ( 2 isolate ) , 4 ) lyta ( 50 isolates).conclusion : cpsa gene was specific for detection of s. pneumoniae isolates which were colonized in nasopharynx . 
 the lyta gene was the most frequent gene among the s. pneumoniae isolates , and combination of rrga , lyta was the most observed pattern . 
 thus , it is important for future monitoring of vaccine formulation in our country . 