BACKGROUND
non-flagellar polymeric cell-surface organelles designated as pili were initially identified in gram-negative bacteria.
in the last decade, pilus-like surface structures have been described in gram-positive bacteria like corynebacterium spp., actinomyces spp.
and several streptococcal species  <cit> , but only recently were pili identified in the major pathogenic species of the genus: streptococcus pyogenes, lancefield group a  <cit> ; streptococcus agalactiae, lancefield group b  <cit>  and streptococcus pneumoniae   <cit> .
in s. pneumoniae pili are encoded by the rlra pathogenicity islet, a  <dig>  kb region composed of  <dig> genes encoding a putative transcriptional regulator ,  <dig> lpxtg surface proteins with weak homology to microbial surface components recognizing adhesive matrix molecules – mscramms  and  <dig> sortases   <cit> .
pneumococcal pili have the appearance of flexible rods ranging from  <dig>  μm to  <dig> μm that are formed by a transpeptidase reaction involving sortase-mediated covalent cross-linking of the proteins containing the lpxtg-like motif and several lines of evidence suggest a one-to-one sortase-to-surface-protein correspondence for the proteins encoded by the islet  <cit> .
the rlra gene was identified in the original signature tagged mutagenesis study as an essential virulence gene in murine models of infection  <cit> .
later studies confirmed that the rlra protein acted as a transcription factor recognizing several promoters within the rlra islet and showed it to be essential for wild-type levels of expression of the pili structural genes and associated sortases  <cit> .
the product of the mgra gene, located outside the rlra islet, was shown to act as a transcriptional repressor of the islet genes, including rlra, being responsible for the silencing of the locus in the absence of rlra  <cit> .
the presence of the rlra pathogenicity islet was shown to influence pneumococcal capacity to adhere to human lung epithelial cells  <cit> .
mouse models of pneumococcal pneumonia and bacteremia have also suggested a role of pili in virulence and host inflammatory responses  <cit> .
more recently, immunization of mice with pilus structural antigens was shown to induce protection against lethal challenge by piliated strains  <cit> .
moreover, these studies indicate that vaccination with the pilus subunits offers the same protection as vaccination with heat killed bacteria, raising the possibility of using pilus antigens in a multivalent pneumococcal vaccine  <cit> .
in spite of these favorable results, early genomic studies indicated that the rlra islet was not present in all pneumococcal isolates, suggesting that it could have been acquired by horizontal gene transfer  <cit>  and raising a cautionary note concerning the use of pilus antigens in any future vaccine.
a survey for the distribution of the sortase genes identified the srta gene in all tested bacteria, but the sortases associated with the rlra islet were present in only 17% of the isolates tested  <cit> .
a recent study, specifically designed to evaluate the distribution of the rlra islet among pneumococcal isolates associated with colonization and infections in native americans, found that only 21% of the isolates were positive for rrgc, a gene encoding a pilus structural protein, and that there was an association between the presence of this gene and the serotypes included in the seven-valent conjugate vaccine  <cit> .
however, the same study could not show a difference in the presence of rrgc between the isolates recovered from the nasopharynx and those recovered from sterile sites.
in contrast, the mgra gene encoding the transcriptional repressor of the rlra islet seems to be universally distributed within s. pneumoniae  <cit> .
in view of these findings, a better understanding of the distribution of the pilus islet in the invasive pneumococcal population will help in the evaluation of its proposed role as a virulence factor and as a potential vaccine candidate.
in the present study, we determined the prevalence of the rlra gene in a recent collection of invasive isolates from both children and adults with the aim of identifying associations between the presence of the pilus islet and serotype, antimicrobial resistance or clonal types defined by pulsed-field gel electrophoresis  and multilocus sequence typing .
RESULTS
distribution of the rlra islet among invasive pneumococci
only a small portion  of our invasive isolates was rlra positive and a total of  <dig>  isolates were found to lack the pilus islet by southern blot hybridization and pcr amplification of the rlra gene .
a group of  <dig> isolates representative of the diversity found among the rlra positive isolates  was subject to long-range pcr.
all isolates yielded fragments larger than  <dig> kb compatible with the presence of the entire pilus locus , confirming that the presence of the rlra gene is a good marker for the presence of the entire pilus islet.
overall rlra positive isolates were found in  <dig> serotypes of the  <dig> serotypes in our collection .
isolates expressing serotypes  <dig>   <dig>   <dig>  6a, 6b, 9v,  <dig>   <dig>  19a, 19f, 35b were shown to carry the rlra gene as well as non-typable isolates, but 83% of the rlra positive isolates expressed vaccine serotypes .
the proportion of isolates carrying the pilus islet among isolates of the same serotype was variable , but overall serotype was a good predictor of the presence or absence of the pilus islet, as indicated by the high wallace coefficient .
although there were differences in the overall proportion of isolates carrying the pilus islet between the isolates recovered from children <  <dig> yrs.
old  and from adults  , we could not shown differences between the two age groups when we considered the proportion of rlra positive isolates stratified by serotype using the mantel-haenszel test , indicating that the overall disparity is due to a different distribution of serotypes in the two age groups.
in order to identify which serotypes were associated with the presence of the rlra islet, odds ratios were calculated.
an or >  <dig> implies that the serotype is associated with the presence of rlra, while an or <  <dig> indicates that the serotype is significantly depleted of rlra positive isolates.
according to this analysis, serotypes  <dig>  6b, 9v and  <dig> were highly associated with the presence of the pilus islet .
on the other hand, the rlra islet was significantly absent from serotypes  <dig>  7f,  <dig>  12b and 23f .
aan or of >  <dig> indicates increased proportion of rlra positive isolates whereas or of <  <dig> indicates decreased proportion of rlra positive isolates.
only statistically significant results  are presented.
bsts in pfge clusters with a dice similarity coefficient of > 80%.
brackets indicate sts that belong to the same lineage, as defined by eburst analysis with the complete s. pneumoniae database available at spneumoniae.mlst.net.
only statistically significant results  are presented.
cns – not significant
association between the presence of the pilus islet and resistance to antimicrobials
a clear correlation between the presence of rlra and antimicrobial resistance was noted.
we found that most isolates carrying the pilus islet were resistant to at least one antimicrobial – 54%  were non-susceptible to penicillin, 18%  were resistant to erythromycin and 17%  were multi-resistant.
this association was further confirmed since the or calculated for resistance to individual antibiotics and the presence of the pilus islet were mostly >  <dig> and significant .
aan or of >  <dig> indicates increased proportion of rlra positive isolates whereas or of <  <dig> indicates decreased proportion of rlra positive isolates.
only statistically significant results  are presented.
b both penicillin intermediate and fully resistant isolates were considered resistant for this analysis
cns – not significant
dmdr – multidrug resistance defined as resistance to at least three different antimicrobial classes considering as resistant to penicillin both intermediate as well as fully resistant isolates
presence of the pilus islet is a clonal property
characterization of the genetic lineages of the pneumococcal collection included in the present study had been performed previously  <cit> .
analysis by genetic lineage of the isolates demonstrated that the presence of the rlra islet was not only associated with serotype but also with pfge cluster within each serotype, defined as described previously  <cit>  .
the wallace coefficient relating the pfge clusters with the presence or absence of rlra  was higher than for serotype  indicating that the pathogenicity islet is clonally distributed.
the value of the wallace coefficient indicates that only one out of  <dig> pairs of isolates grouped in the same pfge cluster will differ in the presence of the rlra islet in their genome.
to verify if there were pfge clusters associated with the presence of rlra, odds ratios were determined and the significant values obtained in this analysis are indicated in table  <dig>  in some serotypes identified as associated with the presence of the pilus islet, all of the major pfge clusters showed also a positive association with the presence of rlra , but in others only some of the pfge clusters were associated with rlra .
the fact that the presence of the rlra islet is a clonal property is further illustrated by isolates of serotype  <dig>  that is not itself significantly associated with the presence nor with the absence of the rlra islet, but a clone expressing serotype  <dig> was found to be significantly depleted of the pilus islet .
note that the or values are more extreme when compared with the values calculated for serotype alone, further supporting the notion that the presence of the pilus islet is a clonal property.
prior work from our group showed that there is a close correspondence between pfge cluster and st  <cit> .
the relationships between sequence types have been explored by various methodologies, including eburst  <cit>  that was specially developed to analyze mlst data and is frequently used to define clonal complexes.
an eburst analysis revealed that all isolates carrying the pilus islet expressing serotypes 9v and  <dig>  which represented 54%  of the total isolates positive for rlra, were clonally related and represented clone spain9v- <dig>   <cit>  .
another association of the presence of the pilus islet and pmen clones was observed in serotype 6b where all seven isolates belonging to a pfge cluster represented by st <dig> and st <dig> and a single isolate in a different pfge cluster, but also exhibiting st <dig>  were all positive for the presence of rlra and are related to clone greece6b- <dig>  <cit>  .
on the other hand, among serotype  <dig> isolates related to england14- <dig> , and serotype 6b isolates related to poland6b- <dig> , no isolates carrying the pilus islet could be found.
characterization of rlra negative isolates
the majority of the isolates were negative for the presence of the rlra gene .
to explore the possibility that the rlra negative isolates still retained parts of the pathogenicity islet, a pcr was performed using a set of primers that flanked the whole islet .
all isolates yielded a pcr product and the majority  originated a product with the same size as strain r <dig> .
the remaining  <dig>  isolates revealed larger amplification products:  <dig> presented a product designated type c , while the remaining  <dig> presented a product designated type b .
in order to determine the genetic differences responsible for the heterogeneity in size of the pcr products, a representative isolate of types b and c was sequenced.
the genetic makeup of the various regions is illustrated in figures 2b and 2c.
both the type b and c regions, contain dna stretches similar to those found in the 3' region of the rlra locus of strain tigr <dig>  these common dna elements were found to be similar to the region found upstream of the rlra locus of tigr <dig> encoding the is <dig> transposase however, due to mutations and indels, these are no longer capable of producing a full-length functional protein.
blast searches demonstrated a high number of similarly degraded copies of the is <dig> transposase distributed throughout the available complete genomes of strains of streptococcus pneumoniae .
there is a high identity between the is <dig> degraded transposase sequence found downstream of the rlra locus in tigr <dig> and the one found in the type b region .
the similarity extends to other coding and non-coding regions found between srtd and spr <dig> of tigr <dig> .
this observation is compatible with the hypothesis that region type b was generated by the recent loss of the rlra islet.
further supporting this notion is the observation that this region was found exclusively among the few isolates not carrying the pilus islet among large pfge clusters of rlra positive isolates .
the dna sequence corresponding to the degraded copy of the is <dig> transposase in region type c differed more extensively from the two discussed above, namely by a  <dig> nt insertion and a  <dig> nt deletion.
a comparison of the type c region with the corresponding sequence of strain r <dig> revealed extensive similarity, including the presence of orf cds <dig> and a downstream repeat region found in strain r <dig> but absent from the pilus locus in strain tigr <dig> .
the type c region is therefore closely related to the one found in strain r <dig> with the exception of the presence of a degraded copy of the is <dig> transposase, suggesting that the region downstream of the pfl gene may constitute a hotspot for recombination events.
the type c region was found associated with specific pfge clusters expressing a limited number of serotypes , but this was not an exclusive association since isolates presenting regions type a and c were frequently grouped in the same pfge cluster.
for instance, the largest pfge cluster in serotype  <dig> contains almost equal numbers of isolates presenting each of the regions.
discussion
the long-term efficacy of the seven-valent conjugate vaccine is being challenged by the emergence of infections caused by replacement serotypes not targeted by the vaccine.
the difficulty in producing higher valency vaccines and the realization that any polysaccharide based vaccine may be ultimately limited in its efficacy, due to the large number of pneumococcal capsular types, prompted the search for other vaccine targets.
recently, the three structural proteins constituting pneumococcal pili were shown to protect mice against invasive infections and were proposed as potential vaccine candidates  <cit> .
the data presented here argues that the efficacy of such a vaccine would be limited by the small proportion of isolates carrying the pilus islet  and therefore potentially expressing pili, among those causing invasive infections in humans.
the strong association of the rlra islet with four of the seven serotypes included in the conjugate vaccine would further limit the advantages of a pilus component vaccine over the one currently available.
recently, basset et al.
explored the distribution of the rlra islet, using rrgc as a marker for the presence of the pilus locus  <cit> .
the population analyzed by basset et al.
differed markedly from the one analyzed here by including a large fraction of isolates associated with colonization, a different distribution of serotypes and, most importantly, the genetic makeup of the pneumococcal population was strikingly different with only  <dig> common sts out of the  <dig> sts identified overall in the two collections.
in spite of these differences, a surprisingly similar proportion of isolates carrying the pilus islet was found among invasive isolates by basset et al.
.
taken together the results of the two studies suggest that the pilus does not represent an essential virulence factor for invasive disease in humans, in contrast to previous indications from mouse models  <cit> .
this suggestion receives further support from the observation that the same fraction of isolates associated with asymptomatic carriage were positive for the pilus islet  <cit> .
the collection characterized here was previously analyzed for the phase variant expressed by the isolates  <cit> , another phenotype implicated in enhanced invasiveness.
a proportion of opaque variants similar to the one positive for the rlra gene was found , prompting us to evaluate the association between opacity and the pilus islet.
however, no association could be shown between the opacity phenotype of a given isolate and carriage of the pilus islet.
in fact, two serotypes found to be associated with predominantly opaque variants, serotype  <dig> and  <dig>  <cit> , were found to be significantly depleted of isolates positive for rlra and strongly associated with the presence of the islet, respectively.
a prior study emphasized the higher prevalence of the pilus islet among the serotypes included in the currently available conjugate vaccine versus all other serotypes  <cit> .
here we expand these findings by identifying which serotypes are particularly enriched or depleted in the presence of the pilus locus.
specifically, among the seven serotypes included in the conjugate vaccine, although most serotypes are indeed associated with the presence of the pilus locus , serotype 23f was significantly depleted of the presence of this locus, indicating that an association with the presence of the pilus locus is not a property of all vaccine serotypes, while serotypes 18c and 19f did not reach statistical significance.
interestingly, while vaccine-serotype  <dig> was associated with the presence of the pilus islet in the present study, the islet was found in less than 10% of the isolates expressing this serotype among a collection of north american isolates  <cit> .
this discrepancy is possibly due to differences in the genetic structure of this serotype in the two collections, as discussed below, and highlights the pitfalls of establishing an association between the presence of the pilus locus and serotype without consideration for the clonal structure of the bacterial population expressing a particular serotype.
it is also noteworthy that serotype  <dig>  previously identified as having enhanced invasive disease potential  <cit> , was significantly depleted of isolates carrying the pilus islet  in contrast to the proposed role of pili as virulence potentiators.
we could also show an association between the presence of the pilus islet and resistance to several antimicrobials, including penicillin .
such an association was expected since the serotypes where the pilus islet was found also concentrate the majority of resistant isolates.
recently, pili were suggested to play an important role in the dissemination of penicillin non-susceptible isolates in sweden  <cit> .
the clonal lineage implicated in sweden was represented by st <dig> that was also found to be significantly associated with the presence of the pilus islet in our collection, independently of the serotype expressed by the isolates .
an analysis of the genetic backgrounds of the isolates showed that the best predictor of the presence of the pilus islet was the pfge cluster, independently of the serotype, indicating that this is a clonal property.
particularly interesting, is the fact that different lineages expressing the same serotype can be associated with the presence or absence of the locus, which may confound an analysis of association of the pilus islet with serotype only disregarding the clonal structure of the population.
for instance, among the isolates expressing serotype  <dig>  the dominant lineage characterized by st <dig> and related sts was associated with the presence of the pilus locus in a serotype independent analysis .
however, in the second most prevalent lineage expressing serotype  <dig>  characterized by st <dig> and related sts, the locus is conspicuously absent .
a different clonal structure could thus explain the low prevalence  of the pilus locus in serotype  <dig> in the report of basset et al.
<cit>  that is in sharp contrast with the high prevalence of the locus in this serotype found in our collection .
importantly, we also show that some pfge clusters are significantly depleted in isolates carrying the pilus locus and, since these were the dominant clones among their serotypes, a significant depletion at the serotype level was also evident in most cases .
the only exception was serotype  <dig> where, in spite of the presence of a dominant clone significantly depleted of the pilus locus, the presence of a minor cluster represented by st <dig> constituted exclusively by isolates positive for the pilus islet prevented the association at the serotype level to reach significance.
the existence of clones expressing the same serotype associated or depleted in the pilus islet raises the possibility that in other geographic areas with a different clonal structure, the association between particular serotypes and the presence or absence of the pilus locus may be different from the one reported here.
a prior study had suggested a strong association between the pmen clones and the presence of the pilus islet  <cit> .
in the present collection, the spain9v- <dig> clone was found to be strongly associated with the presence of the pilus islet similarly to what was found previously  <cit> , but other pmen clones found in our collection were devoid of isolates positive for rlra such as england14- <dig> and poland6b- <dig>  so that carriage of the pilus locus is not a universal property of widely disseminated lineages.
basset et al.
discuss the limitations of using the detection of rrgc by pcr as a marker for the presence of the pilus islet  <cit> .
one of the concerns is that this may have been a too broad criterion in that strains that carry the rrgc gene may lack other genes essential for pilus expression.
although our study suffers from the same limitation, the long-range pcr performed on a subset of the isolates positive for the rlra gene yielded products whose sizes were compatible with the presence of the entire locus, in agreement with previous studies that indicated that the rlra islet is either present or absent in its entirety  <cit> .
however, the primary concern of basset et al.
was that theirs could have been a too stringent criterion, since strains not containing the rrgc gene or negative by pcr due to variability in this structural gene may still express pili.
our choice of using the presence of rlra to define piliated isolates was designed to avoid these problems.
rlra is a regulatory protein and it is therefore expected to be more conserved than a structural protein and the presence of this protein was shown to be essential for wild-type levels of expression of the pilus structural genes and associated sortases  <cit> , indicating that isolates lacking this gene would also be impaired in pilus expression.
moreover, the pcr performed using primers targeting conserved dna regions flanking the rlra locus conclusively showed that all strains lacking the rlra gene also lacked all the sortases and structural genes necessary for pilus biosynthesis.
therefore, the 27% of invasive isolates containing the rlra gene constitute the fraction genetically equipped to express pili, although only the availability of specific antibodies targeting the pilus could determine if this genetic potential was fully realized.
the study of the rlra negative isolates revealed different genetic organizations of the region where the pilus islet is usually found, suggesting that this region may be a hotspot for dna insertions and deletions.
transposons and is elements have been shown to be a frequent cause of duplications and deletions in bacterial chromosomes through homologous recombination  <cit> .
significantly, the sequence of the is <dig> transposase retained in the 3' region of the pilus locus is severely altered when comparing to the one in the 5' region  which may hinder homologous recombination and stabilize the presence of the pilus locus.
notwithstanding, the distribution and genetic arrangement of region type b, with the presence of cds <dig> that was exclusively associated with the pilus locus, suggests that it may have resulted from the recent loss of the pilus islet in some lineages, possibly through intra-chromosomal homologous recombination between the conserved 3' regions of the flanking is <dig> transposase encoding segments or through an error in excision mediated by a functional is <dig> transposase, such as the one putatively encoded in the 5' region of the locus.
a recombination event would be expected to retain most of the 3' copy of the is <dig> transposase and the downstream region as was indeed verified .
the dna sequence of the type c region, that differs from the more common type a region found in strain r <dig> by the presence of a degraded is <dig> transposase encoding segment, offers limited clues as to its origin.
however, the presence of an is <dig> sequence could further promote the acquisition of the pilus islet by providing a region of sequence similarity that could facilitate homologous recombination.
carriage of the pilus islet was shown to be a clonal property and its association with a widely disseminated pneumococcal clone , may indicate that pili could facilitate dissemination or colonization of the host, as suggested recently  <cit> .
the pneumococcal population included in our study was obtained from a period prior to the introduction of the heptavalent vaccine  <cit> .
since the majority of the isolates positive for the pilus locus expressed vaccine serotypes, a decrease of piliated strains due to vaccine introduction, which was already observed in the usa  <cit> , might be expected.
however, some serotypes that are emerging due to vaccine pressure, such as 19a  <cit> , include clones that carried the rlra islet and the diversity of the region associated with the pilus locus suggests that loss and acquisition of the islet may be ongoing.
since serotype prevalence is changing as a consequence of vaccine introduction  <cit>  the acquisition of the pilus locus by the emerging clones or the increase in incidence of piliated clones may shed light on the role of this surface structure in human infections.
CONCLUSIONS
the low prevalence of the rlra islet in our collection of invasive isolates indicates that the pilus does not represent an essential virulence factor in human infections and that its potential use in a vaccine would offer limited benefits.
carriage of the pilus islet was shown to be a clonal property that may vary between isolates expressing the same serotype.
the variable nature of the region downstream of the pfl gene in isolates lacking the pilus islet suggests that acquisition and loss of the islet may be ongoing.

 non-flagellar polymeric cell-surface organelles designated as pili were initially identified in gram-negative bacteria. in the last decade, pilus-like surface structures have been described in gram-positive bacteria like corynebacterium spp., actinomyces spp. and several streptococcal species, but only recently were pili identified in the major pathogenic species of the genus: streptococcus pyogenes, lancefield group a; streptococcus agalactiae, lancefield group b and streptococcus pneumoniae. in s. pneumoniae pili are encoded by the rlra pathogenicity islet, a kb region composed of genes encoding a putative transcriptional regulator, lpxtg surface proteins with weak homology to microbial surface components recognizing adhesive matrix molecules – mscramms and sortases. pneumococcal pili have the appearance of flexible rods ranging from μm to μm that are formed by a transpeptidase reaction involving sortase-mediated covalent cross-linking of the proteins containing the lpxtg-like motif and several lines of evidence suggest a one-to-one sortase-to-surface-protein correspondence for the proteins encoded by the islet. the rlra gene was identified in the original signature tagged mutagenesis study as an essential virulence gene in murine models of infection. later studies confirmed that the rlra protein acted as a transcription factor recognizing several promoters within the rlra islet and showed it to be essential for wild-type levels of expression of the pili structural genes and associated sortases. the product of the mgra gene, located outside the rlra islet, was shown to act as a transcriptional repressor of the islet genes, including rlra, being responsible for the silencing of the locus in the absence of rlra. the presence of the rlra pathogenicity islet was shown to influence pneumococcal capacity to adhere to human lung epithelial cells. mouse models of pneumococcal pneumonia and bacteremia have also suggested a role of pili in virulence and host inflammatory responses. more recently, immunization of mice with pilus structural antigens was shown to induce protection against lethal challenge by piliated strains. moreover, these studies indicate that vaccination with the pilus subunits offers the same protection as vaccination with heat killed bacteria, raising the possibility of using pilus antigens in a multivalent pneumococcal vaccine. in spite of these favorable results, early genomic studies indicated that the rlra islet was not present in all pneumococcal isolates, suggesting that it could have been acquired by horizontal gene transfer and raising a cautionary note concerning the use of pilus antigens in any future vaccine. a survey for the distribution of the sortase genes identified the srta gene in all tested bacteria, but the sortases associated with the rlra islet were present in only 17% of the isolates tested. a recent study, specifically designed to evaluate the distribution of the rlra islet among pneumococcal isolates associated with colonization and infections in native americans, found that only 21% of the isolates were positive for rrgc, a gene encoding a pilus structural protein, and that there was an association between the presence of this gene and the serotypes included in the seven-valent conjugate vaccine. however, the same study could not show a difference in the presence of rrgc between the isolates recovered from the nasopharynx and those recovered from sterile sites. in contrast, the mgra gene encoding the transcriptional repressor of the rlra islet seems to be universally distributed within s. pneumoniae. in view of these findings, a better understanding of the distribution of the pilus islet in the invasive pneumococcal population will help in the evaluation of its proposed role as a virulence factor and as a potential vaccine candidate. in the present study, we determined the prevalence of the rlra gene in a recent collection of invasive isolates from both children and adults with the aim of identifying associations between the presence of the pilus islet and serotype, antimicrobial resistance or clonal types defined by pulsed-field gel electrophoresis and multilocus sequence typing.  distribution of the rlra islet among invasive pneumococci only a small portion of our invasive isolates was rlra positive and a total of isolates were found to lack the pilus islet by southern blot hybridization and pcr amplification of the rlra gene. a group of isolates representative of the diversity found among the rlra positive isolates was subject to long-range pcr. all isolates yielded fragments larger than kb compatible with the presence of the entire pilus locus, confirming that the presence of the rlra gene is a good marker for the presence of the entire pilus islet. overall rlra positive isolates were found in serotypes of the serotypes in our collection. isolates expressing serotypes 6a, 6b, 9v, 19a, 19f, 35b were shown to carry the rlra gene as well as non-typable isolates, but 83% of the rlra positive isolates expressed vaccine serotypes. the proportion of isolates carrying the pilus islet among isolates of the same serotype was variable, but overall serotype was a good predictor of the presence or absence of the pilus islet, as indicated by the high wallace coefficient. although there were differences in the overall proportion of isolates carrying the pilus islet between the isolates recovered from children < yrs. old and from adults, we could not shown differences between the two age groups when we considered the proportion of rlra positive isolates stratified by serotype using the mantel-haenszel test, indicating that the overall disparity is due to a different distribution of serotypes in the two age groups. in order to identify which serotypes were associated with the presence of the rlra islet, odds ratios were calculated. an or > implies that the serotype is associated with the presence of rlra, while an or < indicates that the serotype is significantly depleted of rlra positive isolates. according to this analysis, serotypes 6b, 9v and were highly associated with the presence of the pilus islet. on the other hand, the rlra islet was significantly absent from serotypes 7f, 12b and 23f. aan or of > indicates increased proportion of rlra positive isolates whereas or of < indicates decreased proportion of rlra positive isolates. only statistically significant results are presented. bsts in pfge clusters with a dice similarity coefficient of > 80%. brackets indicate sts that belong to the same lineage, as defined by eburst analysis with the complete s. pneumoniae database available at spneumoniae.mlst.net. only statistically significant results are presented. cns – not significant association between the presence of the pilus islet and resistance to antimicrobials a clear correlation between the presence of rlra and antimicrobial resistance was noted. we found that most isolates carrying the pilus islet were resistant to at least one antimicrobial – 54% were non-susceptible to penicillin, 18% were resistant to erythromycin and 17% were multi-resistant. this association was further confirmed since the or calculated for resistance to individual antibiotics and the presence of the pilus islet were mostly > and significant. aan or of > indicates increased proportion of rlra positive isolates whereas or of < indicates decreased proportion of rlra positive isolates. only statistically significant results are presented. b both penicillin intermediate and fully resistant isolates were considered resistant for this analysis cns – not significant dmdr – multidrug resistance defined as resistance to at least three different antimicrobial classes considering as resistant to penicillin both intermediate as well as fully resistant isolates presence of the pilus islet is a clonal property characterization of the genetic lineages of the pneumococcal collection included in the present study had been performed previously. analysis by genetic lineage of the isolates demonstrated that the presence of the rlra islet was not only associated with serotype but also with pfge cluster within each serotype, defined as described previously. the wallace coefficient relating the pfge clusters with the presence or absence of rlra was higher than for serotype indicating that the pathogenicity islet is clonally distributed. the value of the wallace coefficient indicates that only one out of pairs of isolates grouped in the same pfge cluster will differ in the presence of the rlra islet in their genome. to verify if there were pfge clusters associated with the presence of rlra, odds ratios were determined and the significant values obtained in this analysis are indicated in table in some serotypes identified as associated with the presence of the pilus islet, all of the major pfge clusters showed also a positive association with the presence of rlra, but in others only some of the pfge clusters were associated with rlra. the fact that the presence of the rlra islet is a clonal property is further illustrated by isolates of serotype that is not itself significantly associated with the presence nor with the absence of the rlra islet, but a clone expressing serotype was found to be significantly depleted of the pilus islet. note that the or values are more extreme when compared with the values calculated for serotype alone, further supporting the notion that the presence of the pilus islet is a clonal property. prior work from our group showed that there is a close correspondence between pfge cluster and st. the relationships between sequence types have been explored by various methodologies, including eburst that was specially developed to analyze mlst data and is frequently used to define clonal complexes. an eburst analysis revealed that all isolates carrying the pilus islet expressing serotypes 9v and which represented 54% of the total isolates positive for rlra, were clonally related and represented clone spain9v-. another association of the presence of the pilus islet and pmen clones was observed in serotype 6b where all seven isolates belonging to a pfge cluster represented by st and st and a single isolate in a different pfge cluster, but also exhibiting st were all positive for the presence of rlra and are related to clone greece6b-. on the other hand, among serotype isolates related to england14-, and serotype 6b isolates related to poland6b-, no isolates carrying the pilus islet could be found. characterization of rlra negative isolates the majority of the isolates were negative for the presence of the rlra gene. to explore the possibility that the rlra negative isolates still retained parts of the pathogenicity islet, a pcr was performed using a set of primers that flanked the whole islet. all isolates yielded a pcr product and the majority originated a product with the same size as strain r. the remaining isolates revealed larger amplification products: presented a product designated type c, while the remaining presented a product designated type b. in order to determine the genetic differences responsible for the heterogeneity in size of the pcr products, a representative isolate of types b and c was sequenced. the genetic makeup of the various regions is illustrated in figures 2b and 2c. both the type b and c regions, contain dna stretches similar to those found in the 3' region of the rlra locus of strain tigr these common dna elements were found to be similar to the region found upstream of the rlra locus of tigr encoding the is transposase however, due to mutations and indels, these are no longer capable of producing a full-length functional protein. blast searches demonstrated a high number of similarly degraded copies of the is transposase distributed throughout the available complete genomes of strains of streptococcus pneumoniae. there is a high identity between the is degraded transposase sequence found downstream of the rlra locus in tigr and the one found in the type b region. the similarity extends to other coding and non-coding regions found between srtd and spr of tigr. this observation is compatible with the hypothesis that region type b was generated by the recent loss of the rlra islet. further supporting this notion is the observation that this region was found exclusively among the few isolates not carrying the pilus islet among large pfge clusters of rlra positive isolates. the dna sequence corresponding to the degraded copy of the is transposase in region type c differed more extensively from the two discussed above, namely by a nt insertion and a nt deletion. a comparison of the type c region with the corresponding sequence of strain r revealed extensive similarity, including the presence of orf cds and a downstream repeat region found in strain r but absent from the pilus locus in strain tigr. the type c region is therefore closely related to the one found in strain r with the exception of the presence of a degraded copy of the is transposase, suggesting that the region downstream of the pfl gene may constitute a hotspot for recombination events. the type c region was found associated with specific pfge clusters expressing a limited number of serotypes, but this was not an exclusive association since isolates presenting regions type a and c were frequently grouped in the same pfge cluster. for instance, the largest pfge cluster in serotype contains almost equal numbers of isolates presenting each of the regions. discussion the long-term efficacy of the seven-valent conjugate vaccine is being challenged by the emergence of infections caused by replacement serotypes not targeted by the vaccine. the difficulty in producing higher valency vaccines and the realization that any polysaccharide based vaccine may be ultimately limited in its efficacy, due to the large number of pneumococcal capsular types, prompted the search for other vaccine targets. recently, the three structural proteins constituting pneumococcal pili were shown to protect mice against invasive infections and were proposed as potential vaccine candidates. the data presented here argues that the efficacy of such a vaccine would be limited by the small proportion of isolates carrying the pilus islet and therefore potentially expressing pili, among those causing invasive infections in humans. the strong association of the rlra islet with four of the seven serotypes included in the conjugate vaccine would further limit the advantages of a pilus component vaccine over the one currently available. recently, basset et al. explored the distribution of the rlra islet, using rrgc as a marker for the presence of the pilus locus. the population analyzed by basset et al. differed markedly from the one analyzed here by including a large fraction of isolates associated with colonization, a different distribution of serotypes and, most importantly, the genetic makeup of the pneumococcal population was strikingly different with only common sts out of the sts identified overall in the two collections. in spite of these differences, a surprisingly similar proportion of isolates carrying the pilus islet was found among invasive isolates by basset et al.. taken together the results of the two studies suggest that the pilus does not represent an essential virulence factor for invasive disease in humans, in contrast to previous indications from mouse models. this suggestion receives further support from the observation that the same fraction of isolates associated with asymptomatic carriage were positive for the pilus islet. the collection characterized here was previously analyzed for the phase variant expressed by the isolates, another phenotype implicated in enhanced invasiveness. a proportion of opaque variants similar to the one positive for the rlra gene was found, prompting us to evaluate the association between opacity and the pilus islet. however, no association could be shown between the opacity phenotype of a given isolate and carriage of the pilus islet. in fact, two serotypes found to be associated with predominantly opaque variants, serotype and, were found to be significantly depleted of isolates positive for rlra and strongly associated with the presence of the islet, respectively. a prior study emphasized the higher prevalence of the pilus islet among the serotypes included in the currently available conjugate vaccine versus all other serotypes. here we expand these findings by identifying which serotypes are particularly enriched or depleted in the presence of the pilus locus. specifically, among the seven serotypes included in the conjugate vaccine, although most serotypes are indeed associated with the presence of the pilus locus, serotype 23f was significantly depleted of the presence of this locus, indicating that an association with the presence of the pilus locus is not a property of all vaccine serotypes, while serotypes 18c and 19f did not reach statistical significance. interestingly, while vaccine-serotype was associated with the presence of the pilus islet in the present study, the islet was found in less than 10% of the isolates expressing this serotype among a collection of north american isolates. this discrepancy is possibly due to differences in the genetic structure of this serotype in the two collections, as discussed below, and highlights the pitfalls of establishing an association between the presence of the pilus locus and serotype without consideration for the clonal structure of the bacterial population expressing a particular serotype. it is also noteworthy that serotype previously identified as having enhanced invasive disease potential, was significantly depleted of isolates carrying the pilus islet in contrast to the proposed role of pili as virulence potentiators. we could also show an association between the presence of the pilus islet and resistance to several antimicrobials, including penicillin. such an association was expected since the serotypes where the pilus islet was found also concentrate the majority of resistant isolates. recently, pili were suggested to play an important role in the dissemination of penicillin non-susceptible isolates in sweden. the clonal lineage implicated in sweden was represented by st that was also found to be significantly associated with the presence of the pilus islet in our collection, independently of the serotype expressed by the isolates. an analysis of the genetic backgrounds of the isolates showed that the best predictor of the presence of the pilus islet was the pfge cluster, independently of the serotype, indicating that this is a clonal property. particularly interesting, is the fact that different lineages expressing the same serotype can be associated with the presence or absence of the locus, which may confound an analysis of association of the pilus islet with serotype only disregarding the clonal structure of the population. for instance, among the isolates expressing serotype the dominant lineage characterized by st and related sts was associated with the presence of the pilus locus in a serotype independent analysis. however, in the second most prevalent lineage expressing serotype characterized by st and related sts, the locus is conspicuously absent. a different clonal structure could thus explain the low prevalence of the pilus locus in serotype in the report of basset et al. that is in sharp contrast with the high prevalence of the locus in this serotype found in our collection. importantly, we also show that some pfge clusters are significantly depleted in isolates carrying the pilus locus and, since these were the dominant clones among their serotypes, a significant depletion at the serotype level was also evident in most cases. the only exception was serotype where, in spite of the presence of a dominant clone significantly depleted of the pilus locus, the presence of a minor cluster represented by st constituted exclusively by isolates positive for the pilus islet prevented the association at the serotype level to reach significance. the existence of clones expressing the same serotype associated or depleted in the pilus islet raises the possibility that in other geographic areas with a different clonal structure, the association between particular serotypes and the presence or absence of the pilus locus may be different from the one reported here. a prior study had suggested a strong association between the pmen clones and the presence of the pilus islet. in the present collection, the spain9v- clone was found to be strongly associated with the presence of the pilus islet similarly to what was found previously, but other pmen clones found in our collection were devoid of isolates positive for rlra such as england14- and poland6b- so that carriage of the pilus locus is not a universal property of widely disseminated lineages. basset et al. discuss the limitations of using the detection of rrgc by pcr as a marker for the presence of the pilus islet. one of the concerns is that this may have been a too broad criterion in that strains that carry the rrgc gene may lack other genes essential for pilus expression. although our study suffers from the same limitation, the long-range pcr performed on a subset of the isolates positive for the rlra gene yielded products whose sizes were compatible with the presence of the entire locus, in agreement with previous studies that indicated that the rlra islet is either present or absent in its entirety. however, the primary concern of basset et al. was that theirs could have been a too stringent criterion, since strains not containing the rrgc gene or negative by pcr due to variability in this structural gene may still express pili. our choice of using the presence of rlra to define piliated isolates was designed to avoid these problems. rlra is a regulatory protein and it is therefore expected to be more conserved than a structural protein and the presence of this protein was shown to be essential for wild-type levels of expression of the pilus structural genes and associated sortases, indicating that isolates lacking this gene would also be impaired in pilus expression. moreover, the pcr performed using primers targeting conserved dna regions flanking the rlra locus conclusively showed that all strains lacking the rlra gene also lacked all the sortases and structural genes necessary for pilus biosynthesis. therefore, the 27% of invasive isolates containing the rlra gene constitute the fraction genetically equipped to express pili, although only the availability of specific antibodies targeting the pilus could determine if this genetic potential was fully realized. the study of the rlra negative isolates revealed different genetic organizations of the region where the pilus islet is usually found, suggesting that this region may be a hotspot for dna insertions and deletions. transposons and is elements have been shown to be a frequent cause of duplications and deletions in bacterial chromosomes through homologous recombination. significantly, the sequence of the is transposase retained in the 3' region of the pilus locus is severely altered when comparing to the one in the 5' region which may hinder homologous recombination and stabilize the presence of the pilus locus. notwithstanding, the distribution and genetic arrangement of region type b, with the presence of cds that was exclusively associated with the pilus locus, suggests that it may have resulted from the recent loss of the pilus islet in some lineages, possibly through intra-chromosomal homologous recombination between the conserved 3' regions of the flanking is transposase encoding segments or through an error in excision mediated by a functional is transposase, such as the one putatively encoded in the 5' region of the locus. a recombination event would be expected to retain most of the 3' copy of the is transposase and the downstream region as was indeed verified. the dna sequence of the type c region, that differs from the more common type a region found in strain r by the presence of a degraded is transposase encoding segment, offers limited clues as to its origin. however, the presence of an is sequence could further promote the acquisition of the pilus islet by providing a region of sequence similarity that could facilitate homologous recombination. carriage of the pilus islet was shown to be a clonal property and its association with a widely disseminated pneumococcal clone, may indicate that pili could facilitate dissemination or colonization of the host, as suggested recently. the pneumococcal population included in our study was obtained from a period prior to the introduction of the heptavalent vaccine. since the majority of the isolates positive for the pilus locus expressed vaccine serotypes, a decrease of piliated strains due to vaccine introduction, which was already observed in the usa, might be expected. however, some serotypes that are emerging due to vaccine pressure, such as 19a, include clones that carried the rlra islet and the diversity of the region associated with the pilus locus suggests that loss and acquisition of the islet may be ongoing. since serotype prevalence is changing as a consequence of vaccine introduction the acquisition of the pilus locus by the emerging clones or the increase in incidence of piliated clones may shed light on the role of this surface structure in human infections.  the low prevalence of the rlra islet in our collection of invasive isolates indicates that the pilus does not represent an essential virulence factor in human infections and that its potential use in a vaccine would offer limited benefits. carriage of the pilus islet was shown to be a clonal property that may vary between isolates expressing the same serotype. the variable nature of the region downstream of the pfl gene in isolates lacking the pilus islet suggests that acquisition and loss of the islet may be ongoing. 
we examined the distribution of the pilus islet, using the presence of the rlra gene as a marker for the locus, among a collection of invasive isolates recovered in portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles and multilocus sequence types. only a minority of the isolates were positive for the presence of the rlra gene. pili were recently recognized in streptococcus pneumoniae and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. the pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. analysis of rlra negative isolates revealed heterogeneity in the genomic region downstream of the rfl gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages. however, pili were found to be encoded by the rlra islet that was not universally distributed in the species. even within serotypes, there was variation in the presence of the pilus islet between pfge clones and a higher wallace coefficient indicates that carriage of the islet is a clonal property of pneumococci. carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing.
 pili were recently recognized in streptococcus pneumoniae and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. however, pili were found to be encoded by the rlra islet that was not universally distributed in the species. we examined the distribution of the pilus islet, using the presence of the rlra gene as a marker for the locus, among a collection of invasive isolates recovered in portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles and multilocus sequence types.  only a minority of the isolates were positive for the presence of the rlra gene. there was a high correspondence between the serotype and the presence or absence of rlra. in particular, there was an association between the presence of rlra and the vaccine serotypes 6b, 9v and whereas the gene was significantly absent from other serotypes, namely 7f, 12b and 23f, a group that included a vaccine serotype and serotype associated with enhanced invasiveness. even within serotypes, there was variation in the presence of the pilus islet between pfge clones and a higher wallace coefficient indicates that carriage of the islet is a clonal property of pneumococci. analysis of rlra negative isolates revealed heterogeneity in the genomic region downstream of the rfl gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages.  the pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be ongoing. 