BACKGROUND
streptococcus agalactiae  can colonize the gastrointestinal and genitourinary tracts of healthy individuals without any symptoms of disease  <cit> .
nevertheless, this bacterium can cause life-threatening invasive diseases in pregnant women, neonates or non-pregnant adults.
colonized women, during pregnancy or the postpartum period, are usually asymptomatic, but gbs may cause bacteremia, urinary tract infections, chorioamnionitis, endometritis, puerperal sepsis and, occasionally meningitis and septic thrombophlebitis  <cit> .
gbs colonization among pregnant women also increases the risk of premature delivery and perinatal transmission of the microorganism to newborns, which can cause fatal sepsis and meningitis  <cit> .
a successful perinatal disease prevention strategy based on intrapartum chemoprophylaxis for pregnant women at risk  <cit>  leads to a significant decrease in gbs infections in neonates  <cit> .
however, in the last decades, gbs have been increasingly associated with invasive disease in non-pregnant adults, mainly in the elderly, immunocompromised and those with diabetes mellitus and cancer  <cit> .
most importantly, mortality associated with these patients is frequently higher than for newborns  <cit> .
these data draw attention to the need for prevention strategies against gbs infections among adults.
penicillin has been established as a first-line antimicrobial for the prophylaxis and treatment of gbs infections.
moreover, clindamycin and erythromycin have been used as alternatives in penicillin-allergic individuals.
however, resistance to these antimicrobials among gbs isolated from pregnant and non-pregnant individuals has been described in several countries  <cit> , raising concerns about their use in the treatment of gbs infections.
resistance to penicillin is frequently associated with mutation of penicillin-binding proteins  2x and 2b  <cit> .
overall, the mechanisms that confer resistance to erythromycin include the post-transcriptional methylation of the adenine residues of 23s ribosomal rna mediated by erm genes and efflux of the antibiotic mediated by a membrane-bound protein encoded by mef genes.
the expression of erm genes results in the mlsb phenotype, responsible for generating cross-resistance to macrolides, lincosamides and streptogramin b  <cit> .
on the other hand, phenotype m, encoded by mef genes, confers resistance only to 14- and 15-membered ring macrolides   <cit> .
according to the immunologic reactivity of sialic acid-rich capsular polysaccharide, gbs are divided into ten serotypes, ia, ib, ii-viii  <cit>  and ix  <cit> .
different surveys all over the world have shown the prevalence of serotypes ia, ib, ii, iii and v as major streptococcal disease-causing agents  <cit> .
the diverse array of clinical manifestations caused by gbs reflects an efficient adaptability of bacteria to different host environments.
gbs may express virulence factors, allowing the colonization and invasion of epithelial barriers, leading to resistance to immune clearance and persistence in host tissues, which contribute to the pathogenesis of infection.
besides defining gbs serotypes, the cell wall-anchored polysaccharide capsule has been recognized as important virulence factor of this bacterium.
it prevents the deposition of alternative complement pathway factor c3b on the bacterial surface, resulting in decreased phagocytosis by macrophages and neutrophils  <cit> .
in the last decade, a pilus-like structure was identified in gbs  <cit>  and shown to play an important role in the adhesion to and invasion of host cells  <cit> , biofilm formation  <cit>  and resistance to phagocyte killing  <cit> .
extracellular β-hemolysin/cytolysin  is a pore-forming toxin encoded by the chromosomal cyle gene  <cit> , which is toxic to a broad range of eukaryotic cells, resulting in cell invasion  <cit>  and evasion of phagocytosis  <cit> .
the expression of cyle is also associated with the production of an orange carotenoid pigment that contributes to the protection of bacteria against the toxic effects of reactive oxygen species generated by the oxidative burst mechanism of phagocytic killing of macrophages  <cit> .
the product of gene hylb, a secreted hyaluronate lyase, can hydrolyze hyaluronan polymers, which are components of the extracellular matrix of human tissues, suggesting that this enzyme can facilitate the spread of bacteria during infection  <cit> .
in the study described here, gbs isolated from women at reproductive age with no clinical evidence of streptococcal infection were characterized by phenotypic and molecular methods.
all isolates were tested for capsular type, hemolysis and carotenoid pigment production.
in addition, the in vitro susceptibility pattern of the isolates to antimicrobial agents, the genetic relatedness and the occurrence of virulence determinant genes were also investigated.
RESULTS
patients, gbs capsular types and multiple locus variable number of tandem repeat analysis  genotypes
a total of  <dig> isolates of gbs from women with no clinical evidence of streptococcal infection were enrolled in this study.
these isolates were taken from the bacterial collection of the laboratory of clinical microbiology of university hospital of londrina, the major referral center for healthcare management that serves londrina city, besides several localities of paraná, são paulo and mato grosso do sul states, in brazil.
the age of the patients ranged from  <dig> to 58 years .
gbs isolates were distributed in five capsular types according to the multiplex-pcr method, and type ia  was the most frequent, followed by type v , type iii  and type ii .
one each of type ix  and nt  was identified among isolates.
the genetic relatedness of gbs isolates was assessed by mlva.
by using a cutoff value of 85% similarity, a total of  <dig> different mlva types  were identified among the isolates, and overall the diversity index obtained with this method was  <dig> .
the largest groups of similar mlva profiles consisted of  <dig>  and  <dig>  isolates.
thirty five isolates were grouped in six mts, one with four , eight , and seven  isolates each, and three with five  isolates each.
the other seven  had unique mlva profiles.
most gbs capsular type ia were grouped in mt <dig> , and the other  <dig> isolates were distributed in seven distinct mlva types.
the gbs capsular types v and iii were distributed in seven and three mlva types respectively, whereas all isolates displaying the capsular type ii were grouped in mt <dig>  and all the isolates except one had an identical mlva profile .
phenotypic and genotypic characterization of antimicrobial susceptibility
all isolates were susceptible to penicillin, ampicillin, cefepime, cefotaxime, chloramphenicol, levofloxacin and vancomycin.
resistance to erythromycin and clindamycin was detected in  <dig>  and  <dig>  isolates, respectively.
all isolates resistant to clindamycin were also resistant to erythromycin, and among them only one had a constitutive macrolide-lincosamide-streptogramin b  phenotype  and harbored the ermb gene.
of the  <dig> isolates displaying the indutible mlsb  phenotype, seven carried the erma gene, whereas one isolate carried the ermb gene and two both genes.
all isolates  resistant only to erythromycin showed phenotype m and carried the mefa/e gene.
resistance to both erythromycin and clindamycin was detected among isolates belonging to serotypes v  and iii , which were grouped in mts  <dig>   <dig>   <dig>   <dig> and  <dig>  all isolates resistant only to erythromycin belonged to serotype ia and mt <dig> .
streptococcus agalactiae
: distribution of capsular type, mlva genotypes and antimicrobials resistance features
a
b
c
d
e
erma
ermb
mefa/e
athe genetic diversity was assessed by mlva typing  <cit> .
a cutoff value of 85% similarity was applied to define mlva types.
bthe capsular type was identified by multiplex-pcr  <cit> .
cerythromycin resistance phenotype was determined by the double-disk diffusion method  <cit> .
dthe presence of specified gene was determined by pcr.
presence;  absence.
ethe minimum inhibitory concentrations  were determined by the agar-dilution method.
clindamycin ; erythromycin .
detection of potential virulence traits of commensal gbs
the presence of the nucleotide sequences corresponding to the cyle, hylb and pilus islands pi- <dig>  pi-2a and pi-2b was detected by pcr.
all isolates harbored the cyle and hylb genes and at least one pilus island.
four  of the  <dig> gbs isolates did not produce a hemolytic halo around the bacterial colonies .
concomitantly, these isolates were not able to produce the orange carotenoid pigment in granada medium.
most of the isolates harbored pi-2a alone  or in combination with pi- <dig> .
pi-2a was distributed in all capsular types identified in this study, including the type ix and nt isolates.
however, the presence of this pilus island alone or in combination with pi- <dig> was found mainly in capsular type ia and v, respectively.
besides, pi- <dig> was also found in combination with pi-2b  and all isolates belonged to capsular type iii.
the presence of pi-2b alone was observed in seven isolates  and all belonged to capsular type ia.
all isolates grouped in mts  <dig> ,  <dig> ,  <dig>  and  <dig>  harbored pi- <dig> and pi-2a islands.
in addition, these pili were also detected in isolates belonged to mts  <dig>   <dig>   <dig> and  <dig>  all isolates belonging to mts  <dig> ,  <dig>   <dig> and  <dig>  and one isolate  of mt <dig> harbored the pi-2a island.
pi- <dig> and pi-2b was detected only in isolates of mt <dig> , whereas the pi-2b island was detected in isolates of mts  <dig> ,  <dig> and  <dig>  .
the isolates displaying the mlsb phenotype harbored the pilus islands pi- <dig> and pi-2a, whereas the isolates showing the m phenotype harbored only the pi-2a.
discussion
in this study, five capsular types  were identified and, except for type ix, all are frequently associated with gbs infections worldwide  <cit> .
the serotypes identified in this study were also detected in different surveys that were performed with brazilian isolates among pregnant and non-pregnant adults.
in those studies, the serotypes ib  <cit>  iv  <cit> , vi  <cit>  and viii  <cit>  were also identified.
the genetic diversity of gbs isolates were assessed by mlva  <cit> , which is based on the amplification of polymorphic tandem repeat sequences .
it is easy to use, displays shorter time of execution, can be applied to a small or large numbers of isolates and has been employed successfully for the typing of different bacteria species.
in addition, it has higher discriminatory power than multi locus sequencing typing , the reference method for genotyping streptococcus spp.
<cit> .
the diversity index obtained with mlva for this bacterial population was  <dig> , lower than observed by others  <cit> .
however, despite the close relatedness of several isolates, as judged by the capsular type and presence of pili islands, this genotyping scheme discriminated the gbss in this study.
in fact, a total of  <dig> different genetic groups were identified among these isolates.
sag <dig>  sag <dig> and sag <dig> presented higher degree of the variation in the number of repeats among the isolates, corroborating the data of haguenoer et al.
<cit> .
although no statistical correlation was performed, it was observed that isolates belonging to the capsular type ii were confined to mt <dig>  indicating that the genetic background of this serotype may be well conserved.
higher number of isolates may corroborate these findings.
all isolates were susceptible to the antimicrobials evaluated in this study, except erythromycin and clindamycin.
although it was not an epidemiological investigation, the overall rate of erythromycin resistance among the isolates analyzed was  <dig> %.
previous epidemiological and bacterial collection data from brazilian gbs isolates showed that erythromycin resistance ranged from  <dig> to 14%  <cit> .
a higher incidence rate was observed in other regions, where erythromycin resistance up to 40% among gbs isolates was detected in europe  <cit>  and usa  <cit> .
in this study, resistance to both erythromycin and clindamycin was observed in gbs isolates of capsular types iii and v, whereas the isolates displaying resistance only to erythromycin were exclusively found in the ia capsular type.
similar results were previously obtained by other authors  <cit> ; however, resistant isolates for both antimicrobials were also observed among the ib, ii, iv, vi and viii capsular serotypes  <cit> .
the mechanisms of macrolide resistance are mediated by erma, ermb and mefa/e, and the distribution of these genes among gbs isolates in this study were in accordance with the macrolide-resistance phenotypes.
these results were also observed by others  <cit> .
the increasing numbers of isolates showing macrolide resistance together with the description of reduced susceptibility to penicillin emphasize the need for continued monitoring of antimicrobial susceptibility profile to identify the emergence of resistance among gbs isolates.
data of the potential virulence of gbs isolates from brazil are limited.
three genomic islands encoding the structurally distinct types of pili  were identified in gbs.
these pili are organized in two different loci, where pi-2a and pi-2b are located at the same chromosomal locus, with these being mutually exclusive  <cit> .
to our knowledge, this is the first study describing the prevalence of the pilus island in brazilian gbs isolates, and at least one pilus type was detected among the isolates, supporting their use as an antigen for vaccine development.
the combination of pi- <dig> and pi-2a was the most prevalent among the gbs isolates, and this result is in agreement with previous reports  <cit> .
in addition, the presence of this combination was correlated with maternal colonization and invasive disease in adults  <cit> .
the cyl locus of gbs consists of a cluster of twelve genes  <cit> , and some of them can modulate cyle expression and secretion  <cit> , which is crucial for β-h/c activity.
this toxin can promote the invasion of epithelial or endothelial cells  <cit> , facilitate their persistence and intracellular survival  <cit> , and enable their dissemination at host sites.
besides, acting as a virulence factor, cyle is associated with the characteristic translucent halo around gbs colonies grown on blood agar plates and production of orange carotenoid pigment on specific chromogenic agar, features that are used for presumptive identification of s. agalactiae.
in this study, four gbs isolates were non-hemolytic and simultaneously non-pigment producers.
indeed, approximately 3% of gbs isolates are non-hemolytic  <cit> , emphasizing the need to develop new methods that combine identification and detection of antimicrobial resistance for these bacteria.
the role of hyaluronidase in the pathogenesis of gbs infections is still unclear, but it is postulated that this enzyme can facilitate the invasion and dissemination of gbs during infection.
the expression of this enzyme has been associated with gbs isolated from invasive infections  <cit> ; however, hyaluronidase activity has also been detected in commensal gbs isolates from women’s genital tract  <cit> .
CONCLUSIONS
in conclusion, we identified the predominant occurrence of capsular types ia, ii, iii and v among commensal gbss isolated from women at reproductive age seen at university hospital of londrina, paraná.
the gbs isolates harbored at least one pilus island.
our findings are in agreement with a higher proportion of capsular types and distribution of pili previously reported among gbs isolated from different countries.
these data support the notion of developing of a vaccine globally effective against this opportunistic bacterium.
we also detected resistance to erythromycin and clindamycin and the occurrence of the genes encoding virulence determinants cyle and hylb among these isolates, reinforcing the need for continued monitoring of gbs to prevent the development of infections.
in addition, a total of  <dig> different genetic groups were identified, and isolates belonging to the capsular type ii were confined to mt <dig>  besides, resistance only to erythromycin was observed in gbs isolates belonging to capsular type ia and mt <dig>  whereas isolates resistant to both erythromycin and clindamycin were distributed over various capsular and mlva types.
higher number of isolates may corroborate these findings.

 streptococcus agalactiae can colonize the gastrointestinal and genitourinary tracts of healthy individuals without any symptoms of disease. nevertheless, this bacterium can cause life-threatening invasive diseases in pregnant women, neonates or non-pregnant adults. colonized women, during pregnancy or the postpartum period, are usually asymptomatic, but gbs may cause bacteremia, urinary tract infections, chorioamnionitis, endometritis, puerperal sepsis and, occasionally meningitis and septic thrombophlebitis. gbs colonization among pregnant women also increases the risk of premature delivery and perinatal transmission of the microorganism to newborns, which can cause fatal sepsis and meningitis. a successful perinatal disease prevention strategy based on intrapartum chemoprophylaxis for pregnant women at risk leads to a significant decrease in gbs infections in neonates. however, in the last decades, gbs have been increasingly associated with invasive disease in non-pregnant adults, mainly in the elderly, immunocompromised and those with diabetes mellitus and cancer. most importantly, mortality associated with these patients is frequently higher than for newborns. these data draw attention to the need for prevention strategies against gbs infections among adults. penicillin has been established as a first-line antimicrobial for the prophylaxis and treatment of gbs infections. moreover, clindamycin and erythromycin have been used as alternatives in penicillin-allergic individuals. however, resistance to these antimicrobials among gbs isolated from pregnant and non-pregnant individuals has been described in several countries, raising concerns about their use in the treatment of gbs infections. resistance to penicillin is frequently associated with mutation of penicillin-binding proteins 2x and 2b. overall, the mechanisms that confer resistance to erythromycin include the post-transcriptional methylation of the adenine residues of 23s ribosomal rna mediated by erm genes and efflux of the antibiotic mediated by a membrane-bound protein encoded by mef genes. the expression of erm genes results in the mlsb phenotype, responsible for generating cross-resistance to macrolides, lincosamides and streptogramin b. on the other hand, phenotype m, encoded by mef genes, confers resistance only to 14- and 15-membered ring macrolides. according to the immunologic reactivity of sialic acid-rich capsular polysaccharide, gbs are divided into ten serotypes, ia, ib, ii-viii and ix. different surveys all over the world have shown the prevalence of serotypes ia, ib, ii, iii and v as major streptococcal disease-causing agents. the diverse array of clinical manifestations caused by gbs reflects an efficient adaptability of bacteria to different host environments. gbs may express virulence factors, allowing the colonization and invasion of epithelial barriers, leading to resistance to immune clearance and persistence in host tissues, which contribute to the pathogenesis of infection. besides defining gbs serotypes, the cell wall-anchored polysaccharide capsule has been recognized as important virulence factor of this bacterium. it prevents the deposition of alternative complement pathway factor c3b on the bacterial surface, resulting in decreased phagocytosis by macrophages and neutrophils. in the last decade, a pilus-like structure was identified in gbs and shown to play an important role in the adhesion to and invasion of host cells, biofilm formation and resistance to phagocyte killing. extracellular β-hemolysin/cytolysin is a pore-forming toxin encoded by the chromosomal cyle gene, which is toxic to a broad range of eukaryotic cells, resulting in cell invasion and evasion of phagocytosis. the expression of cyle is also associated with the production of an orange carotenoid pigment that contributes to the protection of bacteria against the toxic effects of reactive oxygen species generated by the oxidative burst mechanism of phagocytic killing of macrophages. the product of gene hylb, a secreted hyaluronate lyase, can hydrolyze hyaluronan polymers, which are components of the extracellular matrix of human tissues, suggesting that this enzyme can facilitate the spread of bacteria during infection. in the study described here, gbs isolated from women at reproductive age with no clinical evidence of streptococcal infection were characterized by phenotypic and molecular methods. all isolates were tested for capsular type, hemolysis and carotenoid pigment production. in addition, the in vitro susceptibility pattern of the isolates to antimicrobial agents, the genetic relatedness and the occurrence of virulence determinant genes were also investigated.  patients, gbs capsular types and multiple locus variable number of tandem repeat analysis genotypes a total of isolates of gbs from women with no clinical evidence of streptococcal infection were enrolled in this study. these isolates were taken from the bacterial collection of the laboratory of clinical microbiology of university hospital of londrina, the major referral center for healthcare management that serves londrina city, besides several localities of paraná, são paulo and mato grosso do sul states, in brazil. the age of the patients ranged from to 58 years. gbs isolates were distributed in five capsular types according to the multiplex-pcr method, and type ia was the most frequent, followed by type v, type iii and type ii. one each of type ix and nt was identified among isolates. the genetic relatedness of gbs isolates was assessed by mlva. by using a cutoff value of 85% similarity, a total of different mlva types were identified among the isolates, and overall the diversity index obtained with this method was. the largest groups of similar mlva profiles consisted of and isolates. thirty five isolates were grouped in six mts, one with four, eight, and seven isolates each, and three with five isolates each. the other seven had unique mlva profiles. most gbs capsular type ia were grouped in mt, and the other isolates were distributed in seven distinct mlva types. the gbs capsular types v and iii were distributed in seven and three mlva types respectively, whereas all isolates displaying the capsular type ii were grouped in mt and all the isolates except one had an identical mlva profile. phenotypic and genotypic characterization of antimicrobial susceptibility all isolates were susceptible to penicillin, ampicillin, cefepime, cefotaxime, chloramphenicol, levofloxacin and vancomycin. resistance to erythromycin and clindamycin was detected in and isolates, respectively. all isolates resistant to clindamycin were also resistant to erythromycin, and among them only one had a constitutive macrolide-lincosamide-streptogramin b phenotype and harbored the ermb gene. of the isolates displaying the indutible mlsb phenotype, seven carried the erma gene, whereas one isolate carried the ermb gene and two both genes. all isolates resistant only to erythromycin showed phenotype m and carried the mefa/e gene. resistance to both erythromycin and clindamycin was detected among isolates belonging to serotypes v and iii, which were grouped in mts and all isolates resistant only to erythromycin belonged to serotype ia and mt. streptococcus agalactiae: distribution of capsular type, mlva genotypes and antimicrobials resistance features a b c d e erma ermb mefa/e athe genetic diversity was assessed by mlva typing. a cutoff value of 85% similarity was applied to define mlva types. bthe capsular type was identified by multiplex-pcr. cerythromycin resistance phenotype was determined by the double-disk diffusion method. dthe presence of specified gene was determined by pcr. presence; absence. ethe minimum inhibitory concentrations were determined by the agar-dilution method. clindamycin; erythromycin. detection of potential virulence traits of commensal gbs the presence of the nucleotide sequences corresponding to the cyle, hylb and pilus islands pi- pi-2a and pi-2b was detected by pcr. all isolates harbored the cyle and hylb genes and at least one pilus island. four of the gbs isolates did not produce a hemolytic halo around the bacterial colonies. concomitantly, these isolates were not able to produce the orange carotenoid pigment in granada medium. most of the isolates harbored pi-2a alone or in combination with pi-. pi-2a was distributed in all capsular types identified in this study, including the type ix and nt isolates. however, the presence of this pilus island alone or in combination with pi- was found mainly in capsular type ia and v, respectively. besides, pi- was also found in combination with pi-2b and all isolates belonged to capsular type iii. the presence of pi-2b alone was observed in seven isolates and all belonged to capsular type ia. all isolates grouped in mts,, and harbored pi- and pi-2a islands. in addition, these pili were also detected in isolates belonged to mts and all isolates belonging to mts, and and one isolate of mt harbored the pi-2a island. pi- and pi-2b was detected only in isolates of mt, whereas the pi-2b island was detected in isolates of mts, and. the isolates displaying the mlsb phenotype harbored the pilus islands pi- and pi-2a, whereas the isolates showing the m phenotype harbored only the pi-2a. discussion in this study, five capsular types were identified and, except for type ix, all are frequently associated with gbs infections worldwide. the serotypes identified in this study were also detected in different surveys that were performed with brazilian isolates among pregnant and non-pregnant adults. in those studies, the serotypes ib iv, vi and viii were also identified. the genetic diversity of gbs isolates were assessed by mlva, which is based on the amplification of polymorphic tandem repeat sequences. it is easy to use, displays shorter time of execution, can be applied to a small or large numbers of isolates and has been employed successfully for the typing of different bacteria species. in addition, it has higher discriminatory power than multi locus sequencing typing, the reference method for genotyping streptococcus spp.. the diversity index obtained with mlva for this bacterial population was, lower than observed by others. however, despite the close relatedness of several isolates, as judged by the capsular type and presence of pili islands, this genotyping scheme discriminated the gbss in this study. in fact, a total of different genetic groups were identified among these isolates. sag sag and sag presented higher degree of the variation in the number of repeats among the isolates, corroborating the data of haguenoer et al.. although no statistical correlation was performed, it was observed that isolates belonging to the capsular type ii were confined to mt indicating that the genetic background of this serotype may be well conserved. higher number of isolates may corroborate these findings. all isolates were susceptible to the antimicrobials evaluated in this study, except erythromycin and clindamycin. although it was not an epidemiological investigation, the overall rate of erythromycin resistance among the isolates analyzed was %. previous epidemiological and bacterial collection data from brazilian gbs isolates showed that erythromycin resistance ranged from to 14%. a higher incidence rate was observed in other regions, where erythromycin resistance up to 40% among gbs isolates was detected in europe and usa. in this study, resistance to both erythromycin and clindamycin was observed in gbs isolates of capsular types iii and v, whereas the isolates displaying resistance only to erythromycin were exclusively found in the ia capsular type. similar results were previously obtained by other authors; however, resistant isolates for both antimicrobials were also observed among the ib, ii, iv, vi and viii capsular serotypes. the mechanisms of macrolide resistance are mediated by erma, ermb and mefa/e, and the distribution of these genes among gbs isolates in this study were in accordance with the macrolide-resistance phenotypes. these results were also observed by others. the increasing numbers of isolates showing macrolide resistance together with the description of reduced susceptibility to penicillin emphasize the need for continued monitoring of antimicrobial susceptibility profile to identify the emergence of resistance among gbs isolates. data of the potential virulence of gbs isolates from brazil are limited. three genomic islands encoding the structurally distinct types of pili were identified in gbs. these pili are organized in two different loci, where pi-2a and pi-2b are located at the same chromosomal locus, with these being mutually exclusive. to our knowledge, this is the first study describing the prevalence of the pilus island in brazilian gbs isolates, and at least one pilus type was detected among the isolates, supporting their use as an antigen for vaccine development. the combination of pi- and pi-2a was the most prevalent among the gbs isolates, and this result is in agreement with previous reports. in addition, the presence of this combination was correlated with maternal colonization and invasive disease in adults. the cyl locus of gbs consists of a cluster of twelve genes, and some of them can modulate cyle expression and secretion, which is crucial for β-h/c activity. this toxin can promote the invasion of epithelial or endothelial cells, facilitate their persistence and intracellular survival, and enable their dissemination at host sites. besides, acting as a virulence factor, cyle is associated with the characteristic translucent halo around gbs colonies grown on blood agar plates and production of orange carotenoid pigment on specific chromogenic agar, features that are used for presumptive identification of s. agalactiae. in this study, four gbs isolates were non-hemolytic and simultaneously non-pigment producers. indeed, approximately 3% of gbs isolates are non-hemolytic, emphasizing the need to develop new methods that combine identification and detection of antimicrobial resistance for these bacteria. the role of hyaluronidase in the pathogenesis of gbs infections is still unclear, but it is postulated that this enzyme can facilitate the invasion and dissemination of gbs during infection. the expression of this enzyme has been associated with gbs isolated from invasive infections; however, hyaluronidase activity has also been detected in commensal gbs isolates from women’s genital tract.  in conclusion, we identified the predominant occurrence of capsular types ia, ii, iii and v among commensal gbss isolated from women at reproductive age seen at university hospital of londrina, paraná. the gbs isolates harbored at least one pilus island. our findings are in agreement with a higher proportion of capsular types and distribution of pili previously reported among gbs isolated from different countries. these data support the notion of developing of a vaccine globally effective against this opportunistic bacterium. we also detected resistance to erythromycin and clindamycin and the occurrence of the genes encoding virulence determinants cyle and hylb among these isolates, reinforcing the need for continued monitoring of gbs to prevent the development of infections. in addition, a total of different genetic groups were identified, and isolates belonging to the capsular type ii were confined to mt besides, resistance only to erythromycin was observed in gbs isolates belonging to capsular type ia and mt whereas isolates resistant to both erythromycin and clindamycin were distributed over various capsular and mlva types. higher number of isolates may corroborate these findings. 
all isolates resistant to clindamycin were simultaneously resistant to erythromycin and were distributed in the capsular types iii and v. one isolate showed the constitutive macrolide-lincosamide-streptogramin b phenotype and ten showed the inducible mlsb phenotype. all isolates harbored the gene hylb and at least one pilus variant, pi- pi-2a or pi-2b. our results indicate the potential virulence of commensal gbs isolates, reinforcing the need for continued screening for this bacterium to prevent infections. a total of multiple locus variable number of tandem repeat analysis types were identified among the isolates, seven were singletons and eight were represented by more than four isolates. the isolates displaying resistance only to erythromycin belonged to capsular type ia, and showed the m phenotype, which was mediated by the mefa/e gene. capsular types ia, ii, iii and v were identified in most gbs. this study aimed to characterize gbs isolates from women of reproductive age seen at university hospital of londrina, according to capsular typing, genetic relatedness, antimicrobial susceptibility profile and occurrence of virulence determinants. streptococcus agalactiae or group b streptococci have the ability to access various host sites, which reflects its adaptability to different environments during the course of infection. one isolate each was classified as type ix and non-typeable. this adaptation is due to the expression of virulence factors that are involved with survival, invasion and bacterial persistence in the host.
 streptococcus agalactiae or group b streptococci have the ability to access various host sites, which reflects its adaptability to different environments during the course of infection. this adaptation is due to the expression of virulence factors that are involved with survival, invasion and bacterial persistence in the host. this study aimed to characterize gbs isolates from women of reproductive age seen at university hospital of londrina, according to capsular typing, genetic relatedness, antimicrobial susceptibility profile and occurrence of virulence determinants.  a total of gbs isolates were enrolled in this study. capsular types ia, ii, iii and v were identified in most gbs. one isolate each was classified as type ix and non-typeable. a total of multiple locus variable number of tandem repeat analysis types were identified among the isolates, seven were singletons and eight were represented by more than four isolates. all isolates were susceptible to penicillin, ampicillin, cefepime, cefotaxime, chloramphenicol, levofloxacin and vancomycin. resistance to erythromycin and clindamycin was observed in and % of isolates, respectively. all isolates resistant to clindamycin were simultaneously resistant to erythromycin and were distributed in the capsular types iii and v. one isolate showed the constitutive macrolide-lincosamide-streptogramin b phenotype and ten showed the inducible mlsb phenotype. the mechanism of resistance to erythromycin and clindamycin more prevalent among these isolates was mediated by the gene erma, alone or in combination with the gene ermb. the isolates displaying resistance only to erythromycin belonged to capsular type ia, and showed the m phenotype, which was mediated by the mefa/e gene. all isolates harbored the gene hylb and at least one pilus variant, pi- pi-2a or pi-2b. although cyle was observed in all gbs, four isolates were classified as gamma-hemolytic and carotenoid pigment non-producers.  our results indicate the potential virulence of commensal gbs isolates, reinforcing the need for continued screening for this bacterium to prevent infections. the distribution of capsular and pili antigens, and mlva profiles was also identified, which may contribute to the development of new strategies for the prevention and treatment of gbs infection. group b streptococcuscapsular antigenerythromycin, clindamycin, mlva typingpilus island, virulence traits 