599 online | memorias.ioc.fiocruz.br Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 104(4): 599-603, July 2009 Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil Ana Beatriz de A Corrêa, Ivi Cristina M de Oliveira, Tatiana de CA Pinto, Marcos C de Mattos, Leslie C Benchetrit/ + Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Instituto de Microbiologia Professor Paulo de Góes, Av. Carlos Chagas Filho 373 Bloco I, 21941-902 Rio de Janeiro, RJ, Brasil Group B streptococci (GBS) infections occur worldwide. Although serotyping has been used for epidemiologic purposes, this does not accurately characterize enough members of a genetically heterogeneous bacterial popula- tion. The aims of this work were to evaluate the genetic diversity of 45 type Ia GBS strains isolated in Brazil by pulsed-field gel electrophoresis as well as to evaluate antimicrobial susceptibility profiles and identify virulence genes. Twenty-four strains were assigned to cluster A. All strains under study contained the hylB and scpB genes. The bca gene was detected in only 10 strains and none of the streptococci carried the bac gene. Thirty-nine strains were resistant to tetracycline. Key words: group B streptococci - pulsed-field gel electrophoresis - polymerase chain reaction Financial support: CNPq, FAPERJ, FINEP, MCT, The Thrasher Re- search Fund + Corresponding author: leslie@micro.ufrj.br Received 24 November 2008 Accepted 26 June 2009 Group B streptococci (GBS) are responsible for a large variety of human infections and have been rec- ognized over the last few decades as a leading cause of perinatal disease worldwide (Farley 2001, Weisner et al. 2004). GBS are classified in serotypes (Ia, Ib and II- VIII) that occur in combination with different protein antigens (alpha, beta and rib) (Dmitriev et al. 2001). Currently, serotypes Ia, III and V are the most com- mon in many countries (Duarte et al. 2005, Fluegge et al. 2005, Bergseng et al. 2008, Poyart et al. 2008, von Both et al. 2008). Pulsed-field gel electrophoresis (PFGE), used to ex- amine GBS strains, is a powerful technique employed for the classification of microorganisms after digestion of the genomic DNA by restriction enzymes (Benson & Ferrieri 2001, Oliveira et al. 2005, 2006, von Both et al. 2008). Using polymerase chain reaction (PCR), Franken et al. (2001) suggested that the scpB and lmb genes (en- coding c5a peptidase and laminin binding protein) must exist in GBS strains that infect humans. The hylB gene is frequently detected in these strains (Dore et al. 2003). The bac and bca genes are present in 23% and 100% of type Ia strains, respectively (Maeland et al. 1997, 2000). The lack of information on Ia Brazilian strains and the availability of DNA techniques led us to investigate the genetic make-up of type Ia GBS strains isolated in distinct regions of Brazil. Additionally, antimicrobial susceptibil- ity was examined to better characterize these isolates. MATERIALS AND METHODS Bacterial strains - Forty-five human type Ia GBS strains derived from clinical specimens were obtained in Florianopolis, Santa Catarina (n = 3), a city located in the Southern Region of Brazil, in São Paulo, São Paulo (SP) (n = 1), and in Rio de Janeiro, Rio de Janeiro (n = 40), in the Southeast Region of the country. The source of one strain was unknown. Isolates were obtained from 1981-2002 from public health laboratories, gynaecological clinics, hospitalsand universities. The clinical sources included urine (n = 16), oropharynx (n = 9), vagina (n = 5), anus (n = 2), lung (n = 2), placenta (n = 1), external ear canal (n = 1) and perineum (n = 1). The sources of eight specimens were unknown. A confirmatory identification of serotypes was carried out again by immunoprecipitation in aga- rose using sera produced in the research facilities of the authors and HCl antigenic extracts from the streptococci (Benchetrit et al. 1982). PFGE - PFGE was performed as previously described (Oliveira et al. 2005). DNA was digested with the SmaI restriction enzyme (Amersham) and submitted to elec- trophoresis with a program as follows: switch time of 1-30 sec during 23 h, with a 120º angle, at a temperature of 11.3ºC and a voltage gradient of 6 V/cm. The lambda ladder PFGE marker kit (New England Biolabs) was used as a DNA size marker. Gels were stained with ethidium bromide and photographed under UV light. Criteria for analysis of the PFGE patterns were those originally sug- gested by Tenover et al. (1995) and used in our previous studies (Oliveira et al. 2005, 2006). PCR - DNA extraction was performed according to Sambrook et al. (1989). DNA fragments of the dif- ferent GBS genes were amplified at a temperature of 53ºC. PCR-amplified products were run on agarose gels