Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil

ARTICLES

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

ABSTRACT

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 population. 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

Group B streptococci (GBS) are responsible for a large variety of human infections and have been recognized 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 common 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 examine 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 (encoding 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 susceptibility 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 agarose 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 electrophoresis with a program as follows: switch time of 1-30 sec during 23 h, with a 120° angle, at a temperature of 11.3°C 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 suggested 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 different GBS genes were amplified at a temperature of 53°C. PCR-amplified products were run on agarose gels and stained with ethidium bromide. The 123-bp lambda ladder kit (Invitrogen) was used as a DNA size marker. A metabolic gene encoding the glucose 6-phosphate-isomerase-1 was employed as a positive control in all reactions. Primers were designed by the authors and prepared by Promicro (SP) as follows: 5' -GTACCTTGGTGCAAAAGCAG (forward) and 5' -GAGAAGTTTGCTGATGTAGG (reverse; gene encoding the glucose 6-phosphate-isomerase-1); 5' -CTACAATTCCAGGGAGTGCA (forward) and 5' -ACTTTCTTCCGTCCACTTAG (reverse; bca, encodes alpha protein); 5' -AAGCAACTAGAAGAGGAAGC (forward) and 5' -TTCTGCTCTGGTGTTTTAGG (reverse; bac, encodes beta protein); 5' -CCTGCTAAGACTGCTGATAC (forward) and 5' -CATAAGCATAGTCGTAAGCC (reverse; scpB, encodes C5a peptidase); 5' -CACCAATCCCCACTCTACTA (forward) and 5' -TGTGTCAAACCATCTATCAG (reverse; hylB, encodes hyaluronate lyase). One strain of Streptococcus pyogenes was used as a negative control. Two GBS strains obtained from humans, provided by Dr. L. M. Teixeira (Universidade Federal do Rio de Janeiro), were used as positive controls.

Antimicrobial susceptibility testing - Susceptibility to seven antimicrobial agents was examined by using the single-disk diffusion method and Clinical Laboratory Standards Institute guidelines (CLSI 2006).

RESULTS

After digestion with SmaI, 34 electrophoretic profiles belonging to eight clusters (A - H) were observed for the 45 strains (Fig. 1). Twenty-four of the strains belonged to the A cluster (A1-A16 profiles). Five isolates belonged to the B cluster (B1-B3 profiles), three belonged to the C cluster (C1 and C2 profiles), nine belonged to the D cluster (D1-D9 profiles) and each of the remaining four strains belonged to clusters E, F, G and H, respectively. The A1 profile was identified in a strain isolated in 1984 in Florianopolis and 18 years later in Rio de Janeiro. There is a distance of 1,150 km between these two locations. All strains were bac negative, hylB and scpB positive and 35 were bca negative. As expected, virulence genes were detected in control strains and the metabolic gene was detected in all reactions (Fig. 2). In addition, all strains were sensitive to penicillin, vancomycin, clindamycin, erythromycin and chloramphenicol. Resistance to tetracycline was seen in 39 isolates and sensitivity was observed for five strains. Forty-three GBS strains were sensitive to rifampin. Intermediate susceptibilities to tetracycline and rifampin were observed for one and two strains, respectively. PFGE and virulence profiles, antimicrobial susceptibilities and the clinical sources of the strains are shown in Table.

DISCUSSION

Our data describing the molecular epidemiology of 45 Ia GBS strains isolated in Brazil show a remarkable clustering, with 24 isolates in cluster A. It is important to note that the strains were collected over a period of 21 years. There was no specific local or time clustering, as clusters were spread over the 21-year time period and were from all sources. Other researchers found a more enhanced genetic heterogeneity in strains of serotype Ia, which were isolated during a shorter period of time. Skjaervold et al. (2004) identified 10 different electrophoretic profiles among 12 strains isolated in 1999 and 2000 in Norway. Savoia et al. (2008) identified seven clusters among 16 strains isolated in 2005 and 2006 in Italy and six of these strains were allocated to one single cluster. Martins et al. (2007), however, reported that 59 of 60 serotype Ia strains isolated between 2000-2004 in Portugal belonged to a single cluster.

There was previously no data about the genetic diversity among serotype Ia Brazilian strains. A similar study was conducted by our research group to analyse strains of type II, III and V isolated in humans in the same geographic areas and period of time (Oliveira et al. 2005). A predominant profile was clearly defined for each of these serotypes but was not identified in our Ia isolates, which instead displayed a slightly higher genetic heterogeneity than others.

The hylB and scpB genes were detected in all isolates. According to the literature, the hylB gene is uncommon in bovine GBS strains but is often present in human strains. Some authors even suggest that the presence of the scpB gene in human isolates is mandatory (Dmitriev et al. 2001, 2004, Franken et al. 2001). However, Duarte et al. (2005) detected scpB in 97% of human isolates and Dore et al. (2003) described one hylB negative human strain. Ten of our strains carried the bca gene. According to the literature, between 30-55% of serotype Ia GBS human strains carry this bca gene (Dore et al. 2003, Duarte et al. 2005).

In the present study, none of the 45 streptococci carried the bac gene. This was also observed in the Brazilian isolates studied by Duarte et al. (2005).

Variable susceptibilities of GBS to erythromycin and clindamycin (from 4-40%) are described in the literature (Hsueh et al. 2001, Uh et al. 2001, Díaz et al. 2008). Simões et al. (2007) did not detect resistance to erythromycin and clindamycin among Ia isolates collected between 2003-2004 in São Paulo. We also did not detect resistance to these two antibiotics. Duarte et al. (2005) described some resistance to erythromycin (5%) among Ia strains collected between 2000-2001 in Rio de Janeiro. In the present report, susceptibility to other antimicrobial agents is in agreement with other data in the literature (Hsueh et al. 2001, Uh et al. 2001, Simões et al. 2007).

Our data may suggest a relevant perpetuation and dissemination of cluster A throughout Brazil and may suggest the direction of further studies on this GBS serotype in geographical areas of the Southern Hemisphere, especially in developing countries.

ACKNOWLEDGMENTS

To Dr. R. R. Facklam (CDC, Atlanta, USA) and P. Ferrieri (University of Minnesota, USA), for help in the typing program.

Received 24 November 2008

Accepted 26 June 2009

Financial support: CNPq, FAPERJ, FINEP, MCT, The Thrasher Research Fund

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Publication Dates

• Publication in this collection
  24 Aug 2009
• Date of issue
  July 2009

History

• Received
  24 Nov 2008
• Accepted
  26 June 2009