Phenotypical and genotypical characterization of Bordetella pertussis strains isolated in São Paulo, Brazil, 1988-2002

Caracterização fenotípica e genética de cepas de Bordetella pertussis isoladas em São Paulo, Brasil, 1988-2002

BRIEF COMMUNICATION

Phenotypical and genotypical characterization of Bordetella pertussis strains isolated in São Paulo, Brazil, 1988-2002

Caracterização fenotípica e genética de cepas de Bordetella pertussis isoladas em São Paulo, Brasil, 1988-2002

Célia R. Gonçalves^I; Tânia M. I. Vaz^I; Marta I. C. Medeiros^II; Maria T. F. Castro^III; Marilu M. M. Rocha^IV; Carmo E. A. Melles^I; Kinue Irinoi

^IInstituto Adolfo Lutz, São Paulo, SP, Brasil

^IIInstituto Adolfo Lutz de Ribeirão Preto, SP, Brasil

^IIIInstituto Adolfo Lutz de Santos, SP, Brasil

^IVInstituto Adolfo Lutz de Campinas, SP, Brasil

^{Correspondence to}

SUMMARY

Whooping cough or pertussis was a major cause of childhood morbidity and mortality in the world until the introduction of a whole-cell vaccine in the 1940's. However, since the early 1980's whooping cough cases have increased in many countries, becoming an important problem of public health. This increase may be due to accuracy of laboratory diagnosis and reporting of the disease, a decline in immunity over time, demographic changes, and adaptation of the bacterial population to vaccine-induced immunity. The purpose of this study was to analyze phenotypically and genotypically a collection of 67 Bordetella pertussis isolates recovered during the period 1988-2002 in São Paulo State, Brazil to determine their characteristics and relatedness. All isolates were submitted to susceptibility testing to erythromycin, serotyping, and 56 isolates were analyzed by Pulsed Field Gel Electrophoresis (PFGE). All isolates were susceptible to erythromycin and the majority of them belonged to serotype 1,3. The 56 isolates were classified into 11 PFGE profiles according to the differences in banding patterns. Although more than 60% of the isolates were recovered from patients aged less than three months, almost 15% of them were isolated from adolescents/adults evidencing the increase in the incidence of pertussis among this group of age.

Keywords:Bordetella pertussis; Whooping cough; Serotypes, Genetic profiles.

RESUMO

A coqueluche ou pertussis foi a maior causa de morbidade e mortalidade infantil em todo o mundo até a introdução de uma vacina na década de 1940. Entretanto, desde a década de 1980, a coqueluche tornou-se, em muitos países , um importante problema de saúde pública. Este acontecimento pode ser atribuído à melhoria do diagnóstico laboratorial e da notificação da doença, declínio da imunidade no decorrer do tempo, mudanças demográficas ou adaptação da população bacteriana à imunidade induzida pela vacina. O objetivo deste estudo foi analisar as características fenotípicas e genotípicas de uma coleção de 67 cepas de Bordetella pertussis isoladas no período 1988-2002 em São Paulo, Brasil. Todas as cepas foram submetidas à determinação do perfil de resistência à eritromicina, à sorotipagem e 56 cepas à eletroforese em campo pulsado (PFGE). Todas as cepas foram sensíveis à eritromicina e a maioria delas pertencia ao sorotipo 1,3. As 56 cepas foram classificadas em 11 perfis de PFGE com base nas diferenças no padrão de bandas. Embora mais de 60% das cepas tenham sido isoladas de crianças com menos de três meses de idade, cerca de 15% delas era de adolescentes/adultos evidenciando um aumento da incidência da coqueluche nesse grupo etário.

INTRODUCTION

Although a significant decline in the incidence of whooping cough was observed following the widespread use of pertussis vaccine in the mid-1940s³, the incidence of the disease has increased since the early 1980s in many countries even with high vaccination coverage^3,5. Data concerning the prevalence of pertussis in Brazil have been published since late 1970's but more recently report shows that pertussis cases were substantially reduced after the public use of pertussis vaccine since the 1980´s^2,6,11,12. In Brazil, pertussis became a reportable disease only in 2001¹ making laboratory methods for pertussis diagnosis be implanted and or improved in all State health laboratories in the country. After 2001, laboratory data show that pertussis cases may be underestimated and that Bordetella pertussis strains are still circulating in São Paulo State.

Characterization of B. pertussis is important to understand the dynamic of the disease, to determine the relatedness of the isolates, and also to provide information related to the appropriated vaccine composition to be used in each region. In this study, we analyzed a collection of 67 B. pertussis isolated during the period of 1988-2002 in São Paulo State. Suspected colonies of B. pertussis on Reagan-Lowe medium (RL) were submitted to Gram staining, biochemical tests and nutritional requirements assays following the standard methods¹⁰. Susceptibility of all isolates to erythromycin was determined by disk diffusion testing⁵. For serotyping specific antisera against antigen 1, 2 and 3 were prepared at Instituto Adolfo Lutz, using reference strains and following the methods previously described⁸. Molecular typing was performed using Pulsed Field Gel Electrophoresis (PFGE) as described by GAUTOM⁴.

All B. pertussis isolates were susceptible to erythromycin. The majority of the isolates belonged to serotypes 1,3 (86%) whereas strains belonging to serotypes 1, and 1,2 accounted for 10% and 4%, respectively. PFGE using XbaI generated eigth to twelve bands ranging from approximately 90 to 700 kb. Although few differences were detected in banding patterns, it was possible to discriminate the 56 isolates into 11 genetic profiles (1-11); however, no genetic profile was associated with a particular geographic area, age of patients or serotypes. Strains belonging to profile 8 were the most prevalent.

Table 1

Four strains isolated in the city of Santos, exhibited the same banding pattern, and three of them were recovered from three infants between January-February 1989, suggesting the occurrence of a probable pertussis outbreak in that period. In another city, Ribeirão Preto, a genetically similar profile was also seen among strains isolated from one infant (strain 24/02) and his parents (strains 25/02 and 26/02) suggesting them as the possible source of infection.

Our data show that although more than 60% of the 67 isolates were recovered from patients aged < 3 months, almost 15% were isolated from patients > 12 years of age. This shows the important role of adolescents and adults as reservoir for infections in very young infants^5,7. Laboratory data are important for improving surveillance programs and for accurately estimate pertussis cases, helping to set priorities for immunization strategies to control this re-emerging disease⁵.

ACKNOWLEDGMENTS

We thank the Division of Respiratory Diseases - Epidemiological Surveillance Center "Professor Alexandre Vranjac", Secretary of Health, São Paulo State, Brazil for the assistance during the implementation of laboratory methods for pertussis diagnosis.

Received: 9 January 2006

Accepted: 31 August 2006

• 1. BRASIL. Ministério da Saúde - Portaria Número 1943, de 18 de outubro de 2001. Define a relação de doenças de notificação compulsória para todo o território nacional. Diário Oficial da União, Poder Executivo, 24 de outubro de 2001.
• ²
  BRASIL. Ministério da Saúde - Situação epidemiológica da coqueluche no Brasil. Uma breve descrição. Relatório de Maria da Glória Vicente. Brasília, outubro de 2003.
• 3. CHERRY, J.D. - Pertussis in the preantibiotic and prevaccine era, with emphasis on adult pertussis. Clin. infect. Dis., 28(suppl. 2): S107-S111, 1999.
• 4. GAUTOM, R.K. - Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other Gram-negative organisms in one day. J. clin. Microbiol., 35: 2977-2980, 1997.
• 5. GUIDELINES FOR THE CONTROL OF PERTUSSIS OUTBREAKS. Atlanta, Centers for Disease Control and Prevention, 2000.
• 6. IARIA, S.T. - Isolamento de bactérias do gênero Bordetella e provas sorológicas, a partir de crianças com sintomas de coqueluche, atendidas no Hospital de Isolamento Emílio Ribas de São Paulo.Rev. Saúde públ. (S. Paulo), 7: 409-432, 1973.
• 7. MOOI, F.R.; HALLANDER, H., WIRSING von KONIG, C.H.; HOET, B. & GUISO, N. - Epidemiological typing of Bordetella pertussis isolates: recommendations for a standard methodology. Europ. J. clin. Microbiol. infect. Dis., 19: 174-181, 2000.
• 8. PRESTON, N.W. - Technical problems in the laboratory diagnosis and prevention of whooping-cough. Lab. Pract., 19: 482-486, 1970.
• 9. REGAN, J. & LOWE, F. - Enrichment medium for the isolation of Bordetella J. clin. Microbiol., 6: 303-309, 1977.
• 10. SANDEN, N. & wEYANT, r.s. - Genus III. Bordetella. In: BRENNER, D.J.; KRIEG, N.R. & STALEY, J.T., ed. Bergey´s manual of systematic Bacteriology. New York, Springer, 2005. v. 2.
• 11. UBATUBA, A. - Bacteriologia da coqueluche. Identificação das Bordetellas isoladas em crianças no Rio de Janeiro. Hospital (Rio de J.), 75: 97-107, 1969.
• 12. UBATUBA, A. & SALMITO, A.T. - Bacteriologia da coqueluche. II. Determinação dos fatores aglutinantes "major" nas amostras de Bordetella pertussis isoladas no Rio de Janeiro. Hospital (Rio de J.), 78: 1301-1310, 1970.

Publication Dates

• Publication in this collection
  03 May 2007
• Date of issue
  Apr 2007

History

• Accepted
  31 Aug 2006
• Received
  09 Jan 2006