Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis / Distribuição de biótipos e atividade leucotóxica de Aggregatibacter actinomycetemcomitans isolados de pacientes brasileiros com periodontite crônica

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18 percent) of the periodontal patients and one (2 percent) healthy subject. However, by PCR, this organism was detected in 44 percent of the periodontal patients and in 16 percent of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases

Aggregatibacter actinomycetemcomitans é um importante agente etiológico da periodontite e produz infecções extra-bucais. Neste estudo, foram detectados os biótipos, o gene ltxA associado à produção de leucotoxina e o promotor ltx em A. actinomycetemcomitans de pacientes com e sem periodontite. A atividade leucotóxica sobre células HL-60 também foi avaliada. A atividade leucotóxica foi determinada através do método de exclusão do azul de tripam. A deleção de 530 bp no promotor ltx foi avaliada usando-se o par de iniciadores PRO. A. actinomycetemcomitans foi detectado por cultura e por PCR. Por cultura, A. actinomycetemcomitans foi detectado em nove pacientes com periodontite (18 por cento) e em um indivíduo sadio (2 por cento). Por PCR esse microrganismo foi detectado em 44 por cento dos pacientes com periodontite e em 16 por cento dos saudáveis. Verificou-se diferença estatística entre a detecção do promotor do operon ltx, por PCR, diretamente do biofilme subgengival e do DNA bacteriano. Somente uma amostra clínica apresentou A. actinomycetemcomitans altamente leukotóxico. O biótipo II foi o mais prevalente e não foi observada correlação biótipo-atividade leucotóxica. A expressão da leucotoxina por A. actinomycetemcomitans na doença periodontal e outras doenças infecciosas necessita ser avaliado.

Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis.

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases.