Characterisation and toxic activity of Aggregatibacter actinomycetemcomitans isolates

Abstract

Periodontal diseases are widely distributed in the world and represent a major oral health problem both in developed and in developing countries. These chronic inflammatory diseases are characterized by the destruction of tooth supporting tissues. It is commonly accepted that dental plaque bacteria are the primary etiologic agents of periodontal disease. More than 700 species have been detected in the oral cavity in different individuals. Approximately 400 of these species have been isolated from different subgingival microenvironments. However, only a few species have been associated with the disease. From these bacteria specifically associated with destructive disease, Aggregatibacter actinomycetemcomitans is considered as one of the bacterial species of etiological importance in periodontitis and has contributed to the initiation and/or progression of destructive forms of periodontitis. The aim of this study was to evaluate the occurance characteristics and toxic activity of A. actinomycetemcomitans in the subgingival biofilm in subjects with periodontal health and disease. Periodontal status of all included subjects was evaluated during the initial screening visit. A fullmouth clinical examination was performed in each patient using a manual probe and the following parameters were recorded at six sites per tooth: probing depth (PD in mm), clinical attachment loss (CAL in mm), bleeding on probing (BOP) and plaque index (PI). Pooled samples of subgingival plaque were taken for culture-based identification of microorganisms and further molecular analysis. Colonies suspected to be A. actinomycetemcomitans were selected for molecular identification using 16S rRNA gene sequencing. Genotyping was performed by polymerase chain reactions specific to the ltx promotor region, serotype-specific and cdt region and by sequencing of 16S rRNA. Cytotoxicity was examined on extravillous trophoblast cell line HTR-8/SVneo...

Parodontalna oboljenja su široko rasprostanjena u celom svetu i u velikoj meri narušavaju oralno zdravlje, kako u razvijenim tako i u nerazvijenim zemljama. Parodontopatije su hronična inflamatorna oboljenja potpornog aparata zuba koja usled razaranja dubljih parodontalnih tkiva mogu da rezultiraju gubitkom zuba. Etiologija parodontopatije je polimikrobna po svojoj prirodi. Opisano više od 700 različitih vrsta mikroorganizama koje naseljavaju usnu duplju, od kojih je oko 400 izolovano iz subgingivalne regije. Međutim, samo nekoliko bakterijskih vrsta je dovedeno u vezu sa parodontopatijama, među kojima je i Aggregatibacter actinomycetemcomitans. Ovaj parodontopatogen se smatra veoma važnim u etiologiji parodontopatije, odnosno da doprinosi kako inicijaciji tako i progresiji destrukcije parodontalnih tkiva. Cilj ove studije je bio da se ispita prisustvo, karakteristike i toksična aktivnost bakterije A. actinomycetemcomitans poreklom iz subgingivalnog dentalnog plaka, kod obolelih od parodontopatije kao i kod osoba sa klinički zdravim parodoncijumom. Kod svih ispitanika uključenih u studiju, evidentiran je parodontalni status i nivo oralne higijene verifikacijom kliničkih parametara: dubinom sondiranja (DS u mm), nivoom pripojnog epitla (NPE u mm), krvarenjem na provokaciju (KNP) i plak indeksom (PI). Pulovani uzorci subgingivalnog dentalnog plaka su se koristili za kultivaciju i molekularne analize bakterije A. actinomycetemcomitans. Kultivacijom dobijene kolonije su selektovane u cilju identifikacije bakterije A. actinomycetemcomitans, sekvenciranjem 16S rRNK gena. Korišćene su specifične PCR reakcije u cilju genotipizacije promotorskog operona za leukotoksin, citoletalnog toksina istezanja i serotipizaciju kao i identifikaciju bakterije A. actinomycetemcomitans umnožavanjem dela gena za 16S rRNK. Ispitivan je uticaj bakterije A. actinomycetemcomitans na inhibiciju rasta ekstravilusne trofoblastne ćelijske linije HTR-8/SVneo...