Study of phagocytic ability of human macrophages against different strains of Enterococcus faecalis

• Ano de defesa: 2016
• Autor(a) principal: Ponce, Jose Burgos
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Enterococcus faecalis; Fagocitose; Macrófagos; Macrophages; Phagocytosis
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/25/25150/tde-03122021-122351/

Enterococcus faecalis (E. faecalis) is an microorganism present in persistent endodontic lesions, with greater resistance than other bacteria to the calcium hydroxide, an alkaline intracanal dressing which eliminate several bacterial species during endodontic treatment. The objectives of this study were: (a) to evaluate the response of E. faecalis, isolated from root canal, under alkaline-stress, starvation, antimicrobial resistance/susceptibility and biofilm formation on dentin disks; (b) to evaluate the phagocytic ability and the nitric oxide (NO) concentration of human macrophages against root canal E. faecalis isolates submitted to alkaline stress; (c) to evaluate the intensity of TLR2 and CD14 expression on the surface of macrophages challenged with the different bacterial strains. The bacterial strains used were: ATCC 4083 (CANAL 1) and a clinical strain, obtained by us, from a primary endodontic lesion (CANAL 2), both isolated from pulpless teeth; and ATCC29212, isolated from urine (URINE), was a reference for comparison. All strains were inoculated in alkaline-BHI broth for 4, 24, 48 and 72 hours. The alkalineresistant bacteria were seeded in agar and quantified by CFU/mL. Antimicrobial susceptibility of bacterial strains, stressed or not (control) was determined by the Etest and the biovolume after biofilm formation was quantified by microscopy. To evaluate the phagocytic ability, macrophages obtained by culture of peripheral blood monocyte, were challenged with bacterial strains, stressed or not in BHI-alkaline for 30 minutes at 5:1 ratio (bacteria/macrophages) and stained with Acridine Orange. The total of macrophages with internalized bacteria and also the number of internalized bacteria per cell (<5 and 5) were counted. The NO concentration in the supernatants was measured by Griess reaction and the intensity of TLR2 and CD14 expression on the surface of macrophages was also analyzed by flow cytometry. Results shows less resistance to alkaline stress in root canal strains and less resistance to tested antibiotics when compared with urine enterococci. The lack of nutrient was a determining factor for the bacterial growth in all enterococci strains. The biovolume of biofilm formed by all strains were similar, and were not altered after exposure to an alkaline-BHI. In the presence of alkaline-stressed bacteria, there was a smaller number of macrophages with internalized bacteria, when compared to the control. The NO production or the TLR2 and CD14 expression were not altered. Regardless of the strain or alkaline environment, the number of macrophages that showed 5 internalized bacteria per cell was higher. Without an alkaline-stress the NO production results higher in the urine strain, when compared with the root canal strains, however, was not modificated after the exposure of bacteria to alkalinestress. We conclude that root-canal strains have different features when compared with urine enterococci, with the main differences being evident in their resistance/susceptibility to antibiotics; thus, we suggest that researches with aims directed to interpreting responses to endodontic treatment should be conducted with strains from root-canals. Besides, an alkaline environment associated to a starvation condition can reduce bacterial growth. Additionally, alterations in the structure of bacterial cell wall after alkali-stressing possibly made their recognition difficult, reducing their ability to be phagocytized, but not their ability to activate NO production. Therefore, intracanal medication with calcium hidroxyde dressing and coronal restorations, to prevent infiltration, should be critical in treatments of endodontics infections. However, the impact of alkaline stress, in alkaline-resistant enterococci, can impair the phagocytosis, contributing to their persistence in endodontic disease.