In vitro treatment of Enterococcus faecalis with calcium hydroxide impairs phagocytosis by human macrophages

Abstract

Objective: Monocyte-derived macrophages (MDMs) ability to phagocytize and produce nitric oxide (NO) was tested against root-canal strains of Enterococcus faecalis submitted to alkaline stress. Root-canal strains were also compared with urine Enterococci.

Materials and Methods: Enterococcus faecalis were stressed with alkaline-BHI broth and incubated in vitro at a cell/bacteria ratio of 1:5. Phagocytosis was analyzed by fluorescence microscopy using acridine orange stain, and NO concentration was measured in supernatants.

Results and Conclusions: Alkaline-stress significantly impaired MDMs phagocytosis of E. faecalis strains analyzed, except in ATCC4083 isolated from a pulpless tooth, but NO production was unchanged. Comparison of different strains showed the urine isolate had higher NO levels than root canal strains. Alterations in the bacterial cell wall structures after alkaline-stress possibly made bacteria less recognizable and phagocytized by MDMs but did not affect their ability to activate NO production. Furthermore, root canal strains elicited different responses by immune cells compared with strains from urine. Clinically, impaired phagocytosis of E. faecalis could contribute to their persistence in root canal systems previously treated with calcium hydroxide.

Keywords:

• Calcium hydroxide
• Enterococcus faecalis
• macrophages
• phagocytosis
• nitric oxide

Acknowledgments

The authors thank the Professor José Roberto Pereira Lauris (Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, USP) for the statistical analysis. They also thank Margery Galbraith for valuable English corrections.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) [Finance Code 001] and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [Finance Code 307232/2015-8].