In vitro activity of mycobacteriophages against Mycobacterium tuberculosis biofilms

Abstract

Tolerance of Mycobacterium tuberculosis to antibiotics has been associated with its tendency to grow in cell aggregates known as biofilms. These biofilms consist of layers of cells covered by a lipid-rich extracellular polymeric substance. Biofilms favor the persistence of M. tuberculosis in the body by physically shielding the bacilli from antibiotics; providing a platform for sharing resistance genes and harboring mycolic acids, which are an important carbon source for M. tuberculosis in the ‘persister state’. It is important therefore to target biofilms when treating persistent tuberculosis. The aim of this study, therefore, was to evaluate the activity of mycobacteriophages on M. tuberculosis biofilms. A laboratory-based experimental design was used to carry out the study. Mycobacteriophages were isolated using both direct spot test and enrichment methods from soil and sewage samples collected along the Nakivubo channel in Luzira-Kampala. The phages were characterized based on their plaque morphologies, adsorption rate, burst time, and burst sizes. Mycobacteriophage host range was determined using the spot test on sixty clinical isolates obtained from both the pulmonary and extra-pulmonary niches of the body. Phage susceptible Mtb isolates were grown in 7H9 broth on MBEC plates for one and two weeks to establish active (immature) and mature biofilms respectively. The biofilms were treated with a cocktail of three phages at five multiplicities of infection for one week. The residual biofilm mass was measured as optical density (600nm) of the biofilm-associated dye following crystal violet staining. Phage anti-biofilm activity was expressed as a percentage of reduction in the biofilm mass of the no phage treated cultures. Phages reduced biofilm formation by up to 40% in active biofilms and up to 30% in mature biofilms. The phage anti-biofilm activity was directly proportional to the multiplicity of infection used. There was no significant difference in the anti-biofilm activity among pulmonary and extra-pulmonary M. tuberculosis isolates. However, pulmonary M. tuberculosis isolates were more susceptible to bacteriophage infection compared to the extra-pulmonary isolates. This study demonstrated the potential of mycobacteriophages as anti-biofilm agents. Further research is required to understand the mechanisms through which the phages inhibit and disrupt biofilm formation.