Comparative study on the virulence of mycobacteriophages

Abstract

The global tuberculosis (TB) epidemic affected 10 million people and caused 1.3 million deaths in 2022 alone. Multidrug-resistant TB is successfully treated in less than 60% of cases by long, expensive and aggressive treatments. Mycobacteriophages, viruses that can infect bacteria such as Mycobacterium tuberculosis-the species responsible for TB, have the potential to redefine TB prevention and treatments. However, the development of phage-based products necessitates the assessment of numerous parameters, including virulence and adsorption, to ensure their performance and quality. In this work, we characterized the virulence of three different mycobacteriophages (Fionnbharth, Muddy and D29), alone and as cocktails, against a TB model host (Mycobacterium smegmatis) under planktonic and early-stage biofilm growth conditions. Phage D29 and cocktails containing D29 had the highest virulence under all conditions. Interestingly, phages Fionnbharth and Muddy and their combination showed higher virulence against early-stage biofilm than against the planktonic phenotype. Adsorption assays indicated that all three phages had lower adsorption efficiencies on the early-stage biofilm phenotype than on the planktonic one, suggesting a reduced availability of receptors in the former. Given that, despite these lower adsorption efficiencies, the virulence of the phages and phage cocktails was either unchanged or higher against the early-stage biofilm, this phenotype must display properties that are favorable to other steps of the infection process. These results inform us on the dynamics of mycobacteriophage infections, alone and in cocktail formulations, under different host growth conditions, and serve as a basis for the development of phage products targeting mycobacteria biofilms.

Keywords: Bacteriophages; D29; Fionnbharth; Muddy; Mycobacterium smegmatis; infection dynamics; mycobacteriophages; phage adsorption; phages; virulence; virulence index.

Figure 1.. Schematic representation of the virulence and adsorption assays under different growth and assay conditions.

Virulence (A): A planktonic M. smegmatis culture was transferred to 96-well plates to establish the planktonic (shaking growth condition) and early-stage biofilm (static growth condition) host cultures. Phages were added to perform the adapted virulence index protocol for each phenotype. Adsorption (B): Planktonic cultures (shaking growth condition) were used as the host for the adsorption assay performed under shaking and static conditions. Early-stage biofilm cultures (static growth) were used as host for the adsorption assay under shaking conditions.

Figure 2.. Comparison of the virulence of mycobacteriophages and their cocktail formulations.

Spider diagram depicting the virulence index values of phages Fionnbharth, Muddy, D29 and their combinations infecting planktonic (A) and early-stage biofilm (B) M. smegmatis. The virulence index scale increases from a minimum of 0 (central point of the chart) to a maximum of 1 (outermost circle). Each axis corresponds to a phage (labelled on the outside of the chart) or their combinations (interposed axes). The virulence index values of the different combinations are indicated by dots in the spider diagram (orange: individual phage; green: two-phage cocktails; red: three-phage cocktail) and listed in the insert tables (average ± standard deviation, n ≥ 3).

Figure 3.. Virulence in planktonic and early-stage biofilm M. smegmatis.

Virulence curves for phages Fionnbharth (A), Muddy (B), D29 (C), the two-phage cocktails (Fionnbharth + Muddy (D), Fionnbharth + D29 (E), Muddy + D29 (F)), and the three-phage cocktail (Fionnbharth + Muddy + D29 (G)) infecting M. smegmatis in planktonic (filled circle) and early-stage biofilm (empty circle) growth conditions. The results are compiled for planktonic (H) and early-stage biofilm (I) for comparison purposes. Values are reported as average ± standard deviation (n ≥ 3).

Figure 4.. Adsorption to M. smegmatis.

The free phage concentration P relative to the initial phage concentration P₀ of Fionnbharth (A), Muddy (B), and D29 (C) adsorbing to planktonic or early-stage biofilm M. smegmatis. Adsorption to the planktonic phenotype was measured under shaking (filled circle) and static (empty circle) conditions; adsorption to early-stage biofilm phenotype was measured under shaking conditions (filled diamonds). All data is shown as average ± standard deviation, n ≥ 3).