Toward a Phage Cocktail for Tuberculosis: Susceptibility and Tuberculocidal Action of Mycobacteriophages against Diverse Mycobacterium tuberculosis Strains

Abstract

The global health burden of human tuberculosis (TB) and the widespread antibiotic resistance of its causative agent Mycobacterium tuberculosis warrant new strategies for TB control. The successful use of a bacteriophage cocktail to treat a Mycobacterium abscessus infection suggests that phages could play a role in tuberculosis therapy. To assemble a phage cocktail with optimal therapeutic potential for tuberculosis, we have explored mycobacteriophage diversity to identify phages that demonstrate tuberculocidal activity and determined the phage infection profiles for a diverse set of strains spanning the major lineages of human-adapted strains of the Mycobacterium tuberculosis complex. Using a combination of genome engineering and bacteriophage genetics, we have assembled a five-phage cocktail that minimizes the emergence of phage resistance and cross-resistance to multiple phages, and which efficiently kills the M. tuberculosis strains tested. Furthermore, these phages function without antagonizing antibiotic effectiveness, and infect both isoniazid-resistant and -sensitive strains.IMPORTANCE Tuberculosis kills 1.5 million people each year, and resistance to commonly used antibiotics contributes to treatment failures. The therapeutic potential of bacteriophages against Mycobacterium tuberculosis offers prospects for shortening antibiotic regimens, provides new tools for treating multiple drug-resistant (MDR)-TB and extensively drug-resistant (XDR)-TB infections, and protects newly developed antibiotics against rapidly emerging resistance to them. Identifying a suitable suite of phages active against diverse M. tuberculosis isolates circumvents many of the barriers to initiating clinical evaluation of phages as part of the arsenal of antituberculosis therapeutics.

Keywords: Mycobacterium tuberculosis; bacteriophage therapy; bacteriophages; tuberculosis.

FIG 1

Phage susceptibility of M. tuberculosis H37Rv. Phage lysates, shown on the left, were 10-fold serially diluted and 3 μl of the 10⁻¹ to 10⁻⁸ dilutions were spotted onto top agar overlays containing M. smegmatis mc²155 or M. tuberculosis H37Rv. Phage cluster/subcluster designation are shown on the right.

FIG 2

Phage infection of strains from different M. tuberculosis lineages. Phage lysates, as indicated on the left, were spotted onto lawns of M. smegmatis mc²155, M. tuberculosis H37Rv, and six M. tuberculosis clinical isolates. The lineage (i.e., L1, L2, L3, or L4) of each M. tuberculosis strain is shown in parentheses. A summary of phage infections of a larger panel of strains is shown in Table 3.

FIG 3

Expanded host range mutants of phage Muddy. (A) A map of the Muddy genome shows genes as colored boxes above a genome marker. The direction of transcription (horizontal arrows) and locations of head, tail, and lysis genes are indicated; DNA polymerase (Pol) and RecA (Rec) genes are also shown. Below is an expanded view of tail gene 24 showing predicted secondary structure motifs (α, alpha helix; β, beta sheets; CC, coiled coil). The positions of amino acid substitutions conferring an expanded host range phenotype are shown above gene 24. (B) Lysates of WT Muddy and host range mutant derivatives (as shown) were serially diluted and spotted onto lawns of mycobacterial strains as indicated. The lineage of each M. tuberculosis strain is shown in parentheses.

FIG 4

Phage resistance of M. tuberculosis strains. (A) Approximately 10⁷ CFU of each M. tuberculosis strain (as indicated above with lineage shown in parentheses) was challenged with 10⁷ to 10⁸ PFU of phage in liquid medium for 1 week and plated onto solid medium. Plates were incubated for 4 weeks. (B) Engineering of Fred313_cpm. On the left is shown a map of part of the Fred313_cpm genome with genes shown as colored boxes with the gene name within each box. Genes shown above and below the genome rule are transcribed rightward and leftward, respectively. The position of the BRED substrate is indicated, and below is the structure of the Fred313_cpmΔ33 mutant in which the integrase gene has been removed. On the right is shown (top) PCR amplification of primary plaques recovered from BRED, all of which contain the wild-type allele (wt) and one also containing the mutant (mut) corresponding to the predicted size. After replating the indicated plaque for purification, secondary plaques were screened by PCR (bottom), one of which (asterisk) is homogenous for the desired mutation. The complete genome was sequenced to confirm the desired construction.

FIG 5

Cross resistance of phage-resistant mutants. Phage-resistant mutants CG20, CG21, CG22, CG23, CG24, and CG25 were purified and plated onto agar lawns. (A) Cross resistance was assessed by spotting phage dilutions onto strains CG20 and CG21 as shown in Fig. 1 and 2. (B) Cross resistance to other phages was determined by spotting 5 μl of single 10⁻¹ dilutions (∼5 × 10⁶ to 5 × 10⁷ PFU) onto agar lawns of resistant mutants CG22, CG23, CG23, and CG25. A numbered coordinate grid (right) indicates which phage was plated as follows: 1, Fred313_cpmΔ33; 2, FionnbharthΔ45Δ47; 3, AdephagiaΔ41Δ43; 4, Isca_cpm; 5, Muddy HRM⁰⁰⁵²-1; 6, ZoeJΔ45; 7, DS6A; 8, D29. (C) Tabulated summary of cross-resistance observed for all resistance mutants; S, sensitive; R, resistant.

FIG 6

Killing efficiencies of individual phages and the five-phage cocktail for M. tuberculosis lineages. (A) A 10-fold dilution series of each of five M. tuberculosis strains (with lineages shown in parentheses) were prepared with the least dilute on the left at ∼10⁷ CFU total and incubated in liquid medium for 7 days with phages (as indicated on left) each at a total of 10⁷ PFU. Aliquots of 3 μl (∼3 × 10⁴ CFU at 10⁻¹ dilution) were then plated onto solid medium and incubated for 4 weeks at 37°C. (B) Dilutions of M. tuberculosis strains were prepared as in panel A and incubated in liquid culture with a five-phage cocktail containing equal amounts of AdephagiaΔ41Δ43, Fred313_cpmΔ33, FionnbharthΔ45Δ47, Muddy_HRM^N0157-1 (gp24 G487W), and D29. The top rows contain a total of 10⁷ PFU, and below are shown 10-fold serial dilutions of the phage input.

FIG 7

Phage and antibiotic interactions. (A) Controls of input M. tuberculosis H37Rv in the experiment. The left and right panels show plating of 100 μl of an undiluted culture of M. tuberculosis H37Rv and a 10⁻⁵ dilution, respectively. (B) Aliquots (100 μl) of an undiluted culture of M. tuberculosis H37Rv were plated directly onto solid medium containing either rifampin or isoniazid at the final concentrations indicated, or onto plates on which 10⁹ PFU of Fionnbharth had been added and spread over the agar surface. Plates were incubated for 4 weeks.

FIG 8

Phage infection of M. tuberculosis mc²4977. Ten-fold serial dilutions of phages as shown on the left were spotted onto lawns of M. smegmatis mc²155, M. tuberculosis H37Rv, and M. tuberculosis mc²4977, which is isoniazid resistant due to deletion of the katG gene.

FIG 9

Fionnbharth resistance escape mutants. (A) Alignment of the tail gene segments of Adephagia, ZoeJ, and Fionnbharth (subclusters K1, K2, and K4, respectively) genomes shows the location of Fionnbharth gene 26, coding for a putative phage tail protein. Genes are shown as colored boxes with gene numbers within the boxes, with coloring reflecting similar phamilies of protein sequences. Spectrum-colored shading between the genomes reflects nucleotide sequence similarity, with violet being the most similar, and red the least similar above a threshold E value of 10⁻⁴ (64). (B) An expanded view of Fionnbharth gene 26 showing the locations of two mutations conferring substitutions (G93R and G93D) in the resistance escape mutants REM-1 and REM-2, respectively. (C) Phage infections of Fionnbharth and CG-REM-1 and CG-REM2 mutants on lawns of M. smegmatis mc²155, CG20, and CG21.