Enhanced Antibacterial Activity of Repurposed Mitomycin C and Imipenem in Combination with the Lytic Phage vB_KpnM-VAC13 against Clinical Isolates of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harboring OXA-245 β-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by optical density at 600 nm (OD₆₀₀) measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an in vivo infection model (Galleria mellonella). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both in vitro and in vivo. The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harboring OXA-245 β-lactamase. Interestingly, the combinations decreased the emergence of in vitro resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage-mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harboring OXA-245 β-lactamase.

Keywords: Klebsiella pneumoniae; bacteriophage therapy; drug repurposing; persistence; resistance; synergy.

FIG 1

Optical density of K2534 (a) and K3325 (b) and number of CFU per milliliter of K2534 (c) and K3325 (d) in the presence of the lytic phage alone at an MOI of 10 (black), mitomycin C alone (dark red), and the combination of both (dark gray). Light gray symbols/lines represent the control in the absence of any drug. *, P < 0.05; **, P < 0.01. Absence of asterisk indicates no statistically significant difference.

FIG 2

Optical density of K2534 (a) and K3325 (b) and number of CFU per milliliter of K2534 (c) and K3325 (d) in the presence of the lytic phage alone at an MOI of 10 (black), imipenem alone (dark red), and the combination of both (dark gray). Light gray represents the control in the absence of any drug. *, P < 0.05; **, P < 0.01. Absence of asterisk indicates no statistically significant difference.

FIG 3

Frequency of mutants of K2534 (a and c) and K3325 (b and d) resistant to the lytic phage vB_KpnM-VAC13 or the tested drugs (mitomycin C [MitC] and imipenem [IMP]), alone or in combination with the phage over 24 h. *, P < 0.05; **, P < 0.01. Absence of asterisk indicates the result was not statistically significant.

FIG 4

Percentage of survival of G. mellonella larvae infected with K2534 or K3325. For each group, 15 larvae were included. *, P < 0.05; **, P < 0.01. Absence of asterisk indicates no statistically significant difference.