Phage cocktail amikacin combination as a potential therapy for bacteremia associated with carbapenemase producing colistin resistant Klebsiella pneumoniae

Abstract

The increasing occurrence of hospital-associated infections, particularly bacteremia, caused by extensively drug-resistant (XDR) carbapenemase-producing colistin-resistant Klebsiella pneumoniae highlights a critical requirement to discover new therapeutic alternatives. Bacteriophages having host-specific bacteriolytic effects are promising alternatives for combating these pathogens. Among 12 phages isolated from public wastewater in Thailand, two phages-vB_kpnM_05 (myovirus) and vB_kpnP_08 (podovirus) showed broad-host range, producing bacteriolytic activities against 81.3% (n = 26) and 78.1% (n = 25) of 32 XDR carbapenemase-producing colistin-resistant K. pneumoniae, with capsular types-K15, K17, K50, K51, K52/wzi-50 and K2/wzi-2. Both phages showed short replication times, large burst sizes with rapid adsorptions. They exhibited significant stability under various environmental conditions. Genomic analysis revealed that both phages are genetically distinct phages from Myoviridae and Podoviridae family, with the lack of toxin, virulence, lysogeny and antibiotic resistance genes. These characteristics highlighted their promising potential for utilizing in phage therapy for combating XDR K. pneumoniae. Although phage cocktail combining vB_kpnM_05 and vB_kpnP_08 provided significant bacteriolysis for longer duration (8 h) than its monophage (6 h), bacterial regrowth was observed which suggested an evitable development of phage resistance under phages' selection pressures. Future study will be undertaken to elucidate the precise mechanisms by which these XDR K. pneumoniae developed phage resistance and their associated fitness cost. Remarkably, combining phage cocktail with amikacin at their sub-inhibitory concentrations produced potent synergy by completely suppressing bacterial regrowth in vitro. Our study demonstrated the significant therapeutic and prophylactic effectiveness of a phage cocktail-amikacin combination as a promising alternative strategy for overcoming bacteremia associated with XDR K. pneumoniae having carbapenemase and colistin resistance in vivo.

Keywords: Klebsiella pneumoniae; Bacteremia; Carbapenemase; Colistin resistance; Extensively drug-resistant; Phage cocktail; Phage cocktail-antibiotic combination.

Fig. 1

Heatmap illustrating the host range of isolated phages (n = 12) by determination of phages bacteriolytic activities against XDR carbapenemase-producing colistin-resistant K. pneumoniae clinical isolates (n = 32) collected between 2016 and 2021. Blue: Isolates with high sensitivity to phage’s bacteriolytic activities; white: Isolates with low sensitivity to phage’s bacteriolytic activities.

Fig. 2

Morphological, and biological characteristics of phage. Plaque morphologies and electron micrographs of (a) vB_kpnM_05 and (b) vB_kpnP_08; (c) optimal MOIs of vB_kpnM_05 and vB_kpnP_08; (d) adsorption of vB_kpnM_05 and vB_kpnP_08, (e) one-step growth curve of vB_kpnM_05, and (f) one-step growth curve of vB_kpnP_08.

Fig. 3

Stability of phages under different environmental conditions. (a) Thermostability of vB_kpnM_05 and vB_kpnP_08; (b) stability of vB_kpnM_05 and vB_kpnP_08 under different pH, (c) stability of vB_kpnM_05 and vB_kpnP_08 under UV exposure; (d) stability of vB_kpnM_05 and vB_kpnP_08 for long-term storage; (e) stability of vB_kpnM_05 and vB_kpnP_08 under chloroform and ethanol exposure.

Fig. 4

(a) Genomic structure of vB_kpnM_05, (b) genomic structure of vB_kpnP_08; (c) phylogenetic tree of vB_kpnM_05, (d) phylogenetic tree of vB_kpnP_08.

Fig. 5

(a) Bacteriolytic activities of vB_kpnM_05 as monophage therapy at different MOIs (0.01–100); (b) bacteriolytic activities of vB_kpnP_08 as monophage therapy at different MOIs (0.01–100): (c) bacteriolytic activities of vB_kpnM_05 and vB_kpnP_08 as monophage and combined phage cocktail at MOI-10 in vitro.

Fig. 6

(a) Checkerboard screening assay showing the synergistic activities of a phage cocktail-amikacin combination (FICI < 0.5); Time-kill confirmation assay showing the synergistic activity of a phage cocktail-amikacin combination for reducing the (b) bacterial growth (OD600nm), and (c) bacterial viability (CFU/ml) in vitro.

Fig. 7

Therapeutic effectiveness of phage cocktail and amikacin as monotherapy and combination therapy in bacteremia by determination of (a) bacterial burden in internal organs (blood, kidney, liver, and spleen), (b) survival of treated mice, and (c) viable titre of phage in internal organs (blood, kidney, liver, and spleen) of mice receiving different treatments in vivo.

Fig. 8

Prophylactic effectiveness of phage cocktail and amikacin as monotherapy and combination therapy in bacteremia by determination of (a) bacterial burden in internal organs (blood, kidney, liver, and spleen), (b) survival of treated mice, and (c) viable titre of phage in organs (blood, kidney, liver, and spleen) of mice receiving different treatments in vivo.