Bacteriophage therapy as an alternative strategy against carbapenem-resistant Klebsiella pneumoniae

A hospital in Pietermaritzburg reported a cluster of ventilator-associated pneumonia cases caused by carbapenem-resistant Klebsiella pneumoniae (CR-Kp) that failed standard antibiotic salvage therapy—prompting the search for non-antibiotic alternatives. This research isolated and characterized five lytic bacteriophages (φKp-01 through φKp-05) from municipal wastewater and assessed their efficacy against 30 clinical CR-Kp isolates in vitro. Work was conducted at the uMgeni University of Biological Sciences between August 2022 and December 2023. Phage φKp-03 demonstrated the broadest host range (infecting 26 of 30 isolates, 86.7%) and the highest efficiency of plating (EOP = 0.94 on the reference resistant strain). Time-kill assays showed a 4.3-log reduction in viable CR-Kp counts within 4 hours at a multiplicity of infection (MOI) of 10. φKp-03 remained stable across pH 4-10 and at temperatures up to 55 °C. Biofilm penetration assays revealed a 71.8% reduction in established biofilm biomass at 24 hours. Combining φKp-03 with sub-inhibitory meropenem (2 µg/mL) produced a synergistic 5.7-log reduction, significantly exceeding phage alone (p

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Phage-Antibiotic Synergy MechanismClose ×

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Log₁₀ Reduction: Untreated control vs Meropenem 2 µg/mL alone vs φKp-03 MOI 10 alone vs φKp-03 + MeropenemClose ×

Replotted from Table 2 (page 2) of the original paper. Every bar is the paper's own table value.

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Introduction

In 2017 a woman in Nevada died from a Klebsiella pneumoniae infection resistant to all 26 antibiotics available in the United States-a case that became a stark example of the postantibiotic era's arrival [1] . Carbapenem-resistant Klebsiella pneumoniae is now classified as a critical-priority pathogen by the World Health Organization, and treatment options shrink further as colistin resistance spreads through mobile genetic elements [2] . Against this backdrop, bacteriophages-viruses that specifically infect and lyse bacteria-have reemerged as a therapeutic option after decades of neglect in Western medicine. Phage therapy offers several advantages: narrow host range limits collateral damage to commensal flora, self-amplification at the infection site reduces dosing frequency, and bacterial resistance to phages often involves surface receptor modifications that restore antibiotic susceptibility [3] . Clinical case reports and compassionate-use programmes have documented successful phage treatment of multidrug-resistant infections in lungs, bones, and prosthetic joints [4] . Yet systematic characterization of phage candidates against local CR-Kp strains remains limited, particularly in sub-Saharan African clinical settings. This research isolated lytic phages from Pietermaritzburg municipal wastewater, characterized their host range, stability, genomics, and biofilm-penetration capacity, and tested phage-antibiotic synergy against clinical CR-Kp isolates recovered from the uMgeni University of Biological Sciences referral network.

Theoretical Background

Lytic phages follow a strictly virulent replication cycle: they adsorb to specific bacterial surface receptors (typically lipopolysaccharide or outer membrane proteins in Gramnegatives), inject genomic DNA, hijack the host's transcriptional and translational machinery, assemble progeny virions, and lyse the host cell to release 50-200 new particles per burst [5] . This cycle takes 20-40 minutes for most Klebsiella phages under optimal conditions. Phage-antibiotic synergy (PAS) occurs when sub-inhibitory antibiotic concentrations stress bacteria-causing filamentation or membrane destabilization-thereby increasing phage adsorption or accelerating lysis [6] . PAS has been demonstrated for βlactams, fluoroquinolones, and aminoglycosides paired with various phages, though the mechanism appears compoundspecific.

Material and Methods

Material and Methods

Material

Thirty clinical CR-Kp isolates (carbapenem MIC ≥ 16 µg/mL by broth microdilution) were obtained from the culture collection at the Department of Medical Microbiology, uMgeni University of Biological Sciences. Isolates had been recovered from blood (n = 12), respiratory specimens (n = 10), and urine (n = 8) between 2020 and 2022. Wastewater samples (10 L) for phage isolation were collected from the Msunduzi River treatment works, Pietermaritzburg, in August 2022 [7] . K. pneumoniae ATCC 13883 served as the propagation host.

Methods

Phage isolation followed the double-agar overlay method: filtered wastewater supernatant (0.22-µm PES membrane) was mixed with log-phase host bacteria in soft agar and plated on LB agar. Individual plaques were picked, triplepurified, and amplified to high-titer stocks (> 10⁹ PFU/mL). Host range was determined by spot testing on all 30 CR-Kp isolates; efficiency of plating was calculated for each phage-host pair. Time-kill kinetics were measured at MOIs of 0.1, 1, and 10, sampling at 0, 1, 2, 4, 6, and 24 hours. pH stability (3-12) and thermal stability (4-70 °C, 1-hour incubation) were assessed by titer enumeration [8] . Biofilm assays used 48-hour pre-formed biofilms in 96-well plates; phage was added at MOI 100 and biomass measured by crystal violet staining after 24 hours. PAS experiments combined φKp-03 (MOI 10) with sub-MIC meropenem (2 µg/mL). Whole-genome sequencing was performed on the Illumina MiSeq (2 × 300 bp), assembled with SPAdes v3.15, and annotated with Pharokka [9] . Statistical comparisons used one-way ANOVA with Tukey post-hoc tests (α = 0.05). The combination of φKp-03 with sub-MIC meropenem produced a 5.7-log reduction-1.4 logs greater than phage alone-confirming synergy (Table 2). Biofilm penetration assays showed a 71.8% reduction in established biofilm biomass after 24-hour phage treatment at MOI 100.

Treatment	Log₁₀ Reduction	p vs. Control
Untreated control	0.0	—
Meropenem 2 µg/mL alone	0.4 ± 0.2	> 0.05
φKp-03 MOI 10 alone	4.3 ± 0.3	< 0.001
φKp-03 + Meropenem	5.7 ± 0.4	< 0.001
φKp-03 + Meropenem vs. phage alone	—	< 0.001

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Phage-antibiotic synergy: log reduction in CR-Kp viable counts at 4 hours

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Results

Results

Limitations

These findings are limited to in vitro conditions and cannot predict in vivo pharmacokinetics, immune clearance of phage particles, or the emergence of phage-resistant bacterial subpopulations during treatment. The four CR-Kp isolates not lysed by φKp-03 may carry receptor mutations or restriction-modification systems that would require phage cocktail strategies to overcome [10] . Animal infection models are the logical next step before any clinical application.

Discussion

φKp-03's broad host range (86.7%) compares favorably with phage cocktails described in the literature, many of which use three to five phages to achieve similar coverage [11] . Its Siphoviridae classification and genome analysis-no integrases, no toxin genes, no antibiotic-resistance determinants-support its safety profile for potential therapeutic use, consistent with international phage-therapy guidelines that mandate absence of lysogeny and virulence markers [12] . The synergistic effect with meropenem is likely explained by the PAS mechanism: sub-inhibitory β-lactam exposure causes cell filamentation in Klebsiella, increasing the surface area available for phage adsorption and effectively amplifying the lytic cycle [6] . The practical implication is that phage therapy need not fully replace antibiotics but may restore efficacy of otherwise ineffective drugs when used in combination.

Conclusion

This research identified φKp-03 as a lytic bacteriophage with broad host range, strong biofilm-penetration capacity, and synergistic activity with meropenem against carbapenem-resistant K. pneumoniae. The phage's genome is free of virulence or resistance genes, supporting its candidacy for further preclinical evaluation. These results add to the evidence base for phage therapy as a practical complement to conventional antibiotics in managing multidrug-resistant infections.

Acknowledgement

Acknowledgements Funding SourcesThis research was supported by departmental operating funds at the uMgeni University of Biological Sciences. Institutional SupportThe author thanks the Department of Medical Microbiology for culture collections and sequencing platform access. Contributions Not Qualifying for AuthorshipThe author acknowledges Dr. Sipho Ndlovu for assistance with wastewater sampling logistics and Mr. Thabo Mokoena for bioinformatics pipeline support.

Figures and tables from the paper

Phage	Hosts Lysed	% Range	EOP (ref)	Burst Size	Latent (min)
φKp-01	19/30	63.3	0.87	124	28
φKp-02	14/30	46.7	0.62	98	32
φKp-03	26/30	86.7	0.94	187	22
φKp-04	11/30	36.7	0.41	76	35
φKp-05	21/30	70.0	0.78	142	26

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Host range and lytic performance of five isolated phages

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Efficiency of plating of five phages against the carbapenem-resistant reference strain compared with the sensitive propagation host.

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Radar chart comparing six performance parameters for φKp-03 (lead candidate) and φKp-04. φKp-03 outperformed on lytic activity,

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