Abstract
BACKGROUND: Colistin resistance significantly constrains available treatment options and results in the emergence of pandrug-resistant (PDR) strains. Treating PDR infections is a major public health issue. A promising solution lies in using colistin-based combinations. Despite the availability of in vitro data evaluating these combinations, the in vivo studies remain limited. RESULTS: Thirty colistin-resistant Klebsiella pneumoniae (ColRKp) isolates were collected from hospitalized patients. Colistin resistance was detected using broth microdilution, and antimicrobial susceptibility was tested using the Kirby-Bauer method against 18 antibiotics. Extremely high resistance levels were detected, with 17% of the isolates being PDR. Virulence profiling, assessed using Anthony capsule staining, the string test, and the crystal violet assay, indicated the predominance of non-biofilm formers and non-hypermucoid strains. The isolates were screened for mcr genes using polymerase chain reaction. Whole-genome sequencing (WGS) and bioinformatics analysis were performed to characterize the genomes of PDR isolates. No plasmid-borne mcr genes were detected, and WGS analysis revealed that PDR isolates belonged to the high-risk clones: ST14 (n = 1), ST147 (n = 2), and ST383 (n = 2). They carried genes encoding extended-spectrum β-lactamases and carbapenemases, bla(CTX-M-15) and bla(NDM-5), on conjugative IncHI1B/IncFIB plasmids, illustrating the convergence of virulence and resistance genes. The most common mechanism of colistin resistance involved alterations in mgrB. Furthermore, deleterious amino acid substitutions were also detected within PhoQ, PmrC, CrrB, ArnB, and ArnT. Seven colistin-containing combinations were compared using the checkerboard experiment. Synergy was observed when combining colistin with tigecycline, doxycycline, levofloxacin, ciprofloxacin, sulfamethoxazole/trimethoprim, imipenem, or meropenem. The efficacy of colistin combined with either doxycycline or levofloxacin was assessed in vitro using a resistance modulation assay, and in vivo, using a murine infection model. In vitro, doxycycline and levofloxacin reversed colistin resistance in 80% and 73.3% of the population, respectively. In vivo, the colistin + doxycycline combination demonstrated superiority over colistin + levofloxacin, rescuing 80% of infected animals, and reducing bacterial bioburden in the liver and kidneys while preserving nearly intact lung histology. CONCLUSIONS: This study represents the first comparative in vitro and in vivo investigation of the efficacy of colistin + doxycycline and colistin + levofloxacin combinations in clinical PDR ColRKp isolates characterized at a genomic level.