Insect-Derived Spinigerin as an Imipenem Adjuvant: Combinatorial Activity against Clinical MDR Klebsiella pneumoniae Isolates

Abstract

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a global health threat, rendering imipenem and other last-line antibiotics increasingly ineffective.. While antimicrobial peptides from insects have shown promise, the synergistic potential of termite-derived spinigerin with imipenem against clinical MDR K. pneumoniae isolates remains largely unexplored. This study investigated the combinatorial activity of spinigerin and imipenem against clinical MDR K. pneumoniae isolates. Three molecularly identified K. pneumoniae strains (KPA2, KPK6, KPDD) were isolated from urine samples. Minimum inhibitory concentrations (MICs) of spinigerin and imipenem, alone and in combination at nine ratios (9:1 to 1:9), were determined using broth microdilution. Spinigerin alone exhibited MICs of 0.25–0.5, while imipenem alone showed uniform MICs of 0.125. Combination therapy produced ratio-dependent MIC reductions, with maximal potentiation at 3:7 and 2:8 ratios (spinigerin:imipenem). KPK6 MIC decreased from 0.25 to 0.008 (31.3-fold reduction), KPA2 from 0.5 to 0.063 (7.9-fold), and KPDD from 0.5 to 0.125 (4-fold). One-way ANOVA confirmed significant MIC reductions across combination ratios for all isolates (F = 18.6–26.3, df = 10, p < 0.001). Post-hoc analysis revealed that ratios 5:5 to 2:8 produced significantly lower MICs than spinigerin alone (p < 0.01), whereas ratios 9:1 to 7:3 did not (p > 0.05). Spinigerin potentiates imipenem activity against MDR K. pneumoniae in a ratio-dependent manner, significantly reducing effective MICs.. This study provides the first evidence of spinigerin-imipenem synergy against clinical urinary isolates of K. pneumoniae, demonstrating that optimized peptide-antibiotic ratios can resensitize carbapenem-resistant strains.