Dual-species biofilm of MDR Acinetobacter baumannii and Klebsiella pneumoniae are susceptible to colistin-rifamycin combination therapy

BACKGROUND: Co-infections by MDR Acinetobacter baumannii and Klebsiella pneumoniae pose daunting challenges in healthcare settings. This study aimed at investigating the biofilm formation potential of their co-culture and evaluating the effect of combination therapy. METHOD: Spatial distribution of A. baumannii and K. pneumoniae in co-cultured biofilm was analysed by Confocal Laser Scanning Microscopy (CLSM) with Green Fluorescent Protein (GFP) and mCherry-tagged strains and Field Emission Scanning Electron Microscopy (FESEM). The antibiotic combination was selected through checkerboard assay and its antibiofilm activity was assessed against the co-culture of MDR strains. RESULTS: Cell-free supernatant of K. pneumoniae enhanced the planktonic and biofilm growth of A. baumannii. Co-culture of these pathogens revealed interspersed growth in close proximity and significantly higher biofilm than their monocultures (P-value < 0.01). Synergistic combination of colistin (MIC/8) and rifamycin (MIC/4) at Fractional Inhibitory Concentration (FIC) killed both the pathogens in monoculture within 3 h. However, the co-culture exhibited enhanced resistance requiring 24 h for complete eradication. Biofilm formation was inhibited by 77% at 2 × FIC. Whereas the preformed biofilm was eradicated by 40% at 3 × FIC (1/4th of minimum biofilm eradication concentration). CLSM confirmed structural disruption of the biofilm matrix post-treatment at 3 × FIC, with reduction in biofilm thickness from 7 to 4 µm. CONCLUSION: Acinetobacter baumannii and K. pneumoniae co-exist harmoniously, forming enhanced biofilms in co-cultures. Colistin-rifamycin combination proved highly effective against these dual-species biofilms.