Phage–antibiotic synergy restores β-lactam efficacy in MDR Klebsiella quasipneumoniae biofilms and suppresses resistance

Background Biofilms formed by multidrug-resistant (MDR) Klebsiella spp . present a significant clinical challenge due to elevated antibiotic tolerance. Bacteriophages (phages) represent a promising alternative, particularly in combination with antibiotics, where phage–antibiotic synergy (PAS) can increase antibiofilm activity. Evaluating treatment efficacy in these complex structures requires real-time, noninvasive viability analysis. Methods To address this, we used light-sheet fluorescence microscopy (LSFM), a high-resolution, minimally invasive approach, for dynamic tracking of PAS in intact biofilms. To our knowledge, this is the first in vitro application of LSFM for investigating PAS. We studied the combined activity of a virulent phage (vB_KpUKJ_2) and ceftazidime (CAZ) against an extended-spectrum β-lactamase-producing K. quasipneumoniae . Results In planktonic cultures, PAS was strongly affected in a dose-dependent manner. In mature biofilms, LSFM imaging revealed that high-dose phages (10⁸ PFU/mL) combined with CAZ at a 0.25× minimum inhibitory concentration (MIC) induced a rapid and sustained reduction in viability over 24 hours. This regimen significantly outperformed mono-treatments ( p  < 0.01), demonstrating that phage coadministration can reduce the required antibiotic dose. Mechanistically, treatment resulted in phage-mediated degradation of α- and β-polysaccharides within the extracellular polymeric substance. Crucially, while phage mono-treatment led to the emergence of resistant mutants, the combination treatment fully suppressed resistance. Whole-genome sequencing revealed mutations in genes such as fhuA, purA , and rpoC , suggesting diverse resistance mechanisms linked to fitness trade-offs such as impaired biofilm formation. Conclusions Our findings highlight a precision-guided strategy with translational potential for device-associated infections, providing a mechanistic and methodological foundation for optimizing PAS-based therapies.