Rapid Adaptive Evolution under Combination Therapy in <i>Klebsiella pneumoniae</i>

<i>Klebsiella pneumoniae</i> poses a substantial health concern worldwide, with high mortality often associated with its elevated resistance levels. Combination antibiotic therapies have emerged as a viable strategy for addressing infections caused by these highly resistant pathogens, yet the evolutionary routes to resistance under such regimens remain poorly understood. Here, we investigated how resistance can emerge during exposure to combination therapy by conducting an in vitro evolutionary experiment with a clinical <i>K. pneumoniae</i> KPC-producing isolate (KP03, Brazil), which was initially susceptible to both amikacin and polymyxin B (AmkPol). While the combination therapy displayed an additive effect in vitro, subinhibitory exposure rapidly drove resistance. In three independent lineages, bacteria tolerated concentrations nearly 10-fold (polymyxin B) and 5-fold (amikacin) above EuCAST breakpoints, with MICs reaching 128-256 μg·mL^-1 after 45 passages. These high resistance levels persisted for at least 10 days without selective pressure, although resistance lineages exhibited measurable fitness costs. Whole-genome sequencing revealed diverse mutations affecting <i>lapB</i>, <i>phoP</i>, <i>rho</i>, <i>smbA</i>, <i>mlaA</i>, and <i>asmA</i>, while transcriptomics analysis showed upregulation of the <i>arn</i> operon and the <i>aphA</i> alongside with downregulation of envelope- and efflux-associated genes. Cross-resistance was also observed against colistin and certain antimicrobial peptides, raising concern for treatment options beyond the AmkPol combination. Although combination therapy represents an important treatment strategy, our findings demonstrate that <i>K. penumoniae</i> can rapidly evolve stable, high-level resistance under combination therapy, highlighting the need for a deeper understanding of how such regimens influence resistance development and the continued need to develop novel antibiotics strategies.