Synergistic antimicrobial combination of third-generation cephalosporins and polymyxin B against carbapenem-polymyxin-resistant <i>Klebsiella pneumoniae:</i> an <i>in vitro</i> and <i>in vivo</i> analysis

Antibiotic combination therapy is a promising approach to address the urgent need for novel treatment options for infections caused by carbapenem-polymyxin-resistant <i>Klebsiella pneumoniae</i> (CPR-Kp). The present study aimed to investigate the synergistic potential of four cephalosporins in combination with polymyxin B (PMB). A checkerboard assay was performed to evaluate the synergistic effects of cephalexin (CLX), cefixime, cefotaxime (CTX), and cefmenoxime (CMX) in combination with PMB. Subsequently, experiments evaluating the use of CTX or CMX in combination with PMB (CTX-PMB or CMX-PMB, respectively), including growth curve and SynergyFinder analysis, antibiofilm activity assays, cell membrane integrity assays, and scanning electron microscopy, were performed. Safety assessments were also conducted, including hemolysis and toxicity evaluations, using <i>Caenorhabditis elegans</i>. Furthermore, an <i>in vivo</i> model in <i>C. elegans</i> was adopted to assess the treatment efficacy against CPR-Kp infections. CTX-PMB and CMX-PMB exhibited low fractional inhibitory concentration indexes ranging from 0.19 to 0.50 and from 0.25 to 1.5, respectively, and zero interaction potency scores of 37.484 and 15.076, respectively. The two combinations significantly reduced growth and biofilm formation in CPR-Kp. Neither CTX-PMB nor CMX-PMB compromised bacterial cell integrity. Safety assessments revealed a low hemolysis percentage and high survival rates in the <i>C. elegans</i> toxicity evaluations. The <i>in vivo</i> model revealed that the CTX-PMB and CMX-PMB treatments improved the survival rates of <i>C. elegans</i>. The synergistic effects of the CTX-PMB and CMX-PMB combinations, both <i>in vitro</i> and <i>in vivo</i>, indicate that these antibiotic pairings could represent effective therapeutic options for infections caused by CPR-Kp.