Investigation of cephalosporin-based antimicrobial combination strategies for targeting resistant bacterial infections

Abstract

The Gram-negative bacterium Klebsiella pneumoniae represents a major threat to global health due to its high rate of resistance to antimicrobials, leading to treatment limitations. Given this, the present study aimed to investigate the synergism between antibiotic combinations as a therapeutic alternative for infections caused by K. pneumoniae. For this purpose, a checkerboard test was performed with combinations of the following cephalosporins - Cefotaxime, Cefmenoxime, Ceftibuten, Cephalexin, Cefixime - with Polymyxin B and, from this, the fractional inhibitory concentration index (FICI) was calculated. In addition, survival curves were obtained, and analyses were performed with the Synergyfinder software for the studied combinations. The bacterial biofilm inhibition potential of the combinations was also explored. Scanning electron microscopy (SEM) images of K. pneumoniae treated with the combinations were obtained. In addition, the hemolysis test was performed to investigate the combinations hemolytic potential. The combinations, Cefotaxime-Polymyxin B and Cefmenoxime-Polymyxin B were explored in an in vivo model of Caenorhabditis elegans, where toxicity and antibacterial efficacy experiments were performed. In vitro pharmacokinetic/pharmacodynamic (PK/PD) was performed for Ceftibuten-Polymyxin B, using a modified Emax model to elucidate the combination antibacterial potential and efficacy. In vivo assay with Swiss mice was performed with Ceftibuten-Polymyxin B combination. Cefotaxime-Polymyxin B, Cefmenoxime-Polymyxin B and Ceftibuten-Polymyxin B exhibited low FICIs, ranging from 0.19 to 0.5, from 0.25 to 1.5 and from 0.15 to 0.37, respectively, with zero interaction potency (ZIP) scores of 37,484, 15,076 and 45,754, representing synergism. Plus, PK/PD study demonstrated that Ceftibuten-Polymyxin B had enhanced antibacterial activity comparing to treatments alone, based on a lower EC50 dose (the antibiotic concentration required to achieve 50% of the maximum effect) and a higher kmax (bacterial killing constant). The three studied combinations significantly reduced the growth and biofilm formation of carbapenem polymyxin resistant K. pneumoniae, without compromising the bacterial cell integrity, according to SEM images. Safety evaluations demonstrated low percentages of hemolysis for the treatments. In accordance, Cefotaxime-Polymyxin B and Cefmenoxime-Polymyxin B generated high survival rates in toxicity evaluations in C. elegans. In the K. pneumoniae infection in vivo model, Cefotaxime-Polymyxin B and Cefmenoxime Polymyxin B significantly increased the survival of C. elegans nematodes. Furthermore, in vivo studies revealed the efficacy of Ceftibuten-Polymyxin B in significantly reducing the bacterial load of the peritoneal lavage fluid of mice. These results highlight the potential of the combination between the studied cephalosporins and Polymyxin B for thetreatment of infections caused by K. pneumoniae resistant to carbapenems and polymyxins.