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

Abstract

Antibiotic combination therapy is a promising approach to address the urgent need for novel treatment options for infections caused by carbapenem-polymyxin-resistant Klebsiella pneumoniae (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 Caenorhabditis elegans. Furthermore, an in vivo model in C. elegans 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 C. elegans toxicity evaluations. The in vivo model revealed that the CTX-PMB and CMX-PMB treatments improved the survival rates of C. elegans. The synergistic effects of the CTX-PMB and CMX-PMB combinations, both in vitro and in vivo, indicate that these antibiotic pairings could represent effective therapeutic options for infections caused by CPR-Kp.

Keywords: Caenorhabditis elegans; Klebsiella pneumoniae; antimicrobial resistance; cephalosporins; combination therapy.

Fig 1

Growth curves generated for CPR-Kp K18 treated with (A) the CTX-PMB combination and the isolated antibiotics at a 1 × MIC; (B) the CMX-PMB combinations and the isolated antibiotics at a 1 × MIC. Positive (+) and negative (–) controls were also included in the experiments. One-way ANOVA was performed, and the differences were considered significant at P < 0.5. Significant differences are indicated using different letters (a-b; b-c; a-c), while nonsignificant differences are indicated using the same letter (a-a; b-b; c-c).

Fig 2

(A) OD of the CTX-PMB combination at 0, 0.25, 0.5, 1, and 2 × MIC against CPR-Kp K18 and (B) OD of the CMX-PMB combination at 0, 0.25, 0.5, 1, and 2 × MIC CPR-Kp K18. The OD is proportional to microbial growth. One-way ANOVA was performed, and the differences were considered significant at P < 0.5. Significant differences are indicated using different letters (a-b; b-c; a-c), while nonsignificant differences are indicated using the same letter (a-a; b-b; c-c). (C) Multiple concentration combinations of CTX-PMB that inhibited CPR-Kp K18 growth, with the lowest concentration indicated using FICI (0.19). (D) Multiple concentration combinations of CMX-PMB that inhibited CPR-Kp K18 growth, with the lowest concentration indicated using FICI (0.25).

Fig 3

SynergyFinder analysis. (A) Dose-response matrix for the CTX-PMB combination. (B) Dose-response matrix for the CMX-PMB combination. (C) The ZIP synergy score (37.484) demonstrated synergism for the CTX-PMB combination. (D) The ZIP synergy score (15.076) demonstrated synergism for the CMX-PMB combination. The regions depicted in the deepest red indicate better dose combinations related to bacterial growth inhibition (inhibition >80%).

Fig 4

Antibiofilm activity of (A) the CTX-PMB combination and the isolated antibiotics at a concentration of 16 mg/L for each; (B) the CTX-PMB combination and the isolated antibiotics at a concentration of 0.065 mg/L for CMX and 64 mg/L for PMB. Positive (+) and negative (–) controls were also included in the experiments. One-way ANOVA was performed, and the differences were considered significant at P < 0.5. Significant differences are indicated using different letters (a-b; b-c; a-c), while nonsignificant differences are indicated using the same letter (a-a; b-b; c-c). CTX [16], cefotaxime 16 mg/L; CTX [16]-PMB [16], cefotaxime 16 mg/L associated with polymyxin B 16 mg/L; PMB [16], polymyxin B 16 mg/L; CMX [0.065], cefmenoxime 0.065 mg/L; CMX [0.065]-PMB [64], cefmenoxime 0.065 mg/L associated with polymyxin B 64 mg/L; PMB [64], polymyxin B 64 mg/L.

Fig 5

SEM images at 5,000× magnification for the CPR-Kp K18 strain treated with (A) CTX at 8 mg/L in combination with PMB at 4 mg/L; the microbial cell membrane is indicated using an arrow; (B) CMX at 8 mg/L in combination with PMB at 8 mg/L; the microbial cell membrane is indicated using an arrow; (C) PMB at 8 mg/L; (D) CTX at 8 mg/L; (E) CMX at 8 mg/L; (F) BHI broth only, used as the bacterial growth control.

Fig 6

Safety experiment results. (A) Hemolysis rates achieved by the CTX-PMB and CMX-PMX combinations at a concentration of 1 × MIC for each, and for comparison, the hemolysis rates achieved by the isolated antibiotics CTX, CMX, and PMB at a concentration of 64 mg/L for each. Triton was used as the positive control (+), and D-PBS was used as the negative control (–). (B) C. elegans (N2) survival rate when exposed to the CTX-PMB and CMX-PMB combinations and isolated antibiotics at a concentration of 1 × MIC. M9 buffer was used as the positive control (+), and 16 mg/L tigecycline was used as the reference antibiotic. One-way ANOVA was performed, and the differences were considered significant at P < 0.5. Significant differences are indicated using different letters (a-b; b-c; a-c), while nonsignificant differences are indicated using the same letter (a-a; b-b; c-c). TGC, tigecycline.

Fig 7

(A) Kaplan-Meier survival curves for C. elegans (Au37) infected with the CPR-Kp K1 strain and treated with the CTX-PMB combination and isolated antibiotics. (B) Kaplan-Meier survival curves for the CMX-PMB combination and isolated antibiotics for the infection of C. elegans (Au37) with the CPR-Kp K1 strain. Log-rank was made for both curves, and they were considered statistically different (P < 0.5). (C) Microscopy image of the CTX-PMB-treated C. elegans (Au37) group, highlighting numerous eggs, indicated by an arrow, demonstrating the treatment's success. (D) Microscopy image of isolated CMX-treated C. elegans (Au37), with an arrow indicating intestinal obstruction due to CPR-Kp K1 infection (P < 0.5). TGC, tigecycline.