Activity of polymyxin B combined with cefepime-avibactam against the biofilms of polymyxin B-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae in in vitro and in vivo models

Abstract

Bacterial biofilms, often forming on medical devices, can lead to treatment failure due to their increased antimicrobial resistance. Cefepime-avibactam (CFP-AVI) exhibits potent activities against Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) when used with polymyxin B (PMB). However, its efficacy in biofilm-related infections is unknown. The present study aimed to evaluate the activity of PMB combined with CFP-AVI against the biofilms of PMB-resistant Gram-negative bacteria. Five K. pneumoniae strains and three P. aeruginosa strains known to be PMB-resistant and prone to biofilm formation were selected and evaluated. Antimicrobial susceptibility assays demonstrated that the minimal biofilm inhibitory and eradication concentrations of PMB and CFP-AVI for biofilms formed by the eight strains were significantly higher than the minimal inhibitory concentrations of the antibiotics for planktonic cells. The biofilm formation inhibition and eradication assays showed that PMB combined with CFP-AVI cannot only suppress the formation of biofilm but also effectively eradicate the preformed mature biofilms. In a modified in vitro pharmacokinetic/pharmacodynamic biofilm model, CFP-AVI monotherapy exhibited a bacteriostatic or effective activity against the biofilms of seven strains, whereas PMB monotherapy did not have any activity at 72 h. However, PMB combined with CFP-AVI demonstrated bactericidal activity against the biofilms of all strains at 72 h. In an in vivo Galleria mellonella infection model, the 7-day survival rates of larvae infected with biofilm implants of K. pneumoniae or P. aeruginosa were 0-6.7%, 40.0-63.3%, and 46.7-90.0%, respectively, for PMB alone, CFP-AVI alone, and PMB combined with CFP-AVI; the combination therapy increased the rate by 6.7-33.3% (P < 0.05, n = 6), compared to CFP-AVI monotherapy. It is concluded that PMB combined with CFP-AVI exhibits effective anti-biofilm activities against PMB-resistant K. pneumoniae and P. aeruginosa both in vitro and in vivo, and thus may be a promising therapeutic strategy to treat biofilm-related infections.

Keywords: Klebsiella pneumoniae; Pseudomonas aeruginosa; Biofilm; Cefepime-avibactam; Polymyxin B-resistance.

Fig. 1

A: The mean OD595 values of eight experimental strains. The bars represent standard deviations. B: Biofilm formation at different time points of eight experimental strains. The results are expressed as means± standard deviations

Fig. 2

Biofilm inhibitory (A) and eradication (B) effects of polymyxin B (PMB) and cefepime-avibactam (CFP-AVI). The results are expressed as the mean ± standard deviation. *, P < 0.05, compared with all other groups

Fig. 3

CLSM images reveal the inhibitory (A&B) and eradication (C&D) effects of polymyxin B (PMB), cefepime- avibactam (CFP-AVI) on biofilm formation by Klebsiella pneumoniae K002 (A&C) and Pseudomonas aeruginosa P002 (B&D). Scale bar, 100 μm

Fig. 4

The targeted and measured concentrations of polymyxin B (PMB) (A), cefepime (CFP) (B) andavibactam (AVI) (C) in the in vitro biofilm PK/PD model.

Fig. 5

Bacterial killing by different treatments against biofilm-embedded bacteria of K. pneumoniae and P. aeruginosa in the in vitro biofilm PK/PD model after 72 h treatment

Fig. 6

Survival of G. mellonella larvae treated only with polymyxin B (PMB) and cefepime-avibactam (CFP-AVI) without infection. ns, no statistical differences, compared between normal saline and antibiotics

Fig. 7

Survival of G. mellonella larvae infected with biofilm implants, with or without polymyxin B (PMB) and cefepime-avibactam (CFP-AVI) treatment. Experiment was conducted with 30 larvae per group. *, P < 0.05, compared between CFP-AVI monotherapy and PMB combined with CFP-AVI; **, P < 0.001, compared between positive controls and PMB combined with CFP-AVI; ns, no statistical differences, compared between CFP-AVI monotherapy and PMB combined with CFP-AVI