In recent years, the escalating prevalence of drug-resistant gram-negative bacteria has emerged as a major threat to global human health and public environmental problems worldwide (Lee et al., 2017). The threat of antibiotic resistance rendered patients at risk of ineffective treatment and increasing healthcare costs. Carbapenems, including imipenem, ertapenem, and meropenem, have been established as the preferred antibiotics for managing severe infections caused by multidrug-resistant (MDR) Gram-negative pathogens (Bergen et al., 2012; Zhanel et al., 2022). However, irrational utilization of carbapenems has facilitated the emergence of carbapenem-resistant Enterobacteriaceae (CRE), compounding clinical treatment challenges (Zhang et al., 2018; Suay-Garcia and Perez-Gracia, 2019; Wang et al., 2020). Thus, novel antibiotics are urgently needed to combat this challenge.

Polymyxins are generally regarded as the final recourse for the treatment of infections that are induced by CRE (Candan and Aksoz, 2015; Aguayo et al., 2016). Although the precise mechanism of antibacterial action of polymyxins remains unclear, it is widely acknowledged that polymyxin B and colistin (polymyxin E) are generally classified as cationic antimicrobial lipopeptides (CAMPs) that can effectively target a wide range of multidrug-resistant bacteria by disrupting the outer membrane (OM) barrier through lipid A binding (Couet et al., 2012; Gregoire et al., 2017). Although earlier studies suggested that polymyxin B exhibited a low incidence of bacterial resistance, recent evidence points to a marked increase in resistance rates, posing significant challenges to antibiotic treatment options (Cai et al., 2012; Karaiskos et al., 2017). The appearance of plasmid-borne resistant gene mcr-1 and mutations in chromosomal genes, phoPQ and pmrAB, have been implicated in the mechanism of resistance to polymyxin B (Li et al., 2020; Mmatli et al., 2022). Although the resistance rate of polymyxin B has been increasing annually, the combination therapy of polymyxin B with other drugs remains an effective strategy for the treatment of CRE infections (Rigatto et al., 2019; Fedrigo et al., 2021).

Fusidic acid, a natural steroid antibiotic, was initially isolated from the fungus Fusidium coccineum in the early 1960s, exhibiting potent activity against gram-positive bacteria (Fernandes and Pereira, 2011). As is well known, fusidic acid is a well-known inhibitor of elongation factor G (EF-G) function. Fusidic acid can impede bacterial growth through its inhibitory actions (Fernandes, 2016). Gram-negative bacteria are naturally resistant to fusidic acid. It was reported that the use of fusidic acid in combination with other drugs has the potential to be a new treatment strategy (Grohs et al., 2003).

In this study, we aimed to evaluate the potential synergistic effect of combining polymyxin B and fusidic acid against strains of Escherichia coli and Klebsiella pneumoniae that exhibit resistance to polymyxin B.

Alongside the increasing utilization of carbapenem, there is a growing concern about the development of novel resistance to antibiotics used against Gram-negative bacteria. It is imperative to identify new antimicrobials that can effectively combat against CRE. Polymyxin B has been considered as the last-resort treatment option for MDR infections, including infections caused by CRE (Li et al., 2006; Lim et al., 2010). However, the reported epidemiological information suggests that many cases of polymyxin B-resistant strains are emerging worldwide at an alarming rate (Nation and Li, 2009; Moffatt et al., 2019).

Currently, the mechanisms for Polymyxin B resistance have not been clearly elucidated. The previous studies showed the following four principal findings. Firstly, numerous bacteria develop resistance to polymyxins by decreasing the alteration of the head group of lipid A. This modification is originally enabled by electrostatic interactions and is likely mediated by genes located on both the chromosomal and plasmid (Sarkar et al., 2007; Nordmann and Poirel, 2016; Moffatt et al., 2019). Secondly, another mechanism is the induction of efflux pump systems and barriers, which involves increasing the production of capsular polysaccharides, mutations that alter the expression of efflux pumps, and the presence of modified porins that reduce outer membrane permeability, among others (Gregoire et al., 2017; Moffatt et al., 2019). In addition, enzymes produced by bacteria can facilitate the degradation of polymyxin B, leading to the reversal of the resistance phenotype associated with polymyxin B (Sarkar et al., 2007; Moffatt et al., 2019). Finally, the heterogeneity of resistance mechanisms also plays a crucial role in the development and spread of drug resistance (Rigatto et al., 2019).

Several studies have demonstrated that the combinations of polymyxin B and various traditional antibiotics result in increased antibacterial efficacy, highlighting the potential of combination therapy in addressing drug-resistant bacteria (Liu et al., 2020; Allend et al., 2022; Almutairi, 2022). Tian et al. (2021) observed the combination of polymyxin B and tigecycline reduced both of their MICs, indicating that tigecycline combined with polymyxin B may be a promising strategy. Phee et al. (2019) first identified colistin/fusidic acid as a novel strategy against Multidrug-resistant Acinetobacter baumannii (MDR-AB). The combination treatment remains effective even at low concentrations, which is clinically feasible while minimizing drug toxicity. The antibiotic fusidic acid targets EFG, thereby obstructing protein synthesis (Kinoshita et al., 1968). Moreover, fusidic acid exhibits an immunoregulatory effect primarily by impeding cytokine production, eradicating bacteria, and treating various inflammatory responses instigated by bacterial toxins (Kraus and Burnstead, 2011). Fusidic acid has been shown highly effective against staphylococcus, and is usually administrated via oral and parenteral routes (Mlynarczyk-Bonikowska et al., 2022). However, it is notable that gram-negative bacteria have an inherent resistance to fusidic acid.

According to the pharmacokinetics of fusidic acid, administration of a single 500 mg oral dose on an empty stomach results in a blood concentration of approximately 30 μg/mL within 2 to 3 h. For dosing regimens involving oral administration every 8 h over a span of 4 consecutive days, the blood concentration can reach 50–100 μg/mL. Given that large initial doses autoinhibit the clearance of fusidic acid, a steady state can be achieved earlier with dosing regimens that contain higher doses (Bulitta et al., 2013). Therefore, in this study, we tested MIC of polymyxin B against 41 isolates in the presence of 32 μg/mL, 64 μg/mL and 128 μg/mL fusidic acid in order to simulate the drug concentration achieved in human plasma and inhibit the clearance of fusidic acid. Recent pharmacodynamic (PD) and pharmacokinetic (PK) findings concerning polymyxin B indicate that the use of polymyxin B monotherapy is insufficient in achieving consistent and effective plasma concentrations (Garonzik et al., 2011). Notably, monotherapy may lead to resistance development, particularly when a concentration exceeds clinically feasible levels (Bergen et al., 2008). Low levels of resistance evolve repeatedly when low concentration of polymyxin B is applied, but this resistance is reversed after the antibiotic is removed. In contrast, super-MIC levels of polymyxin B (≥4 × MIC) drive the evolution of irreversible resistance. Therefore, combination therapy is recommended to enhance antimicrobial activity and counter resistance (Zhao et al., 2022). In our study, we compared the effectiveness of fusidic acid and polymyxin B in combination therapy and monotherapy for treating E. coli and K. pneumoniae infections. Additionally, a murine model was employed to further assess the efficacy of the combination therapy of polymyxin and fusidic acid.

In our study, E. coli with resistance to polymyxin B were isolated from animals, while K. pneumoniae with resistance to polymyxin B were isolated from humans. We also found that the combined use of polymyxin B and fusidic acid was more effective in bacterial killing than single-drug therapies. Notably, this synergistic effect was observed across various bacterial strains, as evidenced by the low FIC index of less than 0.5 in 5 E. coli and 12 K. pneumoniae strains tested in the chequerboard assay (Table 2). Besides, a higher survival rate was shown in the mice receiving combination therapy compared to those treated with polymyxin B alone.

Our findings suggested that the combination of fusidic acid and polymyxin B has a potential for broad clinical application value. Meanwhile, the drug resistance of polymyxin caused by the usage of polymyxin in animal husbandry deserves attention. Therefore, we urge a decrease in the administration of polymyxin B in animal husbandry.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was approved by Laboratory Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University. The study was conducted in accordance with the local legislation and institutional requirements.

SC, PZ, and CW: conceptualization, data curation, formal analysis, methodology, and writing–original draft. JW and YZ: data curation, methodology, and writing–original draft. JZ and BW: methodology. HZ: software. LR: formal analysis. FY and ML: conceptualization and writing–review and editing. CL: conceptualization, project administration, and writing–review and editing. All authors contributed to the article and approved the submitted version.