Therapeutic Switching of Rafoxanide: a New Approach To Fighting Drug-Resistant Bacteria and Fungi

Abstract

Control and management of life-threatening bacterial and fungal infections are a global health challenge. Despite advances in antimicrobial therapies, treatment failures for resistant bacterial and fungal infections continue to increase. We aimed to repurpose the anthelmintic drug rafoxanide for use with existing therapeutic drugs to increase the possibility of better managing infection and decrease treatment failures. For this purpose, we evaluated the antibacterial and antifungal potential of rafoxanide. Notably, 70% (70/100) of bacterial isolates showed multidrug resistance (MDR) patterns, with higher prevalence among human isolates (73.5% [50/68]) than animal ones (62.5% [20/32]). Moreover, 22 fungal isolates (88%) were MDR and were more prevalent among animal (88.9%) than human (87.5%) sources. We observed alarming MDR patterns among bacterial isolates, i.e., Klebsiella pneumoniae (75% [30/40; 8 animal and 22 human]) and Escherichia coli (66% [40/60; 12 animal and 28 human]), and fungal isolates, i.e., Candida albicans (86.7% [13/15; 4 animal and 9 human]) and Aspergillus fumigatus (90% [9/10; 4 animal and 5 human]), that were resistant to at least one agent in three or more different antimicrobial classes. Rafoxanide had antibacterial and antifungal activities, with minimal inhibitory concentration (MICs) ranging from 2 to 128 μg/mL. Rafoxanide at sub-MICs downregulated the mRNA expression of resistance genes, including E. coli and K. pneumoniae bla_CTX-M-1, bla_TEM-1, bla_SHV, MOX, and DHA, C. albicans ERG11, and A. fumigatus cyp51A. We noted the improvement in the activity of β-lactam and antifungal drugs upon combination with rafoxanide. This was apparent in the reduction in the MICs of cefotaxime and fluconazole when these drugs were combined with sub-MIC levels of rafoxanide. There was obvious synergism between rafoxanide and cefotaxime against all E. coli and K. pneumoniae isolates (fractional inhibitory concentration index [FICI] values ≤ 0.5). Accordingly, there was a shift in the patterns of resistance of 16.7% of E. coli and 22.5% of K. pneumoniae isolates to cefotaxime and those of 63.2% of C. albicans and A. fumigatus isolates to fluconazole when the isolates were treated with sub-MICs of rafoxanide. These results were confirmed by in silico and mouse protection assays. Based on the in silico study, one possible explanation for how rafoxanide reduced bacterial resistance is through its inhibitory effects on bacterial and fungal histidine kinase enzymes. In short, rafoxanide exhibited promising results in overcoming bacterial and fungal drug resistance. IMPORTANCE The drug repurposing strategy is an alternative approach to reducing drug development timelines with low cost, especially during outbreaks of disease caused by drug-resistant pathogens. Rafoxanide can disrupt the abilities of bacterial and fungal cells to adapt to stress conditions. The coadministration of antibiotics with rafoxanide can prevent the failure of treatment of both resistant bacteria and fungi, as the resistant pathogens could be made sensitive upon treatment with rafoxanide. From our findings, we anticipate that pharmaceutical companies will be able to utilize new combinations against resistant pathogens.

Keywords: in silico; mouse protection; rafoxanide; repurposing; resistance; resistance gene downregulation; treatment failure.

FIG 1

Numbers of bacterial (E. coli and K. pneumoniae) and fungal (C. albicans and A. fumigatus) isolates recovered in the current study and those exhibiting multidrug resistance patterns. MDR is defined as resistance to at least one antimicrobial agent in three or more different antimicrobial classes. The MDR percentage was calculated in relation to the total number of recovered isolates from each bacterial and fungal species.

FIG 2

Frequency of resistance to antimicrobials among all recovered E. coli (n = 60), K. pneumoniae (n = 40), C. albicans (n = 15), and A. fumigatus (n = 10) isolates based on Kirby-Bauer disc diffusion and broth microdilution methods using Mueller-Hinton agar and commercial antimicrobial and antifungal disks. Inoculum preparation and data interpretation were conducted in accordance with CLSI recommendations. Both procedures were performed in three biological replicates. AMC, amoxicillin-clavulanic acid; CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; CEF, cefepime; CIP, ciprofloxacin; ATM, aztreonam; C, chloramphenicol; E, erythromycin; CN, gentamicin; SXT, trimethoprim-sulfamethoxazole; TZP, piperacillin-tazobactam; IPM, imipenem; TE, tetracycline; FLZ, fluconazole; ITZ, itraconazole; Ciclo, ciclopirox; AP, amphotericin B; NY, nystatin; CASP, caspofungin; TERB, terbinafine; 5FC, 5-fluorocytosine.

FIG 3

Heat map showing hierarchical clustering and overall distribution of the investigated C. albicans and A. fumigatus isolates based on their antifungal resistance patterns. The presence and absence of resistance to each antifungal drug (red and blue, respectively) were used to construct the heat map using the R environment program (v. 3.6.2). The code numbers on the right refer to C. albicans (C) and A. fumigatus (A) isolates from animal (C1 to C5 and A1 to A4) and human (C6 to C15 and A5 to A10) sources. With the exception of two pairs of isolates (isolates C1 and C15 and isolates A7 and A9), all fungal isolates were distributed in different clones with a high degree of antifungal diversity, reflecting weak clonality and host specificity. This heat map enables the visualization of the distribution of antimicrobial resistance among our fungal isolates. Resistance to antifungal drugs was predominant, as shown by red squares, in contrast to the sensitivity patterns, shown by blue squares. The hierarchical clustering groups similar isolates into a set of clusters, where each cluster is distinct from the others and the isolates within each cluster are similar to each other. FLZ, fluconazole; ITZ, itraconazole; Ciclo, ciclopirox; AP, amphotericin B; NY, nystatin; CASP, caspofungin; TERB, terbinafine; 5FC, 5-fluorocytosine.

FIG 4

Heat map showing hierarchical clustering of the investigated E. coli and K. pneumoniae isolates based on the occurrence of antimicrobial resistance, ESBL and AmpC types, and resistance genes (bla_CTX-M-1, bla_TEM-1, bla_SHV, MOX, and DHA) using the R environment program (v. 3.6.2). In the heat map, red and blue indicate resistance and sensitivity to a particular antimicrobial agent and to the presence and absence of certain beta-lactamase types and resistance genes, respectively. The code numbers on the right refer to E. coli (E) and K. pneumoniae (K) isolates from animal (E1 to E20 and K1 to K12) and human (E21 to E60 and K13 to K40) sources. With the exception of six clusters, including five pairs of isolates (E11 and E36; E47 and K12; E5 and E42; E6 and E27; and E35 and K40) and one group of three isolates (K27, E48, and K7), all isolates were distributed in different lineages, reflecting high diversity and weak clonality within each host. AMC, amoxicillin-clavulanic acid; CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; CEF, cefepime; CIP, ciprofloxacin; ATM, aztreonam; C, chloramphenicol; E, erythromycin; CN, gentamicin; SXT, trimethoprim-sulfamethoxazole; TZP, piperacillin-tazobactam; IPM, imipenem; TE, tetracycline.

FIG 5

Heat map and hierarchical clustering of the investigated C. albicans and A. fumigatus isolates based on the in vitro activity of rafoxanide alone and in combination with fluconazole. Red indicates upregulation of C. albicans ERG11 and A. fumigatus cyp51A resistance genes and high MICs and FICI values, while the darker blue color represents downregulation of resistance genes and low MICs and FICI values. MICs of rafoxanide (MIC_RAF), fluconazole (MIC_FLZ), fluconazole used with sub-MICs of rafoxanide (MIC_FLZ+RAF), and rafoxanide used in combination with fluconazole (MIC_RAF+FLZ) and sub-MICs of rafoxanide are in micrograms per milliliter, and other values represent the expression levels of the resistance genes C. albicans ERG11 and A. fumigatus cyp51A in the untreated and rafoxanide-treated fungal isolates. The code numbers on the right refer to C. albicans (C) and A. fumigatus (A) isolates. MIC_RAF, MIC_RAF+FLZ, and sub-MICs of rafoxanide are divided by 10.

FIG 6

Heat map and hierarchical clustering of the investigated E. coli and K. pneumoniae isolates based on the in vitro activity of rafoxanide alone and in combination with cefotaxime. Red indicates upregulation of bla_CTX-M-1, bla_TEM-1, bla_SHV, MOX, and DHA and high MICs and FICI values, while the darker blue color represents downregulation of resistance genes and low MICs and FICI values. MICs of rafoxanide (MIC_RAF), cefotaxime (MIC_CTX), cefotaxime used with sub-MICs of rafoxanide (MIC_CTX+RAF), and rafoxanide used in combination with cefotaxime (MIC_RAF+CTX), and sub-MICs of rafoxanide are in micrograms per milliliter; other values represent the expression levels of the investigated resistance genes The code numbers on the right refer to E. coli (E) and K. pneumoniae (K) isolates. MIC_RAF and MIC_RAF+CTX are divided by 100.

FIG 7

Binding modes of rafoxanide (A) and citrate anion (B) and 2D interaction diagrams of corresponding ligands with bacterial CitA protein (PDB ID 2J80). Green shading in 2D interaction diagrams represents hydrophobic interactions, gray areas are accessible surfaces, and dotted arrows represent hydrogen bonds.

FIG 8

3D interaction and 3D protein positioning of rafoxanide at the sensory fungal histidine kinase with PDB ID 1OXB.

FIG 9

Survival analysis of albino mice following intraperitoneal and intravenous injection of untreated and treated MDR bacterial (K. pneumoniae [A] and E. coli [B]) and fungal (C. albicans [C] and A. fumigatus [D]) isolates (1 × 10⁸ CFU/mL) with sub-MIC levels of cefotaxime or fluconazole alone or in combination with rafoxanide every 24 h for 3 successive days, with standard errors. The negative-control group remained unchallenged and untreated. The survival rates (percent) of mice (20/group or subgroup) were recorded over 5 days.