Abstract
Antimicrobial combinations have been proven to be a promising approach in the confrontation with multi-drug resistant bacterial pathogens, owing to enhancement of antibacterial efficacy, deceleration of resistance development rate and mitigation of side effects by lowering the doses of two drugs. In the present study, we report that combination of furazolidone (FZ) and other nitrofurans with a secondary bile salt, Sodium Deoxycholate (DOC), generates a profound synergistic effect on growth inhibition and lethality in enterobacteria, including Escherichia coli, Salmonella, Citrobacter gillenii and Klebsiella pneumoniae. Taking E. coli as the model organism to study the mechanism of DOC-FZ synergy, we found that the synergistic effect involves FZ-mediated inhibition of efflux pumps that normally remove DOC from bacterial cells. We further show that the FZ–mediated nitric oxide production contributes to the synergistic effect. This is to our knowledge the first report of nitrofuran-DOC synergy against Gram-negative bacteria.
Introduction
Antimicrobial resistance (AMR) is one of the most serious threats with which humans have been confronted. A UK-Prime-Minister-commissioned report in 2014 estimated that AMR, without appropriate interventions, will cause globally 10 million deaths per annum with a cumulative loss of US $100 trillion by 2050 (1). In this dire context, alternative approaches are urgently needed besides traditional discovery of novel antibiotics, in which antimicrobial combinations have been proven to be a promising approach with some widely accepted advantages, including enhancement of antimicrobial efficacy, deceleration of the rate of resistance and alleviation of side effects (2, 3). Moreover, this approach could amplify the significance of ongoing antimicrobial discovery programs; particularly the advent of any novel antimicrobial compound would bring about a large number of possible double combinations with existing antimicrobial agents to be evaluated, let alone triple and quadruple combinations.
Sodium Deoxycholate (DOC) (Figure 1E) is a facial amphipathic compound in bile, which is secreted into the duodenum to aid lipid digestion and confer some antimicrobial protection (4). Though extensive research has been conducted to elucidate the interaction between DOC, either alone or in bile mixture, and enteric bacteria, the mode of its antimicrobial action remains elusive. It was suggested that DOC could attack multiple cellular targets, including disturbing cell membranes, causing DNA damage, triggering oxidative stress and inducing protein misfolding (4–6). Nonetheless, Gram-negative bacteria such as Escherichia coli and Salmonella are highly resistant to DOC by many mechanisms such as employment of diverse active efflux pumps, down-regulation of outer membrane porins and activation of various stress responses (5, 7-10).
The 5-nitrofurans are an old class of synthetic antimicrobials, clinically introduced in the 1940s and 1950s (11); several are commercially available, including furazolidone (FZ), nitrofurantoin (NIT) and nitrofurazone (NFZ) (Figure 1). FZ is used to treat bacterial diarrhea, giardiasis and as a component in combinatorial therapy for Helicobacter pylori infections; NIT and NFZ are used for urinary tract infections and topical applications, respectively (12). They are prodrugs which require reductive activation mediated largely by two type-I oxygen-insensitive nitroreductases, NfsA and NfsB. These two enzymes perform stepwise 2-electron reduction of the nitro moiety of the compound into the nitroso and hydroxylamino intermediates and biologically inactive amino-substituted product (13, 14). The detailed mechanism of how bacterial cells are killed by the reactive intermediate has yet to be clarified. Nevertheless, it has been proposed that the hydroxylamino derivatives could trigger DNA lesions, disrupt protein structure and arrest RNA and protein biosynthesis (15-19). Some reports also suggested that nitric oxide could be generated during the activation process, thus inhibiting electron transport chain of bacterial cells though clear evidence for that is not available as yet (20, 21).
In this study, we have characterized interaction of DOC with FZ and three other related nitrofurans against a range of enterobacteria. We identified the underlying mechanism of DOC-FZ synergy using E. coli K12 as a model organism.
Results
The synergy between DOC and 5-nitrofurans against enterobacteria
To evaluate the synergy between DOC and FZ, the checkerboard growth inhibition assays were performed for a range of enterobacteria, including Salmonella enterica sv. Typhimurium LT2, Citrobacter gillenii, Klebsiella pneumoniae and two E. coli antibiotic-resistant strains (streptomycin-resistant and streptomycin/ampicillin-resistant). DOC and FZ act synergistically in inhibiting the growth of the microorganisms listed (Figure 2), with FICI ranging from 0.125 in streptomycin-resistant E. coli strain (Figure 2A) to 0.35 in K. pneumoniae (Figure 2F). DOC-FZ synergy was also observed against two E. coli pathogenic strains (E. coli strain O157 and urinary tract infection strain P50; Figure S1). It is worth noting that, when used alone, very high DOC concentrations were required to exert an equivalent effect on inhibiting the growth of these Gram-negative enterobacteria, reflecting the inherent resistance to DOC in these bacteria thanks to their impermeable outer membrane and active efflux pumps, which prevent the intracellular accumulation of toxic xenobiotics.
We also examined the interaction between DOC and other nitrofuran compounds, including NIT, NFZ and CM4 (a 5-nitrofuran compound we found during an antimicrobial synergy screening campaign against E. coli, Figure 1D) in all the bacterial species mentioned above. We found that NIT, NFZ and CM4 were synergistic with DOC in E. coli laboratory strain (Figure 4), Citrobacter gillenii (Figure S2) and Salmonella Typhimurium LT2 (Figure S3) but indifferent in K. pneumoniae isolate (Figure S4).
To elaborate the interaction between DOC and FZ in terms of bactericidal effects, the time-kill assay was employed. Streptomycin-resistant E. coli K12 laboratory strain K1508 and S. enterica serovar Typhimurium strain LT2 were exposed to sub-inhibitory concentrations of DOC (2500 µg/mL) alone, or FZ (0.5 × MIC) alone, or combination of the two drugs at such sub-inhibitory concentrations, over a 24 h period. The sample was taken at different time points and the surviving bacteria were titrated on the antimicrobial-free plates. Centrifugation and resuspension were applied for each sample to eliminate the antimicrobial carryover before plating. After 24 h, the total cell count in the sample treated with the DOC-FZ combination was about six to seven orders of magnitude lower than that in the sample treated with either DOC or FZ alone for both E. coli and Salmonella (Figure 3), demonstrating the synergy in bacterial killing between DOC and FZ.
The role of AcrAB-TolC efflux pump in synergistic interaction between DOC and nitrofurans
One commonly accepted principle is that the synergy between two drugs is a consequence of one drug suppressing bacterial physiological pathways that mediate resistance to the other one. It has been reported that DOC could be expelled out of the cell via a wide range of efflux pumps, in which the tripartite efflux system AcrAB-TolC plays the major role (7, 9). This led us to hypothesize that FZ inhibits the activity of efflux pumps, thus allowing intracellular accumulation of DOC to exert its lethal effect. If this scenario were true, disruption of the function of efflux pumps by mutation was expected to make this activity of FZ redundant, thus reducing the interaction index (FICI) in the mutant strains.
To validate this model in E. coli, the checkerboard assay was performed on the strains containing deletions of individual genes encoding the AcrAB-TolC efflux pump system, ΔtolC and ΔacrA. Deletion of tolC caused a shift from the synergistic interaction between DOC and FZ in the wild type (FICI = 0.125) to indifferent interaction (FICI=0.75; Figure 4A). The ΔacrA mutant exhibited a 3-fold decrease in the FICI index relative to the isogenic wild type strain. Such changes were also observed for the interaction between DOC and other nitrofurans, NIT, NFZ or CM4 (Figure 4BCD).
To confirm that these observations were conferred by direct effect of the tolC and acrA deletion, rather than indirect effects of other genes or proteins, complementation of the corresponding deletion mutations by plasmid-expressed tolC and acrA was performed. To compensate for the multiple copies of plasmid-containing genes, complementation was carried out at a low level of expression, nevertheless it completely restored the strong synergy between DOC and FZ in these complemented strains (Figure 5). These findings collectively support the model that the efflux pumps act as the interacting point for the synergy between DOC and FZ.
An intriguing question to be unraveled is how FZ could negatively influence the action of efflux pumps. We hypothesized that FZ could lower the energy supply to efflux pumps by mediating an increase in concentration of nitric oxide (NO). To verify the proposed model, the interaction between DOC and FZ in the E. coli strain with increased expression of protein Hmp (the E. coli nitric oxide dioxygenase) was inspected. The rationale for this is that overexpression of Hmp protein would increase detoxification of NO by conversion into benign NO3−ions, thus relieving the effect exerted by NO (22). If NO was involved in the mechanism of the interaction between the two drugs, the synergy degree between them was expected to decrease with an increased abundance of Hmp proteins. In agreement with this hypothesis, overexpression of hmp was found to suppress the synergy between DOC and FZ by a factor of 3 (Figure 6). This finding supports the model that NO generated during FZ metabolism participates in the inhibition of electron transport chain (23), with the secondary effect of inhibiting the function of efflux pumps which are dependent on the electron transport chain for their activity.
Discussion
The widespread emergence of antimicrobial drug resistance and the drying pipeline of antibiotics for Gram-negative pathogens imposed an urgency to seek for novel approaches to combat these pathogens. Capitalization on drug combinations is one of the promising approaches to design novel therapies that will allow application of antimicrobials which have heretofore been ineffective against Gram-negative bacteria at concentrations that are acceptable for medical treatments. In the present study, we describe the synergistic interaction between DOC and FZ in a range of enterobacteria in terms of growth inhibition and/or lethality. These findings offer two major implications. Firstly, Gram-negative bacteria, such as E. coli and Salmonella have evolved to be highly resistant to bile salts, including DOC (10); inclusion of an active agent, such as FZ or other 5-nitrofurans could revitalize DOC in the battle against such formidable pathogens. This discovery raises a possibility of using synergistic combinations in enabling use of antimicrobials that are on their own ineffective against Gram-negative bacteria at sub-toxic concentrations, for treatment of infections caused by these resilient organisms.
Secondly, DOC and other bile salts are inherently present at varying concentrations along the gastrointestinal tract. The efficacy of any drug dedicated to treat intestinal infections would depend on physicochemical properties of the local environment in which interaction with bile salts is one important factor. The synergy between DOC and FZ described here partly explains the success of using FZ in curing bacterial diarrhea (12, 24). To further highlight such an interaction, it has also been reported that rifaximin, an RNA synthesis inhibitor, worked more efficiently in treating diarrhea-producing E. coli in the intestine than in the colon, due to the difference in the bile salt concentration (25). From these observations, we propose the co-administration of DOC and FZ to treat bacterial diarrhea for the patients who have low intestinal concentrations of DOC due to malnourishment, disorders in enterohepatic circulation or intestinal absorption (4). Nonetheless, further investigations are required to justify the validity of that proposal.
In this study, we have also provided some insights into the underlying mechanism of the synergy between DOC and FZ in their antibacterial action against E. coli as a model Gram-negative bacterium. We showed that disruption of tolC or acrA gene caused a considerable decrease in the synergy between DOC and FZ in the corresponding mutants. The TolC protein, whose removal disrupts the synergy more strikingly, appears to be the key determinant of synergy.
The observed difference in the susceptibility to DOC/FZ combination between ΔtolC and ΔacrA mutants is in agreement with the fact that the TolC protein is shared by at least seven multidrug efflux pumps, while AcrA protein acts as the periplasmic connecting bridge for only two (26). Thus, deletion of tolC gene is expected to give rise to a more pronounced effect on the loss of efflux activities than deletion of acrA gene.
Of great interest is how FZ could influence the activity of efflux pumps. The obtained findings indicate that more than two efflux pumps (AcrAB-TolC and AcrAD-TolC systems) were affected by FZ. This observation is reminiscent of a common mechanism which could affect a wide range of efflux pumps simultaneously, namely proton motive force. It has been suggested that nitrofuran compounds during reductive activation might generate nitric oxide (NO) which subsequently inhibits the electron transport chain (ETC), diminishing the proton motive force across the cytoplasmic membrane (20, 21, 23, 27). As a result, many efflux pumps would be de-energized, and become less efficient in extruding toxic compounds.
However, NO generation from nitrofurans in bacterial cells remains to be speculative since the trace of NO has yet to be detected using either biochemical or NO-sensing fluorescence methods, possibly due to the detection limit of the used methods or rapid conversion of NO into other compounds (20, 21). In the present work, we provide evidence for the contribution of NO in the interaction between DOC and FZ via the observation that overexpression of NO-detoxifying enzyme Hmp decreased the synergistic interaction between the two agents. Since some DOC-FZ synergy was still retained after NO-detoxification, other mechanisms, including direct inhibition of the ETC by activated FZ, are involved in the efflux pump inhibition.
In conclusion, we report the synergy between FZ and DOC in inhibiting and/or killing different enterobacterial species. In the terms of underlying mechanisms, much evidence supports the model that FZ negatively influences the activity of many efflux pumps such that DOC could accumulate inside the cell to exert its cytotoxic effect. One possible route is via FZ-derived NO which inhibits the electron transport chain, thus dissipating the energy supply for efflux machineries. Nonetheless, other mechanisms might be involved, remaining to be elucidated.
Materials and methods
Bacterial strains, growth conditions and antibiotics
All bacterial strains and plasmids used in this study were described in Table 1 and Table 2. The introduction of the kanR gene deletion mutations into the wild type strain K1508 from the corresponding Keio collection E. coli K12 knock-out strains (28) was performed using phage P1 transduction, according to the standard procedures (29). To eliminate potential polar effects on downstream genes in the operon, the FRT-flanked kanR cassette was then removed using FLP-mediated recombination as previously described (30). Plasmids derived from the pCA24N bearing the gene of interest were purified from E. coli strains of the ASKA collection containing ORF expression constructs derived from this organism (31) using the ChargeSwitch-Pro Plasmid Miniprep Kit (Thermo Fisher Scientific). The plasmid DNA was then chemically transformed into specific E. coli strains for further work (32). Expression from the pCA24N vector is driven from a T5-lac chimeric promoter. In the case of membrane protein expression (TolC and AcrA) the basal expression from uninduced promoter was used in complementation experiments to avoid toxicity of membrane protein overexpression due to the Sec system saturation, whereas expression of Hmp (a cytosolic NO-detoxifying protein) was induced by 1 mM IPTG.
Bacterial culture was grown in 2xYT medium (BD Difco) at 37°C with shaking at 200 rpm. For preparation of exponential phase cells, fresh overnight culture was 100-fold diluted and incubated to reach the OD600nm of about 0.1-0.3. This cell suspension was then diluted to the desirable concentration depending on specific purposes. Sodium Deoxycholate was a kind gift from New Zealand Pharmaceuticals Ltd. Antibiotics used in this study were purchased from GoldBio. CM4 was purchased from Enamine (catalog number Z49681516).
Checkerboard assay
The checkerboard assay for DOC and FZ was performed on the Corning 384-well microtiter plate with the concentration of DOC ranging from 20000 µg/mL to 0 µg/mL and the concentration of FZ ranging from 10 µg/mL to 0 µg/mL, prepared by 2-fold serial dilution. The concentration range could be adjusted depending on the sensitivity of different bacterial strains and the types of nitrofurans to cover at least 2 × MIC to 0.06 × MIC of each drug.
Each well contained the starting inoculum of approximately 106 CFU/mL, 2 % DMSO and the predefined concentration of each drug in the total volume of 50 µL. The wells containing no drugs and 10 µg/mL tetracycline were used as negative controls and positive controls, respectively. After dispensing the reagents, the plate was pulse centrifuged at 1000 × g to eliminate any bubbles. The plate was then incubated at 30°C and the OD600nm of the sample was monitored for every 1 h within 24h using MultiskanTM GO Microplate Spectrophotometer (Thermo Scientific). Each combination was performed in triplicate. The growth inhibition with the cut-off value of 90 % at the time point 24 h was used to define the MIC of the drug used alone or in combination (33). The fractional inhibitory concentration index (FICI) for the two drugs was calculated as follows:
The interaction between two drugs was interpreted as synergistic if FICI was ≤ 0.5, indifferent if it was > 0.5 and ≤ 4, and antagonistic if it was > 4 (34).
Time kill assay
Exponential phase bacterial culture at about 106 CFU/ml was prepared in the final volume of 10 mL containing 2 % DMSO plus DOC at 2500 µg/ml alone or FZ at 0.5 × MIC µg/mL alone or both drugs. The treatments containing no drug were used as negative controls. The samples were incubated at 30°C with shaking at 200 rpm. At the time points of 0 h, 2 h, 4 h, 6 h, 8 h and 24 h, 500 µL were taken from each treatment and centrifuged at 10000 × g for 15 min before being re-suspended in 100 µL maximum recovery diluent (0.1 % peptone, 0.85 % NaCl). 10 µL of serial dilutions was plated on 2xYT agar followed by overnight incubation at 37°C to determine the cell count. Each treatment was performed in triplicate. The antimicrobial interaction was interpreted as synergistic if the combinatorial treatment caused a killing efficiency ≥2 log higher than the most active agent (35).
Funding information
Vuong Van Hung Le has received funding from Callaghan Innovation PhD Scholarship. This work was supported by Massey University, the New Zealand Ministry of Business, Innovation and Employment and New Zealand Pharmaceuticals LTD.
Acknowledgements
We thank Dr. Anne Midwinter, School of Veterinary Sciences, Massey University, for providing a E. coli human O157 isolate and New Zealand Veterinary Pathology Ltd. for an isolate of a canine E. coli UPEC strain (P50). We are grateful to Fraser Glickman from Rockefeller University High Throughput and Spectroscopy Resource Center for hosting and advice on small-molecule drug screen of a synergy screen and to the National BioResource Project (NBRP) via Genetics Strains Research Center, National Institute of Genetics, Japan, for providing the ASKA collection. The Keio Collection was purchased from Dharmacon via ThermoFisher (Australia).