Eur J Clin Microbiol Infect Dis https://doi.org/10.1007/s10096-017-3114-5
ORIGINAL ARTICLE
Repurposing Zidovudine in combination with Tigecycline for treating carbapenem-resistant Enterobacteriaceae infections S. M. S. Ng 1 & J. S. P. Sioson 1 & J. M. Yap 1 & F. M. Ng 1 & H. S. V. Ching 1 & J. W. P. Teo 2 & R. Jureen 2 & J. Hill 1 & C. S. B. Chia 1
Received: 22 August 2017 / Accepted: 27 September 2017 # Springer-Verlag GmbH Germany 2017
Abstract The global emergence of carbapenem-resistant Enterobacteriaceae (CRE) presents a significant clinical concern, prompting the WHO to prioritize CRE as a top priority pathogen in their 2017 global antibiotic-resistant bacteria priority list. Due to the fast-depleting antibiotic arsenal, clinicians are now resorting to using once-abandoned, highly toxic antibiotics such as the polymyxins and aminoglycosides, creating an urgent need for new antibiotics. Drug repurposing, the application of an approved drug for a new therapeutic indication, is deemed a plausible solution to this problem. A total of 1,163 FDA-approved drugs were screened for activity against a clinical carbapenem- and multidrug-resistant E. coli isolate using a single-point 10 μM assay. Hit compounds were then assessed for their suitability for repurposing. The lead candidate was then tested against a panel of clinical CREs, a bactericidal/static determination assay, a time-kill assay and a checkerboard assay to evaluate its suitability for use in combination with Tigecycline against CRE infections. Three drugs were identified. The lead candidate was determined to be Zidovudine (azidothymidine/AZT), an oral anti-viral drug used for HIV treatment. Zidovudine was shown to be the most promising candidate for use in combination with Tigecycline to treat systemic CRE infections. Further experiments should involve the use of animal infection models.
S. M. S. Ng, J. S. P. Sioson and J. M. Yap contributed equally to this work. * C. S. B. Chia [email protected] 1
Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos #03-01, Singapore 138669, Singapore
2
Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore
Keywords Drug repurposing . Antibiotic . Carbapenem-resistant Enterobacteriaceae . Zidovudine
Introduction Escherichia coli and Klebsiella pneumoniae are Enterobacteriaceae responsible for causing various human infections such as bacteremia, meningitis and pneumonia [1, 2]. Standard treatment involve the use of carbapenems, widely considered as last-line antibiotics with favorable safety profiles [3–7]. However, their extensive use has led to the emergence of carbapenem-resistant Enterobacteriaceae (CRE), presenting a global health threat [1, 8–15]. Carbapenem resistance is caused by a variety of mechanisms. Of particular concern are carbapenemase-producing Enterobacteriaceae where their plasmid-borne carbapenemase genes are easily transferred among Enterobacteriaceae, resulting in rapid dissemination [3, 16–18]. This, coupled to the dearth of new antibiotics entering the market has created a global concern, prompting the US CDC and the WHO to redflag CRE as a high-priority pathogen [19, 20]. Treatment options for CRE infections is limited [21–24]. This includes the off-label use of Tigecycline, a tetracycline analog originally indicated for skin and soft tissue infections [21, 22, 25–27]. Since its introduction in 2005, Tigecyclineresistant CRE strains have emerged [28–30] and in 2010, the FDA issued a black box warning due to an increased incidence of deaths in patients on Tigecycline therapy [31–33]. The lack of new antibiotics has prompted clinicians to resort to old antibiotics like the polymyxins (e.g. Colistin) and aminoglycosides (e.g. Gentamicin) which were abandoned in favor of new antibiotics with better safety profiles like the quinolones (e.g. Ciprofloxacin) and the carbapenems (e.g. Meropenem) [22, 24, 26, 34–36]. For example, Colistin,
Eur J Clin Microbiol Infect Dis
introduced in 1958, is nephrotoxic and neurotoxic [21, 23, 26, 37–40], while Gentamicin, introduced in 1963, is nephrotoxic and ototoxic [26, 37, 41–44]. Furthermore, the past use of these old antibiotics has also resulted in the emergence of drug resistance, further amplifying the need for new antibiotics [45–56]. The dearth of novel antibiotics entering the market has been attributed to regulatory hurdles, high research costs and low investment returns [57–60]. A possible solution is drug repurposing: the application of an approved drug for a new therapeutic indication [61–63]. Since the safety, pharmacological profile and manufacturing process of an approved drug is known, it can be rapidly made available for a new disease indication [62]. Based on this, we screened a library of 1,163 FDA-approved drugs for antibacterial activity against carbapenem-resistant E. coli using a single-concentration (10 μM) assay. After identifying hit compounds and removing known antibiotics, the remaining compounds were subjected to a minimum inhibitory concentration (MIC) assay followed by a toxicity and pharmacological assessment to determine their suitability for repurposing as an antibiotic. Next, the most promising candidate was tested against a CRE panel, followed by a bactericidal/ static determination assay and a time-kill assay. Lastly, a combination (checkerboard) assay was conducted with Tigecycline to determine synergism and suitability for repurposing as an antibiotic to treat CRE infections.
Materials and methods
containing the test compounds in duplicates. The final DMSO concentration was kept at 2.5% in each well. After an overnight incubation at 35 °C, optical density (OD600) of e ac h w el l w a s d et er m i n ed u s i n g t h e M i c r o p l a t e Spectrophotometer (Molecular Devices Spectra Max Plus, USA). Minimum inhibitory concentration (MIC) determination assay MICs of antibiotics and test compounds were determined using the microdilution method from the Clinical and Laboratory Standards Institute (CLSI) guidelines [64]. Briefly, bacteria were grown fresh from frozen stock in MH2 agar at 37 °C. After an overnight incubation, five bacteria colonies were selected to grow in cation-adjusted MH2 broth (BD Biosciences) in a shaker incubator at 37 °C and 220 RPM. Cells were grown to an optical density (OD600) of 0.28– 0.30 using a Microplate Spectrophotometer (Molecular Devices Spectra Max Plus), which corresponds to 1– 2 × 108 CFU/mL. Test compounds were constituted into 4 mM DMSO stock solutions and then subjected to two-fold serial dilution in a 96-well plate with concentrations ranging from 100 to 0.2 μM in duplicates. The amount of 50 μL of microbial culture containing ~1–2 × 106 CFU/mL of microbes in the respective broths was introduced into each well containing 50 μL of compound. After an overnight incubation at 35 °C, OD600 measurements were conducted using the Microplate Spectrophotometer. The MIC was defined as the lowest compound concentration (μM) required to stop bacterial growth.
Drug library, antibiotics and bacteria Bactericidal/static determination assay The FDA-approved drug library was purchased from Selleckchem (catalog #L1300). All antibiotics listed in Table 1 were purchased from Sigma-Aldrich. Bacteria were either purchased from ATCC (USA) or obtained from the Singapore National University Hospital (NUH) clinical microbiology unit. Single-concentration bacteria growth inhibition screening assay All bacteria were grown from frozen glycerol stock on Mueller Hinton 2 (MH2) agar overnight at 37 °C. Five colonies were selected from the agar plate and grown in cationadjusted MH2 broth (BD Biosciences) in a shaker incubator at 37 °C and 220 RPM. Cells were grown to 1–2 × 108 CFU/mL and diluted 1:100 in MH2 broth. 50 μL of test compounds pre-dissolved in MH2 broth at 20 μM concentrations were added in duplicates into each well in a 96-well plate. A total of 50 μL of bacteria inoculums containing ~1 × 106 CFU/mL of bacteria in MH2 broth was introduced into each well
After the MICs of test compounds were determined using the microdilution method described earlier, the entire well contents (100 μL) corresponding to 1×, 2×, 4× and 8× the MICs of the compound were transferred and spread on fresh sterile MH2 agar plates. The plates were incubated overnight at 37 °C and the number of CFU were observed the next day. The minimum bactericidal concentration (MBC) of a compound was defined as the lowest compound concentration required to cause ≥99.99% cell death within 24 h and a compound was classified as bactericidal if its MBC lies between 1× to 4× of its MIC [65]. Time-kill assay Bacteria cells were grown to 1 × 108 CFU/mL in MH2 broth and then diluted 200-fold into 5 mL aliquots to a density of 5 × 105 CFU/mL in a flask. Test compounds pre-dissolved in DMSO corresponding to 4× their MICs were then added into each flask with a final DMSO concentration of 2.5% v/v. The
Eur J Clin Microbiol Infect Dis Table 1
MICs (μM) of hit compounds and FDA-approved antibiotics against carbapenem-resistant E. coli (ATCC-BAA-2469)
Hit compounds
Original indication
Administration route(s)
MIC (μM)
MBC (μM)
Azacitidine
Cancer
Intravenous, subcutaneous
6.25
50
Floxuridine
Cancer
Intraarterial
6.25
50
Zidovudine
Anti-viral
Intravenous, oral
3.125
6.25
Colistin
Antibiotic
Intramuscular, intravenous, topical
0.78
0.78
Ciprofloxacin Gentamicin
Antibiotic Antibiotic
Ophthalmic, oral Intramuscular, intravenous, ophthalmic, topical
>100 >100
Not applicable Not applicable
Meropenem Tigecycline
Antibiotic Antibiotic
Intravenous Intravenous
>100 0.78
Not applicable 3.125
Approved antibiotics
negative control was 2.5% DMSO. The flasks were incubated in a shaking incubator (220 RPM) at 37 °C. Samples (100 μL) were taken hourly from the flasks between t = 0 and t = 6 h. The samples were serially diluted in MH2 broth before plating on sterile MH2 agar and incubated overnight at 37 °C. A bacteria count for each plate was conducted the next day. A graph of Log CFU/mL was plotted against time to obtain the time-kill graph. Combination (checkerboard) assay Antibiotic synergism against carbapenem-resistant E. coli (ATCC-BAA-2469) was determined using the checkerboard method described previously [66]. Test compounds were tested over a concentration range: Tigecycline (0.00–6.25 μM), Zidovudine (0.00–12.5 μM). 50 mL of 1 × 106 CFU/mL of E. coli were inoculated into the wells to make a final volume of 100 μL containing 5 × 105 CFU/mL per well followed by an overnight incubation at 37 °C. The fractional inhibitory concentration index (FICI) was calculated for all the clear interface wells using the formula: FICI = FICT + FICZ where FICT is the MIC of Tigecycline used in combination/MIC of Tigecycline alone and FICZ is the MIC of Zidovudine used in combination/MIC of Zidovudine alone. A particular drug combination is considered synergistic when the FICI is ≤0.5; indifferent when the FICI is >0.5 but ≤4; and antagonistic when the FICI is >4 [66].
MIC determination The MICs for all three hit compounds against carbapenemresistant E. coli (ATCC-BAA-2469) ranged between 3.125 and 6.25 μM (Table 1). In comparison, FDA-approved antibiotics Colistin and Tigecycline were found to be equipotent (MICs 0.78 μM), in agreement with a previous report [67] while Ciprofloxacin, Gentamicin and Meropenem were found to be inactive (MICs >100 μM). Bactericidal/static determination assay The three hit compounds were subjected to a bactericidal/ static determination assay with Colistin and Tigecycline as 1x MIC
2x MIC
4x MIC
8x MIC
Azacitidine
Floxuridine
Zidovudine
Colistin
Results Drug library screen
Tigecycline
The 1163 drug library screen yielded 11 hits. After removing known antibiotics, three antibacterial compounds were identified: Azacitidine, Floxuridine and Zidovudine.
Fig. 1 Overnight growth of carbapenem-resistant E. coli (ATCC-BAA2469) after treatment with Azacitidine, Floxuridine, Zidovudine, Colistin and Tigecycline at 1×, 2×, 4× and 8× MIC
Log (CFU/ml)
Eur J Clin Microbiol Infect Dis 9 8 7 6 5 4 3 2 1 0
DMSO
Combination (checkerboard) assay
Tigecycline 4x MIC
Zidovudine 4x MIC Colistin 4x MIC 0
1
2
3
4
5
6
A checkerboard assay was conducted based on an established protocol using Zidovudine and Tigecycline [66]. Colistin was not considered due to its toxic side-effects [21, 26, 37–40]. Results revealed Zidovudine and Tigecycline to be indifferent at various concentrations with FICI values ranging between 0.63 and 1.25 (Fig. 3).
Time (h.)
Fig. 2 Time-kill assay of Colistin, Tigecycline and Zidovudine using carbapenem-resistant E. coli (ATCC-BAA-2469)
Discussion comparators (Fig. 1). The MBCs for Azacitidine and Floxuridine were found to be 50 μM (8× MIC), indicating both were bacteriostatic while Zidovudine was 6.25 μM (2× MIC), indicating that it was bactericidal [65]. The MBCs of Colistin and Tigecycline were 0.78 and 3.125 μM (1× and 4× MIC respectively), indicating both were also bactericidal (Table 1).
By screening a 1163 drug library, 11 FDA-approved drugs were identified to be active against carbapenem-resistant E. coli. After excluding known antibiotics, three drugs: Azacitidine, Floxuridine and Zidovudine were identified for further investigation (MICs 6.25 μM; Table 1). Azacitidine
CRE panel MICs The bioactivity of Zidovudine against a panel of E. coli and K. pneumoniae clinical isolates is summarized in Table 2. Zidovudine, Colistin and Tigecycline were found to be active against all clinical isolates with MICs ranging from 0.39 to 6.25 μM. In contrast, Ciprofloxacin and Gentamicin were inactive (MIC ≥50 μM) against some isolates. Time-kill assay E. coli killing kinetics of Zidovudine was performed using a time-kill assay following a previous protocol [68]. Results revealed Zidovudine to be rapidly bactericidal, achieving a 6-log kill within 3 h (Fig. 2). Table 2
Azacitidine (5-azacytidine/5AZC/Vidaza™) is a nucleoside analogue approved in 2004 for treating various blood cancers [69–71]. The recommended daily dose is 75 mg/m2 (~120 mg for a 60 kg adult) for 7 days [69, 72]. After intravenous administration, Azacitidine is rapidly hydrolysed with a plasma half-life of ~22 min [69, 73]. In comparison, Colistin, in its colistimethate pro-drug form, and Tigecycline have plasma half-lives exceeding 4 and 37 h, respectively [74, 75]. In our view, Azacitidine’s short half-life is unsuitable for repurposing as an antibacterial agent. In addition, the major concern using Azacitidine as an antibacterial agent is its myelosuppressive side-effect manifested by neutropenia which diminishes a patient’s ability to fight bacterial infections [69]. Indeed, the European Medicines Agency (EMA) reported patients on
MICs (μM) of FDA-approved antibiotics and zidovudine against various Enterobacteriaceae clinical isolates
Bacteria
ATCC or NUH number
Ciprofloxacin
Colistin
Gentamicin
Meropenem
Tigecycline
Zidovudine
E. coli E. coli E. coli E. coli E. coli E. coli E. coli K. pneumoniae
BAA-2355 BAA-2340 BAA-2469 BAA-2523 NUH-376 NUH-1289 NUH-5684 700603
>100 >100 >100 0.78 50 >100 25 0.78
0.78 0.78 0.78 0.78 3.125 0.78 0.78 0.78
>100 3.125 >100 >100 >100 1.56 1.56 12.5
0.39 25 >100 3.125 >100 >100 50 0.39
0.78 0.39 0.78 0.78 1.56 1.56 1.56 6.25
3.125 3.125 3.125 1.56 6.25 1.56 0.39 6.25
K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae
BAA-2342 BAA-2524 NUH-34 NUH-2751 NUH-6751
>100 0.2 >100 >100 0.2
1.56 0.39 1.56 1.56 0.78
12.5 0.78 >100 0.78 3.125
>100 25 >100 >100 50
3.125 3.125 3.125 6.25 0.78
3.125 6.25 6.25 6.25 3.125
0.78 0.39
1.06
1.00
0.20
0.75
1.25
0.10
0.63
1.13
0.00
Tigecycline ( M)
1.56
3.13
6.25
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0.00
0.20
0.39
0.78 1.56 Zidovudine ( M)
3.13
6.25
12.5
Fig. 3 Schematic diagram of the Tigecycline-Zidovudine checkerboard assay using carbapenem-resistant E. coli (ATCC-BAA-2469). Grey areas represent cloudy wells (viable bacteria) while white areas represent clear wells. Numbers in bold text represent the FICI value of each individual well. A drug combination is classified synergistic when its FICI is ≤0.5
Azacitidine treatment very commonly contracted pneumonia (≥1 in 10) in their public assessment report [76]. Lastly, Azacitidine was observed to be bacteriostatic against E. coli. (Fig. 1). With these unfavorable characteristics, Azacitidine lacks potential for repurposing as an antibacterial drug. Floxuridine Floxuridine (5-fluorodeoxyuridine/5FDU/FUDR®) is an intraarterially-administered nucleoside analog approved in 1970 for treating colorectal liver metastases and has been reported to act synergistically with Zidovudine against Enterobacteriaceae [77, 78]. Floxuridine inhibits thymidylate synthase, blocking thymidine production and consequently stopping DNA and RNA synthesis, resulting in cell death [77]. The drug is highly toxic with a recommended daily dose of 0.3 mg/kg (18 mg for a 60 kg adult) over 14 days [79] and possesses a very short plasma half-life of ~15 min [80]. Like Azacitidine, it is myelosuppressive and was observed to be bacteriostatic against E. coli. (Fig. 1). Based on these unfavorable characteristics, Floxuridine lacks potential for repurposing as an antibacterial drug. Zidovudine Zidovudine (azidothymidine/AZT/Retrovir®) is an oral or intravenously-administered nucleoside analog approved in 1987 for treating human immunodeficiency virus (HIV) infection [81, 82]. In an infected cell, Zidovudine is phosphorylated
by cellular kinases to form Zidovudine triphosphate which functions as an HIV reverse transcriptase inhibitor, arresting viral DNA production and ultimately viral replication [81–84]. Zidovudine was found to be bactericidal against Enterobacteriaceae with reported MICs against E. coli and K. pneumoniae of 0.0025 to 1.0 μg/mL (0.01 to 3.7 μM) and 0.025 to 3.1 μg/mL (0.1 to 11.6 μM) [85, 86], in agreement with our experimental results (Table 2 and Fig. 1). Zidovudine’s antibacterial activity is thought to be derived from its ability to act as a chain terminator following bacterial DNA incorporation after being phosphorylated by bacterial kinases [85, 86]. Compared to Azacitidine and Floxuridine, Zidovudine has a much better safety profile with a recommended daily dose of 600 mg over a patient’s life-time due to its short plasma halflife (1.1 h) and 100-fold selectivity of its triphosphate analog for HIV reverse transcriptase over human DNA polymerase [81, 82, 84, 87, 88]. The most commonly reported side-effects are anorexia, headaches, myalgia, nausea and vomiting [84]. Although long-term dosing side-effects include anemia, arthralgia, myopathy and neutropenia [84], antibacterial therapy are short-term (< 2 weeks) so these side-effects are unlikely to be of concern. In mice, the median oral lethal dose was found to be 3000 mg/kg [89]. In humans, two cases of oral overdosages of 20 g were reported without adverse effects or bonemarrow suppression [90, 91], suggesting that Zidovudine can be repurposed as an oral antibacterial agent for treating CRE bacteremia. In addition, Zidovudine also penetrates the bloodbrain barrier and can thus potentially be used for treating CRE meningitis [87, 88]. Mouse model E. coli systemic infection studies involving orally-administered Zidovudine (10 mg/kg body weight) showed that the drug was therapeutically efficacious [92]. Zidovudine levels in mouse plasma were found to be approximately 8 μg/mL (30 μM) an hour after oral administration, exceeding the 0.39–6.25 μM in vitro MICs against carbapenem-resistant E. coli and K. pneumoniae (Table 2). This suggests that therapeutically-active concentrations of Zidovudine can be achieved after oral administration [92]. An ideal antibacterial drug should kill bacteria quickly as this reduces the chances of resistance development and can potentially reduce treatment duration. Our time-kill assay (Fig. 2) revealed Zidovudine to be rapidly bactericial against E. coli, achieving a 6-log kill at 4× MIC within 3 h, in agreement with the literature [86]. Although Colistin exhibited faster killing kinetics (6-log kill at 4× MIC) in 1 h, its nephrotoxic and neurotoxic side-effects must be taken into consideration, especially for patients with impaired renal function [37–40]. Drugs that target a specific enzyme are known to be prone to resistance development [93]. Indeed, Zidovudine-resistant E. coli has been reported in HIV patients on Zidovudine
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therapy [94]. In vitro experiments revealed E. coli can become resistant within 24 h due to the loss of thymidine kinase activity which abrogates the bacteria’s ability to activate Zidovudine into the biologically-active triphosphate form [95, 96]. A strategy to circumvent resistance development is to administer Zidovudine in combination with another antibiotic. Earlier antibiotic combination studies have shown that Zidovudine acted synergistically with Colistin or Gentamicin against E. coli [83, 97]. Colistin and Tigecycline became potential combination candidates with Zidovudine as they were the only drugs active against all CRE isolates in Table 2. However, we decided to take Colistin out due to its nephroand neurotoxic side-effects. Thus, Zidovudine was paired with Tigecycline for the checkerboard assay. Results revealed that various Zidovudine-Tigecycline combinations were indifferent (FICI between 0.5 and 4.0) at various concentrations (Fig. 3), suggesting that Zidovudine and Tigecycline can be administered in combination to treat CRE infections. In conclusion, our drug library screen identified Zidovudine as a plausible antibacterial agent that can potentially be repurposed as an antibacterial agent. If so, to reduce the chances of resistance development, we would recommend Zidovudine be used in combination with a drug for which the bacteria has tested susceptible, for example, Tigecycline, when treating infections caused by CREs.
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14. Acknowledgements The authors thank the Agency for Science, Technology and Research (A*STAR) Biomedical Research Council for funding.
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Compliance with ethical standards 16. Ethical statement Not required as no humans or animals were involved in this study. 17. Conflicts of interest The authors of this paper declare no conflicts of interest.
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ORIGINAL ARTICLE
Repurposing Zidovudine in combination with Tigecycline for treating carbapenem-resistant Enterobacteriaceae infections S. M. S. Ng 1 & J. S. P. Sioson 1 & J. M. Yap 1 & F. M. Ng 1 & H. S. V. Ching 1 & J. W. P. Teo 2 & R. Jureen 2 & J. Hill 1 & C. S. B. Chia 1
Received: 22 August 2017 / Accepted: 27 September 2017 # Springer-Verlag GmbH Germany 2017
Abstract The global emergence of carbapenem-resistant Enterobacteriaceae (CRE) presents a significant clinical concern, prompting the WHO to prioritize CRE as a top priority pathogen in their 2017 global antibiotic-resistant bacteria priority list. Due to the fast-depleting antibiotic arsenal, clinicians are now resorting to using once-abandoned, highly toxic antibiotics such as the polymyxins and aminoglycosides, creating an urgent need for new antibiotics. Drug repurposing, the application of an approved drug for a new therapeutic indication, is deemed a plausible solution to this problem. A total of 1,163 FDA-approved drugs were screened for activity against a clinical carbapenem- and multidrug-resistant E. coli isolate using a single-point 10 μM assay. Hit compounds were then assessed for their suitability for repurposing. The lead candidate was then tested against a panel of clinical CREs, a bactericidal/static determination assay, a time-kill assay and a checkerboard assay to evaluate its suitability for use in combination with Tigecycline against CRE infections. Three drugs were identified. The lead candidate was determined to be Zidovudine (azidothymidine/AZT), an oral anti-viral drug used for HIV treatment. Zidovudine was shown to be the most promising candidate for use in combination with Tigecycline to treat systemic CRE infections. Further experiments should involve the use of animal infection models.
S. M. S. Ng, J. S. P. Sioson and J. M. Yap contributed equally to this work. * C. S. B. Chia [email protected] 1
Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos #03-01, Singapore 138669, Singapore
2
Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore
Keywords Drug repurposing . Antibiotic . Carbapenem-resistant Enterobacteriaceae . Zidovudine
Introduction Escherichia coli and Klebsiella pneumoniae are Enterobacteriaceae responsible for causing various human infections such as bacteremia, meningitis and pneumonia [1, 2]. Standard treatment involve the use of carbapenems, widely considered as last-line antibiotics with favorable safety profiles [3–7]. However, their extensive use has led to the emergence of carbapenem-resistant Enterobacteriaceae (CRE), presenting a global health threat [1, 8–15]. Carbapenem resistance is caused by a variety of mechanisms. Of particular concern are carbapenemase-producing Enterobacteriaceae where their plasmid-borne carbapenemase genes are easily transferred among Enterobacteriaceae, resulting in rapid dissemination [3, 16–18]. This, coupled to the dearth of new antibiotics entering the market has created a global concern, prompting the US CDC and the WHO to redflag CRE as a high-priority pathogen [19, 20]. Treatment options for CRE infections is limited [21–24]. This includes the off-label use of Tigecycline, a tetracycline analog originally indicated for skin and soft tissue infections [21, 22, 25–27]. Since its introduction in 2005, Tigecyclineresistant CRE strains have emerged [28–30] and in 2010, the FDA issued a black box warning due to an increased incidence of deaths in patients on Tigecycline therapy [31–33]. The lack of new antibiotics has prompted clinicians to resort to old antibiotics like the polymyxins (e.g. Colistin) and aminoglycosides (e.g. Gentamicin) which were abandoned in favor of new antibiotics with better safety profiles like the quinolones (e.g. Ciprofloxacin) and the carbapenems (e.g. Meropenem) [22, 24, 26, 34–36]. For example, Colistin,
Eur J Clin Microbiol Infect Dis
introduced in 1958, is nephrotoxic and neurotoxic [21, 23, 26, 37–40], while Gentamicin, introduced in 1963, is nephrotoxic and ototoxic [26, 37, 41–44]. Furthermore, the past use of these old antibiotics has also resulted in the emergence of drug resistance, further amplifying the need for new antibiotics [45–56]. The dearth of novel antibiotics entering the market has been attributed to regulatory hurdles, high research costs and low investment returns [57–60]. A possible solution is drug repurposing: the application of an approved drug for a new therapeutic indication [61–63]. Since the safety, pharmacological profile and manufacturing process of an approved drug is known, it can be rapidly made available for a new disease indication [62]. Based on this, we screened a library of 1,163 FDA-approved drugs for antibacterial activity against carbapenem-resistant E. coli using a single-concentration (10 μM) assay. After identifying hit compounds and removing known antibiotics, the remaining compounds were subjected to a minimum inhibitory concentration (MIC) assay followed by a toxicity and pharmacological assessment to determine their suitability for repurposing as an antibiotic. Next, the most promising candidate was tested against a CRE panel, followed by a bactericidal/ static determination assay and a time-kill assay. Lastly, a combination (checkerboard) assay was conducted with Tigecycline to determine synergism and suitability for repurposing as an antibiotic to treat CRE infections.
Materials and methods
containing the test compounds in duplicates. The final DMSO concentration was kept at 2.5% in each well. After an overnight incubation at 35 °C, optical density (OD600) of e ac h w el l w a s d et er m i n ed u s i n g t h e M i c r o p l a t e Spectrophotometer (Molecular Devices Spectra Max Plus, USA). Minimum inhibitory concentration (MIC) determination assay MICs of antibiotics and test compounds were determined using the microdilution method from the Clinical and Laboratory Standards Institute (CLSI) guidelines [64]. Briefly, bacteria were grown fresh from frozen stock in MH2 agar at 37 °C. After an overnight incubation, five bacteria colonies were selected to grow in cation-adjusted MH2 broth (BD Biosciences) in a shaker incubator at 37 °C and 220 RPM. Cells were grown to an optical density (OD600) of 0.28– 0.30 using a Microplate Spectrophotometer (Molecular Devices Spectra Max Plus), which corresponds to 1– 2 × 108 CFU/mL. Test compounds were constituted into 4 mM DMSO stock solutions and then subjected to two-fold serial dilution in a 96-well plate with concentrations ranging from 100 to 0.2 μM in duplicates. The amount of 50 μL of microbial culture containing ~1–2 × 106 CFU/mL of microbes in the respective broths was introduced into each well containing 50 μL of compound. After an overnight incubation at 35 °C, OD600 measurements were conducted using the Microplate Spectrophotometer. The MIC was defined as the lowest compound concentration (μM) required to stop bacterial growth.
Drug library, antibiotics and bacteria Bactericidal/static determination assay The FDA-approved drug library was purchased from Selleckchem (catalog #L1300). All antibiotics listed in Table 1 were purchased from Sigma-Aldrich. Bacteria were either purchased from ATCC (USA) or obtained from the Singapore National University Hospital (NUH) clinical microbiology unit. Single-concentration bacteria growth inhibition screening assay All bacteria were grown from frozen glycerol stock on Mueller Hinton 2 (MH2) agar overnight at 37 °C. Five colonies were selected from the agar plate and grown in cationadjusted MH2 broth (BD Biosciences) in a shaker incubator at 37 °C and 220 RPM. Cells were grown to 1–2 × 108 CFU/mL and diluted 1:100 in MH2 broth. 50 μL of test compounds pre-dissolved in MH2 broth at 20 μM concentrations were added in duplicates into each well in a 96-well plate. A total of 50 μL of bacteria inoculums containing ~1 × 106 CFU/mL of bacteria in MH2 broth was introduced into each well
After the MICs of test compounds were determined using the microdilution method described earlier, the entire well contents (100 μL) corresponding to 1×, 2×, 4× and 8× the MICs of the compound were transferred and spread on fresh sterile MH2 agar plates. The plates were incubated overnight at 37 °C and the number of CFU were observed the next day. The minimum bactericidal concentration (MBC) of a compound was defined as the lowest compound concentration required to cause ≥99.99% cell death within 24 h and a compound was classified as bactericidal if its MBC lies between 1× to 4× of its MIC [65]. Time-kill assay Bacteria cells were grown to 1 × 108 CFU/mL in MH2 broth and then diluted 200-fold into 5 mL aliquots to a density of 5 × 105 CFU/mL in a flask. Test compounds pre-dissolved in DMSO corresponding to 4× their MICs were then added into each flask with a final DMSO concentration of 2.5% v/v. The
Eur J Clin Microbiol Infect Dis Table 1
MICs (μM) of hit compounds and FDA-approved antibiotics against carbapenem-resistant E. coli (ATCC-BAA-2469)
Hit compounds
Original indication
Administration route(s)
MIC (μM)
MBC (μM)
Azacitidine
Cancer
Intravenous, subcutaneous
6.25
50
Floxuridine
Cancer
Intraarterial
6.25
50
Zidovudine
Anti-viral
Intravenous, oral
3.125
6.25
Colistin
Antibiotic
Intramuscular, intravenous, topical
0.78
0.78
Ciprofloxacin Gentamicin
Antibiotic Antibiotic
Ophthalmic, oral Intramuscular, intravenous, ophthalmic, topical
>100 >100
Not applicable Not applicable
Meropenem Tigecycline
Antibiotic Antibiotic
Intravenous Intravenous
>100 0.78
Not applicable 3.125
Approved antibiotics
negative control was 2.5% DMSO. The flasks were incubated in a shaking incubator (220 RPM) at 37 °C. Samples (100 μL) were taken hourly from the flasks between t = 0 and t = 6 h. The samples were serially diluted in MH2 broth before plating on sterile MH2 agar and incubated overnight at 37 °C. A bacteria count for each plate was conducted the next day. A graph of Log CFU/mL was plotted against time to obtain the time-kill graph. Combination (checkerboard) assay Antibiotic synergism against carbapenem-resistant E. coli (ATCC-BAA-2469) was determined using the checkerboard method described previously [66]. Test compounds were tested over a concentration range: Tigecycline (0.00–6.25 μM), Zidovudine (0.00–12.5 μM). 50 mL of 1 × 106 CFU/mL of E. coli were inoculated into the wells to make a final volume of 100 μL containing 5 × 105 CFU/mL per well followed by an overnight incubation at 37 °C. The fractional inhibitory concentration index (FICI) was calculated for all the clear interface wells using the formula: FICI = FICT + FICZ where FICT is the MIC of Tigecycline used in combination/MIC of Tigecycline alone and FICZ is the MIC of Zidovudine used in combination/MIC of Zidovudine alone. A particular drug combination is considered synergistic when the FICI is ≤0.5; indifferent when the FICI is >0.5 but ≤4; and antagonistic when the FICI is >4 [66].
MIC determination The MICs for all three hit compounds against carbapenemresistant E. coli (ATCC-BAA-2469) ranged between 3.125 and 6.25 μM (Table 1). In comparison, FDA-approved antibiotics Colistin and Tigecycline were found to be equipotent (MICs 0.78 μM), in agreement with a previous report [67] while Ciprofloxacin, Gentamicin and Meropenem were found to be inactive (MICs >100 μM). Bactericidal/static determination assay The three hit compounds were subjected to a bactericidal/ static determination assay with Colistin and Tigecycline as 1x MIC
2x MIC
4x MIC
8x MIC
Azacitidine
Floxuridine
Zidovudine
Colistin
Results Drug library screen
Tigecycline
The 1163 drug library screen yielded 11 hits. After removing known antibiotics, three antibacterial compounds were identified: Azacitidine, Floxuridine and Zidovudine.
Fig. 1 Overnight growth of carbapenem-resistant E. coli (ATCC-BAA2469) after treatment with Azacitidine, Floxuridine, Zidovudine, Colistin and Tigecycline at 1×, 2×, 4× and 8× MIC
Log (CFU/ml)
Eur J Clin Microbiol Infect Dis 9 8 7 6 5 4 3 2 1 0
DMSO
Combination (checkerboard) assay
Tigecycline 4x MIC
Zidovudine 4x MIC Colistin 4x MIC 0
1
2
3
4
5
6
A checkerboard assay was conducted based on an established protocol using Zidovudine and Tigecycline [66]. Colistin was not considered due to its toxic side-effects [21, 26, 37–40]. Results revealed Zidovudine and Tigecycline to be indifferent at various concentrations with FICI values ranging between 0.63 and 1.25 (Fig. 3).
Time (h.)
Fig. 2 Time-kill assay of Colistin, Tigecycline and Zidovudine using carbapenem-resistant E. coli (ATCC-BAA-2469)
Discussion comparators (Fig. 1). The MBCs for Azacitidine and Floxuridine were found to be 50 μM (8× MIC), indicating both were bacteriostatic while Zidovudine was 6.25 μM (2× MIC), indicating that it was bactericidal [65]. The MBCs of Colistin and Tigecycline were 0.78 and 3.125 μM (1× and 4× MIC respectively), indicating both were also bactericidal (Table 1).
By screening a 1163 drug library, 11 FDA-approved drugs were identified to be active against carbapenem-resistant E. coli. After excluding known antibiotics, three drugs: Azacitidine, Floxuridine and Zidovudine were identified for further investigation (MICs 6.25 μM; Table 1). Azacitidine
CRE panel MICs The bioactivity of Zidovudine against a panel of E. coli and K. pneumoniae clinical isolates is summarized in Table 2. Zidovudine, Colistin and Tigecycline were found to be active against all clinical isolates with MICs ranging from 0.39 to 6.25 μM. In contrast, Ciprofloxacin and Gentamicin were inactive (MIC ≥50 μM) against some isolates. Time-kill assay E. coli killing kinetics of Zidovudine was performed using a time-kill assay following a previous protocol [68]. Results revealed Zidovudine to be rapidly bactericidal, achieving a 6-log kill within 3 h (Fig. 2). Table 2
Azacitidine (5-azacytidine/5AZC/Vidaza™) is a nucleoside analogue approved in 2004 for treating various blood cancers [69–71]. The recommended daily dose is 75 mg/m2 (~120 mg for a 60 kg adult) for 7 days [69, 72]. After intravenous administration, Azacitidine is rapidly hydrolysed with a plasma half-life of ~22 min [69, 73]. In comparison, Colistin, in its colistimethate pro-drug form, and Tigecycline have plasma half-lives exceeding 4 and 37 h, respectively [74, 75]. In our view, Azacitidine’s short half-life is unsuitable for repurposing as an antibacterial agent. In addition, the major concern using Azacitidine as an antibacterial agent is its myelosuppressive side-effect manifested by neutropenia which diminishes a patient’s ability to fight bacterial infections [69]. Indeed, the European Medicines Agency (EMA) reported patients on
MICs (μM) of FDA-approved antibiotics and zidovudine against various Enterobacteriaceae clinical isolates
Bacteria
ATCC or NUH number
Ciprofloxacin
Colistin
Gentamicin
Meropenem
Tigecycline
Zidovudine
E. coli E. coli E. coli E. coli E. coli E. coli E. coli K. pneumoniae
BAA-2355 BAA-2340 BAA-2469 BAA-2523 NUH-376 NUH-1289 NUH-5684 700603
>100 >100 >100 0.78 50 >100 25 0.78
0.78 0.78 0.78 0.78 3.125 0.78 0.78 0.78
>100 3.125 >100 >100 >100 1.56 1.56 12.5
0.39 25 >100 3.125 >100 >100 50 0.39
0.78 0.39 0.78 0.78 1.56 1.56 1.56 6.25
3.125 3.125 3.125 1.56 6.25 1.56 0.39 6.25
K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae
BAA-2342 BAA-2524 NUH-34 NUH-2751 NUH-6751
>100 0.2 >100 >100 0.2
1.56 0.39 1.56 1.56 0.78
12.5 0.78 >100 0.78 3.125
>100 25 >100 >100 50
3.125 3.125 3.125 6.25 0.78
3.125 6.25 6.25 6.25 3.125
0.78 0.39
1.06
1.00
0.20
0.75
1.25
0.10
0.63
1.13
0.00
Tigecycline ( M)
1.56
3.13
6.25
Eur J Clin Microbiol Infect Dis
0.00
0.20
0.39
0.78 1.56 Zidovudine ( M)
3.13
6.25
12.5
Fig. 3 Schematic diagram of the Tigecycline-Zidovudine checkerboard assay using carbapenem-resistant E. coli (ATCC-BAA-2469). Grey areas represent cloudy wells (viable bacteria) while white areas represent clear wells. Numbers in bold text represent the FICI value of each individual well. A drug combination is classified synergistic when its FICI is ≤0.5
Azacitidine treatment very commonly contracted pneumonia (≥1 in 10) in their public assessment report [76]. Lastly, Azacitidine was observed to be bacteriostatic against E. coli. (Fig. 1). With these unfavorable characteristics, Azacitidine lacks potential for repurposing as an antibacterial drug. Floxuridine Floxuridine (5-fluorodeoxyuridine/5FDU/FUDR®) is an intraarterially-administered nucleoside analog approved in 1970 for treating colorectal liver metastases and has been reported to act synergistically with Zidovudine against Enterobacteriaceae [77, 78]. Floxuridine inhibits thymidylate synthase, blocking thymidine production and consequently stopping DNA and RNA synthesis, resulting in cell death [77]. The drug is highly toxic with a recommended daily dose of 0.3 mg/kg (18 mg for a 60 kg adult) over 14 days [79] and possesses a very short plasma half-life of ~15 min [80]. Like Azacitidine, it is myelosuppressive and was observed to be bacteriostatic against E. coli. (Fig. 1). Based on these unfavorable characteristics, Floxuridine lacks potential for repurposing as an antibacterial drug. Zidovudine Zidovudine (azidothymidine/AZT/Retrovir®) is an oral or intravenously-administered nucleoside analog approved in 1987 for treating human immunodeficiency virus (HIV) infection [81, 82]. In an infected cell, Zidovudine is phosphorylated
by cellular kinases to form Zidovudine triphosphate which functions as an HIV reverse transcriptase inhibitor, arresting viral DNA production and ultimately viral replication [81–84]. Zidovudine was found to be bactericidal against Enterobacteriaceae with reported MICs against E. coli and K. pneumoniae of 0.0025 to 1.0 μg/mL (0.01 to 3.7 μM) and 0.025 to 3.1 μg/mL (0.1 to 11.6 μM) [85, 86], in agreement with our experimental results (Table 2 and Fig. 1). Zidovudine’s antibacterial activity is thought to be derived from its ability to act as a chain terminator following bacterial DNA incorporation after being phosphorylated by bacterial kinases [85, 86]. Compared to Azacitidine and Floxuridine, Zidovudine has a much better safety profile with a recommended daily dose of 600 mg over a patient’s life-time due to its short plasma halflife (1.1 h) and 100-fold selectivity of its triphosphate analog for HIV reverse transcriptase over human DNA polymerase [81, 82, 84, 87, 88]. The most commonly reported side-effects are anorexia, headaches, myalgia, nausea and vomiting [84]. Although long-term dosing side-effects include anemia, arthralgia, myopathy and neutropenia [84], antibacterial therapy are short-term (< 2 weeks) so these side-effects are unlikely to be of concern. In mice, the median oral lethal dose was found to be 3000 mg/kg [89]. In humans, two cases of oral overdosages of 20 g were reported without adverse effects or bonemarrow suppression [90, 91], suggesting that Zidovudine can be repurposed as an oral antibacterial agent for treating CRE bacteremia. In addition, Zidovudine also penetrates the bloodbrain barrier and can thus potentially be used for treating CRE meningitis [87, 88]. Mouse model E. coli systemic infection studies involving orally-administered Zidovudine (10 mg/kg body weight) showed that the drug was therapeutically efficacious [92]. Zidovudine levels in mouse plasma were found to be approximately 8 μg/mL (30 μM) an hour after oral administration, exceeding the 0.39–6.25 μM in vitro MICs against carbapenem-resistant E. coli and K. pneumoniae (Table 2). This suggests that therapeutically-active concentrations of Zidovudine can be achieved after oral administration [92]. An ideal antibacterial drug should kill bacteria quickly as this reduces the chances of resistance development and can potentially reduce treatment duration. Our time-kill assay (Fig. 2) revealed Zidovudine to be rapidly bactericial against E. coli, achieving a 6-log kill at 4× MIC within 3 h, in agreement with the literature [86]. Although Colistin exhibited faster killing kinetics (6-log kill at 4× MIC) in 1 h, its nephrotoxic and neurotoxic side-effects must be taken into consideration, especially for patients with impaired renal function [37–40]. Drugs that target a specific enzyme are known to be prone to resistance development [93]. Indeed, Zidovudine-resistant E. coli has been reported in HIV patients on Zidovudine
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therapy [94]. In vitro experiments revealed E. coli can become resistant within 24 h due to the loss of thymidine kinase activity which abrogates the bacteria’s ability to activate Zidovudine into the biologically-active triphosphate form [95, 96]. A strategy to circumvent resistance development is to administer Zidovudine in combination with another antibiotic. Earlier antibiotic combination studies have shown that Zidovudine acted synergistically with Colistin or Gentamicin against E. coli [83, 97]. Colistin and Tigecycline became potential combination candidates with Zidovudine as they were the only drugs active against all CRE isolates in Table 2. However, we decided to take Colistin out due to its nephroand neurotoxic side-effects. Thus, Zidovudine was paired with Tigecycline for the checkerboard assay. Results revealed that various Zidovudine-Tigecycline combinations were indifferent (FICI between 0.5 and 4.0) at various concentrations (Fig. 3), suggesting that Zidovudine and Tigecycline can be administered in combination to treat CRE infections. In conclusion, our drug library screen identified Zidovudine as a plausible antibacterial agent that can potentially be repurposed as an antibacterial agent. If so, to reduce the chances of resistance development, we would recommend Zidovudine be used in combination with a drug for which the bacteria has tested susceptible, for example, Tigecycline, when treating infections caused by CREs.
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14. Acknowledgements The authors thank the Agency for Science, Technology and Research (A*STAR) Biomedical Research Council for funding.
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Compliance with ethical standards 16. Ethical statement Not required as no humans or animals were involved in this study. 17. Conflicts of interest The authors of this paper declare no conflicts of interest.
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