Abstract
Clostridioides difficile infection (CDI) is a principal cause of hospital-acquired infections and fatilities worldwide. Regrettably, the need for new more potent anticlostridial agents is far from being met. With this in mind, drug repurposing can be utilized as a rapid and cost-efficient method of drug development. The current study evaluates the activity of ronidazole, a veterinary antiprotozoal drug, as a potential treatment for C. difficile infection. Ronidazole inhibited the growth of clinical C. difficile isolates (including NAP1 and toxigenic strains) at a very low concentration (0.125 μg/mL) and showed superior killing kinetics when compared to a known anticlostridial agent from the same chemical category, metronidazole. In addition, ronidazole did not inhibit growth of several commensal organisms naturally present in the human intestine that play a protective role in preventing C. difficile infections. Furthermore, ronidazole was found to be non-toxic to human gut cells and permeated a monolayer of colonic epithelial cells (Caco-2) at a slower rate than metronidazole. Finally, ronidazole outperformed metronidazole, when both were tested at a dose of 1 mg/kg daily, in a mouse model of C. difficile infection. Overall, ronidazole merits further investigation as a potential treatment for C. difficile infections.
Keywords: Clostridioides difficile, Clostridium difficile, nitroimidazoles, ronidazole, anticlostridial, repurposing veterinary medications
1. Introduction
Clostridioides difficile infection (CDI) is a serious infection that is a leading cause of morbidity and mortality, especially in healthcare settings. The U.S. Centers for Disease Control and Prevention (CDC) has recently listed C. difficile infection (CDI) as one of the urgent threats that required rapid action [1]. The recent escalation in the severity of CDIs is mainly due to the emergence of hypervirulent C. difficile strains. Hypervirulent strains produce more robust amounts of toxins at all growth phases. This results in more severe symptoms and death. Additionally, hypervirulent strains can form spores more efficiently and at earlier growth stages than other strains. These spores are considered the vehicle for infection, horizontal transmission and persistence [2].
Unfortunately, the cure rates of current available treatment for CDI are not acceptable. Originally, three antibiotics were exploited for the treatment of CDI, metronidazole, vancomycin and fidaxomicin. Nevertheless, metronidazole was recently removed from the CDI treatment guidelines due to inferiority to vancomycin [3]. In addition, the rate of vancomycin treatment failure and recurrence of infection is nearly 22% [4]. On the other hand, using fidaxomicin has led to in a slight enhancement in the recurrence rate of CDI as compared to other anticlostridials. Yet, this improvement was not observed for infections with hypervirulent strains of C. difficile [5]. Besides, the high cost of both vancomycin and fidaxomicin can be restrictive in developing countries. This is why metronidazole was recommended for CDI treatment only if the other drugs are not available [3, 6]. In conclusion, there is an imperative need for new anticlostridial agents.
Repurposing is a strategy to use FDA-approved drugs or clinical molecules beyond their initial indication. Drug repurposing can significantly cut down the time and cost associated with finding new anticlostridial agents when compared to the conventional de novo method [7, 8]. In a recent study, we screened almost 3,200 FDA-approved drugs and clinical molecules against C. difficile [9]. As expected, nitroimidazole-containing drugs inhibited the growth of C. difficile in vitro. What was unexpected is finding several drugs, including ronidazole, that inhibited C. difficile growth in vitro more potently than metronidazole [9]. In the current study, we further evaluate the in vitro inhibitory activity of ronidazole, its toxicity to colonic epithelial cells, and its ability to permeate across the gastrointestinal tract. In addition, we investigate the in vivo activity of ronidazole to prolong survival of mice infected with C. difficile.
2. Materials and Methods
2.1. In vitro anticlostridial activity of ronidazole
The minimum inhibitory concentration (MIC) for ronidazole along with the control anticlostridial drugs (metronidazole and vancomycin) was determined using the Clinical and Laboratory Standards Institute (CLSI) approved method for broth microdilution assay with slight modification [10]. Briefly, after 48 hours of anaerobic incubation of the C. difficile strains on brain heart infusion supplemented (BHIS) agar plates at 37° C, the colonies were scraped off and suspended in BHIS broth at a concentration of approximately 105 CFU/ml. The bacterial suspension was incubated with drugs anaerobically at 37° C, for 48 hours. The MICs reported are the lowest concentration of each drug that inhibited growth of the bacteria. MIC50 and MIC90 correspond to the lowest concentration that inhibited 50% and 90% of the tested C. difficile isolates, respectively.
2.2. Killing kinetics of ronidazole against C. difficile
A time-kill assay was utilized to assess the killing kinetics of ronidazole against C. difficile [11]. Approximately 106 CFU/mL of C. difficile ATCC BAA-1870 were mixed with the indicated concentrations of ronidazole or control anticlostridial agents, in triplicates. At each designated time point, samples were obtained from each tube to assess the bacterial colony forming units (CFU). A drug was considered bactericidal if it reduced the initial inoculum by 3-log10 in CFU or more within 24 hours.
2.3. Activity of ronidazole against bacterial species that comprise the normal human gut microflora
The broth microdilution assay was utilized to determine the in vitro activity of ronidazole against bacterial species that are part of the normal human flora. Bacterial colonies were suspended in nutrient broth to achieve a concentration of ~105 CFU/mL. Bacterial suspensions were seeded in 96-well plates with each drug concentration and incubated at 37 °C for 48 hours. For Lactobacillus species, MRS agar and broth were used and the incubation was done in a 5% CO2 atmosphere. For Bacteroides and Bifidobacteria species, BHIS agar and broth were used and bacteria were grown in anaerobic conditions.
2.4. In vitro intestinal permeability assay
Caco-2 permeability analysis was exploited to evaluate the in vitro rate of permeation of ronidazole, metronidazole, and control drugs through a monolayer of human intestinal cells [12]. A known concentration of each drug was applied in the apical compartment of a monolayer of Caco-2 cells and the concentrations of each drug in the basolateral compartment was determined after 1 hour of incubation at 37 °C and the apparent permeability coefficient (Papp) was calculated.
2.5. In vivo activity of ronidazole in a mouse model of CDI
The study was performed following the guidelines of the Purdue Animal Care and Use Committee (PACUC). Female C57BL/6 mice, six weeks old, were divided into groups of five and placed in individually ventilated cages. After one week of acclimatization, mice were sensitized for C. difficile infection by adding an antibiotic cocktail into their sterile drinking water for five days pre-infection. The antibiotic cocktail contained kanamycin (400 mg/L), gentamicin (35 mg/L), colistin (42 mg/L), metronidazole (215 mg/L), and vancomycin (45 mg/L) [13]. Two days later, mice were intraperitoneally injected with clindamycin (10 mg/kg) then they were orally infected with 8 × 105 CFU/mL of C. difficile ATCC 43255 spores on the next day. Two hours post-infection, mice were orally treated with ronidazole (1 or 10 mg/kg), metronidazole (1 or 10 mg/kg), or vancomycin (10 mg/kg) once daily for six days while the negative control group was treated using sterile PBS. Animals were monitored for signs of CDI and euthanized upon showing moribund state. On the seventh day post-infection, mice were humanely euthanized using CO2 inhalation.
3. RESULTS
3.1. In vitro antibacterial activity of ronidazole against clinical C. difficile strains
As depicted in Table 1, ronidazole potently inhibited the growth of all 24 clinical isolates of C. difficile tested. The MICs of ronidazole ranged from 0.0625 to 0.25 μg/mL, while the MIC50 and MIC90 values were 0.125 μg/mL. Vancomycin inhibited C. difficile growth at concentrations that ranged from 0.25 to 2 μg/mL. The MIC50 and MIC90 for vancomycin were 0.25 and 2 μg/mL, respectively. Similarly, metronidazole inhibited the growth of the tested strains between 0.125 and 1 μg/mL, while the MIC50 and MIC90 were 0.25 μg/mL. Finally, fidaxomicin inhibited C. difficile growth at a concentration that ranged from 0.0156 – 0.125 μg/mL. The MIC50 and MIC90 of fidaxomicin were 0.0312 and 0.0625 μg/mL, respectively.
Table 1:
Minimum inhibitory concentration (MICs, μg/mL) of ronidazole and control antibiotics against bacterial isolates.
| Strain | Ronidazole | Metronidazole | Vancomycin | Fidaxomicin |
|---|---|---|---|---|
| C. difficile ATCC 43255 | 0.125 | 0.5 | 1 | 0.0312 |
| C. difficile ATCC BAA1870 | 0.0625 | 0.25 | 1 | 0.0625 |
| C. difficile HM-88 | 0.0625 | 0.125 | 0.25 | 0.0312 |
| C. difficile NR-13427 | 0.125 | 0.5 | 2 | 0.0312 |
| C. difficile NR-13431 | 0.125 | 0.25 | 0.25 | 0.0156 |
| C. difficile NR-13436 | 0.25 | 0.25 | 0.25 | 0.0625 |
| C. difficile NR-32885 | 0.125 | 0.25 | 0.25 | 0.0312 |
| C. difficile NR-32887 | 0.0625 | 0.25 | 0.25 | 0.0312 |
| C. difficile NR-32888 | 0.125 | 0.125 | 0.25 | 0.0312 |
| C. difficile NR-32890 | 0.0625 | 0.125 | 1 | 0.0156 |
| C. difficile NR-32891 | 0.0625 | 0.25 | 0.25 | 0.0312 |
| C. difficile NR-32895 | 0.0625 | 0.25 | 2 | 0.0156 |
| C. difficile NR-32896 | 0.125 | 0.25 | 2 | 0.0312 |
| C. difficile NR-32897 | 0.125 | 0.25 | 0.25 | 0.0156 |
| C. difficile NR-49277 | 0.125 | 0.125 | 0.5 | 0.0312 |
| C. difficile NR-49278 | 0.125 | 1 | 0.25 | 0.0625 |
| C. difficile NR-49281 | 0.125 | 0.125 | 0.25 | 0.0312 |
| C. difficile NR-49283 | 0.0625 | 0.25 | 0.25 | 0.0156 |
| C. difficile NR-49285 | 0.0625 | 0.25 | 0.25 | 0.0625 |
| C. difficile NR-49286 | 0.125 | 0.25 | 0.25 | 0.0625 |
| C. difficile NR-49288 | 0.125 | 0.25 | 0.25 | 0.0625 |
| C. difficile NR-49289 | 0.125 | 0.25 | 0.25 | 0.0312 |
| C. difficile NR-49290 | 0.125 | 0.25 | 0.5 | 0.0625 |
| C. difficile NR-49291 | 0.0625 | 0.25 | 0.5 | 0.125 |
| MIC50 | 0.125 | 0.25 | 0.25 | 0.0312 |
| MIC90 | 0.125 | 0.5 | 2 | 0.0625 |
| Bacteroides dorei HM-29 | ≤1 | ≤1 | 16 | >128 |
| Bacteroides dorei HM-719 | ≤1 | ≤1 | 16 | >128 |
| Bacteroides fragilis HM-710 | ≤1 | ≤1 | 16 | >128 |
| Bacteroides fragilis HM-714 | ≤1 | ≤1 | 16 | >128 |
| Bifidobacterium breve HM-1120 | >128 | >128 | 0.5 | ≤1 |
| Bifidobacterium breve HM-411 | >128 | >128 | 0.5 | ≤1 |
| Bifidobacterium breve HM-412 | >128 | >128 | 0.5 | ≤1 |
| Bifidobacterium breve HM-856 | >128 | >128 | 0.5 | >128 |
| Bifidobacterium longum HM-845 | ≤1 | 2 | 0.25 | ≤1 |
| Bifidobacterium longum HM-847 | ≤1 | 2 | 0.25 | ≤1 |
| Lactobacillus casei ATCC 334 | >128 | >128 | >16 | 4 |
| Lactobacillus crispatus HM-375 | 32 | >128 | 0.5 | ≤1 |
| Lactobacillus crispatus HM-422 | 64 | >128 | 1 | ≤1 |
| Lactobacillus gasseri HM-398 | >128 | >128 | 1 | ≤1 |
| Lactobacillus gasseri HM-399 | >128 | >128 | 1 | ≤1 |
| Lactobacillus gasseri HM-400 | 32 | >128 | 1 | ≤1 |
| Lactobacillus gasseri HM-404 | >128 | >128 | 1 | ≤1 |
| Lactobacillus gasseri HM-407 | >128 | >128 | 2 | ≤1 |
| Lactobacillus gasseri HM-409 | >128 | >128 | 1 | ≤1 |
| Lactobacillus gasseri HM-410 | >128 | >128 | 1 | ≤1 |
3.2. Time-kill assay of ronidazole evaluated against C. difficile
As presented in Figure 1, ronidazole diminished the bacterial count to below the detection limit after 8 hours of incubation at both concentrations. Ronidazole, at 5 × MIC, reduced the initial inoculum by more than three-log10 (Figure 1A). However, at a higher concentration (20 × MIC), ronidazole reduced the burden of C. difficile by three-log10 in less than four hours (Figure 1B). This indicates ronidazole exhibits concentration-dependent bactericidal activity against C. difficile. In addition, bacteria did not regrow when exposed to 20 × MIC of ronidazole. In contrast, at 5 × MIC ronidazole, bacterial regrowth was observed, albeit at a lower CFU compared to the initial inoculum used, after 12 hours. Metronidazole, at 5 × MIC, did not reduce the initial bacterial inoculum and the bacteria grew to a similar total CFU count as the untreated control after 12 hours. However, at 20 × MIC of metronidazole, bacterial killing (>3-log reduction) was achieved in less than four hours. The bacterial count was reduced below the detection limit after eight hours exposure to 20 × MIC of metronidazole.
Figure 1: Killing kinetics and apparent permeability of ronidazole.
A & B. Ronidazole and metronidazole were anaerobically incubated with C. difficile ATCC BAA1870 (~106 CFU/mL) at 37 °C. Two different concentrations were used, 5 × MIC (A) and 20 × MIC (B). Samples were taken at the indicated time points and bacterial count was evaluated in each sample through serial dilution and plating on BHIS agar. The mean and standard deviation (error bars) of three technical replicates is presented. C. The permeability of ronidazole through a monolayer of Caco-2 cells was measured and compared to metronidazole. Data are presented as mean apparent permeability (Papp) for each drug transported from the apical to basolateral compartment. Error bars correspond to standard deviation values from duplicate samples for each drug.
3.3. Activity of ronidazole against human normal microflora
Ronidazole showed a pattern of activity similar to that of metronidazole. Against anaerobes, both ronidazole and metronidazole inhibited the growth of Bacteroides dorei, Bacteroides fragilis and Bifidobacterium longum at a concentration of 1 μg/mL or less. However, both drugs did not inhibit the growth of Bifidobacterium breve up to a concentration of 128 μg/mL. On the contrary, ronidazole and metronidazole did not potently inhibit the growth of facultative anaerobes including Lactobacillus casei, Lactobacillus crispatus and Lactobacillus gasseri. Vancomycin inhibited the growth of all tested isolates except Lactobacillus casei. The inhibition was at a higher concentration (16 μg/mL) for Bacteroides dorei and Bacteroides fragilis, while vancomycin exhibited more potent antibacterial activity (≤ 2 μg/mL) against strains of Bifidobacterium breve, Bifidobacterium longum, Lactobacillus crispatus, and Lactobacillus gasseri. Lastly, fidaxomicin did not inhibit growth of strains of Bacteroides dorei, Bacteroides fragilis, and Bifidobacterium breve HM-856 up to a test concentration of 128 μg/mL. However, fidaxomicin inhibited growth of Lactobacillus casei at 4 μg/mL while the drug inhibited growth of all other strains tested at a concentration of 1 μg/mL or less (Table 1).
3.4. Permeability of ronidazole through human intestinal cells
Although ronidazole was highly permeable, it passed through the monolayer of Caco-2 cells at a lower rate than metronidazole. The mean apparent rate of permeability of ronidazole was 56.6 × 10−6 cm/sec whereas for metronidazole it was 69.9 × 10−6 cm/sec (Figure 1C). In addition, results indicate that neither drugs is a substrate for efflux transporters, like P-glycoprotein (data not shown).
3.5. Activity of ronidazole in an animal model of CDI
As depicted in Figure 2, 60% of mice died two days after infection and 80% of the untreated mice died within the first six days of the infection. In contrast, 100% of mice treated with vancomycin (10 mg/kg, the positive control) survived seven days post-infection. As per ronidazole and metronidazole, both drugs protected 60% of the infected mice at a concentration of 10 mg/kg (Figure 2A). Additionally, ronidazole also protected 60% of the mice at 1 mg/kg. Notably, metronidazole at 1 mg/kg was ineffective as all mice in this group died two days after infection (Figure 2B).
Figure 2: Antibacterial activity of ronidazole in a CDI mice model.
The protective effect of ronidazole was evaluated in a mice model of CDI and compared to metronidazole and vancomycin. Antibiotic-primed mice were infected with C. difficile ATCC 43255 spores (8 × 105 CFU/mL). Treatment with either A- 10 mg/kg of ronidazole, metronidazole or vancomycin, or B- 1 mg/kg of ronidazole or metronidazole was started two hours post-infection and was administered orally once daily for six days. Untreated mice received PBS. Mice were checked several times per day and euthanized when exhibited signs of morbidity.
4. DISCUSSION
As mentioned previously, only two anticlostridial agents (vancomycin and fidaxomicin) are currently recommended for the treatment of CDI [3]. Considering the great number of causalities and the potential for C. difficile strains to develop resistance to currently used therapeutics, there is an immense need for developing new anticlostridial agents. One way to minimize the time, cost, and risk associated with drug discovery is repurposing FDA-approved drugs or clinical molecules [14]. We screened two libraries consisting of nearly 3200 FDA-approved drugs and clinical molecules to find molecules with antibacterial activity against C. difficile in vitro [9]. Out of the active hits, we selected ronidazole to be evaluated further against C. difficile both in vitro and in vivo. Ronidazole is a veterinary antiparasitic drug used as a treatment for several different animal species [15]. Importantly, ronidazole is effective against metronidazole-resistant parasitic strains [16]. Given this information, we hypothesized that ronidazole would exhibit better activity than metronidazole against C. difficile.
In the current study, ronidazole’s antibacterial activity was initially evaluated against 24 clinical strains of C. difficile. As observed against parasites, ronidazole (MIC values ranged from 0.0625 to 0.25 μg/mL) exhibited more potent in vitro antibacterial activity than metronidazole (MIC values ranged from 0.125 to 1 μg/mL) against C. difficile. Additionally, a time-kill assay revealed ronidazole exhibited bactericidal activity at a lower concentration relative to metronidazole. Both drugs exhibited a similar profile inhibiting the growth of certain species of anaerobic bacteria. Contrarily, Lactobacillus species and some Bifidobacterium species growth were inhibited by vancomycin and fidaxomicin but not by ronidazole or metronidazole. Although evidence regarding the health benefits of these two genera is contradicting, sparing species of bifidobacteria and lactobacilli from antibiotic insult and conserving the bacterial diversity of the gut is a preferred trait in an anticlostridial agent [17].
We next evaluated the safety of ronidazole towards human gut cells. Ronidazole did not reduce HRT-18 cell viability up to a concentration of 256 μg/mL (> 2000-fold higher than its MIC against C. difficile, data not shown). Additionally, ronidazole demonstrated a slower rate of permeation (Papp) across the Caco-2 monolayer relative to metronidazole. This slower rate of permeability may allow ronidazole to stay in contact with C. difficile at the site of infection (intestinal tract) for longer time and result in more efficient bacterial killing.
In the final step of our study, we evaluated the antibacterial activity of ronidazole in a mouse model of CDI. Ronidazole showed equal activity to ronidazole at a concentration of 10 mg/kg as treatment with either drug prolonged survival for 60% of mice until the seventh day post-infection. However, ronidazole outperformed metronidazole when both drugs were evaluated at a lower concentration (1 mg/kg). Indeed, ronidazole at 1 mg/kg protected 60% of the mice which was equivalent to the effect of metronidazole at 10 mg/kg. In contrast, all mice treated with 1 mg/kg metronidazole died only two days post-infection. The superior activity of ronidazole over metronidazole at a lower concentration might be connected to the bactericidal activity of ronidazole observed at a lower concentration (5 × MIC) in the time-kill assay. Additionally, the increased potency and reduced absorption of ronidazole from the intestinal tract (as determined by the Caco-2 permeability assessment) may play a role in the improved activity of ronidazole observed in the mouse model of CDI.
One limitation of using ronidazole in the treatment of CDI in humans is its presumed toxicity after systemic administration. The major side effect observed with ronidazole administration is reversible neurotoxicity. However, neurotoxicosis is associated with higher doses of ronidazole and is observed only in some, not all, cases [18]. Moreover, ronidazole is anecdotally reported to be carcinogenic and teratogenic after prolonged administration. Nevertheless, administration of ronidazole to pregnant Albino rats throughout the duration of pregnancy, and at a dose that is six times greater than the maximum therapeutic dose, did not cause any teratogenic or embryogenic effects [19]. Notably, CDI treatment takes between 10 to 20 days and hence, short-term studies are required to evaluate the potential toxic effect of ronidazole during this period.
Taken altogether, nitroimidazoles represent an attractive scaffold for anticlostridial drug development. In this study ronidazole was shown to possess superior in vitro and in vivo activities against C. difficile when compared to metronidazole. Per results from this study, ronidazole represents a potential candidate for the treatment of CDI either solely or therapying combination with currently approved drugs and warrants further investigation.
Highlights.
Ronidazole is a potent inhibitor of Clostridioides difficile growth in vitro
Ronidazole is a rapid bactericidal agent at a low concentration
Ronidazole spared some of the protective bacteria of the gut microbiota
Ronidazole permeates intestinal cells at lower rate than metronidazole in vitro
Ronidazole is more effective than metronidazole in vivo
Acknowledgments
Funding: This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R01AI130186.
Footnotes
Competing Interests: The authors declare no competing financial interests.
Declarations
Ethical Approval: Not required
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