Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for the treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug.
KEYWORDS: Clostridium difficile infections (CDIs), spores, toxins, synergy, diiodohydroxyquinoline
ABSTRACT
Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for the treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug. DIHQ exhibited potent activity against 39 C. difficile isolates, inhibiting growth of 50% and 90% of these isolates at concentrations of 0.5 μg/ml and 2 μg/ml, respectively. In a time-kill assay, DIHQ was superior to vancomycin and metronidazole, reducing a high bacterial inoculum by 3 log10 within 6 h. Furthermore, DIHQ reacted synergistically with vancomycin and metronidazole against C. difficile in vitro. Moreover, at subinhibitory concentrations, DIHQ was superior to vancomycin and metronidazole in inhibiting two key virulence factors of C. difficile, toxin production and spore formation. Additionally, DIHQ did not inhibit the growth of key species that compose the host intestinal microbiota, such as Bacteroides, Bifidobacterium, and Lactobacillus spp. Collectively, our results indicate that DIHQ is a promising anticlostridial drug that warrants further investigation as a new therapeutic for CDIs.
INTRODUCTION
Clostridioides difficile is a Gram-positive, spore-forming obligate anaerobic bacterium (1). The incidence of C. difficile infections (CDIs) has increased dramatically and is now the leading cause of antibiotic- and health care-associated diarrhea in the United States (2, 3). In addition, community-onset CDIs are on the rise. CDI is being increasingly recognized in the community, in younger individuals, and in patients lacking the CDI traditional risk factors, such as hospitalization, age, and antibiotic exposure (4, 5).
Fidaxomicin was the only new antibiotic that has been approved in the last 30 years for the treatment of CDIs. Currently, only two drugs are recommended for treatment of both nonsevere and severe CDIs, vancomycin and fidaxomicin. According to the Infectious Diseases Society of America (IDSA) and the Society for Healthcare Epidemiology of America (SHEA) guidelines, metronidazole, which was previously recommended as a first-line therapeutic option for CDIs in adults, is no longer recommended, and its use is now restricted to nonsevere cases of CDI when patients are unable to obtain or be treated with vancomycin or fidaxomicin (6). The available treatment options are inadequate in efficacy and associated with high recurrence rates (7–9). Moreover, it was reported that about 22% of patients treated with metronidazole and 14% of those treated with vancomycin will experience treatment failure, and about 25 to 30% of the patients treated with either metronidazole or vancomycin will go through CDI recurrence (10). Moreover, resistance or reduced susceptibility to these antibiotics is emerging (8, 9). Taken together, there is a critical and an unmet need for new effective drugs against C. difficile.
Drug discovery is a time-consuming and expensive venture. Developing a new drug can take 10 to 15 years from early stage discovery to receiving regulatory approval and can cost more than $2 billion (11). Repurposing FDA-approved drugs, particularly those that are off patent, for new indications represents a promising approach that can significantly reduce the cost and time associated with drug innovation (12–14). Antiparasitic drugs are a class of medications used for the treatment of parasitic diseases caused by parasites such as helminths, amoebae, and protozoans (15). Most of them are poorly absorbed from the gastrointestinal (GI) tract, which is ideal for targeting intestinal pathogens such as C. difficile. Consequently, in this study, we screened a small panel of antiparasitic drugs against C. difficile, with the aim of discovering a new anticlostridial drug. From the initial screening against C. difficile, diiodohydroxyquinoline (DIHQ) emerged as the most potent candidate. DIHQ was subsequently evaluated against a wider panel of C. difficile clinical isolates, examined for the ability to eliminate a high inoculum of C. difficile in a time-kill assay, investigated for the ability to inhibit C. difficile toxin production and spore formation, and tested for its ability to be used in combination with both vancomycin and metronidazole. Finally, we evaluated DIHQ’s effect on the growth of key bacterial species that comprise the human intestinal microbiota that help curb C. difficile colonization.
RESULTS
Screening a small panel of antiparasitic drugs against C. difficile.
A small panel of poorly absorbed antiparasitic drugs was initially screened against two clinical C. difficile strains to determine their anticlostridial activities. As presented in Table 1, the antiparasitic drugs were inactive (up to 128 μg/ml) against both C. difficile strains, with the exception of DIHQ. DIHQ exhibited potent activity against both C. difficile strains (MIC range, 0.5 to 1 μg/ml). It was as potent as vancomycin, the drug of choice for the treatment of C. difficile infections.
TABLE 1.
Initial MIC screening of antiparasitic drugs against C. difficile ATCC BAA-1870 and C. difficile ATCC 43255
| Drug name | MIC (μg/ml) for C. difficile strain: |
|
|---|---|---|
| ATCC-BAA-1870 | ATCC 43255 | |
| Albendazole | >128 | >128 |
| Mebendazole | >128 | >128 |
| Ricobendazole | >128 | >128 |
| Thiabendazole | >128 | >128 |
| Cambendazole | >128 | >128 |
| Praziquantel | >128 | >128 |
| Pyrantel pamoate | >128 | >128 |
| Paromomycin | >128 | >128 |
| Pyrimethamine | >128 | >128 |
| DIHQ | 1 | 0.5 |
| Vancomycin | 1 | 1 |
| Metronidazole | 0.125 | 0.25 |
Antibacterial susceptibility testing of DIHQ against additional C. difficile clinical strains.
After the initial testing against two C. difficile strains, we evaluated the anticlostridial activity of DIHQ against a wider panel of C. difficile clinical isolates. As presented in Table 2, DIHQ inhibited the growth of all 39 clinical isolates at concentrations ranging from 0.06 μg/ml to 4 μg/ml. DIHQ inhibited 50% of the tested isolates (MIC50) at the concentration of 0.5 μg/ml and inhibited 90% of the isolates (MIC90) at the concentration of 2 μg/ml. Interestingly, DIHQ’s MIC values were comparable to the MIC values of vancomycin, which inhibited 50% and 90% of the strains at 0.5 μg/ml and 1 μg/ml, respectively. Metronidazole, the drug of choice for anaerobic bacterial infections (16), was effective at a range of 0.06 to 0.5 μg/ml, with MIC50 and MIC90 values of 0.125 μg/ml and 0.25 μg/ml, respectively. On the other hand, fidaxomicin inhibited 50% and 90% of the tested strains at concentrations of 0.015 μg/ml and 0.06 μg/ml, respectively.
TABLE 2.
MIC and MBC values of DIHQ and control anticlostridial drugs against clinical Clostridioides difficile isolates
| C. difficile strain | MIC or MBC (μg/ml) by druga |
|||||||
|---|---|---|---|---|---|---|---|---|
| DIHQ |
Vancomycin |
Metronidazole |
Fidaxomicin |
|||||
| MIC | MBC | MIC | MBC | MIC | MBC | MIC | MBC | |
| ATCC BAA-1870 | 1 | 1 | 1 | 1 | 0.125 | 0.125 | 0.03 | 0.03 |
| ATCC 43255 | 0.5 | 0.5 | 1 | 1 | 0.25 | 0.25 | 0.015 | 0.015 |
| ATCC 43598 | 1 | 1 | 1 | 1 | 0.125 | 0.125 | 0.015 | 0.015 |
| ATCC 9689 | 0.125 | 0.25 | 0.5 | 1 | 0.125 | 0.25 | 0.03 | 0.03 |
| ATCC 1801 | 2 | 4 | 1 | 1 | 0.25 | 0.25 | 0.06 | 0.125 |
| ATCC 700057 | 0.5 | 0.5 | 0.5 | 0.5 | 0.125 | 0.125 | 0.007 | 0.007 |
| I1 | 1 | 1 | 1 | 1 | 0.125 | 0.125 | 0.007 | 0.007 |
| I2 | 2 | 2 | 1 | 1 | 0.125 | 0.125 | 0.007 | 0.007 |
| I4 | 1 | 1 | 0.5 | 0.5 | 0.125 | 0.125 | 0.015 | 0.015 |
| I6 | 0.5 | 0.5 | 0.5 | 0.5 | 0.25 | 0.25 | 0.06 | 0.06 |
| I9 | 0.06 | 0.06 | 0.5 | 0.5 | 0.06 | 0.06 | 0.03 | 0.03 |
| I10 | 1 | 1 | 0.5 | 1 | 0.25 | 0.25 | 0.015 | 0.03 |
| I11 | 1 | 1 | 1 | 1 | 0.25 | 0.25 | 0.015 | 0.015 |
| I13 | 2 | 2 | 1 | 1 | 0.25 | 0.5 | 0.03 | 0.03 |
| P1 | 0.5 | 0.5 | 0.5 | 0.5 | 0.125 | 0.125 | 0.003 | 0.007 |
| P2 | 0.25 | 0.25 | 1 | 1 | 0.125 | 0.25 | 0.03 | 0.03 |
| P3 | 2 | 2 | 1 | 1 | 0.25 | 0.25 | 0.015 | 0.03 |
| P5 | 2 | 4 | 0.5 | 0.5 | 0.25 | 0.25 | 0.03 | 0.03 |
| P6 | 0.5 | 0.5 | 1 | 1 | 0.25 | 0.25 | 0.003 | 0.003 |
| P7 | 0.5 | 0.5 | 0.5 | 0.5 | 0.125 | 0.125 | 0.06 | 0.06 |
| P8 | 0.125 | 0.125 | 1 | 1 | 0.125 | 0.125 | 0.015 | 0.015 |
| P11 | 0.25 | 0.25 | 1 | 1 | 0.125 | 0.25 | 0.03 | 0.03 |
| P13 | 0.5 | 0.5 | 0.5 | 0.5 | 0.125 | 0.25 | 0.015 | 0.015 |
| P15 | 1 | 1 | 1 | 1 | 0.25 | 0.25 | 0.06 | 0.06 |
| P19 | 0.5 | 1 | 1 | 1 | 0.25 | 0.25 | 0.03 | 0.06 |
| P20 | 1 | 1 | 1 | 1 | 0.25 | 0.25 | 0.015 | 0.015 |
| P24 | 1 | 2 | 0.5 | 0.5 | 0.125 | 0.25 | 0.015 | 0.03 |
| P30 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.007 | 0.007 |
| HM-88 | 4 | 8 | 0.5 | 0.5 | 0.125 | 0.125 | 0.03 | 0.03 |
| HM-89 | 1 | 1 | 1 | 1 | 0.125 | 0.125 | 0.03 | 0.03 |
| HM-745 | 1 | 1 | 1 | 1 | 0.5 | 0.5 | 0.06 | 0.125 |
| NR-49277 | 0.5 | 0.5 | 1 | 1 | 0.125 | 0.125 | 0.03 | 0.03 |
| NR-49278 | 0.25 | 0.5 | 0.25 | 0.5 | 0.25 | 0.5 | 0.007 | 0.015 |
| NR-49281 | 0.25 | 0.25 | 0.25 | 0.25 | 0.125 | 0.125 | 0.007 | 0.007 |
| NR-49284 | 0.125 | 0.125 | 0.25 | 0.5 | 0.125 | 0.125 | 0.015 | 0.015 |
| NR-49285 | 1 | 1 | 0.5 | 0.5 | 0.25 | 0.5 | 0.015 | 0.03 |
| NR-49286 | 0.25 | 0.5 | 0.25 | 0.5 | 0.125 | 0.125 | 0.007 | 0.015 |
| NR-49288 | 0.25 | 0.5 | 0.5 | 1 | 0.25 | 0.25 | 0.007 | 0.015 |
| NR-49290 | 0.25 | 0.5 | 0.5 | 1 | 0.125 | 0.25 | 0.015 | 0.015 |
The MIC50s were 0.5 μg/ml for DIHQ, 0.5 μg/ml for vancomycin, 0.125 μg/ml for metronidazole, and 0.015 μg/ml for fidaxomicin. The MIC90s were 2 μg/ml for DIHQ, 1 μg/ml for vancomycin, 0.25 μg/ml for metronidazole, and 0.06 μg/ml for fidaxomicin.
To determine if DIHQ exhibits bacteriostatic or bactericidal activity against C. difficile, we also determined the minimum bactericidal concentrations (MBCs) against all 39 isolates. The MBC values for DIHQ were equal to or 2-fold higher than the corresponding MIC values for all 39 isolates, indicating that DIHQ is a bactericidal agent. A similar result was observed for vancomycin and metronidazole.
Evaluation of the killing kinetics of DIHQ and control anticlostridial drugs against C. difficile.
In order to confirm the bactericidal activity of DIHQ against C. difficile, we examined how rapidly the drug reduced the burden of a high inoculum of C. difficile via a standard time-kill assay. As presented in Fig. 1, DIHQ exerted rapid bactericidal activity against C. difficile. DIHQ required only 6 h to generate a 3-log10 reduction in CFU/ml and completely eradicated the bacteria (below the detection limit, 100 CFU/ml) within 24 h. Interestingly, DIHQ was superior to both metronidazole and vancomycin in the in vitro time-kill assay. Metronidazole produced a 3-log10 reduction in C. difficile CFU/ml after 12 h. Vancomycin exhibited a slow reduction in C. difficile count and only reduced the bacterial burden by 1.7 log10 CFU/ml within 24 h. The time-kill kinetics results against C. difficile NR-49277 (a hypervirulent strain) are included in Fig. 1S in the supplemental material.
FIG 1.
Time-kill analysis of DIHQ, metronidazole, and vancomycin (at 5× the MIC) against Clostridioides difficile ATCC BAA-1870 over a 24-h incubation period at 37°C. Dimethyl sulfoxide (DMSO) (solvent of the drugs) served as a negative control. The error bars represent standard deviation values obtained from triplicate samples used for each drug studied.
Interactions between DIHQ and metronidazole and vancomycin against C. difficile clinical isolates.
Combination therapy is one of the most effective therapeutic choices for different infections to increase the therapeutic outcomes and decrease relapse rates. To ascertain whether DIHQ has potential to be combined with standard anticlostridial drugs against C. difficile, the checkerboard assay was used. As depicted in Tables 3 and 4, DIHQ exhibited a synergistic interaction with metronidazole more frequently than with vancomycin. When combined with metronidazole, DIHQ possessed a synergistic relationship against 7 out of 9 tested strains, with a fractional inhibitory concentration (FIC) index that ranged from 0.18 to 0.37 (Table 4). On the other hand, DIHQ exhibited a synergistic interaction with vancomycin against 5 out of 9 tested strains, with an FIC index range similar to that of metronidazole (Table 5).
TABLE 3.
Interactions between DIHQ and metronidazole against C. difficile clinical isolates
| C. difficile strain | MIC (μg/ml) by drug treatment |
ΣFIC indexa | Interpretationb | |||
|---|---|---|---|---|---|---|
| Metronidazole |
DIHQ |
|||||
| Alone | Combined with DIHQ | Alone | Combined with metronidazole | |||
| ATCC BAA-1870 | 0.125 | 0.015 | 1 | 0.125 | 0.25 | SYN |
| ATCC 43255 | 0.125 | 0.015 | 1 | 0.25 | 0.375 | SYN |
| ATCC 9689 | 0.25 | 0.03 | 0.125 | 0.06 | 0.62 | ADD |
| ATCC 1801 | 0.25 | 0.03 | 4 | 1 | 0.37 | SYN |
| P11 | 0.25 | 0.03 | 0.5 | 0.125 | 0.37 | SYN |
| P19 | 0.5 | 0.06 | 0.5 | 0.06 | 0.24 | SYN |
| P30 | 0.5 | 0.06 | 1 | 0.06 | 0.185 | SYN |
| I6 | 0.25 | 0.03 | 2 | 0.125 | 0.188 | SYN |
| I9 | 0.125 | 0.015 | 0.06 | 0.06 | 1.125 | ADD |
ΣFIC, fractional inhibitory concentration summation.
An ΣFIC index of ≤0.5 is considered synergistic (SYN); an ΣFIC index of >0.5 to 1.25 is considered additive (ADD); an ΣFIC index of >1.25 to 4 is considered indifferent (IND); and an ΣFIC index of >4 is considered antagonistic.
TABLE 4.
Interactions between DIHQ and vancomycin against C. difficile clinical isolates
| C. difficile strains | MIC (μg/ml) by drug treatment |
ΣFIC indexa | Interpretationb | |||
|---|---|---|---|---|---|---|
| Vancomycin |
DIHQ |
|||||
| Alone | Combined with DIHQ | Alone | Combined with vancomycin | |||
| ATCC BAA-1870 | 1 | 0.125 | 1 | 0.06 | 0.185 | SYN |
| ATCC 43255 | 1 | 0.125 | 1 | 0.125 | 0.25 | SYN |
| ATCC 9689 | 1 | 0.125 | 0.125 | 0.06 | 0.625 | ADD |
| ATCC 1801 | 0.5 | 0.06 | 4 | 2 | 0.625 | ADD |
| P11 | 1 | 0.125 | 0.5 | 0.5 | 1.125 | ADD |
| P19 | 1 | 0.125 | 0.5 | 0.125 | 0.375 | SYN |
| P30 | 1 | 0.125 | 1 | 0.125 | 0.25 | SYN |
| I6 | 0.5 | 0.06 | 2 | 0.5 | 0.375 | SYN |
| I9 | 1 | 0.125 | 0.06 | 0.06 | 1.125 | ADD |
ΣFIC, fractional inhibitory concentration summation.
An ΣFIC index of ≤0.5 is considered synergistic (SYN); an ΣFIC index of >0.5 to 1.25 is considered additive (ADD); an ΣFIC index of >1.25 to 4 is considered indifferent (IND); and an ΣFIC index of >4 is considered antagonistic.
TABLE 5.
Antibacterial activity (MIC in μg/ml) of DIHQ and control anticlostridial drugs against human normal gut microbiota
| Bacterial strain | MIC (μg/ml) for drug: |
|||
|---|---|---|---|---|
| DIHQ | Vancomycin | Metronidazole | Fidaxomicin | |
| Lactobacillus reuteri HM-102 | >128 | >128 | >128 | >128 |
| Lactobacillus casei ATCC 334 | 16 | >128 | 16 | >128 |
| Lactobacillus crispatus HM-371 | 64 | 4 | >128 | >128 |
| Bacteroides dorei HM-719 | >128 | 64 | <1 | 128 |
| Bacteroides dorei HM-717 | >128 | 128 | <1 | >128 |
| Bacteroides dorei HM-718 | >128 | 128 | <1 | >128 |
| Bacteroides dorei HM-29 | >128 | 64 | <1 | >128 |
| Bacteroides fragilis HM-711 | >128 | 128 | <1 | >128 |
| Bacteroides fragilis HM-709 | >128 | 64 | 2 | >128 |
| Bacteroides fragilis HM-710 | >128 | 64 | 2 | >128 |
| Bacteroides fragilis HM-714 | >128 | 128 | <1 | >128 |
| Bifidobacterium adolescentis HM-633 | 128 | <1 | <1 | <1 |
| Bifidobacterium angulatum HM-1189 | 128 | <1 | <1 | <1 |
| Bifidobacterium breve HM-411 | >128 | <1 | 2 | 2 |
| Bifidobacterium breve HM-1120 | >128 | <1 | <1 | <1 |
| Bifidobacterium longum HM-845 | 64 | <1 | <1 | <1 |
| Bifidobacterium longum HM-847 | 128 | <1 | <1 | <1 |
DIHQ inhibits C. difficile toxin production.
Toxins are the main virulence factor of C. difficile. As a result, inhibition of toxin production will contribute to effective treatment of CDI. Therefore, we tested the toxin-inhibitory activity of DIHQ against a toxigenic C. difficile strain. DIHQ exhibited a dose-dependent inhibition of C. difficile toxins A and B and was effective at subinhibitory concentrations. As shown in Fig. 2, DIHQ inhibited nearly 17.8% and 27% of the total toxin production at 1/4× the MIC and 1/2× the MIC, respectively. Fidaxomicin, which is known to inhibit toxin production (17), inhibited 31.9% and 46.2% of the toxin production at 1/4× the MIC and 1/2× the MIC, respectively. No toxin inhibition was observed with either vancomycin or metronidazole, in agreement with previous reports (10, 18). The toxin inhibition results against 2 other C. difficile strains are included in Fig. 2S and 3S.
FIG 2.
Toxin inhibition activity of DIHQ and control anticlostridial drugs (vancomycin, metronidazole, and fidaxomicin) against C. difficile ATCC BAA-1870. Drugs, at concentrations of 1/4× the MIC or 1/2× the MIC, were incubated with the hypervirulent toxigenic strain C. difficile ATCC BAA-1870. The bacterial counts (represented by gray bars) were determined for each sample, and the toxin levels (represented by the connected lines) were assessed in the supernatant using an enzyme-linked immune fluorescent assay (ELISA). Error bars represent the standard deviation values from triplicate samples for each treatment. The data were analyzed via one-way analysis of variance (ANOVA) with post hoc Dunnett’s test for multiple comparisons. Asterisks (*) denote statistical significant difference between the results obtained in fidaxomicin- or DIHQ-treated samples compared to the untreated samples.
DIHQ inhibits C. difficile spore formation.
Spore formation is a key virulence factor of C. difficile that is responsible for both the rapid spread and high recurrence rate of infection (19, 20). We investigated the efficacy of DIHQ to inhibit C. difficile ATCC BAA-1870 spore formation. As shown in Fig. 3, DIHQ-treated bacteria displayed a significantly reduced spore count by nearly 0.74 log10 and 1.24 log10 at 1/2× the MIC and 1× the MIC, respectively. Fidaxomicin, which is capable of inhibiting spore formation (21), significantly reduced C. difficile spore formation by nearly 1.02 log10 (at 1/2× the MIC) and 1.8 log10 (at 1× the MIC). On the other hand, almost no reduction in the spore count was observed when vegetative cells were exposed to either vancomycin or metronidazole. Spore inhibition results against 2 other C. difficile strains are included in Fig. 4S and 5S.
FIG 3.
Spore inhibition activity of DIHQ against C. difficile ATCC BAA-1870 compared to the control anticlostridial drugs, fidaxomicin, vancomycin, and metronidazole. DMSO (solvent of the drugs) served as a negative control. Drugs (1/2× the MIC and 1× the MIC) were incubated with bacteria for 5 days, followed by serial dilution and plating to determine both the total bacterial count and spore count. Error bars represent standard deviation values from triplicate samples for each treatment. Asterisks (*) denote significant differences between the total count and the spore count for each test agent analyzed via a two-way ANOVA with post hoc Dunnett’s test for multiple comparisons (P < 0.05).
In vitro antimicrobial evaluation of DIHQ against normal microflora.
The administration of antibiotics, particularly those that are broad spectrum, can lead to an alteration of the normal intestinal microbial composition, which results in gut colonization by opportunistic pathogens like C. difficile (22). Consequently, it was imperative to determine whether DIHQ may possess a deleterious effect on commensal organisms that are part of the normal gut microbiota. We tested the antibacterial activity of DIHQ and the control anticlostridial drugs against representative bacteria that comprise the human gut microbiome, including species of Lactobacillus, Bacteroides, and Bifidobacterium. As presented in Table 5, DIHQ did not inhibit the growth of species of Bacteroides and Bifidobacterium up to the maximum tested concentration of 128 μg/ml (except Bifidobacterium longum [MIC, 64 to 128 μg/ml] and Bifidobacterium adolescentis and Bifidobacterium angulatum [MIC, 128 μg/ml]). This was in contrast to metronidazole, which inhibited the growth of all species of Bacteroides and Bifidobacterium tested at concentrations that ranged from ≤1 μg/ml up to 2 μg/ml. DIHQ exhibited weak antibacterial activity against both Lactobacillus casei (MIC, 16 μg/ml) and Lactobacillus crispatus (MIC = 64 μg/ml) and did not inhibit growth of Lactobacillus reuteri (MIC, >128 μg/ml). Fidaxomicin did not inhibit growth of Lactobacillus and Bacteroides strains tested (with the exception of B. dorei HM-719) up to a concentration of 128 μg/ml. However, it exhibited a similar activity to metronidazole against Bifidobacterium strains.
DISCUSSION
The current treatment options for CDI are limited and still result in unsatisfactory outcomes. Only three drugs are currently available for treatment, vancomycin, metronidazole, and fidaxomicin. Metronidazole is no longer recommended as a first-line therapy for CDI due to a high recurrence rate and increased resistance. However, metronidazole is still recommended for use in treating nonsevere cases of CDI where patients have limited access to, or cannot successfully be treated with, vancomycin or fidaxomicin (6). In addition, the available treatment options suffer from several limitations, such as increasing recurrence rates and treatment failure, in addition to the increased risk of the emergence of resistant mutants and that they promote the overgrowth of other opportunistic enteric pathogens like vancomycin-resistant enterococci (VRE) (10, 23). Fidaxomicin has an efficacy comparable to that of vancomycin but suffers from the same pitfall of recurrence (although it occurs at a lower rate) (24). Fecal microbiota transplantation (FMT) has been evaluated as a nonantibiotic treatment option for CDI, yet it has many restrictions and strict regulations. Additionally, the significantly higher cost associated with FMT makes it unaffordable for the majority of CDI patients (25). Without a doubt, there is an urgent need to identify new effective drugs to treat CDI.
Ideally, an effective anticlostridial drug should exhibit potent bactericidal activity against C. difficile with limited activity against intestinal microbiota and experience limited absorption from the GI tract (when administered orally) to accumulate at a sufficient concentration at the target site. Consequently, we screened a small panel of antiparasitic drugs against C. difficile. We chose these drugs because they are commercially available, are administered orally, are poorly absorbed from the GI tract, and were not screened before against C. difficile. Out of these drugs, DIHQ, an antiamoebic drug that was first introduced for use in humans in the early 1960s, possesses desirable qualities as an anticlostridial drug. DIHQ is a poorly absorbed halogenated hydroxyquinoline, acting as a chelator for ferrous ions that are essential for amoebic metabolism (26). DIHQ acts as a luminal amoebicide, but its exact mechanism of action is unknown (27, 28).
In this study, we evaluated DIHQ against a large panel of 39 C. difficile strains, including hypervirulent (NAP1, ribotype 027) and clinical toxigenic isolates. DIHQ inhibited the growth of C. difficile isolates at very low, clinically achievable concentrations. Interestingly, DIHQ was as potent as vancomycin against the C. difficile isolates tested, with an MIC50 of 1 μg/ml. Antibiotics exhibiting bactericidal activity are hypothesized to contribute to better clinical outcomes than the bacteriostatic agents (29). Thus, we determined the MBC for DIHQ against C. difficile. DIHQ was found to be bactericidal, similar to the standard anticlostridial drugs vancomycin and metronidazole. Next, we examined how rapidly DIHQ can reduce the burden of a high C. difficile inoculum via a time-kill kinetics assay. DIHQ exhibited a more rapid bactericidal activity against C. difficile than those of metronidazole and vancomycin. Similar to previous reports, metronidazole exhibited bactericidal activity against C. difficile, generating a 3-log10 CFU/ml reduction after 12 h (30). Vancomycin, in agreement with previous reports (30, 31), exhibited a slow reduction in C. difficile count (1.7 log10 CFU/ml) after 24 h. Rapid bactericidal activity contributes to reducing the emergence of bacterial resistance to antibacterial drugs (32), and it is highly desirable for C. difficile infections to prevent the progression of the disease.
Combination therapy has become a standard in several diseases. This strategy is now often used in the health care setting to gain the advantages of combined drugs, such as different mechanisms of action, lower toxicity, potential synergism, and less probability of development of resistance to both agents (33, 34). In 1987, Buggy et al. used a combination of vancomycin plus rifampin for the treatment of CDI patients and found that this combination resolved infections and decreased the rate of recurrence (35). The vancomycin-metronidazole combination was also proposed for CDI, although it was as effective as vancomycin alone (36). Nevertheless, combination therapy is a promising strategy for treating CDI, particularly for severe infections where one agent may not be effective. A previous study found that the therapeutic outcomes of the DIHQ plus metronidazole combination is better than outcomes with metronidazole alone for the treatment of intestinal amoebiasis (37). This encouraged us to evaluate the combination of DIHQ with standard anticlostridial drugs, vancomycin and metronidazole, against nine different C. difficile strains. Interestingly, DIHQ exhibited a synergistic relationship in 7 out of 9 tested strains (when combined with metronidazole) and in 5 out of 9 tested strains (when combined with vancomycin). Since the treatment course of CDI is long, combination therapy is highly desirable to decrease the probability of developing resistance, to lower the toxicity potential of the administered drugs, and to curb the recurrence of infection.
After confirming DIHQ’s in vitro activity against C. difficile, both alone and in combination with metronidazole and vancomycin, we moved to investigate if DIHQ can interfere with the expression of key virulence factors used by C. difficile to promote infection. First, we evaluated the ability of DIHQ to inhibit C. difficile toxin production. Toxigenic C. difficile strains are capable of inducing inflammation and provoking pseudomembranous colitis. TcdA and TcdB can inactivate host GTPases, including Rac, Rho, and CDC42, resulting in rearrangement of the actin cytoskeleton, intense inflammation, enormous fluid secretion, disruption of mucosal layer barrier function, and, finally, necrosis and apoptosis of the colonic mucosal layer (38, 39). Therefore, agents capable of inhibiting C. difficile toxin production may contribute to an effective treatment of CDI by limiting damage to the host’s intestinal mucosa. Fidaxomicin is the only currently available anticlostridial drug with antitoxin activity (17). We tested the toxin inhibition activity of DIHQ, at subinhibitory concentrations, against a hypervirulent toxigenic C. difficile strain. DIHQ (at 1/2× the MIC) inhibited the total toxin production of C. difficile by 27%. A similar effect was observed with fidaxomicin (46% reduction in toxin production at 1/2× the MIC) but not with vancomycin or metronidazole.
As DIHQ exhibited an ability to partially inhibit C. difficile toxin production, we moved to investigate the drug’s ability to inhibit a second key virulence factor, spore formation. Spores are metabolically dormant and highly resistant to standard disinfection procedures. That is why they can persist for long periods and spread in the environment. Once ingested by susceptible hosts, spores germinate, in response to bile acids in the small intestine, into vegetative cells that produce toxins and cause disease (40). In addition, persistent spores in the intestine are the reason behind recurrence, where they can germinate in the intestine after the conclusion of treatment (41). The ability of DIHQ to inhibit C. difficile spore formation was tested. DIHQ partially inhibited C. difficile spore formation at subinhibitory concentrations. Neither vancomycin nor metronidazole reduced the spore count. This result suggests that DIHQ may contribute to lower CDI recurrence rates by inhibiting C. difficile spore formation, though this needs to be confirmed in appropriate CDI animal models.
Finally, we sought to investigate DIHQ’s effect on growth of major bacterial species that compose the healthy intestinal flora. Disrupting intestinal microbiota increases the susceptibility of hosts to C. difficile colonization. Thus, it is critically important for new anticlostridial drugs to show minimal activity against the normal microbiome. Unlike vancomycin, fidaxomicin, and metronidazole, DIHQ exhibited limited activity against the tested representative members of the human normal intestinal microbiota.
It is worth mentioning that the recommended dose of DIHQ for treatment of amoebiasis in adults is 650 mg/kg of body weight, 3 times daily for 20 days, which is a much greater course than what would be expected for C. difficile treatment (28). It can also be used for children at a dosage of 30 to 40 mg/kg daily in divided doses, for 20 days, with a maximum of 2 g/day without reported toxicity; however, it is not well tolerated in children and should be avoided if possible (42). Moreover, DIHQ has rare, mild, and self-limiting side effects, such as nausea, vomiting, stomach upset, abdominal cramps, diarrhea, headache, and dizziness. Reported toxicity cases of DIHQ are rare and are associated with administering high doses for a prolonged period. Seizures and encephalopathy were reported in a 9-year-old boy treated with a very high dose of DIHQ (420 mg, 3 times daily, for 20 days) (43). Furthermore, few cases of neuropathy and optic atrophy in children after prolonged administration of high doses of hydroxyquinolines (namely, clioquinol) for prolonged periods were reported (44).
In conclusion, the current study highlights that DIHQ, an FDA-approved antiamoebic drug, has potent in vitro antibacterial activity against C. difficile and exhibits a more rapid bactericidal activity than that with both metronidazole and vancomycin. DIHQ interacted synergistically with both vancomycin and metronidazole against most strains of C. difficile tested in vitro. In addition to its antibacterial activity, DIHQ also exhibited antivirulence properties, namely, partial inhibition of both toxin production and spore formation by C. difficile. Furthermore, DIHQ exhibited a minimal effect against important commensal organisms that comprise the intestinal microbiome. Future studies will need to be conducted to validate the in vitro findings in vivo in suitable animal models of CDI in order to further develop DIHQ as a novel anticlostridial drug.
MATERIALS AND METHODS
Bacterial strains, chemicals, and media.
All experiments were performed following relevant guidelines and regulations of the Purdue University Institutional Biosafety Committee. C. difficile isolates were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) and the American Type Culture Collection (ATCC) (Table 1S). DIHQ, albendazole, mebendazole (Tokyo Chemical Industry), praziquantel, cambendazole, ricobendazole, thiabendazole (Cayman Chemicals), paromomycin sulfate (Alfa Aesar), pyrantel pamoate (Acros Organics), pyrimethamine (MP Biomedicals), fidaxomicin (Cayman Chemicals), metronidazole (Alfa Aesar), and vancomycin hydrochloride (Gold Biotechnology) were purchased from commercial vendors. Brain heart infusion broth and de Man-Rogosa-Sharpe (MRS) broth were purchased from Becton, Dickinson and Company, and phosphate-buffered saline (PBS) was purchased from Fisher Scientific. Yeast extract, l-cysteine, vitamin K, and hemin were all obtained from Sigma-Aldrich.
Screening a small panel of antiparasitic drugs against C. difficile.
The MICs of all drugs were determined against two C. difficile clinical strains, as previously described (45–47). Briefly, 0.5 McFarland bacterial solution was prepared and diluted in brain heart infusion supplemented (BHIS) broth (inoculum size, ∼5 × 105 CFU/ml). Drugs were added and serially diluted before plates were incubated anaerobically at 37°C for 48 h. The MICs reported are the lowest drug concentration that completely suppressed the growth of bacteria, as observed visually.
Antibacterial activity of DIHQ against a wide panel of C. difficile strains.
The MICs of DIHQ and control antibiotics (vancomycin, metronidazole, and fidaxomicin) were determined against 39 clinical isolates of C. difficile using the broth microdilution method (45). MIC50 and MIC90 are the minimum concentrations of each agent that inhibited growth of 50% and 90% of the tested isolates, respectively. The minimum bactericidal concentration (MBC) of these drugs was tested by plating 5 μl from wells with no growth onto BHIS agar plates. The MBC was categorized as the lowest concentration that reduced bacterial growth by 99.9% (48).
Time-kill assay.
To examine the killing kinetics of DIHQ, a time-kill assay against C. difficile ATCC BAA-1870 and C. difficile NR-49277 was performed, as described previously (49). C. difficile cells in logarithmic-growth phase were diluted to ∼106 CFU/ml and exposed to concentrations equivalent to 5× the MIC of DIHQ, metronidazole, or vancomycin (in triplicate) in BHIS broth. Aliquots (100 μl) were collected from each treatment after 0, 4, 6, 8, 12, and 24 h for determination of the viable CFU per milliliter.
Combination testing of DIHQ and standard anticlostridial drugs (metronidazole and vancomycin) against C. difficile.
To evaluate the interactions between DIHQ and vancomycin or metronidazole, a standard checkerboard assay was utilized (50, 51). The fractional inhibitory concentration index (ΣFIC) was calculated for each interaction against nine C. difficile clinical isolates. Interactions where the ΣFIC index was ≤0.5 were categorized as synergistic (SYN). An ΣFIC value of >0.5 to 1.25 was considered additive (ADD), and an ΣFIC value of >1.25 to 4 was considered to represent indifference. ΣFIC values of >4 were categorized as antagonistic (52).
Effect of DIHQ on C. difficile toxin inhibition.
To investigate DIHQ’s effect on C. difficile toxin production, toxin A and toxin B levels were measured, as described previously (17, 45). Briefly, drug concentrations equivalent to 1/4× the MIC and 1/2× the MIC were added to a late-exponential-phase culture of a hypervirulent toxigenic strains (C. difficile ATCC BAA-1870, C. difficile ATCC 43255, and C. difficile NR-49277) and incubated anaerobically at 37°C for 8 h. The total concentration of C. difficile toxins A and B was measured in the supernatant of each tube using an enzyme-linked immunosorbent assay (ELISA) kit (tgcBIOMICS, Dunwoody, GA), following the manufacturer’s instructions. The optical density at 450 nm and 620 nm (OD450 and OD620) values, corresponding to the toxin concentration, were determined for DIHQ and control drugs.
Effect of DIHQ on C. difficile spore formation.
The spore inhibition assay was performed as described in previous reports (45, 53). Briefly, log-phase cultures of C. difficile strains (ATCC BAA-1870, ATCC 43255, and NR-49277) were diluted in BHIS to an initial density of ∼106 CFU/ml. Afterwards, the bacterial suspension was split into microcentrifuge tubes, and drugs were added (in triplicate) at concentrations equal to 1/2× the MIC or 1× the MIC. The tubes were then incubated anaerobically for 5 days at 37°C. Thereafter, an aliquot from each tube was diluted and plated on BHIS agar plates supplemented with 0.1% taurocholic acid to count the total bacterial counts (vegetative bacteria plus spores). The remaining solution was centrifuged, and the pellet was suspended in PBS, stored overnight at 4°C, and subsequently shock heated at 70°C for 30 min to kill the vegetative cells. The resulting solution was serially diluted and plated to determine the heat-resistant spore counts.
In vitro antibacterial evaluation of DIHQ against normal microflora.
The broth microdilution assay was utilized to determine the MICs of DIHQ against commensal organisms that compose the human gut microflora, as described elsewhere (54, 55). A bacterial solution equivalent to 0.5 McFarland standard was prepared and diluted in BHIS broth (for Bacteroides and Bifidobacterium spp.) or in MRS broth (for Lactobacillus spp.) to achieve a bacterial concentration of about 5 × 105 CFU/ml. Drugs were added and serially diluted with medium containing bacteria. Plates were incubated for 48 h at 37°C before recording the MIC by visual inspection of growth.
Supplementary Material
ACKNOWLEDGMENTS
We thank Haroon Mohammad for grammatical check and English editing of the manuscript and Ahmed AbdelKhalek for his kind help in a part of the work.
This work was supported by the National Institutes of Health (grant R01AI130186).
We declare no conflicts of interest.
Footnotes
Supplemental material is available online only.
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