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Clostridium difficile infection (CDI) is a primary cause of antibiotic-associated diarrhoeal illness. Current therapies are insufficient as relapse rates following antibiotic treatment range from 25% for initial treatment to 60% for treatment of recurrence. In this study, we looked at the efficacy of SQ641 in a murine model of CDI. SQ641 is an analogue of capuramycin, a naturally occurring nucleoside-based compound produced by Streptomyces griseus.
In a series of experiments, C57BL/6 mice were treated with a cocktail of antibiotics and inoculated with C. difficile strain VPI10463. Animals were treated orally with SQ641 for 5 days at a dose range of 0.1–300 mg/kg/day, 20 mg/kg/day vancomycin or drug vehicle. Animals were monitored for disease severity, clostridial shedding and faecal toxin levels for 14 days post-infection.
Five day treatment of CDI with SQ641 resulted in higher 14 day survival rates in mice compared with either vancomycin or vehicle alone. CDI survival rates were 100% (13 of 13) and 94% (32 of 34), respectively, in the 1 and 10 mg/kg/day SQ641 treatment groups, 37% (7 of 19) with vancomycin treatment at 20 mg/kg/day and 32% (14 of 44) in the vehicle-only control group. Secondary measures of efficacy, such as prevention of weight loss, decreased disease severity, decreased C. difficile shedding and decreased toxin in faeces, were observed with SQ641 and vancomycin treatment.
SQ641 is effective for CDI treatment with prevention of relapse in the murine model of CDI.
There is currently a great need for alternative therapies for Clostridium difficile infection (CDI), the leading nosocomial-acquired infection in the United States with incidence rates approaching 8.75 cases per 1000 hospital discharges in 2008.1 Current therapies are insufficient as relapse rates following initial treatment approach 25% and succeeding treatment up to more than 60%.2,3 The Society for Healthcare Epidemiology of America (SHEA) and the IDSA and ESCMID recommend oral metronidazole (500 mg) administration three times daily for 10–14 days for an initial episode of mild to moderate CDI and oral vancomycin treatment (125 mg) four times daily or fidaxomicin treatment (200 mg) twice daily for the same duration for an initial episode of severe CDI.2,4 Treatment of the first recurrence of CDI is usually with the same drug used for the initial episode. Fidaxomicin, a new macrocyclic antibiotic approved for CDI treatment in 2011, shows similar initial cure rates to vancomycin and a lower relapse rate (25% recurrence rate in vancomycin-treated patients compared with 15% in fidaxomicin-treated patients); however, there were no significant differences between recurrence rates in patients infected with the epidemic NAP1/BI/027 strain of C. difficile treated with either vancomycin or fidaxomicin.5
In this paper, we describe the effects of SQ641 treatment on CDI in a murine model. SQ641 is an analogue of capuramycin, a nucleoside-based compound produced by the bacterium Streptomyces griseus.6 Its primary mechanism of action is the inhibition of translocase (TL1), a key regulator of cell wall synthesis in Gram-positive bacteria.7 SQ641 has shown in vitro efficacy against C. difficile while having a minimal effect on other potentially beneficial commensal flora.8 Additionally, SQ641 has very low bioavailability following oral administration, making it a good candidate for the treatment of CDI.9 Our aim in this study was to determine the efficacy of SQ641 in an established murine model of CDI.10 To determine efficacy, we evaluated SQ641 dose–response curves, tested two drug formulations for oral delivery and compared effectiveness with vancomycin as a standard antibiotic treatment for CDI. The primary endpoint was survival at 14 days, while secondary endpoints included disease severity score, weight loss and clostridial shedding and toxin in faeces.
All animal experiments were performed under a protocol approved by the Animal Institutional Care and Use Committee at the Center for Comparative Medicine at the University of Virginia, in line with national standards. Mice were infected with C. difficile using a previously described model.10 Eight week old male C57BL/6 mice from Jackson Laboratories (Farmington, CT, USA) were treated with a pre-infection antibiotic cocktail in drinking water [vancomycin (0.0045 mg/g bodyweight), colistin (0.0042 mg/g), gentamicin (0.0035 mg/g) and metronidazole (0.0215 mg/g)] for 3 days starting 6 days prior to infection. Mice were administered clindamycin (0.032 mg/g) via intraperitoneal injection 1 day prior to infection. Mice were then inoculated with live, vegetative C. difficile strain VPI 10463 (ATCC, Manassas, VA, USA) at 105 cfu by oral gavage and housed singly to prevent cross-contamination from other mice. Mice were monitored for up to 14 days post-infection and checked daily for weight and disease severity scores. Disease severity scoring was based on a 0–20 point scale as previously described, which takes into account weight loss, activity, posture, coat appearance, diarrhoea and eye squint/discharge.10 Moribund animals, animals that had lost more than 25% of their baseline bodyweight post-infection and animals that had a combined clinical score of 14 or higher were euthanized according to protocol.
Caecal and colonic tissue samples were harvested and fixed in Bouin solution for 24 h and then moved to a 70% ethanol solution, paraffin embedded and stained using haematoxylin/eosin (services provided by University of Virginia Research Histology Core). A blinded reader scored the coded slides using published parameters.11 Histopathological scores of both caecum and colon were combined then averaged for each animal in our results.
Faecal samples were taken just prior to infection and on days 1, 2–3, 7–8 and 12 following infection to determine C. difficile shedding. Samples were normalized to faecal weight and run in duplicate. Genomic DNA was extracted from faeces using a QIAamp DNA Stool mini kit (Qiagen, Redwood City, CA, USA) according to the manufacturer's protocol. Quantitative real-time PCR experiments were performed using the CFX96TM Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A total reaction volume of 20 µL per sample was prepared by mixing 10 μL of Kapa SYBR Fast quantitative PCR Master Mix (Kapa Biosystems, Wilmington, MA, USA), 1 μL of each forward and reverse primers (Eurofins MWG Operon, Huntsville, AL, USA) at 10 μM (final concentration 0.4 μM), 4 μL of template DNA and 4 μL of DEPC-treated nuclease-free sterile water (Fisher Scientific, Pittsburgh, PA, USA). tcdB forward primer (5′-GGTATTACCTAATGCTCCAAATAG-3′) and tcdB reverse primer (5′-TTTGTGCCATCATTTTCTAAGC-3′) were used to quantify C. difficile in faeces.12 A two-step amplification reaction consisted of 95°C for 5 min, then 40 cycles of amplification with a denaturating step at 95°C for 10 s with an annealing/extension step at 57°C for 45 s. Following amplification, melting curve analysis was carried out by 0.5°C increments for 5 s starting at 65°C and ending with 95°C to determine the specificity of PCR reactions. The fluorescence signal was measured during the annealing step of each cycle and the Ct values were compared with standards with known concentrations of bacterial DNA (C. difficile strain VPI 10463). Samples were run in duplicate.
To determine toxin production, the C. DIFFICILE TOX A/B II™ test (Techlab, Blacksburg, VA, USA) was used and samples were processed as recommended by the manufacturer. Samples were normalized according to weights using diluent provided in the kit. Samples were diluted 10× and OD readings were taken at 450/620 nm (readings taken at 450 nm, blanked against air at 620 nm).
SQ641 was prepared in either a 10% DMSO/1% methylcellulose solution or a 10% d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) solution and administered once daily starting 24 h post-infection for 5 days via oral gavage at indicated dosages. Drug vehicle and 20 mg/kg/day vancomycin, also administered in drug vehicle, were administered to the control groups using the same method. SQ641 compound and TPGS diluent were provided by Sequella Inc. (Rockville, MD, USA).
Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad software, San Diego, CA, USA). Significance was defined at P≤0.05. Survival data were analysed by log-rank (Mantel–Cox) survival analysis. Scores, weights and assay values were analysed using a one-way or two-way analysis of variance (ANOVA) test where appropriate. A Bonferroni correction post-test followed significant ANOVA results to determine differences between groups.
Two parameters were examined to determine how effective SQ641 was at treating CDI in mice: survival during treatment of the initial acute infection, and survival from relapse following cessation of treatment. Current therapies, including vancomycin and fidaxomicin, are efficacious at treating initial disease but have high relapse rates. In our experiments, we used a treatment course of 5 days followed by monitoring for recovery for a total of 14 days. Based on our previously published models this was enough time for relapse to occur in most animals.10
The data reported in this paper are from a series of experiments and cumulative results are combined in the graphs.
The effects of treatment for 5 days on survival and weight loss during acute CDI were assessed on day 6 post-infection. Survival rates on day 6 (Figure 1a and d) for animals receiving SQ641 at: 0.01 mg/kg/day were 33.3% (2 of 6); 0.1 mg/kg/day, 50.0% (3 of 6); 1 mg/kg/day, 100% (13 of 13); 10 mg/kg/day, 94.1% (32 of 34); 100 mg/kg/day, 89.7% (26 of 29); and 300 mg/kg/day, 85.7% (12 of 14). Survival of mice treated with 20 mg/kg/day vancomycin was 100% (19 of 19) and infected control animals treated with drug vehicle alone was 31.8% (14 of 44) at day 6. Log-rank tests confirmed significant differences in survival between groups receiving SQ641 at doses ≥1 mg/kg day and the infected control mice as well as vancomycin-treated mice and the infected control mice at day 6 post-infection (Figure 1a). There were no significant differences in survival at day 6 post-infection between vancomycin-treated mice and mice receiving SQ641 at doses between 1 and 300 mg/kg/day. No differences in survival were observed in mice receiving SQ641 at doses ≤0.1 mg/kg and the infected control mice at day 6 (Figure 1d). In summary, treatment for 5 days with ≥1 mg/kg/day SQ641 was as effective as vancomycin in preventing mortality at day 6.
Mice receiving SQ641 at doses of 10–300 mg/kg/day and vancomycin at doses of 20 mg/kg/day (Figure 1b) had significantly higher weights at the height of the initial illness (days 2–4 post-infection) compared with infected control mice (P<0.05, two-way ANOVA with Bonferroni correction post-test), demonstrating that mice are better able to maintain bodyweight during treatment of the CDI with these doses. Weight loss in groups treated with SQ641 at 0.01 and 0.1 mg/kg/day (Figure 1e) was not significantly different than the infected control mice on days 2–4 post-infection. Clinical scores of animals receiving vancomycin at doses of 20 mg/kg/day or SQ641 at doses ≥1 mg/kg/day were also significantly lower than infected control mice on days 2–3 post-infection (Figure 1c), while the clinical scores of animals receiving SQ641 at doses ≤0.1 mg/kg/day were not significantly different compared with infected control mice (Figure 1e).
Survival of mice treated with SQ641 formulated in DMSO/methylcellulose was comparable to mice treated with SQ641 formulated in TPGS (Figure S1, available as Supplementary data at JAC Online); there were no significant differences between groups via ANOVA.
Mortality was assessed in mice after treatment was discontinued at days 6–14 post-infection. As shown in Figure 1, the 14 day survival rates for these mice were: 31.8% (14 of 44) for the drug vehicle only; 100% (13 of 13) for 1 mg/kg/day SQ641; 94.1% (32 of 34) for 10 mg/kg/day SQ641; 82.7% (24 of 29) for 100 mg/kg/day SQ641; 71.4% (10 of 14) for 300 mg/kg/day SQ641; and 36.8% (7 of 19) for 20 mg/kg/day vancomycin. Doses of SQ641 ≥1 mg/kg/day had higher 14 day survival rates compared with infected control mice, while mortality in mice treated with SQ641 at doses ≤0.1 mg/kg/day was indistinguishable from infected control mice by day 14. Mortality in vancomycin-treated mice was comparable to the infected control mice by day 14. All deaths in vancomycin-treated mice occurred post-treatment suggesting recurrence of fulminant CDI.
Animals receiving vancomycin at doses of 20 mg/kg/day also had significantly decreased body weight by day 10 post-infection compared with SQ641-treated mice at doses ≥1 mg/kg/day (P<0.05) (Figure 1b). There was also significantly increased clinical scoring in animals receiving vancomycin at doses of 20 mg/kg/day compared with animals receiving SQ641 at doses ≥1 mg/kg/day on days 9–11 post-infection demonstrating more severe CDI in the vancomycin-treated group.
As shown in Figure 2(a), C. difficile shedding in faeces was inhibited with SQ641 drug treatment at doses ≥1 mg/kg/day or with vancomycin treatment at doses of 20 mg/kg/day compared with the infected control mice on day 2 or 3 post-infection (P<0.001, one-way ANOVA with Bonferroni correction post-test) and in mice treated with SQ641 at doses ≥100 mg/kg/day or vancomycin compared with infected control mice on days 7–8 post-infection (P<0.05) suggesting a potential post-antibiotic effect at higher doses of SQ641. There was no difference in bacterial load in faeces between groups in samples collected on days 12–14 post-infection indicating persistence of C. difficile in the gastrointestinal tract, as C. difficile levels had recovered post-treatment.
There were no significant differences in toxin levels in faeces between treatment groups on days 1 or 12 post-infection (Figure 2b). Interestingly, the toxin A/B burden in faeces was significantly less in the groups receiving 300 mg/kg SQ641, 100 mg/kg SQ641 and vancomycin at doses of 20 mg/kg/day compared with the groups receiving 1 or 10 mg/kg SQ641 on days 2–3 and days 7–8 post-infection (P<0.05, one-way ANOVA with Bonferroni correction post-test), even though the most efficacious in terms of outcome were 1 and 10 mg/kg/day. Toxin levels were significantly inhibited by doses of 100 and 300 mg/kg/day SQ641, as well as vancomycin at doses of 20 mg/kg/day, on days 7–8 post-infection; however, all survivors from all treatment groups had high toxin levels in faeces by day 12 post-infection and there were no significant differences between groups.
Colon and caecum tissue were harvested from animals post-euthanasia, either from moribund animals during the course of the experiment or at day 14 after initial infection. In general, the combined histopathological scoring was lower in mice treated with SQ641 at doses ≥1 mg/kg/day as compared with the infected control, as more of the treated mice survived infection. The representative histology of colon tissue collected from animals is shown in Figure 3. Untreated infected intestinal tissues exhibited epithelial erosion, inflammatory cell infiltration, mucosal hypertrophy, submucosal oedema and luminal exudates at the peak of infection (Figure 3b). Intestinal tissues from vancomycin-treated mice at the time of relapse had similar histopathologic changes to infected controls (Figure 3c). Infected tissues from mice treated with SQ641 at 10 mg/kg/day (Figure 3d) had intact epithelium, decreased cellularity and no or minimal luminal exudates, mucosal hypertrophy or oedema, almost similar to uninfected tissues (Figure 3e) at day 14.
There is a great need for therapeutics that effectively treat CDI without high recurrence rates. In this study, we found that mice treated with ≥1 mg/kg/day SQ641 for 5 days had similar survival and disease severity as mice treated with vancomycin at doses of 20 mg/kg/day during initial infection. However, SQ641-treated mice had increased survival, decreased weight loss and milder disease post-treatment compared with mice treated with vancomycin. Overall, C. difficile shedding and toxin in faeces were also decreased in mice treated with the higher doses of SQ641 as compared with drug vehicle alone although these effects were not sustained and, moreover, not observed at lower doses, indicating that other factors such as intestinal microbial factors may play a role. Treatment of mice with SQ641 for 5 days at ≥1 mg/kg/day was associated with a decrease in mortality by day 14 as compared with mice treated with vancomycin or the infected control mice. Mortality by day 14 in mice treated with SQ641 at doses ≤0.1 mg/kg/day was indistinguishable from the infected control mice.
An important limitation in our study is that the intestinal microbiota were not analysed during the experiments. We suspect that delayed mortality in mice treated with higher doses of SQ641 or vancomycin at doses of 20 mg/kg/day for 5 days may involve the antibacterial activity of these antibiotics on resident microbiota leading to increased relapse in these groups. Decreased diversity of the host gut microbiota, primarily from antibiotic therapy, leads to susceptibility to colonization by C. difficile.13 Relapse is primarily driven by two factors: current antibiotic therapies do not completely eliminate C. difficile bacteria or spores, and continued exposure to broad-spectrum antibiotics during CDI treatment prevents key members of the host gut microbiota from repopulating, thus increasing the chance of relapse on cessation of treatment.14 Indeed, the decreased relapse rate associated with fidaxomicin is thought to be related to the drug's ability to specifically inhibit C. difficile while causing minimal impact on members of the resident microbiota compared with vancomycin; this includes clostridial clusters XIVa and IV, Bifidobacterium, Bacteroides fragilis and Bacteroides/Prevotella.15–17 Antibiotic specificity and level of drug exposure in the gut may be important factors in preventing delayed mortality following treatment for 5 days in our murine model of CDI. Antibiotics that allow recovery of the normal intestinal microbiome while specifically inhibiting C. difficile and bacterial toxin production may be critical in preventing death following treatment in our model of infection. For example, although 20 mg/kg/day vancomycin reduced C. difficile shedding and toxin production in a similar fashion to SQ641, it did not improve survival as compared with the infected control mice. Treatment with SQ641 at low doses allowed temporary inhibition of vegetative C. difficile growth and toxin production, enough for the animals to not become lethally sick while still allowing their microbiota to recover, resulting in improved survival at day 14. This observation brings into question current strategies for treatment of recurrent CDI with repeat courses of antibiotics or tapering or pulsing of vancomycin.18 We have previously shown that decreasing the duration of vancomycin treatment in vivo from 5 days down to 1 day decreases the relapse potential in mice infected with CDI while still protecting animals from mortality during the initial disease.10 Exposure to vancomycin will decrease levels of certain groups of gut microbiota, primarily Gram-positive bacteria belonging to the Firmicutes phylum, including butyrate-producing bacteria Clostridium cluster IV and XIVa, which are important for gut barrier function.17,19 Additionally, there are associated increases in bacteria from the Proteobacteria phylum while overall microbial diversity is significantly decreased, which is a risk factor for C. difficile colonization and likely relapse.20 We suspect that avoiding overly high SQ641 exposure in the gut may limit the impact on key members of the host microbiome and lead to decreased recurrent disease and increased survival. Whether or not this would apply to other antibiotics used to treat CDI remains to be seen. In conclusion, we have found that SQ641 at doses ≥1 mg/kg/day are more effective than vancomycin at doses of 20 mg/kg/day in treating CDI and are associated with improved survival at 14 days in mice. Understanding the impact of drugs such as SQ641, vancomycin and fidaxomicin on members of the resident microbiome will help guide the approach to treating CDI while preventing recurrent disease.
Funding for this study was provided by NIH grant 5R01AI094458-05 (M. P.).
E. B., B. N., L. E., A. J. P. and M. P. are employed by Sequella Inc. L. E. owns stocks and A. J. P. and M. P. have stock options in Sequella. All other authors: none to declare.