FXR-Dependent Modulation of the Human Small Intestinal Microbiome by the Bile Acid Derivative Obeticholic Acid.

BACKGROUND & AIMS: Intestinal bacteria can modify the composition of bile acids and bile acids, which are regulated by the farnesoid X receptor, affect the survival and growth of gut bacteria. We studied the effects of obeticholic acid (OCA), a bile acid analogue and farnesoid X receptor agonist, on the intestinal microbiomes of humans and mice. METHODS: We performed a phase I study in 24 healthy volunteers given OCA (5, 10, or 25 mg/d for 17 days). Fecal and plasma specimens were collected at baseline (day 0) and on days 17 (end of dosing) and 37 (end of study). The fecal specimens were analyzed by shotgun meta-genomic sequencing. A Uniref90 high-stringency genomic analysis was used to assign specific genes to the taxonomic signature of bacteria whose abundance was associated with OCA. Male C57BL/6 mice were gavage fed daily with water, vehicle, or OCA (10 mg/kg) for 2 weeks. Small intestine luminal contents were collected by flushing with saline and fecal pellets were collected at baseline and day 14. Mouse samples were analyzed by 16S-tagged sequencing. Culture experiments were performed to determine the taxonomic-specific effects of bile acids and OCA on bacterial growth. RESULTS: Suppression of endogenous bile acid synthesis by OCA in subjects led to a reversible induction of gram-positive bacteria that are found in the small intestine and are components of the diet and oral microbiota. We found that bile acids decreased proliferation of these bacteria in minimum inhibitory concentration assays. In these organisms, there was an increase in the representation of microbial genomic pathways involved in DNA synthesis and amino acid metabolism with OCA treatment of subjects. Consistent with these findings, mice fed OCA had lower endogenous bile acid levels and an increased proportion of Firmicutes, specifically in the small intestine, compared with mice fed water or vehicle. CONCLUSIONS: In studying the effects of OCA in humans and mice, we found evidence for interactions between bile acids and features of the small intestinal microbiome. These findings indicate that farnesoid X receptor activation alters the intestinal microbiota and could provide opportunities for microbiome biomarker discovery or new approaches to engineering the human microbiome. ClinicalTrials.gov, NCT01933503.

Comparative microbiological studies of transcription inhibitors fidaxomicin and the rifamycins in Clostridium difficile.

Fidaxomicin (FDX) is a narrow-spectrum antibiotic for the treatment of Clostridium difficile-associated diarrhea. While FDX and rifamycins share the same target (RNA polymerase), FDX exhibits a unique mode of action distinct from that of rifamycins. In comparative microbiological studies with C. difficile, FDX interacted synergistically with rifamycins, demonstrated a lower propensity for the development of resistance to rifamycins, and exhibited no cross-resistance with rifamycins. These results highlight differences in the mechanisms of action of FDX and rifamycins.

Whole-genome sequencing demonstrates that fidaxomicin is superior to vancomycin for preventing reinfection and relapse of infection with Clostridium difficile.

Whole-genome sequencing was used to determine whether the reductions in recurrence of Clostridium difficile infection observed with fidaxomicin in pivotal phase 3 trials occurred by preventing relapse of the same infection, by preventing reinfection with a new strain, or by preventing both outcomes. Paired isolates of C. difficile were available from 93 of 199 participants with recurrences (28 were treated with fidaxomicin, and 65 were treated with vancomycin). Given C. difficile evolutionary rates, paired samples ≤2 single-nucleotide variants (SNVs) apart were considered relapses, paired samples >10 SNVs apart were considered reinfection, and those 3-10 SNVs apart (or without whole-genome sequences) were considered indeterminate in a competing risks survival analysis. Fidaxomicin reduced the risk of both relapse (competing risks hazard ratio [HR], 0.40 [95% confidence interval {CI}, .25-.66]; P = .0003) and reinfection (competing risks HR, 0.33 [95% CI, 0.11-1.01]; P = .05).

Fidaxomicin inhibits toxin production in Clostridium difficile.

OBJECTIVES: Fidaxomicin, which was recently approved for the treatment of Clostridium difficile-associated diarrhoea, demonstrates narrow-spectrum bactericidal activity via inhibition of RNA polymerase. In this study we evaluated its inhibitory activity versus C. difficile toxin gene expression and toxin production by quantifying toxin mRNA and protein. METHODS: The effects of fidaxomicin, its major metabolite (OP-1118), vancomycin and metronidazole on toxin A and toxin B production were determined by assaying culture supernatants of two C. difficile isolates (ATCC 43255, a high-level toxin-producing strain, and UK-14, a NAP1/027/BI epidemic strain) using a commercial ELISA. The effects of the drugs on toxin gene expression were assessed in stationary-phase cells of C. difficile strain UK-1 (NAP1/027/BI type epidemic strain) and in the closely related non-epidemic strain CD196 by quantitative RT-PCR. RESULTS: Subinhibitory levels (1/4× MIC) of fidaxomicin or OP-1118 (but not vancomycin or metronidazole) strongly suppressed toxin production in C. difficile (≥ 60%) through at least 1 week of culture. Additionally, transcripts from the pathogenicity loci (tcdR, tcdA and tcdB) were nearly completely inhibited by both fidaxomicin (2× MIC) and OP-1118 (2.5× MIC), but not vancomycin (2.5× MIC). CONCLUSIONS: Both fidaxomicin and OP-1118 are able to inhibit toxin production in vitro, which may explain prior post-treatment observations of less frequent detectable toxin in fidaxomicin-treated patients (27 subjects) than those treated with vancomycin (8 patients).

Both fidaxomicin and vancomycin inhibit outgrowth of Clostridium difficile spores.

Fidaxomicin (FDX) is approved to treat Clostridium difficile-associated diarrhea and is superior to vancomycin in providing a sustained clinical response (cure without recurrence in the subsequent 25 days). The mechanism(s) behind the low recurrence rate of FDX-treated patients could be multifactorial. Here, we tested effects of FDX, its metabolite OP-1118, and vancomycin on spore germination and determined that none affected the initiation of spore germination but all inhibited outgrowth of vegetative cells from germinated spores.

Reduced acquisition and overgrowth of vancomycin-resistant enterococci and Candida species in patients treated with fidaxomicin versus vancomycin for Clostridium difficile infection.

Fidaxomicin causes less disruption of anaerobic microbiota during treatment of Clostridium difficile infection (CDI) than vancomycin and has activity against many vancomycin-resistant enterococci (VRE). In conjunction with a multicenter randomized trial of fidaxomicin versus vancomycin for CDI treatment, we tested the hypothesis that fidaxomicin promotes VRE and Candida species colonization less than vancomycin. Stool was cultured for VRE and Candida species before and after therapy. For patients with negative pretreatment cultures, the incidence of VRE and Candida species acquisition was compared. For those with preexisting VRE, the change in concentration during treatment was compared. The susceptibility of VRE isolates to fidaxomicin was assessed. Of 301 patients, 247 (82%) had negative VRE cultures and 252 (84%) had negative Candida species cultures before treatment. In comparison with vancomycin-treated patients, fidaxomicin-treated patients had reduced acquisition of VRE (7% vs 31%, respectively; P < .001) and Candida species (19% vs 29%, respectively; P = .03). For patients with preexisting VRE, the mean concentration decreased significantly in the fidaxomicin group (5.9 vs 3.8 log(10) VRE/g stool; P = .01) but not the vancomycin group (5.3 vs 4.2 log(10) VRE/g stool; P = .20). Most VRE isolates recovered after fidaxomicin treatment had elevated fidaxomicin minimum inhibitory concentrations (MICs; MIC(90), 256 µg/mL), and subpopulations of VRE with elevated fidaxomicin MICs were common before therapy. Fidaxomicin was less likely than vancomycin to promote acquisition of VRE and Candida species during CDI treatment. However, selection of preexisting subpopulations of VRE with elevated fidaxomicin MICs was common during fidaxomicin therapy.

Antimicrobial activities of fidaxomicin.

Fidaxomicin is bactericidal against Clostridium difficile. The combined results of 8 in vitro studies of 1323 C. difficile isolates showed the minimum inhibitory concentration (MIC) range of fidaxomicin to be ≤ 0.001-1 µg/mL, with a maximum MIC for inhibition of 90% of organisms (MIC(90)) of 0.5 µg/mL. Isolates from 2 phase III clinical trials demonstrated that fidaxomicin MICs of baseline isolates did not predict clinical cure, failure, or recurrence of C. difficile infections. No resistance to fidaxomicin developed during treatment in either study, although a single strain recovered from a cured patient had an elevated MIC of 16 µg/mL at the time of recurrence. For 135 strains, OP-1118, a major metabolite, had an MIC for inhibition of 50% of organisms of 4 µg/mL and an MIC(90) of 8 µg/mL. Changes in inoculum size (10(2)-10(5) colony-forming units/spot) or cation concentrations of calcium or magnesium appeared to have no effect on fidaxomicin MICs. Fidaxomicin has little or no activity against gram-negative aerobes and anaerobes or yeast.

Fidaxomicin inhibits spore production in Clostridium difficile.

Fidaxomicin (FDX) is a novel antimicrobial agent with narrow-spectrum and potent bactericidal activity against Clostridium difficile. In recent clinical trials, FDX was superior to vancomycin in preventing recurrences of C. difficile infection. A possible mechanism of reducing recurrence may be through an inhibitory effect on sporulation. The effect of FDX and its major metabolite, OP-1118, on C. difficile growth and sporulation kinetics was compared with that of vancomycin, metronidazole, and rifaximin. Drugs at subminimum inhibitory concentrations (sub-MICs) were added to cells at an early stationary phase of growth; this was followed by collection of cells at various intervals for quantitation of total viable cell and heat-resistant spore counts on taurocholate-containing media. The effect of the drugs at 2-2.5× MIC on the expression of sporulation genes in C. difficile was also compared using quantitative reverse-transcriptase polymerase chain reaction. Both FDX and OP-1118 (1/4× MIC) inhibited sporulation when added to early-stationary-phase cells in C. difficile strains, including the epidemic NAP1/BI/027 strain. In contrast, vancomycin, metronidazole, and rifaximin (at similar sub-MICs) did not inhibit sporulation. The number of spores following treatment with comparator drugs increased to the same level as the no-drug control treatment. Expression of mother cell-specific (spoIIID) and forespore-specific (spoIIR) sporulation genes also was inhibited by FDX and OP-1118 but not significantly by vancomycin. Both FDX and OP-1118 (unlike vancomycin, rifaximin, and metronidazole) effectively inhibited sporulation by C. difficile. The inhibitory effect of FDX on C. difficile sporulation may contribute to its superior performance in sustaining clinical response and reducing recurrences and may also be beneficial in decreasing shedding and transmission of this pathogen.

Decreased cure and increased recurrence rates for Clostridium difficile infection caused by the epidemic C. difficile BI strain.

BACKGROUND: An epidemic strain of Clostridium difficile designated by restriction endonuclease analysis (REA) as group BI has caused multiple outbreaks of severe C. difficile infection (CDI). The treatment response of patients infected with this strain is uncertain. METHODS: Clostridium difficile isolates were collected from 2 phase 3 clinical trials comparing fidaxomicin to vancomycin and typed using REA. Clinical cure and recurrence outcomes were analyzed by strain type of the infecting organism, BI and non-BI, using both univariate and multivariate analyses. RESULTS: From 999 patients, 719 isolates were available for typing (356 fidaxomicin treated and 363 vancomycin treated). BI was the most common REA group (34% of isolates). Patients infected with BI had lower cure rates (86.6%; 214 of 247) than those infected with non-BI strains (94.3%; 445 of 472) (P < .001). The cure rate difference between the BI and non-BI patients was significant for both vancomycin (P = .02) and fidaxomicin (P = .007). BI patients had a recurrence rate of 27.4% (51 of 186), compared with a recurrence rate of 16.6% (66 of 397) in non-BI patients (P = .002). By multivariate analysis, BI infection was statistically significant as a risk factor for reduced cure (odds ratio [OR], 0.48; 95% confidence interval [CI], .27-.85; P = .030) and for increased recurrence (OR, 1.57; 95% CI, 1.01-2.45; P = .046). CONCLUSIONS: The clinical cure rate of patients infected with the epidemic BI C. difficile strain is lower than the cure rate of those infected with non-BI strains whether treated with fidaxomicin or vancomycin. Similarly, the CDI recurrence rate is increased in patients with the BI strain compared with patients with other C. difficile strains.

Comparative susceptibilities to fidaxomicin (OPT-80) of isolates collected at baseline, recurrence, and failure from patients in two phase III trials of fidaxomicin against Clostridium difficile infection.

A 10-day course of oral fidaxomicin (200 mg twice a day [b.i.d.]), a potent new macrocyclic drug, was compared to vancomycin (125 mg four times a day [q.i.d.]) in 1,164 adults (1,105 in the modified intent-to-treat [mITT] population) with Clostridium difficile infection in two phase III randomized, double-blind trials at sites in North America and 7 European countries. Of 1,105 mITT patients, 792 (71.7%), including 719/999 (72.0%) in the per-protocol (PP) population, provided a C. difficile strain at baseline, of whom 356 received fidaxomicin with 330 cures (92.7%) and 363 received vancomycin with 329 cures (90.6%). The susceptibilities (MIC(90)) of baseline isolates did not predict clinical cure, failure, or recurrence for fidaxomicin (MIC(90), 0.25 µg/ml for both; range, ≤ 0.007 to 1 µg/ml), but there was a one-dilution difference in the MIC(90) (but not the MIC(50)) for vancomycin (MIC(90), 2 µg/ml [range, 0.25 to 8 µg/ml] for cure and 4.0 µg/ml [range, 0.5 to 4 µg/ml] for failures). A total of 65 (7.9%) "rifaximin-resistant" (MIC > 256 µg/ml) strains were isolated in both treatment groups on enrollment, which increased to 25% for failures at the end of therapy. No resistance to either fidaxomicin or vancomycin developed during treatment in either of the phase III studies, although a single strain isolated from a cured patient had an elevated fidaxomicin MIC of 16 µg/ml at the time of recurrence. All isolates were susceptible to ≤ 4 µg/ml of metronidazole. When analyzed by restriction endonuclease analysis (REA) type, 247/719 (34.4%) isolates were BI group isolates, and the MICs were generally higher for all four drugs tested (MIC(90)s: fidaxomicin, 0.5; vancomycin, 2.0; metronidazole, 2.0; and rifaximin, >256 µg/ml) than for the other REA types. There was no correlation between the MIC of a baseline clinical isolate and clinical outcome. MIC(90)s were generally low for fidaxomicin and vancomycin, but BI isolates had higher MICs than other REA group isolates.

Postantibiotic effect of fidaxomicin and its major metabolite, OP-1118, against Clostridium difficile.

Fidaxomicin (FDX), a narrow-spectrum antibiotic recently shown to be superior to vancomycin in providing sustained clinical response to Clostridium difficile infection, was investigated along with its major metabolite, OP-1118, with regard to their postantibiotic effects (PAE). FDX was found to have a prolonged PAE (10 h versus ATCC strains and 5.5 h versus a clinical isolate), and OP-1118's PAE was longer than that of the standard comparator, vancomycin (3 versus 0 to 1.5 h, respectively).

Killing kinetics of fidaxomicin and its major metabolite, OP-1118, against Clostridium difficile.

The kinetics of bacterial killing for fidaxomicin and its major metabolite, OP-1118, were investigated against Clostridium difficile strains, including two clinical strains belonging to the restriction endonuclease analysis group BI (ORG 1687 and 1698), the ATCC 43255 strain and two laboratory-derived mutant strains with decreased susceptibility to fidaxomicin (ORG 919 and 1620). Both fidaxomicin and OP-1118 demonstrated time-dependent killing of C. difficile strains. Fidaxomicin (at 4× MIC) reduced bacterial counts of the ATCC 43255 strain, clinical strain ORG 1687 and the two laboratory-generated mutant strains by ≥3 logs within 48 h of exposure. The other BI strain, ORG 1698, was tested at 2× MIC fidaxomicin with bacterial counts decreasing 1 log in 48 h. Exposure to OP-1118 (at 4× MIC) also resulted in a ≥3 log drop in c.f.u. counts for the ATCC 43255 strain, the clinical BI strain ORG 1687 and the mutant strain ORG 919. Higher concentrations of OP-1118 (32× MIC) were required for a 3 log reduction in c.f.u. counts for the other BI strain, ORG 1698. In summary, the results indicate that both fidaxomicin and its major metabolite, OP-1118, are bactericidal against C. difficile strains, including the hypervirulent restriction endonuclease analysis group BI strains, at concentrations that are many fold below the detected faecal concentrations of these compounds after oral administration of fidaxomicin.