Susceptibility of Clostridium difficile isolates from a Phase 2 clinical trial of cadazolid and vancomycin in C. difficile infection.

OBJECTIVES: The aim of this study was to evaluate the susceptibilities of Clostridium difficile isolates to cadazolid, a novel antibiotic for the treatment of C. difficile infection. METHODS: Ribotyping and susceptibilities were determined for C. difficile isolates from a multicentre, double-blind, Phase 2 study of oral cadazolid in patients with C. difficile infection (NCT01222702, ClinicalTrials.gov; EudraCT 2010-020941-29, European Clinical Trials Database). Patients were randomized to receive 250, 500 or 1000 mg of cadazolid twice daily or 125 mg of vancomycin four times daily, for 10 days. MICs of cadazolid, vancomycin, fidaxomicin, linezolid and moxifloxacin were determined at baseline for all patients and post-baseline for patients with clinical failure or recurrence, using the agar dilution method. RESULTS: Seventy-eight of 84 patients had an evaluable toxigenic C. difficile isolate at baseline. The most frequent PCR ribotype was 027 (15.4%). Cadazolid MICs for baseline isolates (including epidemic strain 027) ranged from 0.06 to 0.25 mg/L. Baseline cadazolid MICs were similar to those of fidaxomicin and lower than those of vancomycin, linezolid and moxifloxacin. For each clinical outcome group (clinical cure, clinical failure, sustained clinical response and clinical failure or recurrence), the baseline cadazolid MIC range was 0.06-0.25 mg/L. Mean (min-max) cadazolid faecal concentration (µg/g) on day 5 was 884 (101-2710), 1706 (204-4230) and 3226 (1481-12 600) for the doses 250, 500 and 1000 mg, respectively. CONCLUSIONS: For all cadazolid doses, the faecal concentration was in excess of several thousand-fold the MIC90 for C. difficile. The MIC of cadazolid for all C. difficile isolates, including epidemic strains, was low and in the same narrow range regardless of treatment outcome.

In vitro activity of cadazolid against clinically relevant Clostridium difficile isolates and in an in vitro gut model of C. difficile infection.

OBJECTIVES: We investigated the in vitro activity of cadazolid against 100 Clostridium difficile isolates and its efficacy in a simulated human gut model of C. difficile infection (CDI). METHODS: MICs of cadazolid, metronidazole, vancomycin, moxifloxacin and linezolid were determined using agar incorporation for 100 C. difficile isolates, including 30 epidemic strains (ribotypes 027, 106 and 001) with reduced metronidazole susceptibility, 2 linezolid-resistant isolates and 2 moxifloxacin-resistant isolates. We evaluated the efficacy of two cadazolid dosing regimens (250 versus 750 mg/L twice daily for 7 days) to treat simulated CDI. Microflora populations, C. difficile total viable counts and spores, cytotoxin titres, possible emergence of cadazolid, linezolid or quinolone resistance, and antimicrobial concentrations were monitored throughout. RESULTS: Cadazolid was active against all (including linezolid- and moxifloxacin-resistant) C. difficile strains (MIC90 0.125, range 0.03-0.25 mg/L). The cadazolid geometric mean MIC was 152-fold, 16-fold, 9-fold and 7-fold lower than those of moxifloxacin, linezolid, metronidazole and vancomycin, respectively. Both cadazolid dosing regimens rapidly reduced C. difficile viable counts and cytotoxin with no evidence of recurrence. Cadazolid levels persisted at 50-100-fold supra-MIC for 14 days post-dosing. Cadazolid inhibition of enumerated gut microflora was limited, with the exception of bifidobacteria; Bacteroides fragilis group and Lactobacillus spp. counts were unaffected. There was no evidence for selection of strains resistant to cadazolid, quinolones or linezolid. CONCLUSIONS: Cadazolid activity was greater than other tested antimicrobials against 100 C. difficile strains. Cadazolid effectively treated simulated CDI in a gut model, with limited impact on the enumerated gut microflora and no signs of recurrence or emergence of resistance within the experimental timeframe.

4-Toluene sulfonate methyl-monooxygenase from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

Comamonas testosteroni T-2 synthesizes an inducible enzyme system that oxygenates 4-toluene sulfontate (TS) to 4-sulfobenzyl alcohol when grown in TS-salts medium. We purified this TS methyl-monooxygenase system (TSMOS) and found it to consist of two components. A monomeric, iron-sulfur flavoprotein (component B), which has been shown to act as a reductase in the 4-sulfobenzoate dioxygenase system of this organism (H. H. Locher, T. Leisinger, and A. M. Cook, Biochem. J. 274:833-842, 1991), carried electrons from NADH to component M, an oxygenase. This oxygenase had the UV-visible spectral characteristics of an iron-sulfur protein. Mrs of about 152,000 for the native oxygenase and of 43,000 under denaturing conditions indicated a homotri- or homotetrameric enzyme, whose N-terminal amino acids and amino acid composition were determined. The activity of the purified enzyme was enhanced about fivefold by the addition of Fe2+. In the presence of O2 and NADH, components B and M together catalyzed the stoichiometric transformation of TS or p-toluate to the corresponding alcohol. The reaction was confirmed as oxygenation of the methyl group by observation of an oxygen atom from 18O2 in carboxybenzyl alcohol. The substrate range of TSMOS included carboxylated analogs of TS (p- and m-toluates and 4-ethylbenzoate), whereas p-xylene, toluene, and p-cresol were not substrates. TSMOS also catalyzed demethylation; 4-methoxybenzoate was transformed to 4-hydroxybenzoate and formaldehyde.

4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2.

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

Degradation of p-toluenesulphonic acid via sidechain oxidation, desulphonation and meta ring cleavage in Pseudomonas (Comamonas) testosteroni T-2.

Pseudomonas (Comamonas) testosteroni T-2 completely converted p-toluenesulphonic acid (TS) or p-sulphobenzoic acid (PSB) to cell material, CO2 and sulphate, with growth yields of about 5 g protein (mol C)-1. PSB and sulphite were excreted as transient intermediates during growth in TS-salts medium. All reactions of a catabolic pathway involving sidechain oxidation and cleavage of the sulphonate moiety as sulphite were measurable in the soluble portion of cell extracts. Degradation of TS and PSB was inducible and apparently involved at least two regulons. TS was converted to p-sulphobenzyl alcohol in a reaction requiring NAD(P)H and 1 mol O2 (mol TS)-1. This alcohol was in an equilibrium (in the presence of NAD+) with p-sulphobenzaldehyde, which was converted to PSB in an NAD(P)+-dependent reaction. PSB was desulphonated to protocatechuic acid in a reaction requiring NAD(P)H and 1 mol O2 (mol PSB)-1. Experiments with 18 O2 confirmed involvement of a dioxygenase, because both atoms of this molecular oxygen were recovered in protocatechuate. Protocatechuate was converted to 2-hydroxy-4-carboxymuconate semialdehyde by a 4.5-dioxygenase.

Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2.

The mechanism of transport of the xenobiotic 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. Rapid uptake of TS was observed only in cells grown with TS or 4-methylbenzoate as a carbon and energy source. Initial uptake rates under aerobic conditions showed substrate saturation kinetics, with an apparent affinity constant (Kt) of 88 microM and a maximal velocity (Vmax) of 26.5 nmol/min/mg of protein. Uptake of TS was inhibited completely by uncouplers and only marginally by ATPase inhibitors and the phosphate analogs arsenate and vanadate. TS uptake was also studied under anaerobic conditions, which prevented intracellular TS metabolism. TS was accumulated under anaerobic conditions in TS-grown cells upon imposition of an artificial transmembrane pH gradient (delta pH, inside alkaline). Uptake of TS was inhibited by structurally related methylated and chlorinated benzenesulfonates and benzoates. The results provide evidence that the first step in the degradation of TS by C. testosteroni T-2 is uptake by an inducible secondary proton symport system.

Antibacterial activities of epiroprim, a new dihydrofolate reductase inhibitor, alone and in combination with dapsone.

Epiroprim (EPM; Ro 11-8958) is a new selective inhibitor of microbial dihydrofolate reductase. EPM displayed excellent activity against staphylococci, enterococci, pneumococci, and streptococci which was considerably better than that of trimethoprim (TMP). EPM was also active against TMP-resistant strains, although the MICs were still relatively high. Its combination with dapsone (DDS) was synergistic and showed as in vitro activity superior to that of the TMP combination with sulfamethoxazole (SMZ). The EPM-DDS (ratio, 1:19) combination inhibited more than 90% of all important gram-positive pathogens at a concentration of 2 + 38 micrograms/ml. Only a few highly TMP-resistant staphylococci and enterococci were not inhibited. EPM was also more active than TMP against Moraxella catarrhalis, Neisseria meningitidis, and Bacteroides spp., but it was less active than TMP against all other gram-negative bacteria tested. Atypical mycobacteria were poorly susceptible to EPM, but the combination with DDS was synergistic and active at concentrations most probably achievable in biological fluids (MICs from 0.25 +/- 4.75 to 4 + 76 micrograms/ml). EPM and the EPM-DDS combination were also highly active against experimental staphylococcal infections in a mouse septicemia model. The combination EPM-DDS has previously been shown to exhibit activity in Pneumocystis carinii and Toxoplasma models and, as shown in the present study, also shows good activity against a broad range of bacteria including many strains resistant to TMP and TMP-SMZ.

3-nitrobenzenesulfonate, 3-aminobenzenesulfonate, and 4-aminobenzenesulfonate as sole carbon sources for bacteria.

Aerobic, carbon-limited, enrichment cultures containing 3-aminobenzenesulfonate or 3-nitrobenzenesulfonate as the sole source of carbon and energy yielded growth and complete substrate disappearance. Pure cultures of putative pseudomonads were isolated which utilized these compounds quantitatively. Degradation was compared with that of 2- and 4-aminobenzenesulfonate.