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 4¾ 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 2¾ MIC fidaxomicin with bacterial counts decreasing 1 log in 48 h. Exposure to OP-1118 (at 4¾ 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 (32¾ 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.

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).Clostridium difficile infections (CDI) continue to be a major cause of diarrhea in hospitalized patients undergoing antibiotic treatment and among the elderly in long-term-care facilities. Current standard therapies for CDI are inadequate and often result in disease recurrence that can lead to multiple episodes in some individuals (12,16). In contrast, fidaxomicin (FDX), a new narrow-spectrum antibacterial agent with no cross-resistance to known antibiotics (1,2,6,7,8,9,10,13,18), has been demonstrated to be superior to vancomycin in sustaining a cure without recurrence of CDI (11). Following oral administration (400 mg/day) for 10 days, FDX and its major metabolite, OP-1118 (derived by hydrolysis of the isobutyryl ester located at the 4Љ position of fidaxomicin), are detected abundantly (in mg/g concentrations and in approximately a 2:1 ratio, respectively) in stool samples (17). Like FDX, OP-1118 demonstrates narrow-spectrum activity, albeit with a 32-fold increase in MIC relative to that of FDX, for 90% of C. difficile strains (MIC 90 ) tested from phase 3 trials. This study investigates the postantibiotic effects (PAE) of both FDX and OP-1118 in comparison to those of vancomycin and metronidazole against C. difficile ATCC strains (ATCC 9689 and ATCC 43255) and a clinical isolate (LC3).In order to use drug concentrations above the MIC for the PAE experiments, the MIC values were determined via the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method, which more closely represents the conditions of PAE experiments (4). Lysed blood, which obscures the color of media, was omitted from brucella broth so that the redox indicator resazurin could be used to monitor the anaerobicity. Briefly, microtiter plates with serially diluted drugs were equilibrated inside an anaerobic glove box for a minimum of 3 h. C. difficile was added to each well (10 5 CFU/well), and following 48 h of incubation at 35°C, the plates were examined for growth. The MIC was determined as the drug concentration where no growth or the most significant reduction of growth was observed. Drug MIC values for 3 strains of C. difficile, shown in Table 1 Using the standard viable-cell plate count method described by Craig and Gudmundsson (5), the PAE for all antibiotics was determined under anaerobic conditions. Briefly, each organism, grown to log phase, was incubated with an ϳ4-fold MIC of antibiotic for 1 h at 35°C. Prior to and after three washing steps (via centrifugation and supernatant removal), samples were withdrawn at different time...

The effects of the inoculum, pH, cation concentrations, and different lots of commercial media on the in vitro susceptibility of Clostridium difficile to fidaxomicin were examined. Of the factors evaluated, only pH alterations influenced the activity of fidaxomicin against C. difficile, noticeably reducing its activity at higher pH (>7.9).In recent years, the epidemiology of Clostridium difficile infections (CDIs) has been changing, and there are increasing numbers of CDI cases being reported each year (2,3,5,7,9,12,13,15,17,19,20,21). While standard therapies for CDIs reduce the rates of morbidity and mortality, they are known to lead to high rates of recurrence, with 15% to 30% of patients demonstrating relapses in symptoms in the first few weeks after treatment is discontinued (9). Fidaxomicin (formerly known as OPT-80 and PAR-101) is a novel and narrow-spectrum macrocyclic compound (1, 6, 10) that is in clinical development for the treatment of CDIs. In a recent phase 3 trial, fidaxomicintreated patients demonstrated better clinical outcomes; namely, they had significantly lower rates of recurrences than subjects who were treated with vancomycin (16).The site of action of C. difficile is the large intestine, a milieu filled with vast numbers of different species of anaerobic flora that, through their metabolites, maintain a physiological pH ranging from 5.5 to 7 (11). Disruption of the gut flora by antibiotic therapy can therefore lead to pH changes, which can affect the pH-dependent activities of many antibiotics (4,8,21). Other environmental variables, such as divalent cation concentrations (including calcium and magnesium) and bacterial density, can also influence the antimicrobial activities of compounds. The dependence of the antibacterial activity on these factors is an important consideration, particularly for an unabsorbed antibiotic such as fidaxomicin that localizes and targets bacteria in the gut, where these parameters can vary greatly with diet and disease state.The in vitro activities of antimicrobial compounds (expressed as the MICs) under conditions with such environmental variables are also important factors to be considered when a methodology for future in vitro testing is designed. Brucella agar, which is recommended by the Clinical and Laboratory Standards Institute (CLSI) (18) for use for MIC determination, is not standardized, and the consistency of the divalent cation concentrations has not been established. Moreover, the pH of the medium used under anaerobic conditions in a glove box may also vary with different gas mixtures, as the CO 2 concentration in the gas mixture has the propensity to acidify the medium and can thus be a significant source of variability. Macrolides, as an example, show elevated MICs in the presence of CO 2 (8). The inoculum size may also be difficult to standardize, given the variety of atmospheric conditions available for anaerobic susceptibility testing (H 2 /CO 2 generator, evacuation/replacement method, or anaerobic chamber) and the duration of organism ex...

After injection of living tetrahymena into the hemocoels of male cockroaches, recovered hemolymph immobilizes washed ciliates. The immobilizing material is sensitive to heat and acid, and can be separated with ammonium sulfate. Hemolymph from immune animals confers protection in another animal into which it is injected. Immobilizing activity of the hemolymph from immune animals is associated with a nonreactive, normal protein component present in hemolymph from nonimmunized insects.