Following alcohol shock (50% EtOH for 1 hour), samples were again diluted and inoculated on CCEYL agar to enumerate spores. Total viable counts were enumerated on CCEYL agar (as described above) following dilution (10-fold series) in peptone water. Samples were taken at 0, 3, 6, 24 and 48 hours post inoculation (triplicate biological and technical replicates for each strain, n = 9). Outgrowth and toxin production following germination of antibiotic-exposed, washed sporesįor the three strains investigated in more detail (RT027, RT078, RT005), antibiotic-exposed, FF washed spores were inoculated into pre-reduced Brain Heart Infusion broth (BHI) alongside a non antibiotic-exposed, FF washed control. The residual activity of samples following washing was determined using the calibration curve of the relevant exposure antimicrobial. Limits of detection refer to the lowest concentration that creates a measurable zone of inhibition. The vancomycin bioassay was performed on Muller Hinton Agar with Staphylococcus aureus (ATCC 29 213) indicator organism and a 2–128 mg/L calibration series (limit of detection ~1mg/L). The fidaxomicin bioassay was performed on Wilkins-Charlgren agar with Kocuria rhizophila (ATCC 9341) indicator organism, and a calibration series ranging from 2–128 mg/L (limit of detection ~0.5mg/L). All samples were assayed in triplicate against a calibration curve of both fidaxomicin and vancomycin. Calibration lines were plotted from squared zone diameters, and active antimicrobial concentration was calculated from the calibration lines. After 24 h growth at 37☌, zones of inhibition were measured using callipers accurate to 0.1mm. Washed spore samples (20 μL) were inoculated into wells alongside a doubling dilution calibration series of known antibiotic concentration. Once set, plates were dried, and 25x 9 mm wells dug in the agar (no. difficile spores, and demonstrate that this persistence affects spore recovery, spore outgrowth, vegetative cell growth and toxin production.ĭetermination of active antimicrobial concentration by bioassayīioassay agar (100mL) was sterilised by autoclave, cooled to 50☌, seeded with 1mL indicator organism (0.5 MacFarlane standard suspension in PBS) and pored into 245 mm x 245 mm bioassay dishes. In this study, we have quantified the persistence of fidaxomicin activity on C. We have termed this ‘persistence of activity’. difficile spores, which persists despite washing. Similar prolonged detection of antimicrobial activity has been observed for both ramoplanin and oritavancin, and has correlated with an association of antimicrobial activity on C. We have previously observed that fidaxomicin activity continues to be detected for prolonged periods of time in an in vitro gut model, whereas vancomycin activity quickly washes out. In phase III clinical trials fidaxomicin was non inferior to vancomycin for initial clinical cure, but was superior in preventing recurrence and sustained clinical sure. However, the recent introduction of fidaxomicin offers a therapeutic alternative. difficile population following successful initial treatment.įor many years, treatment options were limited to vancomycin and metronidazole. These are unaffected by standard antimicrobial therapy, and provide a key reservoir for recurrent disease, allowing re-establishment of a vegetative C. difficile spores in the intestinal lumen (or mucosal associated biofilm) following treatment. A key contributing factor to the high rates of recurrence associated with CDI is the persistence of C. Morbidity and mortality, particularly associated with recurrent disease, remain problematic, with ~25% of patients experiencing a recurrence of symptoms following treatment. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.Ĭlostridium difficile infection (CDI) continues to be a leading infective cause of antibiotic-associated diarrhoea, placing substantial burdens on healthcare systems worldwide. There are no patents, products in development or marketed products to declare. Chris Longshaw contributed to this study as a full-time employee of Astellas Pharma Europe Ltd, the funder of this study. MW has received research funding from Actelion, Astellas, Biomerieux, Cubist, Pfizer, Summit and The Medicines Company and consultancies and/or lecture honoraria from Actelion, Astellas, Astra-Zeneca, Bayer, Cubist, Durata, J&J, Merck, Nabriva, Novacta, Novartis, Optimer, Pfizer, Sanofi-Pasteur, The Medicines Company, VH Squared, Viropharma. GC has received support to attend meetings from Astellas. In the past 2 years, CC has received research funding from Astellas Pharma EMEA, Paratek Pharmaceuticals, Cubist and Da Volterra, and support to attend meetings/lecture honoraria from Astellas. Competing Interests: We have the following interests.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |