Efficacy of Topical Vancomycin- and Gentamicin-Loaded Calcium Sulfate Beads or Systemic Antibiotics in Eradicating Polymicrobial Biofilms Isolated from Diabetic Foot Infections within an In Vitro Wound Model.

Diabetic foot ulcers are notoriously difficult to heal, with ulcers often becoming chronic, in many cases leading to amputation despite weeks or months of antibiotic therapy in addition to debridement and offloading. Alternative wound biofilm management options, such as topical rather than systemic delivery of antimicrobials, have been investigated by clinicians in order to improve treatment outcomes. Here, we collected blood and tissue from six subjects with diabetic foot infections, measured the concentrations of antibiotics in the samples after treatment, and compared the microbiota within the tissue before treatment and after 7 days of antibiotic therapy. We used an in vitro model of polymicrobial biofilm infection inoculated with isolates from the tissue we collected to simulate different methods of antibiotic administration by simulated systemic therapy or topical release from calcium sulfate beads. We saw no difference in biofilm bioburden in the models after simulated systemic therapy (representative of antibiotics used in the clinic), but we did see reductions in bioburden of between 5 and 8 logs in five of the six biofilms that we tested with topical release of antibiotics via calcium sulfate beads. Yeast is insensitive to antibiotics and was a component of the sixth biofilm. These data support further studies of the topical release of antibiotics from calcium sulfate beads in diabetic foot infections to combat the aggregate issues of infectious organisms taking the biofilm mode of growth, compromised immune involvement, and poor systemic delivery of antibiotics via the bloodstream to the site of infection in patients with diabetes.

Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model.

BACKGROUND: Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. OBJECTIVES: We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. METHODS: C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. RESULTS: DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P < 0.005). CONCLUSIONS: DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.

Potential of lactoferrin to prevent antibiotic-induced Clostridium difficile infection.

OBJECTIVES: Clostridium difficile infection (CDI) is a global healthcare problem. Recent evidence suggests that the availability of iron may be important for C. difficile growth. This study evaluated the comparative effects of iron-depleted (1% Fe(3+) saturated) bovine apo-lactoferrin (apo-bLf) and iron-saturated (85% Fe(3+) saturated) bovine holo-lactoferrin (holo-bLf) in a human in vitro gut model that simulates CDI. METHODS: Two parallel triple-stage chemostat gut models were inoculated with pooled human faeces and spiked with C. difficile spores (strain 027 210, PCR ribotype 027). Holo- or apo-bLf was instilled (5 mg/mL, once daily) for 35 days. After 7 days, clindamycin was instilled (33.9 mg/L, four times daily) to induce simulated CDI. Indigenous microflora populations, C. difficile total counts and spores, cytotoxin titres, short chain fatty acid concentrations, biometal concentrations, lactoferrin concentration and iron content of lactoferrin were monitored daily. RESULTS: In the apo-bLf model, germination of C. difficile spores occurred 6 days post instillation of clindamycin, followed by rapid vegetative cell proliferation and detectable toxin production. By contrast, in the holo-bLf model, only a modest vegetative cell population was observed until 16 days post antibiotic administration. Notably, no toxin was detected in this model. In separate batch culture experiments, holo-bLf prevented C. difficile vegetative cell growth and toxin production, whereas apo-bLf and iron alone did not. CONCLUSIONS: Holo-bLf, but not apo-bLf, delayed C. difficile growth and prevented toxin production in a human gut model of CDI. This inhibitory effect may be iron independent. These observations suggest that bLf in its iron-saturated state could be used as a novel preventative or treatment strategy for CDI.

Efficacy of alternative fidaxomicin dosing regimens for treatment of simulated Clostridium difficile infection in an in vitro human gut model.

BACKGROUND: Fidaxomicin treatment reduces the risk of recurrent Clostridium difficile infection (CDI) compared with vancomycin. Extending duration of fidaxomicin therapy may further reduce recurrence. We compared the efficacy of four extended fidaxomicin regimens in an in vitro model of CDI. METHODS: Four gut models were primed with human faeces, spiked with C. difficile spores (PCR ribotype 027) and clindamycin instilled (33.9 mg/L, four-times daily, 7 days) to induce simulated CDI. Four extended fidaxomicin treatment regimens were evaluated: model 1, 20 days, 200 mg/L twice daily; model 2, 5 days 200 mg/L twice daily, 5 days rest, 5 days 200 mg/L twice daily; model 3, 5 days 200 mg/L twice daily, 5 days rest, 10 days 200 mg/L once daily; and model 4, 5 days 200 mg/L twice daily, 20 days 200 mg/L once every other day. C. difficile populations, toxin, gut microbiota and antimicrobial levels were monitored daily. RESULTS: All fidaxomicin regimens successfully resolved simulated CDI without recurrence. Five days of fidaxomicin instillation was barely sufficient to resolve CDI (models 2-4). A second pulse or tapered dosing further reduced C. difficile and toxin detection. All regimens were sparing of microbiota, affecting only enterococci and bifidobacteria. Pulsed or tapered regimens allowed greater bifidobacteria recovery than the extended (20 day) regimen. Bioactive fidaxomicin persisted throughout the experiment in all models at concentrations inhibitory to C. difficile. CONCLUSIONS: Pulsed or tapered fidaxomicin regimens may enhance suppression of C. difficile whilst allowing microbiota recovery; clinical studies are required to ascertain the potential of this approach in further reducing recurrent CDI.

SMT19969 as a treatment for Clostridium difficile infection: an assessment of antimicrobial activity using conventional susceptibility testing and an in vitro gut model.

OBJECTIVES: We investigated the efficacy of the novel antimicrobial agent SMT19969 in treating simulated Clostridium difficile infection using an in vitro human gut model. METHODS: Concentrations of the predominant cultivable members of the indigenous gut microfloras and C. difficile (total and spore counts) were determined by viable counting. Cytotoxin titres were determined using cell cytotoxicity and expressed as log10 relative units (RU). Clindamycin was used to induce simulated C. difficile PCR ribotype 027 infection. Once high-level cytotoxin titres (≥ 4 RU) were observed, SMT19969 was instilled for 7 days. Two SMT19969 dosing regimens (31.25 and 62.5 mg/L four times daily) were evaluated simultaneously in separate experiments. MICs of SMT19969 were determined against 30 genotypically distinct C. difficile ribotypes. RESULTS: SMT19969 was 7- and 17-fold more active against C. difficile than metronidazole and vancomycin, respectively, against a panel of genotypically distinct isolates (P < 0.05). Both SMT19969 dosing regimens demonstrated little antimicrobial activity against indigenous gut microflora groups except clostridia. SMT19969 inhibited C. difficile growth and repressed C. difficile cytotoxin titres in the gut model. CONCLUSIONS: These data suggest that SMT19969 is a narrow-spectrum and potent antimicrobial agent against C. difficile. Additional studies evaluating SMT19969 in other models of C. difficile infection are warranted, with human studies to place these gut model observations in context.

Efficacy of surotomycin in an in vitro gut model of Clostridium difficile infection.

OBJECTIVES: We investigated the efficacy of the cyclic lipopeptide surotomycin in treating clindamycin-induced Clostridium difficile infection (CDI) using an in vitro gut model. METHODS: Two three-stage chemostat gut models were inoculated with human faeces, spiked with C. difficile spores (∼10(7) cfu/mL, PCR ribotype 027 or 001). Clindamycin (33.9 mg/L, four times daily for 7 days) was dosed to induce CDI. Following high-level toxin production, surotomycin (250 mg/L, twice daily for 7 days) was instilled. Microflora populations, C. difficile vegetative cells and spores, cytotoxin titres and antimicrobial levels (LC-MS/MS and bioassay) were determined. The emergence of C. difficile and enterococci with reduced susceptibility to surotomycin was monitored on breakpoint agar (4 × MIC). RESULTS: Counts of viable C. difficile were reduced to near the limit of detection on Days 1 and 3 of surotomycin instillation, and cytotoxin was undetectable on Days 3 and 4 of surotomycin instillation in the 027 and 001 models, respectively. Recurrence of vegetative growth and toxin production occurred 11 days (001 model) and 15 days (027 model) after surotomycin instillation had ceased, and remained for the duration of the experiment. Surotomycin instillation decreased populations of bifidobacteria, clostridia, enterococci and lactobacilli, but was sparing of Bacteroides fragilis group populations. All enumerated organisms had recovered to steady-state levels by 3 weeks post-surotomycin instillation. No evidence of the emergence of reduced susceptibility to surotomycin was observed. CONCLUSIONS: Surotomycin successfully reduced C. difficile vegetative cell counts and toxin levels in the gut model and was sparing of B. fragilis group populations. There was no evidence of decreased susceptibility to surotomycin during exposure or post-exposure.

Successful treatment of simulated Clostridium difficile infection in a human gut model by fidaxomicin first line and after vancomycin or metronidazole failure.

OBJECTIVES: Fidaxomicin reduces the risk of recurrent Clostridium difficile infection (CDI) compared with vancomycin. We investigated fidaxomicin primary or secondary treatment efficacy using a gut model. METHODS: Four triple-stage chemostat gut models were inoculated with faeces. After clindamycin induction of CDI, fidaxomicin (200 mg/L twice daily), vancomycin (125 mg/L four times daily) or metronidazole (9.3 mg/L three times daily) was administered for 7 days. Following failure/CDI recurrence, fidaxomicin (200 mg/L twice daily, 7 days) was instilled. C. difficile (CD) total viable counts (TVC), spore counts (SP), toxin titres (CYT), gut bacteria counts and antimicrobial concentrations were measured throughout. RESULTS: Fidaxomicin instillation reduced CD TVC/SP and CYT below the limit of detection (LOD) after 2 and 4 days, respectively, with no CDI recurrence. Metronidazole instillation failed to decrease CD TVC or CYT. Vancomycin instillation reduced CD TVC and CYT to LOD by day 4, but SP persisted. Recurrence occurred 13 days after vancomycin instillation; subsequent fidaxomicin instillation reduced CD TVC/SP/CYT below the LOD from day 2. CD was isolated sporadically, with no evidence of spore recrudescence or toxin production. Fidaxomicin had a minimal effect on the microflora, except for bifidobacteria. Fidaxomicin was detected for at least 21 days post-instillation, whereas other antimicrobials were undetectable beyond ∼4 days. CONCLUSIONS: Fidaxomicin successfully treated simulated primary and recurrent CDI. Fidaxomicin was superior to metronidazole in reducing CD TVC and SP, and superior to vancomycin in reducing SP without recurrence of vegetative cell growth. Fidaxomicin, but not vancomycin or metronidazole, persisted in the gut model for >20 days after instillation.

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.

Evaluation of the effect of oritavancin on Clostridium difficile spore germination, outgrowth and recovery.

OBJECTIVES: Previous work suggests oritavancin may be inhibitory to Clostridium difficile spores. We have evaluated the effects of oritavancin exposure on C. difficile spore germination, outgrowth and recovery. METHODS: Germination and outgrowth of C. difficile spores exposed to different concentrations of oritavancin, vancomycin, or metronidazole (0.1-10 mg/L) were monitored at 0, 2, 4, 6, 24 and 48 h using phase-contrast microscopy. Recovery of antimicrobial-exposed spores was determined by viable counting on Brazier's modified CCEYL agar. Persistence of oritavancin activity on spores after washing was determined by measuring activity against a Staphylococcus aureus lawn. RESULTS: Oritavancin, vancomycin and metronidazole exposure did not prevent germination of phase-bright spores to phase-dark spores, but did inhibit further outgrowth into vegetative cells. The inhibitory effect of oritavancin persisted after washing, whereas the inhibitory effects of vancomycin and metronidazole did not. Oritavancin exposure affected spore recovery; fewer spores were recovered after washing following oritavancin exposure than vancomycin exposure. The extent of this effect was dependent on PCR ribotype, with recovery of ribotype 078 spores completely prevented, but recovery of ribotype 001 spores only slightly affected. Spores exposed to oritavancin, but not vancomycin, retained antimicrobial activity after washing, indicating adherence of oritavancin, but not vancomycin, to the spore surface CONCLUSIONS: Oritavancin may adhere to spores, potentially causing early inhibition of germinated cells, preventing subsequent vegetative outgrowth and spore recovery. This may prevent some recurrences of symptomatic C. difficile infection that are due to germination of residual spores following antibiotic therapy.

Effectiveness of a short (4 day) course of oritavancin in the treatment of simulated Clostridium difficile infection using a human gut model.

OBJECTIVES: We previously demonstrated that 7 days of oritavancin instillation effectively treats Clostridium difficile infection (CDI) in a human gut model. Oritavancin may be more effective than vancomycin due to apparently increased activity against spores. We compared the efficacy of shortened dosing duration (4 days) of oritavancin and vancomycin for CDI treatment using the gut model. METHODS: Clindamycin induced CDI in two triple-stage chemostat gut models primed with pooled human faeces and C. difficile ribotype 027 spores. Oritavancin (64 mg/L twice daily) or vancomycin (125 mg/L four times daily) was instilled for 4 days and the effects on C. difficile proliferation and toxin production, and gut microflora were determined. RESULTS: Both oritavancin and vancomycin reduced toxin to undetectable levels. Recurrent C. difficile germination occurred 20 days after vancomycin instillation, with high-level toxin production. Oritavancin reduced C. difficile counts to around the detection limit for the remainder of the experiment, with spores undetectable from day 1 of instillation. Toxin production was reduced to below detectable levels, but was sporadically seen later, despite no evidence of germination. Both oritavancin and vancomycin instillation led to only modest effects on gut microflora. CONCLUSIONS: Shortened courses of oritavancin and vancomycin effectively treated CDI in a human gut model, but evidence of recurrence was observed following vancomycin instillation. Oritavancin exposure inhibited the recovery of C. difficile spores, as previously described. Shortened antibiotic exposure minimizes disruption to the gut microflora. These data indicate the possible value of a 4 day oritavancin dosing regimen for CDI treatment.