Ambush of Clostridium difficile Spores by Ramoplanin: Activity in an In Vitro Model

Abstract

bClostridium difficile infection (CDI) is a gastrointestinal disease caused by C. difficile, a spore-forming bacterium that in its spore form is tolerant to standard antimicrobials. Ramoplanin is a glycolipodepsipeptide antibiotic that is active against C. difficile with MICs ranging from 0.25 to 0.50 g/ml. The activity of ramoplanin against the spores of C. difficile has not been well characterized; such activity, however, may hold promise, since posttreatment residual intraluminal spores are likely elements of disease relapse, which can impact more than 20% of patients who are successfully treated. C. difficile spores were found to be stable in deionized water for 6 days. In vitro spore counts were consistently below the level of detection for 28 days after even brief (30-min) exposure to ramoplanin at concentrations found in feces (300 g/ml). In contrast, suppression of spore counts was not observed for metronidazole or vancomycin at human fecal concentrations during treatment (10 g/ml and 500 g/ml, respectively). Removal of the C. difficile exosporium resulted in an increase in spore counts after exposure to 300 g/ml of ramoplanin. Therefore, we propose that rather than being directly sporicidal, ramoplanin adheres to the exosporium for a prolonged period, during which time it is available to attack germinating cells. This action, in conjunction with its already established bactericidal activity against vegetative C. difficile forms, supports further evaluation of ramoplanin for the prevention of relapse after C. difficile infection in patients. Clostridium difficile infection (CDI) remains a clinically important disease, with increasing incidence and mortality rates in the United States; it now surpasses methicillin-resistant Staphylococcus aureus as the most common health care-associated infection (HAI) and results in an annual cost burden to the health care system of $4.8 billion (1-3). A prolonged cure for treated patients remains elusive, as evidenced by the high rates of relapse (same ribotype within 30 days) and reinfection (different ribotype within 30 days). Recurrence rates have ranged from 15% to 40% (4-6) and are most commonly associated with the ribotype 027 epidemic strain (7). One contributing factor for high relapse rates is the presence of spores that persist within the intestinal lumen and evade elimination by standard anti-CDI treatments (8, 9). C. difficile spores are tolerant to standard antimicrobial therapy; hence, antimicrobial interventions targeting vegetative cells alone are unlikely to eradicate this disease reservoir (10).Ramoplanin is a glycolipodepsipeptide antibiotic produced from Actinoplanes, which inhibits peptidoglycan biosynthesis by limiting lipid II availability (11); it is bactericidal to many Grampositive aerobic and anaerobic bacteria, including C. difficile and vancomycin-resistant Enterococcus (11). Ramoplanin has known bactericidal activities against wild-type C. difficile and strains with reduced susceptibilities to vancomycin and has been studied in an open-labe...

Microbiologic factors affecting Clostridium difficile recurrence

“…Persistent fidaxomicin activity prevented vegetative outgrowth and toxin production in batch culture, and similar observations were also made for ramoplanin [93] and oritavancin [95]. It is likely that fidaxomicin adheres to the exosporium of C. difficile (as for ramoplanin [96]), potentially as a result of electrostatic charges resulting from cross-linkages on the spore surfaces. The presence of the exosporium can increase hydrophobicity of C. difficile spores, affecting adherence to cells [84].…”

The Synergistic Effect of High Pressure CO2 and Nisin on Inactivation of Bacillus subtilis Spores in Aqueous Solutions

“…Theoretically, nisin→HPCD should not enhance the inactivation of the spores since nisin at room temperature cannot act on the spores. Possible explanation for nisin→HPCD enhanced inactivation was probably due to the adherence of nisin to the surface of spores after nisin treatment ( Chilton et al, 2013 ; Kraus et al, 2015 ), and the remaining nisin played an enhanced inactivation of the spores. Its action mode was similar to that of HPCD + nisin.…”

Novel therapies and preventative strategies for primary and recurrent Clostridium difficile infections

“…It has been shown to be nonabsorbable, and to bind to spores and kill vegetative C. difficile cells in vitro . This may indicate a potential use for ramoplanin as a preventative measure to reduce risk of initial disease or reduce recurrence by binding to spores and then killing the ones that germinate . A phase II trial comparing treatment using ramoplanin versus vancomycin for CDI showed similar clinical response rates with 400 mg ramoplanin (71% versus 78%) and similar sustained clinical response rates (83% 200 mg ramoplanin, 85.2% 400 mg ramoplanin, and 85.7% vancomycin).…”