In vitro assessment of Clostridium difficile PCR ribotype 002: the most prevalent C. difficile ribotype in the United Kingdom.

Abstract

Background: Clostridium difficile infection (CDI) causes substantial morbidity and healthcare expenditure across Europe. UK prevalence of C. difficile PCR ribotype 027 (NAP1) has declined dramatically recently and other ribotypes have emerged, including ribotype 002 (CD002); now the most prevalent UK ribotype. CD002 is also responsible for CDI in many countries across Europe, including: France, Germany, Ireland, and The Netherlands. We assessed the in vitro phenotypic characteristics of CD002 from across Europe to determine traits that may contribute to its increasing clinical prevalence. Material/methods: Sixty CD002 were studied: UK isolates from 2007-2008 (geographically distinct, N=15), UK isolates from 2011-2013 (19 locations, N=22), and non-UK European isolates from 2012-2014 (N=23, 20 locations). Antimicrobial susceptibilities (13 antimicrobials) were evaluated using an agar incorporation method. Maximum specific growth rates (μmax) were calculated and cytotoxin titres (log10-relative units, RU) determined using Vero cell cytotoxicity assays. Biofilm formation was quantified using 96-well microtitre plate assays and sporulation capacities assessed in liquid culture by quantifying spore-formation over 120 h (CFU/mL). Results: All isolates were susceptible metronidazole, vancomycin, tetracycline and linezolid (MICs ≤2 mg/L). Clindamycin resistance (MIC ≥8 mg/L) was more common in non-UK CD002 (30%) than UK strains (5-13%). Resistance to erythromycin, clarithromycin, nitrofurantoin, chloramphenicol, and moxifloxacin was uncommon (5-7%). MICs for penicillin’s remained below resistance breakpoints, regardless of origin, in all but one isolate (ampicillin MIC 2 mg/L). All CD002 were resistant to trimethoprim (MICs >128 mg/L) and ciprofloxacin (MICs ≥8 mg/L). One MDR strain (UK, 2007) was observed that was macrolide, fluoroquinolone, ampicillin, and nitrofurantoin resistant. Significantly faster μmax was seen in non-UK CD002 (0.92 ±0.058 h-1) than recent/older UK strains (0.76 ±0.063/0.69 ±0.028 h-1 respectively) (P<0.001). Cytotoxin production did not differ significantly (median titres 2-3 RU) between CD002 groups. Recent UK/non-UK CD002 formed significantly greater biofilms by 3 days than asynchronous UK CD002 (P<0.001). Sporulation studies demonstrated that recent UK/non-UK CD002 sporulated more at 24 h than older UK CD002; 18.6-fold/31.2-fold respectively (P<0.05), but by 120 h sporulation did not differ. Conclusions: Recent CD002 from diverse European locations were assessed for traits that may help to explain emergence of CD002 in the UK and compared to asynchronous CD002. Previous studies demonstrated elevated CD002 μmax compared to hypervirulent ribotypes 027/078; and the present study demonstrated that recent non-UK CD002 μmax were significantly further elevated vs. UK isolates. Non-UK CD002 were more clindamycin resistant, but other antimicrobial susceptibilities were similar between CD002 groups. Recent CD002 demonstrated significantly increased sporulation capacities at 24 h and more extensive 3 day biofilm formation compared to asynchronous UK CD002, which could enhance their survival and transmission early in an episode CDI. Further phenotypic and genetic studies are required to evaluate further characteristics of CD002 that may be associated with its emergence in the UK.