Characterisation of Clinical Clostridium difficile PCR Ribotype 002 Isolates from Different Time Lineages

Clostridium difficile (C. difficile) is a leading cause of healthcare-associated infections (HAIs) and an important public health threat. Recently, the prevalence C. difficile PCR ribotype 002 in the UK has been noted, yet the drivers for this increased prevalence remain unclear. The aim of this study was to characterise C. difficile PCR ribotype 002 (CD002) isolates from different time lineages and assess any phenotypic and genotypic traits that may help explain the emergence of this ribotype. A total of 60 clinical isolates of CD002 (isolated between 2007-2014 in the UK and across Europe) were used in this study. Antimicrobial susceptibilities to a range of antimicrobial agents were assessed using the agar dilution method. Maximum specific growth rate (µmax) was measured by batch culture growth curves, and cytotoxin production (log10 relative units (RU)) was evaluated in a Vero cell cytotoxicity assay. Factors associated with C. difficile persistence: sporulation capacity, spore adherence capability, and biofilm formation capacity were characterised using standard techniques. For selected strains, phenotypic microarrays (PMs) were used to elucidate nutrient utilisation profiles, and competition for glucose between isolates from different CD002 lineages was investigated in a single-stage fermenter. Using 1D SDS gel electrophoresis, followed by Liquid Chromatography-Tandem Mass spectrometry (LC-MS/MS), the whole cell proteomes of three CD002 isolates were compared. All CD002 were susceptible to metronidazole, vancomycin, fidaxomicin, chloramphenicol, linezolid, tetracycline (MICs ≤ 2 mg/L), and resistant to trimethoprim (MICs >128 mg/L) and ciprofloxacin (MICs ≥8 mg/L). Resistance to clindamycin (27% n=16), erythromycin (3.3%, n=2), moxifloxacin, nitrofurantoin and rifampicin (1.7%), was present. All but one C. difficile isolate demonstrated intermediate resistance to ampicillin and penicillin (MICs >1mg/L). One UK isolate of the UK 2007-8 lineage was classified as multidrug-resistant (MDR). The µmax of non-UK 2012-14 strains was significantly higher (0.92 h-1, p<0.001) than that of strains from the UK (2007-2014). Cytotoxin titres did not differ significantly between lineages (median titres 2-3 RU). The sporulation formation capacities for recent CD002 (UK and Non- UK) were significantly higher (p<0.001) than those of the older isolates. Spore adherence capability did not differ significantly between CD002 lineages. Recent CD002 (UK 2011-13 & Non-UK 2012-14) strains formed significantly more profuse biofilms in vitro than the older strains (p<0.001). The recent CD002 (UK 2011-13 & Non-UK 2012-14) appeared to have more expanded nutrient utilisation profiles than older CD002 isolates, and one recent UK strain outcompeted a recent Non-UK strain for glucose. Analysis of whole-cell proteomes revealed similarities and differences between strains that suggest a minimal adaptation of the proteome in CD002 has occurred over time. To conclude, the study uncovered some differences between the different lineages of CD002. The increased sporulation, higher µmax, greater biofilm formation, abundance of spores in mature biofilms, and the utilisation of several nutrient substrates, demonstrated by recent C. difficile PCR ribotype 002, suggests that they may have a competitive advantage over other ribotypes, therefore increasing their prevalence in recent years. However, whether these factors, have a greater in vivo implication for this ribotype, remains to be determined

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

Simon Baines, Iye Ameh, Jane Freeman, W.N. Fawley, M.H. Wilcox, ‘In vitro assessment of Clostridium difficile PCR ribotype 002: the most prevalent C. difficile ribotype in the United Kingdom’, poster presented at the 25th European Congress on Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 25-28 August, 2015.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.Peer reviewedFinal Published versio