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

Abstract

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.