Molecular and epidemiologic study of Clostridium difficile reveals unusual heterogeneity in clinical strains circulating in different regions in Portugal

Abstract

Clostridium difficile infection (CDI) represents a great healthcare burden in developed countries. The emergence of the epidemic PCR ribotype (RT) 027 and its acquired fluoroquinolones resistance have accentuated the need for an active surveillance of CDI. Here we report the first countrywide study of CDI in Portugal with the characterization of 498 C. difficile clinical isolates from 20 hospitals in four regions in Portugal regarding RT, virulence factors and antimicrobial susceptibility. We identified 96 RTs with marked variations between and within regions, as only six RTs appeared in all four regions. RT027 was the most frequent RT overall (18.5%) and among healthcare facility-associated isolates (19.6%), while RT014 was the most common among community-associated isolates (12%). The north showed a high RT diversity among isolates and a low moxifloxacin (MXF) resistance rate (11.9%), being the only region in which RT027 was not predominant. In contrast, the isolates from the centre presented the highest RT027 frequency, and 53.4% were resistant to MXF. Overall, MXF resistance (33.2%) was associated (p <0.001) with the presence of binary toxin genes and mutations in tcdC regardless of the RT. Both traits appeared in almost 30% of the strains. RT027 showed a reduced susceptibility to metronidazole (p <0.01), and RT126 had higher minimum inhibitory concentrations to vancomycin (p = 0.03) compared to other RTs. The present study highlights an unusual heterogeneity of RTs in Portugal, with a high frequency of hypervirulent RTs and the emergence of virulence factors in non-027 RTs, emphasizing the need for a surveillance system for CDI in Portugal.

Imipenem Resistance in Clostridium difficile Ribotype 017, Portugal

“…Strains of other ribotypes, including RT078 and RT017, which have enhanced virulence, have emerged ( 6 ). In particular, RT017, the most common toxin A–negative, toxin B–positive ribotype, is widespread in Asia and is common in Europe ( 7 – 9 ). In a pan-European study of ≈900 C. difficile strains, the overall rate of resistance to imipenem, an antimicrobial drug of the carbapenem class, currently widely used as a last-line drug to treat infections by gram-negative bacteria, was found to be 7.41%, and the geometric mean (GM) MIC of imipenem for RT017 strains was 5.91 mg/L ( 8 ).…”

Overview of Clostridium difficile Infection: Life Cycle, Epidemiology, Antimicrobial Resistance and Treatment

“…The rates of resistance vary considerably between countries; however, this is likely due to differences in ribotype diversity, since the countries with higher diversity of ribotypes tend to exhibit lower rates of fluoroquinolones resistance and vice versa [146]. This same trend was observed in Portugal when comparing ribotype diversity and resistance rates between regions [194]. Fluoroquinolone resistance in C. difficile, as a result of antibiotics use selective pressure, is associated with well-known mutations in the quinolone-resistance determining region of DNA gyrase subunits gyrA and gyrB.…”

“…In Portugal, CDI surveillance based on a network of sentinel hospitals has been carried out since 2010, and showed that RT017 is one the most common ribotypes circulating in the country [194]. In particular, one RT017 clone was shown to be endemic in a hospital from 2012 until today, and a different RT017 clone has emerged in another hospital in the beginning of 2016 (our unpublished data).…”

“…In a recent study, rifampicin resistance (13.4%) was reported in 17/22 European countries, and although it was observed in several RTs, it was mostly associated with frequent RTs, such as RT027, RT018 and RT356 [146]. Also, in Portugal, all the RT017 strains, a predominant type [194], show high resistance to rifampicin (unpublished data). It has been demonstrated that in vitro susceptibility to rifampicin is predictive of rifaximin susceptibility [228] and the emergence of rifamycin resistance during a recurrent C. difficile infection following rifaximin treatment has been clearly demonstrated [232].…”

“…Fluoroquinolone resistance in C. difficile, as a result of antibiotics use selective pressure, is associated with well-known mutations in the quinolone-resistance determining region of DNA gyrase subunits gyrA and gyrB. Mutations causing a T82I substitution in GyrA are the most commonly identified in C. difficile, and are found in ribotypes such as RT027 and RT017 [194,218,226]. Importantly, resistance to fluoroquinolones is likely to be maintained even without antibiotic pressure, as it does not impose a fitness cost in C. difficile [227].…”