High prevalence and diversity of tcdA-negative and tcdB-positive, and non-toxigenic, Clostridium difficile in Thailand

Studies on the prevalence and diversity of Clostridium difficile in Thailand have been limited to those derived from a few tertiary hospitals in Central Thailand. In this study, 145 C. difficile isolates collected in 13 provinces in Thailand during 2006-2018 were characterized by ribotyping and detection of toxin genes. Minimum inhibitory concentrations of eight antimicrobial agents were determined also for all 100 C. difficile strains collected from 2006 until 2015. Of the 145 strains of C. difficile, 71 (49%) were non-toxigenic, 46 (32%) were toxin A-negative, toxin B-positive (A-B+) and 28 (19%) were A+B+. No binary toxin-positive strain was found. The most common ribotype (RT) was RT 017 (A-B+CDT-, 19%, 28/145). Besides RT 017, 20 novel non-toxigenic and A-B+ ribotyping profiles, which may be related to RT 017 by the similarity of ribotyping profile, were identified. All C. difficile strains remained susceptible to metronidazole and vancomycin, however, a slight increase in MIC for metronidazole was seen in both toxigenic and non-toxigenic strains (overall MIC50/90 0.25/0.25 mg/L during 2006 – 2010 compared to overall MIC50/90 1.0/2.0 mg/L during 2011 – 2015). There was a high rate of fluoroquinolone resistance among RT 017 strains (77%), but there was little resistance among non-toxigenic strains. These results suggest that RT 017 is endemic in Thailand, and that the misuse of fluoroquinolones may lead to outbreaks of RT 017 infection in this country. Further studies on non-toxigenic C. difficile are needed to understand whether they have a role in the pathogenesis of C. difficile infection in Asia

Global evolutionary dynamics and resistome analysis of Clostridioides difficile ribotype 017

Clostridioides difficile PCR ribotype (RT) 017 ranks among the most successful strains of C. difficile in the world. In the past three decades, it has caused outbreaks on four continents, more than other ‘epidemic’ strains, but our understanding of the genomic epidemiology underpinning the spread of C. difficile RT 017 is limited. Here, we performed high-resolution phylogenomic and Bayesian evolutionary analyses on an updated and more representative dataset of 282 non-clonal C. difficile RT 017 isolates collected worldwide between 1981 and 2019. These analyses place an estimated time of global dissemination between 1953 and 1983 and identified the acquisition of the ermB-positive transposon Tn6194 as a key factor behind global emergence. This coincided with the introduction of clindamycin, a key inciter of C. difficile infection, into clinical practice in the 1960s. Based on the genomic data alone, the origin of C. difficile RT 017 could not be determined; however, geographical data and records of population movement suggest that C. difficile RT 017 had been moving between Asia and Europe since the Middle Ages and was later transported to North America around 1860 (95 % confidence interval: 1622–1954). A focused epidemiological study of 45 clinical C. difficile RT 017 genomes from a cluster in a tertiary hospital in Thailand revealed that the population consisted of two groups of multidrug-resistant (MDR) C. difficile RT 017 and a group of early, non-MDR C. difficile RT 017. The significant genomic diversity within each MDR group suggests that although they were all isolated from hospitalized patients, there was probably a reservoir of C. difficile RT 017 in the community that contributed to the spread of this pathogen

Esculin hydrolysis negative and TcdA ‐only producing strains of clostridium (Clostridiodes) difficile from the environment in Western Australia

Background and Aims Clostridium (Clostridiodes) difficile clade 3 ribotype (RT) 023 strains that fail to produce black colonies on bioMérieux ChromID agar have been reported, as well as variant strains of C. difficile that produce only toxin A. We have recently isolated strains of C. difficile from the environment in Western Australia (WA) with similar characteristics. The objective of this study was to characterize these strains. It was hypothesized that a putative β-glucosidase gene was lacking in these strains of C. difficile, including RT 023, leading to white colonies. Methods and Results A total of 17 environmental isolates of C. difficile from garden soil and compost, and gardening shoe soles in Perth, WA, failed to produce black colonies on ChromID agar. MALDI-TOF MS analysis confirmed these strains as C. difficile. Four strains contained only a tcdA gene (A+B−CDT−) by PCR and were a novel RT (QX 597). All isolates were susceptible to all antimicrobials tested except one with low-level resistance to clindamycin (MIC = 8 mg/L). The four tcdA-positive strains were motile. All isolates contained neither bgl locus but only bgl K or a putative β-glucosidase gene by PCR. Whole-genome sequencing showed the 17 strains belonged to novel multi-locus sequence types 632, 848, 849, 850, 851, 852 and 853, part of the evolutionarily divergent clade C-III. Four isolates carried a full-length tcdA but not tcdB nor binary toxin genes. Conclusions ChromID C. difficile agar is used for the specific detection of C. difficile in the samples. To date, all strains except RT 023 strains from clinical samples hydrolyse esculin. This is the first report to provide insights into the identification of esculin hydrolysis negative and TcdA-only producing (A+B−CDT−) strains of C. difficile from environmental samples. Significance and Impact of the Study White colonies of C. difficile from environmental samples could be overlooked when using ChromID C. difficile agar, leading to false-negative results, however, whether these strains are truly pathogenic remains to be proven

A species-wide genetic atlas of antimicrobial resistance in Clostridioides difficile

Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI), the leading healthcare-related gastrointestinal infection in the world. An association between AMR and CDI outbreaks is well documented, however, data is limited to a few ‘epidemic’ strains in specific geographical regions. Here, through detailed analysis of 10 330 publicly-available C. difficile genomes from strains isolated worldwide (spanning 270 multilocus sequence types (STs) across all known evolutionary clades), this study provides the first species-wide snapshot of AMR genomic epidemiology in C. difficile. Of the 10 330 C. difficile genomes, 4532 (43.9 %) in 89 STs across clades 1–5 carried at least one genotypic AMR determinant, with 901 genomes (8.7 %) carrying AMR determinants for three or more antimicrobial classes (multidrug-resistant, MDR). No AMR genotype was identified in any strains belonging to the cryptic clades. C. difficile from Australia/New Zealand had the lowest AMR prevalence compared to strains from Asia, Europe and North America (P<0.0001). Based on the phylogenetic clade, AMR prevalence was higher in clades 2 (84.3 %), 4 (81.5 %) and 5 (64.8 %) compared to other clades (collectively 26.9 %) (P<0.0001). MDR prevalence was highest in clade 4 (61.6 %) which was over three times higher than in clade 2, the clade with the second-highest MDR prevalence (18.3 %). There was a strong association between specific AMR determinants and three major epidemic C. difficile STs: ST1 (clade 2) with fluoroquinolone resistance (mainly T82I substitution in GyrA) (P<0.0001), ST11 (clade 5) with tetracycline resistance (various tet-family genes) (P<0.0001) and ST37 (clade 4) with macrolide-lincosamide-streptogramin B (MLSB) resistance (mainly ermB) (P<0.0001) and MDR (P<0.0001). A novel and previously overlooked tetM-positive transposon designated Tn6944 was identified, predominantly among clade 2 strains. This study provides a comprehensive review of AMR in the global C. difficile population which may aid in the early detection of drug-resistant C. difficile strains, and prevention of their dissemination worldwide

Can sequencing improve the diagnosis and management of Clostridioides difficile infection?

Clostridioides difficile is a bacterium of great public health importance, responsible for half of the hospital-acquired gastrointestinal infections in Europe [1], as well as approximately 13,000 deaths and losses of one billion dollars in healthcare-related costs annually in the United States [2]..

Genetically related Clostridium difficile from water sources and human CDI cases revealed by whole‐genome sequencing

Clostridium difficile isolates from the environment are closely related to those from humans, indicating a possible environmental transmission route for C. difficile infection (CDI). In this study, C. difficile was isolated from 47.3% (53/112) of lake/pond, 23.0% (14/61) of river, 20.0% (3/15) of estuary and 0.0% (0/89) of seawater samples. The most common toxigenic strain isolated was C. difficile PCR ribotype (RT) 014/020 (10.5%, 8/76). All water isolates were susceptible to fidaxomicin, metronidazole, rifaximin, amoxicillin/clavulanic acid, moxifloxacin and tetracycline. Resistance to vancomycin, clindamycin, erythromycin and meropenem was detected in 5.3% (4/76), 26.3% (20/76), 1.3% (1/76) and 6.6% (5/76) of isolates, respectively. High-resolution core-genome analysis was performed on RT 014/020 isolates of water origin and 26 clinical RT 014/020 isolates from the same year and geographical location. Notably, both human and water strains were intermixed across three sequence types (STs), 2, 13 and 49. Six closely related groups with ≤10 core-genome single nucleotide polymorphisms were identified, five of which comprised human and water strains. Overall, 19.2% (5/26) of human strains shared a recent genomic relationship with one or more water strains. This study supports the growing hypothesis that environmental contamination by C. difficile plays a role in CDI transmission

Antimicrobial resistance in Clostridium difficile ribotype 017

Introduction: Antimicrobial resistance (AMR) played an important role in the initial outbreaks of Clostridium difficile infection (CDI) in the 1970s. C. difficile ribotype (RT) 017 has emerged as the major strain of C. difficile in Asia, where antimicrobial use is poorly regulated. This strain has also caused CDI outbreaks around the world for almost 30 years. Many of these outbreaks were associated with clindamycin and fluoroquinolone resistance. AMR and selective pressure is likely to be responsible for the success of this RT and may drive future outbreaks. Areas covered: This narrative review summarizes the prevalence and mechanisms of AMR in C. difficile RT 017 and transmission of these AMR mechanisms. To address these topics, reports of outbreaks due to C. difficile RT 017, epidemiologic studies with antimicrobial susceptibility results, studies on resistance mechanisms found in C. difficile and related publications available through Pubmed until September 2019 were collated and the findings discussed. Expert opinion: Primary prevention is the key to control CDI. This should be achieved by developing antimicrobial stewardship in medical, veterinary and agricultural practices. AMR is the key factor that drives CDI outbreaks, and methods for the early detection of AMR can facilitate the control of outbreaks

Clostridioides difficile colonisation and infection in a cohort of Australian adults with cystic fibrosis

Background Little is known about Clostridioides difficile infection (CDI) in patients with cystic fibrosis (CF). The aim of this study was to investigate the prevalence, molecular epidemiology and risk factors for CDI in asymptomatic and symptomatic adults with CF in Western Australia. Methods Faecal samples from symptomatic and asymptomatic patients were prospectively collected and tested for the presence of C. difficile by toxigenic culture. Ribotyping was performed by established protocols. Logistic regression analysis was performed to analyse the risk factors for C. difficile colonization and infection. Extensive environmental sampling was performed within the CF clinic in Perth. Results The prevalence rates of asymptomatic toxigenic and non-toxigenic C. difficile colonization were 30% (14/46 patients) and 24% (11/46 patients), respectively. Fifteen ribotypes (RTs) of C. difficile were identified, of which non-toxigenic RT 039 was the most common. Among the symptomatic patients, the prevalence of toxigenic CDI was 33% (11/33 patients). Impaired glucose tolerance/diabetes mellitus and duration of intravenous antibiotic use in the past 12 months were significantly associated with increased risk of asymptomatic toxigenic C. difficile carriage and CDI. A trend towards higher CF transmembrane conductance regulator modulator treatment was observed in the CDI group. Extensive environmental sampling showed no evidence of toxigenic C. difficile contamination within the CF clinic. Conclusions A high prevalence of asymptomatic carriage of toxigenic C. difficile was observed in adults with CF, comparable with that observed in the symptomatic CF population. There was no evidence of direct person-to-person transmission