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

Clostridium difficile ribotype 017 – characterization, evolution and epidemiology of the dominant strain in Asia

Clostridium difficile ribotype (RT) 017 is an important toxigenic C. difficile RT which, due to a deletion in the repetitive region of the tcdA gene, only produces functional toxin B. Strains belonging to this RT were initially dismissed as nonpathogenic and circulated largely undetected for almost two decades until they rose to prominence following a series of outbreaks in the early 2000s. Despite lacking a functional toxin A, C. difficile RT 017 strains have been shown subsequently to be capable of causing disease as severe as that caused by strains producing both toxins A and B. While C. difficile RT 017 strains can be found in almost every continent today, epidemiological studies suggest that the RT is endemic in Asia and that the global spread of this MLST clade 4 lineage member is a relatively recent event. C. difficile RT 017 transmission appears to be mostly from human to human with only a handful of reports of isolations from animals. An important feature of C. difficile RT 017 strains is their resistance to several antimicrobials and this has been documented as a possible factor driving multiple outbreaks in different parts of the world. This review summarizes what is currently known regarding the emergence and evolution of strains belonging to C. difficile RT 017 as well as features that have allowed it to become an RT of global importance

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]..

Genomic basis of antimicrobial resistance in non-toxigenic Clostridium difficile in Southeast Asia

Despite being incapable of causing Clostridium difficile infection, non-toxigenic C. difficile (NTCD) may still be relevant. This study explored the role of NTCD as a reservoir of accessory antimicrobial resistance (AMR) genes in NTCD from Southeast Asia. This region has high rates of antimicrobial use, a high prevalence of NTCD and phenotypic AMR in such strains. More than half of the 28 NTCD strains investigated had at least one accessory AMR gene on mobile genetic elements (MGEs) which were similar to the elements found in other bacteria, including Erysipelothrix rhusiopathiae and Streptococcus suis, both of which are found in the pig gut. Thus, C. difficile may facilitate the movement of AMR genes between different hosts within a wide range of pathogenic bacteria. C. difficile β-lactamases were not located on MGEs and were unlikely to be transferred. Concordance between the MLSB resistance genotype and phenotype was low, suggesting multiple resistance mechanisms, many of which remain unknown. On the contrary, there was a high concordance between resistance genotype and phenotype for both fluoroquinolones and rifaximin. From an epidemiological perspective, NTCD populations in Southeast Asia comprised members of evolutionary clades 1 and 4, which are thought to have originated from Europe and Asia, respectively. This population structure reflects the close relationship between the people of the two regions

Mild or malign: Clinical characteristics and outcomes of clostridium difficile infection in Thailand

Little is known about the clinical characteristics of Clostridium difficile infection (CDI) in Asia in general, and Thailand specifically, with a few studies suggesting that the disease may be milder than elsewhere. This study aimed to describe CDI in Thailand, evaluate treatment options and their outcomes, and explore possible protective factors responsible for any unique disease characteristics. From 2015 to 2018, 469 patients were included in the study. All patients had their stools tested for the tcdB gene by direct PCR and detection of toxigenic C. difficile by culture. C. difficile isolates were subjected to toxin gene profiling and ribotyping, and patient medical records were reviewed retrospectively. There were 248 and 221 patients included in CDI and control groups, respectively. The CDI group had a higher overall 30-day mortality rate than the control group (21% versus 14%, P = 0.046), but only 2 deaths (1%) were directly attributable to CDI. Metronidazole treatment was not inferior to vancomycin in this population, and vancomycin was associated with a higher 30-day mortality rate (P = 0.047). The prevalence of severe CDI and disease outcomes were not different between patients infected with A–B+ C. difficile and A+B+ C. difficile strains or between patients with and without colonization by nontoxigenic C. difficile. Besides C. difficile-specific tests, neither a single laboratory result nor a combination of results was predictive of CDI. In conclusion, CDI in Thailand was relatively mild, and metronidazole remained an effective treatment option for these mild infections

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