COMPARISON OF WHOLE GENOME SEQUENCE-BASED METHODS AND PCR RIBOTYPING FOR SUBTYPING OF Clostridioides difficile

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary-electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better and provide standardized and interlaboratory exchangeable data. Using a well-characterized collection of diverse strains (N=630; 100 unique ribotypes (RTs)), we compared the discriminatory power of core genome multilocus sequence typing (cgMLST) (SeqSphere & EnteroBase), whole genome MLST (wgMLST) (EnteroBase) and single nucleotide polymorphism (SNP) analysis. A unique cgMLST profile (>6 allele differences) was observed in 82/100 RTs, indicating that cgMLST could distinguish most, but not all, RTs. Application of cgMLST in two outbreak settings with RT078 and RT181 (known with a low intra-RT allele difference) showed no distinction between outbreak- and non-outbreak strains, in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize and offers higher discrimination. However, adjusted cut-off thresholds and epidemiological data are necessary to recognize outbreaks of some specific RTs. We propose to use an allelic threshold 3 alleles to identify outbreaks.

Diagnostic Methods of Clostridioides difficile Infection and Clostridioides difficile Ribotypes in Studied Sample

Background: Clostridioides (Clostridium) difficile is the most common nosocomial pathogen and antibiotic-related diarrhea in health-care facilities. Over the last few years, there was an increase in the incidence rate of C. difficile infection cases in Slovakia. In this study, the phenotypic (toxigenicity, antimicrobial susceptibility) and genotypic (PCR ribotypes, genes for binary toxins) patterns of C. difficile isolates from patients with CDI were analyzed, from July to August 2016, taken from hospitals in the Horne Povazie region of northern Slovakia. The aim of the study was also to identify hypervirulent strains (e.g., the presence of RT027 or RT176). Methods: The retrospective analysis of biological samples suspected of CDI were analyzed by GDH, anaerobic culture, enzyme immunoassay on toxins A/B, multiplex “real-time” PCR and PCR capillary-based electrophoresis ribotyping, and by MALDI TOF MS. Results: C. difficile isolates (n = 44) were identified by PCR ribotyping, which revealed five different ribotypes (RT001, 011, 017, 081, 176). The presence of hypervirulent RT027 was not identified. The C. difficile isolates (RT001, 011, 081, 176) were susceptible to metronidazole and vancomycin. One isolate RT017 had reduced susceptibility to vancomycin. A statistically significant difference between the most prevalent PCR ribotypes, RT001 and RT176, regarding variables such as albumin, CRP, creatinine, the length of hospitalization (p = 0.175), and glomerular filtration (p = 0.05) was not found. Conclusion: The results of PCR capillary-based electrophoresis ribotyping in the studied samples showed a high prevalence of RT176 and 001.

Performance of Core Genome Multilocus Sequence Typing Compared to Capillary-Electrophoresis PCR Ribotyping and SNP Analysis of Clostridioides difficile

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary-electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better, but backward compatibility with CE-PCR ribotyping must be assessed. Using a well-characterized collection of diverse strains (N=630; 100 unique ribotypes [RTs]), we aimed to investigate PCR ribotyping prediction from core genome multilocus sequence typing (cgMLST). Additionally, we compared the discriminatory power of cgMLST (SeqSphere & EnteroBase) and whole genome MLST (wgMLST) (EnteroBase) with single nucleotide polymorphism (SNP) analysis). A unique cgMLST profile (>6 allele differences) was observed in 82/100 ribotypes, indicating sufficient backward compatibility. Intra-RT allele difference varied per ribotype and MLST clade. Application of cg/wgMLST and SNP analysis in two outbreak settings with ribotypes RT078 and RT181 (known with a low intra-ribotype allele difference) showed no distinction between outbreak- and non-outbreak strains, in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize and offers higher discrimination. However, in some ribotype complexes adjusted cut-off thresholds and epidemiological data are necessary to recognize outbreaks. We propose to decrease the current threshold of 6 to 3 alleles to better identify outbreaks.

Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile.

Rapid Classification of Clostridioides difficile Strains Using MALDI-TOF MS Peak-Based Assay in Comparison with PCR-Ribotyping

Typing methods are needed for epidemiological tracking of new emerging and hypervirulent strains because of the growing incidence, severity and mortality of Clostridioides difficile infections (CDI). The aim of this study was the evaluation of a typing Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS (T-MALDI)) method for the rapid classification of the circulating C. difficile strains in comparison with polymerase chain reaction (PCR)-ribotyping results. Among 95 C. difficile strains, 10 ribotypes (PR1–PR10) were identified by PCR-ribotyping. In particular, 93.7% of the isolates (89/95) were grouped in five ribotypes (PR1–PR5). For T-MALDI, two classifying algorithm models (CAM) were tested: the first CAM involved all 10 ribotypes whereas the second one only the PR1–PR5 ribotypes. Better performance was obtained using the second CAM: recognition capability of 100%, cross-validation of 96.6% and agreement of 98.4% (60 correctly typed strains, limited to PR1–PR5 classification, out of 61 examined strains) with PCR-ribotyping results. T-MALDI seems to represent an alternative to PCR-ribotyping in terms of reproducibility, set up time and costs, as well as a useful tool in epidemiological investigation for the detection of C. difficile clusters (either among CAM included ribotypes or out-of-CAM ribotypes) involved in outbreaks.

Retrospective Definition of Clostridioides difficile PCR Ribotypes on the Basis of Whole Genome Polymorphisms: A Proof of Principle Study

Clostridioides difficile is a cause of health care-associated infections. The epidemiological study of C. difficile infection (CDI) traditionally involves PCR ribotyping. However, ribotyping will be increasingly replaced by whole genome sequencing (WGS). This implies that WGS types need correlation with classical ribotypes (RTs) in order to perform retrospective clinical studies. Here, we selected genomes of hyper-virulent C. difficile strains of RT001, RT017, RT027, RT078, and RT106 to try and identify new discriminatory markers using in silico ribotyping PCR and De Bruijn graph-based Genome Wide Association Studies (DBGWAS). First, in silico ribotyping PCR was performed using reference primer sequences and 30 C. difficile genomes of the five different RTs identified above. Second, discriminatory genomic markers were sought with DBGWAS using a set of 160 independent C. difficile genomes (14 ribotypes). RT-specific genetic polymorphisms were annotated and validated for their specificity and sensitivity against a larger dataset of 2425 C. difficile genomes covering 132 different RTs. In silico PCR ribotyping was unsuccessful due to non-specific or missing theoretical RT PCR fragments. More successfully, DBGWAS discovered a total of 47 new markers (13 in RT017, 12 in RT078, 9 in RT106, 7 in RT027, and 6 in RT001) with minimum q-values of 0 to 7.40 × 10−5, indicating excellent marker selectivity. The specificity and sensitivity of individual markers ranged between 0.92 and 1.0 but increased to 1 by combining two markers, hence providing undisputed RT identification based on a single genome sequence. Markers were scattered throughout the C. difficile genome in intra- and intergenic regions. We propose here a set of new genomic polymorphisms that efficiently identify five hyper-virulent RTs utilizing WGS data only. Further studies need to show whether this initial proof-of-principle observation can be extended to all 600 existing RTs.

An Outbreak of Clostridium (Clostridioides) difficile Infections within an Acute and Long-Term Care Wards Due to Moxifloxacin-Resistant PCR Ribotype 176 Genotyped as PCR Ribotype 027 by a Commercial Assay

We aimed to characterize Clostridioides difficile isolates cultured during a six-month single-center study from stool samples of patients with C. difficile infection (CDI) genotyped by the Xpert®C. difficile/Epi assay by polymerase chain reaction (PCR) ribotyping, toxin genes’ detection and multi-locus variable number tandem repeats analysis (MLVA). The susceptibility to metronidazole, vancomycin and moxifloxacin was determined by agar dilution. In addition, the presence of Thr82Ile in the GyrA and a single nucleotide deletion at position (Δ117) in the tcdC gene were investigated. Between January 1 and June 30, 2016, of 114 CDIs, 75 cases were genotyped as presumptive PCR ribotype (RT) 027 infections using a commercial assay. C. difficile isolates cultured from presumptive RT027 stool samples belonged to RT176. These isolates carried genes for toxin A (tcdA), B (tcdB), binary (cdtA/B) and had Δ117 in the tcdC gene. Using MLVA, the 71/75 isolates clustered into two clonal complexes (CCs). Of these, 39 isolates (54.9%) were from patients hospitalized in acute care and 32 isolates (45.1%) were isolated from patients hospitalized in the long-term care department. All isolates were susceptible to metronidazole and vancomycin, and 105 isolates were resistant to moxifloxacin (92%) carrying Thr83Ile in the GyrA. An outbreak of RT176 CDIs, suspected as RT027, was recognized in a Slovakian hospital. In order to monitor the emergence and spread of RT027-variants, the identification of a presumptive RT027 CDI should be confirmed at a strain level by PCR ribotyping.

Characterization of Non-Toxigenic Clostridioides difficile Strains Isolated from Preterm Neonates and In Vivo Study of Their Protective Effect

In a previous monocentric study in preterm neonates (PN), we described a high Clostridioides difficile colonization rate (74%) with two uncommon non-toxigenic strains (NTCD) belonging to PCR-ribotype (RT) (CE)847 and (CE)032. To determine the extent of carriage of both NTCD in other spatio-temporal settings, strains isolated in PN stools from two multicenter cohorts were characterized by PCR-ribotyping, MLVA and MLST. We also evaluated the protective role of two NTCD from these RT against C. difficile infection in a hamster caecitis model. Animals were administered either each NTCD alone (n = 7), or followed by a 027 strain (n = 9). A control group received only the 027 strain (n = 8). Clinical activity and colonization by C. difficile in stools were monitored daily until death or sacrifice at D20. We isolated 18 RT(CE)032 (ST-83) strains and 2 RT(CE)847 (ST-26) strains among 247 PN from both cohorts. Within each RT, strains were genetically related. The survival rate was significantly increased when animals received a RT(CE)847 or (CE)032 strain before the 027 strain (4/9 deaths, p = 0.029; 1/9 death, p = 0.0004, respectively). We describe two predominant uncommon NTCD strains, in a PN population from different healthcare facilities. Both NTCD provide a potential protection against C. difficile infection.

Molecular characterisation of, and antimicrobial resistance in, Clostridioides difficile from Thailand, 2017-2018

BackgroundAntimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI). Many antimicrobials, such as fluoroquinolones, have been associated with outbreaks of CDI globally.ObjectivesThis study aimed to characterise AMR among clinical C. difficile strains in Thailand, a country where the use of antimicrobials remains inadequately regulated.MethodsStool samples were screened for tcdB and positives were cultured. C. difficile isolates were characterised by toxin profiling and PCR ribotyping. Antimicrobial susceptibility testing was performed using an agar incorporation method, and whole-genome sequencing and AMR genotyping performed on a subset of strains.ResultsThere were 321 C. difficile strains isolated from 326 stool samples. The most common toxigenic ribotype (RT) was RT 017 (18%), followed by RTs 014 (12%) and 020 (7%). There was a high resistance prevalence (≥ 10%) to clindamycin, erythromycin, moxifloxacin and rifaximin, and resistance prevalence was greatest among RT 017 strains. AMR genotyping revealed a strong correlation between resistance genotype and phenotype for moxifloxacin and rifampicin. The presence of erm-class genes was associated with high-level clindamycin and erythromycin resistance. Point substitutions on the penicillin-binding proteins (PBP1 and PBP3) were not sufficient to confer meropenem resistance, however, a Y721S substitution in PBP3 was associated with a slight increase in meropenem MIC. No resistance to metronidazole, vancomycin or fidaxomicin was observed.ConclusionThere was a large proportion of C. difficile RT 017 in Thailand and a high AMR prevalence among these strains. The concordance between AMR phenotype and genotype was strong.

Burden of Clostridium difficile Infection (CDI) Across Whole Healthcare Economies and European Borders; COMBACTE-CDI Results

Background: The burden of C. difficile infection (CDI) on healthcare facilities is well recognized. However, studies focusing on inpatient settings, in addition to ascertainment bias in general, have led to a paucity of data on the true burden of CDI across whole healthcare economies. Methods: Sites testing both inpatient and community samples were recruited from 12 European countries (1 site per 3 million population). On 2 selected days, all diarrheal fecal samples (regardless of tests requested) were sent to the European Coordinating Laboratory (ECL) for C. difficile toxin testing and culture. The CDI results and tests not requested at each submitting site were compared with the ECL results to determine the number of missed CDIs. Contemporaneous C. difficile isolates from food and animal sources were collected. All isolates underwent PCR ribotyping and toxinotyping; prevalences of ribotypes among regions of Europe and reservoir settings were compared. Results: Overall, 3,163 diarrheal fecal samples were received from 119 sites. The burden of CDI varied by country (positivity rates, 0–15.8%) and by European region; the highest positivity rate in Eastern Europe was 13.1%. The testing and positivity rates in community samples were 29.6% and 1.4% vs 74.9% and 5.0% in hospital samples; 16% and 55% of samples positive for CDI at ECL were not diagnosed in hospitals and the community. The most common C. difficile ribotypes from hospital samples were 027 (11%), 181 (12%), and 014 (8%), although prevalence varied by country. The highest prevalence of toxinotype IIIb (ribotypes 027, 181, and 176) was seen in Eastern Europe (55% of all isolates), which also had the lowest testing rate. For hospital samples, the proportion of toxinotype IIIb was inversely related to the testing rate (r = −0.79) (Fig. 1). The most common ribotypes from food sources were 078 (23%) and 126 (13%) (toxinotype V), and most common ribotypes from community samples were 078 (9%) and 039 (9%). Overall, 106 different ribotypes were identified: 25 in both the hospital and community and 16 in the hospital, community, and food chain. Conclusions: The diagnosed burden of CDI varies markedly among countries in both hospital and community settings. Reduced sampling/testing in Eastern Europe is inversely related to the proportion of toxinotype IIIb strains identified, suggesting that lack of suspicion leads to underdiagnosis and outbreaks of infection. The proportion of missed CDIs in the community was ~3.5× higher than in hospitals, indicating major underrecognition in the former setting. There were marked differences in ribotypes in different reservoir settings, emphasizing the complex epidemiology of C. difficile.Funding: Proprietary organization: COMBACTE-CDI is an EU funded (Horizon2020) consortium of academic and EFPIA partners (bioMerieux, GSK, Sanofi Pasteur, Astra Zeneca, Pfizer, Da Volterra) with additional Funding: from the EFPIA partners.Disclosures: Submitter: Kerrie Davies; the work presented is funded via the EU and EFPIA (commercial) partners in a consortium.