A two-step approach for the investigation of a Clostridium difficile outbreak by molecular methods

Krutova, Marcela; Wilcox, Mark H.; Kuijper, Ed J.

doi:10.1016/j.cmi.2019.07.022

Cited by 9 publications

(3 citation statements)

References 10 publications

(17 reference statements)

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“…As such a transition phase may take time, important outbreak clones, as well as novel introductions of animal-related C. difficile strains into the human population may be missed due to the lacking or hampered comparability of the results obtained by different typing approaches. Therefore, a two-step approach, consisting of first, PCR-ribotyping and second, performing WGS to investigate suspected outbreak-clusters, as has recently been suggested ( Krutova et al., 2019 ), may be reasonable in such a transition phase.…”

Section: Discussionmentioning

confidence: 99%

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

Seth-Smith

Biggel

Roloff

et al. 2021

Front. Cell. Infect. Microbiol.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Seth-Smith

Biggel

Roloff

et al. 2021

Front. Cell. Infect. Microbiol.

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“…Surprisingly, the characterization of C. difficile isolates by ribotyping, as recommended by the ECDC [6], identified that C. difficile isolates cultured from the presumptive ribotype 027 CDIs belong to ribotype 176 (Figure 2 shows the difference in ribotyping profiles of RTs 027 and 176). Using MLVA for the differentiation of highly related and non-related C. difficile isolates of the same ribotype [17], the clonal relatedness of C. difficile RT176 was detected between isolates from cases in acute care and long-term care wards.…”

Section: Discussionmentioning

confidence: 99%

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

Nováková

Kotlebova

Gryndlerova

et al. 2020

JCM

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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.

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“…The PCR capillary-based electrophoresis ribotyping patterns of investigated ribotypes and fragment analysis were performed by a DNA analyzer and by Gene Mapper v5.0 which were kindly provided by the Department of Microbiology of 2nd Faculty of Medicine of Charles University, and Motol University Hospital in Prague. This method is performed in CDI epidemics to identify individual strains, and to perform surveillance for the spread of CDI [ 33 , 34 ].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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.

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A two-step approach for the investigation of a Clostridium difficile outbreak by molecular methods

Cited by 9 publications

References 10 publications

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

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

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

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

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