Dataset Information

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains.

ABSTRACT: Restriction endonuclease analysis (REA) and PCR ribotyping are two typing systems that have been frequently utilized for molecular epidemiologic characterization of Clostridioides (Clostridium) difficile. To correlate typing data obtained from each method, we performed both REA and PCR ribotyping on a large and diverse set of historical and contemporary C. difficile infection clinical isolates. Eighty isolates were selected from each reference laboratory in the United States (Microbiology Reference Laboratory, Hines VA Medical Center) and United Kingdom (Clostridium difficile Network for England and Northern Ireland laboratory, University of Leeds). The 160 isolates were assigned to 82 unique ribotypes and 51 unique REA groups (116 unique REA types). In general, concordance between typing methods was good. Dendrogram analysis of PCR ribotype band patterns demonstrated close genetic relationships among strain types with discordant REA and ribotype assignments. While REA typing was more discriminatory, several REA types in this study were further discriminated by PCR ribotyping, indicating that discriminatory value of these typing methods may be strain dependent. These data will assist with molecular epidemiologic surveillance of strains identified by these two commonly used C. difficile typing systems.

SUBMITTER: Kociolek LK

PROVIDER: S-EPMC6924008 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains.

Kociolek Larry K LK Perdue Eric R ER Fawley Warren N WN Wilcox Mark H MH Gerding Dale N DN Johnson Stuart S

Anaerobe 20180803

Restriction endonuclease analysis (REA) and PCR ribotyping are two typing systems that have been frequently utilized for molecular epidemiologic characterization of Clostridioides (Clostridium) difficile. To correlate typing data obtained from each method, we performed both REA and PCR ribotyping on a large and diverse set of historical and contemporary C. difficile infection clinical isolates. Eighty isolates were selected from each reference laboratory in the United States (Microbiology Refere ...[more]

PMID: 30009944

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Project description: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.

Project description:Clostridioides difficile is an important organism causing healthcare-associated infections. It has been documented that specific strains caused multiple outbreaks globally, and patients infected with those strains are more likely to develop severe C. difficile infection (CDI). With the appearance of a variant strain, BI/NAP1 ribotype 027, responsible for several outbreaks and high mortality rates worldwide, the epidemiology of the CDI changed drastically in the United States, Europe, and some Latin American countries. Although the epidemic strain 027 was not yet detected in Brazil, there are ribotypes exclusively found in the country, such as, 131, 132, 133, 135, 142 and 143, which are responsible for outbreaks in Brazilian hospitals and nursing homes. Although PCR-ribotyping is the most used method in epidemiology studies of C. difficile, it is not available in Brazil. This study aimed to develop and validate an in-house database for detecting C. difficile ribotypes, usually involved in CDI in Brazilian hospitals, by using MALDI-TOF MS. A database with 19 different ribotypes, 13 with worldwide circulation and 6 Brazilian-restricted, was created based on 27 spectra readings of each ribotype. After BioNumerics analysis, neighbor-joining trees revealed that spectra were distributed in clusters according to ribotypes, showing that MALDI-TOF MS could discriminate all 19 ribotypes. Moreover, each ribotype showed a different profile with 42 biomarkers detected in total. Based on their intensity and occurrence, 13 biomarkers were chosen to compose ribotype-specific profiles, and in silico analysis showed that most of these biomarkers were uncharacterized proteins or well-conserved peptides, such as ribosomal proteins. A double-blind assessment using the 13 biomarkers correctly assigned the ribotype in 73% of the spectra analyzed, with 94%-100% of correct hits for 027 and for Brazilian ribotypes. Although further analyses are required, our results show that MALDI-TOF MS might be a reliable, fast and feasible alternative for epidemiological surveillance of C. difficile in Brazil.

Dataset Information

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains.

Publications

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains.

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