Dataset Information

Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort.

ABSTRACT: Whole-genome sequencing (WGS) is a highly sensitive method for identifying genetic relatedness and transmission of Clostridioides difficile strains. Previous studies suggest that as few as 3 core genome single-nucleotide variants (SNVs) discriminate between genetically distinct isolates. Because a single C. difficile colony is selected from culture for WGS, significant within-host genetic diversity could preclude identification of transmission events. To evaluate the likelihood of missed transmission events using WGS of single colonies from culture, we examined within-host genetic diversity among C. difficile isolates collected from children. We performed WGS using an Illumina MiSeq instrument on 8 C. difficile colonies randomly selected from each culture performed on stool collected from 10 children (8 children diagnosed with C. difficile infection and 2 children with asymptomatic carriage); 77/80 (96%) isolate sequences were successfully assembled. Among 8/10 (80%) children, all isolates were the same sequence type (ST). The other 2 children each had mixed infection with two STs, although one ST predominated. Among 9/10 (90%) children, isotypic isolates differed by ≤2 SNVs; an isotypic isolate in the remaining child differed by 3 to SNVs relative to the other isolates from that child. Overall, among the 77 isolates collected from 10 stool cultures, 74/77 (96%) were clonal (i.e., same ST and ≤2 core genome SNVs) to other isolates in stool culture. In summary, we identified rare C. difficile within-host genetic diversity in children, suggesting that WGS of a single colony from stool is likely to appropriately characterize isolate clonality and putative transmission events in the majority of cases.

SUBMITTER: Balaji A

PROVIDER: S-EPMC6711918 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort.

Balaji Aakash A Ozer Egon A EA Kociolek Larry K LK

Journal of clinical microbiology 20190826 9

Whole-genome sequencing (WGS) is a highly sensitive method for identifying genetic relatedness and transmission of <i>Clostridioides difficile</i> strains. Previous studies suggest that as few as 3 core genome single-nucleotide variants (SNVs) discriminate between genetically distinct isolates. Because a single <i>C. difficile</i> colony is selected from culture for WGS, significant within-host genetic diversity could preclude identification of transmission events. To evaluate the likelihood of ...[more]

PMID: 31315950

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Project description:Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well understood. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer variants, following infection with the S-layer-null strain, FM2.5. These variants carry either correction of the original point mutation, or sequence modifications which restored the reading frame, and translation of slpA. Selection of these variant clones was rapid in vivo, and independent of toxin production, with up to 90% of the recovered C. difficile population encoding modified slpA sequence within 24 h post infection. Two variants, subsequently named FM2.5varA and FM2.5varB, were selected for study in greater detail. Structural determination of SlpA from FM2.5varB indicated an alteration in the orientation of protein domains, resulting in a reorganisation of the lattice assembly, and changes in interacting interfaces, which might alter function. Interestingly, variant FM2.5varB displayed an attenuated, FM2.5-like phenotype in vivo compared to FM2.5varA, which caused disease severity more comparable to that of R20291. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates revealed large changes in gene expression between R20291 and FM2.5. Downregulation of tcdA/tcdB and several genes associated with sporulation and cell wall integrity may account for the reported attenuated phenotype of FM2.5 in vivo. RNA-seq data correlated well with disease severity with the more virulent variant, FM2.5varA, showing s similar profile of gene expression to R20291 in vitro, while the attenuated FM2.5varB showed downregulation of many of the same virulence associated traits as FM2.5. Cumulatively, these data add to a growing body of evidence that the S-layer contributes to C. difficile pathogenesis and disease severity.

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.

Dataset Information

Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort.

Publications

Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort.

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