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ToolsDjeghout, Bilal, Le-Viet, Thanh, Martins, Leonardo de Oliveira, Savva, George M., Evans, Rhiannon, Baker, David, Page, Andrew, Elumogo, Ngozi, Wain, John and Janecko, Nicol (2024) Capturing clinically relevant Campylobacter attributes through direct whole genome sequencing of stool. Microbial Genomics, 10 (8). ISSN 2057-5858
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Abstract
Campylobacter is the leading bacterial cause of infectious intestinal disease, but the pathogen typically accounts for a very small proportion of the overall stool microbiome in each patient. Diagnosis is even more difficult due to the fastidious nature of Campylobacter in the laboratory setting. This has, in part, driven a change in recent years, from culture-based to rapid PCRbased diagnostic assays which have improved diagnostic detection, whilst creating a knowledge gap in our clinical and epidemiological understanding of Campylobacter genotypes – no isolates to sequence. In this study, direct metagenomic sequencing approaches were used to assess the possibility of replacing genome sequences with metagenome sequences; metagenomic sequencing outputs were used to describe clinically relevant attributes of Campylobacter genotypes. A total of 37 diarrhoeal stool samples with Campylobacter and five samples with an unknown pathogen result were collected and processed with and without filtration, DNA was extracted, and metagenomes were sequenced by short-read sequencing. Culture-based methods were used to validate Campylobacter metagenome-derived genome (MDG) results. Sequence output metrics were assessed for Campylobacter genome quality and accuracy of characterization. Of the 42 samples passing quality checks for analysis, identification of Campylobacter to the genus and species level was dependent on Campylobacter genome read count, coverage and genome completeness. A total of 65% (24/37) of samples were reliably identified to the genus level through Campylobacter MDG, 73% (27/37) by culture and 97% (36/37) by qPCR. The Campylobacter genomes with a genome completeness of over 60% (n=21) were all accurately identified at the species level (100%). Of those, 72% (15/21) were identified to sequence types (STs), and 95% (20/21) accurately identified antimicrobial resistance (AMR) gene determinants. Filtration of stool samples enhanced Campylobacter MDG recovery and genome quality metrics compared to the corresponding unfiltered samples, which improved the identification of STs and AMR profiles. The phylogenetic analysis in this study demonstrated the clustering of the metagenome-derived with culture-derived genomes and revealed the reliability of genomes from direct stool sequencing. Furthermore, Campylobacter genome spiking percentages ranging from 0 to 2% total metagenome abundance in the ONT MinION sequencer, configured to adaptive sequencing, exhibited better assembly quality and accurate identification of STs, particularly in the analysis of metagenomes containing 2 and 1% of Campylobacter jejuni genomes. Direct sequencing of Campylobacter from stool samples provides clinically relevant and epidemiologically important genomic information without the reliance on cultured genomes.
| Item Type: | Article |
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| Additional Information: | Availability of Data and Materials: All supporting data, code and protocols are included in the article or are available as supplementary data files. The online version of this article contains three supplementary figures (Figs S1–S3) and five supplementary tables (Tables S1–S5). All sequenced Campylobacter jejuni isolate data are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the Bioproject accession numbers PRJNA1046283, PRJNA797426 and PRJNA1049393. Sequence read archive (SRA) accession numbers and associated metadata can be found in the supplementary material of this study (Table S1). All data analysis codes, scripts and pipelines are deposited on GitHub (https://github.com/quadram-institute-bioscience/2024-campymags/tree/main). Funding Information: This project was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant Microbes in the Food Chain BB/R012504/1 and BBSRC Institute Strategic Programme Microbes and Food Safety BB/X011011/1 and the constituent project(s) BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain) and BBS/E/F/000PR10349 (Theme 2, Microbial Survival in the Food Chain) and BBS/E/F/000PR13635 (Theme 2, Microbial Survival in established and evolving food systems). |
| Uncontrolled Keywords: | campylobacter,clinical samples,metagenome-derived genomes,epidemiology,microbiology,molecular biology,genetics ,/dk/atira/pure/subjectarea/asjc/2700/2713 |
| Faculty \ School: | Faculty of Medicine and Health Sciences > School of Health Sciences Faculty of Medicine and Health Sciences > Norwich Medical School |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 23 Oct 2024 15:30 |
| Last Modified: | 25 Oct 2024 15:30 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/97156 |
| DOI: | 10.1099/mgen.0.001284 |
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