通过对粪便进行直接全基因组测序,捕捉与临床相关的弯曲杆菌属性。

IF 4 2区 生物学 Q1 GENETICS & HEREDITY Microbial Genomics Pub Date : 2024-08-01 DOI:10.1099/mgen.0.001284
Bilal Djeghout, Thanh Le-Viet, Leonardo de Oliveira Martins, George M Savva, Rhiannon Evans, David Baker, Andrew Page, Ngozi Elumogo, John Wain, Nicol Janecko
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引用次数: 0

摘要

弯曲状杆菌是引起肠道感染性疾病的主要细菌,但这种病原体通常只占每位患者粪便微生物群的很小一部分。由于弯曲菌在实验室环境中的耐受性很差,因此诊断更加困难。这在一定程度上促使近年来的诊断方法发生了变化,从基于培养的诊断方法转变为基于 PCR 的快速诊断方法,从而提高了诊断检测率,但同时也给我们对弯曲杆菌基因型的临床和流行病学了解造成了知识空白--没有分离物可测序。本研究采用直接元基因组测序方法来评估用元基因组测序取代基因组测序的可能性;元基因组测序结果用于描述弯曲杆菌基因型的临床相关属性。共收集了 37 份带有弯曲杆菌的腹泻粪便样本和 5 份病原体结果未知的样本,经过过滤和不过滤处理,提取了 DNA,并通过短线程测序对元基因组进行了测序。采用基于培养的方法验证弯曲杆菌元基因组(MDG)结果。对弯曲杆菌基因组质量和特征描述准确性的序列输出指标进行了评估。在通过质量检查的 42 份分析样本中,弯曲状杆菌的属种鉴定取决于弯曲状杆菌基因组读数数、覆盖率和基因组完整性。通过弯曲状杆菌 MDG,共有 65%(24/37)的样本被可靠地鉴定为属级;通过培养,73%(27/37)的样本被可靠地鉴定为种级,通过 qPCR,97%(36/37)的样本被可靠地鉴定为属级。基因组完整性超过 60% 的弯曲杆菌基因组(n=21)均被准确鉴定为种一级(100%)。其中,72%(15/21)的序列类型(STs)得到了鉴定,95%(20/21)的抗菌药耐药性(AMR)基因决定因子得到了准确鉴定。与未经过滤的相应样本相比,过滤粪便样本提高了弯曲杆菌 MDG 的回收率和基因组质量指标,从而改进了 STs 和 AMR 图谱的鉴定。本研究中的系统发育分析表明了元基因组与培养基基因组的聚类,并揭示了粪便直接测序基因组的可靠性。此外,在ONT MinION测序仪中,弯曲状杆菌基因组的加标百分比范围为元基因组总丰度的0%至2%,配置为自适应测序,可获得更好的组装质量和ST的准确鉴定,尤其是在分析含有2%和1%空肠弯曲杆菌基因组的元基因组时。直接对粪便样本中的弯曲杆菌进行测序,无需依赖培养基因组,就能提供与临床相关且具有流行病学意义的基因组信息。
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Capturing clinically relevant Campylobacter attributes through direct whole genome sequencing of stool.

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 PCR-based 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.

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来源期刊
Microbial Genomics
Microbial Genomics Medicine-Epidemiology
CiteScore
6.60
自引率
2.60%
发文量
153
审稿时长
12 weeks
期刊介绍: Microbial Genomics (MGen) is a fully open access, mandatory open data and peer-reviewed journal publishing high-profile original research on archaea, bacteria, microbial eukaryotes and viruses.
期刊最新文献
Longitudinal genomic surveillance of a UK intensive care unit shows a lack of patient colonisation by multi-drug-resistant Gram-negative bacterial pathogens. Characterization of psychrotrophic and thermoduric bacteria in raw milk using a multi-omics approach. Chromosome architecture as a determinant for biosynthetic diversity in Micromonospora. Genomic diversity of Campylobacter jejuni and Campylobacter coli isolates recovered from human and poultry in Australia and New Zealand, 2017 to 2019. Identifying gene-level mechanisms of successful dispersal of Vibrio parahaemolyticus during El Niño events.
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