Gallichan Sarah, Forrest Sally, Picton-Barlow Esther, McKeown Claudia, Moore Maria, Heinz Eva, Feasey Nicholas A, Lewis Joseph M, Graf Fabrice E
Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Microbiol Spectr. 2025 Jan 7;13(1):e0105824. doi: 10.1128/spectrum.01058-24. Epub 2024 Nov 22.
Understanding transmission pathways of important opportunistic, drug-resistant pathogens, such as extended-spectrum beta-lactamase (ESBL)-producing , is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing (ESBL-EC) between two sources, single-nucleotide resolution of strains, as well as diversity within and between samples, is required. However, the microbiological methods to best track these pathogens are unclear. Here, we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high-quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-h pre-enrichment in Buffered Peptone Water, plating on cefotaxime-supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole-genome sequencing. We conclude that our optimized workflow can be applied for single-nucleotide variant analysis of an ESBL-EC from stool.IMPORTANCEDrug-resistant infections are increasingly difficult to treat with antibiotics. Preventing infections is thus highly beneficial. To do this, we need to understand how drug-resistant bacteria spread to take action to stop infection and transmission. This requires us to accurately trace these bacteria between different sources. In this study, we compared different laboratory methods to see which worked best for detecting extended-spectrum beta-lactamase (ESBL)-producing , a common cause of urinary tract or bloodstream infections, from human stool samples. We found that enriching stool in a nutrient broth for 4 h, then plating the bacterial suspension on antibiotic-selective MacConkey agar, and finally extracting DNA from the bacteria using a specific DNA purification kit resulted in improved recovery of ESBL and high-quality DNA. Sequencing multiple isolates from stool allowed us to distinguish unambiguously and at high resolution between different variants of ESBL present in stool.
了解重要的机会性耐药病原体（如产超广谱β-内酰胺酶[ESBL]的病原体）的传播途径，对于实施针对性的预防策略以阻断传播并减少感染数量至关重要。为了将两个来源之间产ESBL的大肠杆菌（ESBL-EC）的传播联系起来，需要对菌株进行单核苷酸分辨率分析，以及样本内部和样本之间的多样性分析。然而，最佳追踪这些病原体的微生物学方法尚不清楚。在此，我们比较了微生物学工作流程中的不同步骤，以确定不同的预富集肉汤、预富集孵育时间、预富集选择、选择性平板接种和DNA提取方法对从人类粪便样本中回收ESBL-EC的影响，目的是获取用于测序和基因组流行病学研究的高质量DNA。我们证明，在缓冲蛋白胨水中进行4小时的预富集，接种于补充头孢噻肟的麦康凯琼脂平板上，并使用Lucigen MasterPure DNA纯化试剂盒提取DNA，可提高从人类粪便中回收ESBL-EC的效率，并产生用于全基因组测序的高质量DNA。我们得出结论，我们优化的工作流程可应用于对粪便中ESBL-EC进行单核苷酸变异分析。
重要性
耐药感染越来越难以用抗生素治疗。因此，预防感染非常有益。为此，我们需要了解耐药细菌如何传播，以便采取行动阻止感染和传播。这要求我们在不同来源之间准确追踪这些细菌。在本研究中，我们比较了不同的实验室方法，以确定哪种方法最适合从人类粪便样本中检测产超广谱β-内酰胺酶（ESBL）的大肠杆菌，这是尿路感染或血流感染的常见病因。我们发现，将粪便在营养肉汤中富集4小时，然后将细菌悬液接种于抗生素选择性麦康凯琼脂平板上，最后使用特定的DNA纯化试剂盒从细菌中提取DNA，可提高ESBL的回收率并获得高质量的DNA。对粪便中的多个分离株进行测序，使我们能够在粪便中存在的不同ESBL变体之间进行明确且高分辨率的区分。
