NEW YORK – Researchers from the Wellcome Sanger Institute, University of Oslo, and elsewhere have developed a new whole-genome sequencing-based approach that promises to identify multiple common pathogens linked to hospital-associated infections (HAIs) in one assay.

纽约——奥斯陆大学惠康桑格研究所（WellcomeSanger Institute）和其他地方的研究人员开发了一种新的基于全基因组测序的方法，有望在一次检测中鉴定出与医院相关感染（HAI）相关的多种常见病原体。

Published in Lancet Microbe last month, the study demonstrates the potential utility of culture-based, pan-pathogen WGS for HAI surveillance and prevention, though more studies are still needed to further validate the method's cost, accuracy, and implementation feasibility.

该研究发表在上个月的《柳叶刀》杂志上，证明了基于培养的泛病原体WGS在HAI监测和预防中的潜在效用，尽管仍需要更多的研究来进一步验证该方法的成本，准确性和实施可行性。

Traditionally, sequencing-based pathogen surveillance methods rely on analyzing single-colony bacterial isolates. While this approach can offer greater information on the target bacteria strains, it fails to account for the diversity of microorganisms present within a clinical sample, said Harry Thorpe, a researcher from the University of Oslo and the first author of the study.

传统上，基于测序的病原体监测方法依赖于分析单菌落细菌分离株。奥斯陆大学研究员、该研究的第一作者哈里·索普（HarryThorpe）说，虽然这种方法可以提供有关目标细菌菌株的更多信息，但它无法解释临床样本中存在的微生物的多样性。

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On the other hand, shotgun metagenomic sequencing allows researchers to analyze all microbial diversity within a sample, Thorpe noted, but it generally cannot offer adequate sequencing depth to achieve strain-level resolution for epidemiology and transmission analysis.

另一方面，鸟枪宏基因组测序允许研究人员分析样本中的所有微生物多样性，索普指出，但它通常不能提供足够的测序深度来实现流行病学和传播分析的菌株水平分辨率。

'We wanted to design a method that is intermediate between the single colony method and the metagenomics method,' he added.

他补充道：“我们想设计一种介于单菌落法和宏基因组学方法之间的方法。”。

The method developed by Thorpe and his team starts with sample collection from patients. For the Lancet Microbe paper, they retrospectively collected samples from 256 patients in an Italian hospital between April 7 and May 10, 2020. Of the participants, 169 were men and 87 were women. About half of the patients were hospitalized in the ordinary wards, while the other half were admitted in the ICUs..

索普和他的团队开发的方法始于从患者身上采集样本。对于《柳叶刀》微生物论文，他们回顾性地收集了2020年4月7日至5月10日期间意大利一家医院256名患者的样本。参与者中，男性169人，女性87人。大约一半的患者在普通病房住院，另一半在ICU住院。。

From these patients, the researchers collected 1,130 clinical samples, which included 497 rectal swabs, 439 nasal swabs, and 194 respiratory samples to represent bacteria in the gut, upper airways, and lungs. The collected samples were cultured using six different media to select various pathogen species of interest.

研究人员从这些患者中收集了1130份临床样本，其中包括497份直肠拭子、439份鼻拭子和194份呼吸道样本，以代表肠道、上呼吸道和肺部的细菌。使用六种不同的培养基培养收集的样品，以选择各种感兴趣的病原体种类。

Instead of only picking one colony as with traditional methods, they collected DNA en masse from each plate with positive growth, Thorpe said. .

索普说，他们不是像传统方法那样只采集一个菌落，而是从每个生长呈阳性的平板上收集DNA。

After DNA extraction and library preparation, the samples then underwent deep whole-genome sequencing using the Illumina NovaSeq 6000 platform with the 150 bp paired-end chemistry. The sequencing data were subsequently aligned against the reference datasets to identify the organisms present in each of the samples and their abundance.

DNA提取和文库制备后，样品然后使用Illumina NovaSeq 6000平台进行深度全基因组测序，具有150 bp的配对末端化学。。

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Overall, the analysis identified 52 different bacteria species present in the patient samples. Of these species, 66 percent comprised various strains of the seven most common bacterial infections seen in hospitals: Acinetobacter baumannii, Escherichia coli, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.

总体而言，该分析确定了患者样品中存在的52种不同的细菌种类。在这些物种中，66%包括医院中常见的七种最常见细菌感染的各种菌株：鲍曼不动杆菌，大肠杆菌，屎肠球菌，粪肠球菌，肺炎克雷伯菌，铜绿假单胞菌和金黄色葡萄球菌。

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The authors further investigated the association between each pathogen species and the ward type. There, the results indicated that A. baumannii, K. pneumoniae, P. aeruginosa, and E. faecium were 'significantly associated' with ICU patients for both nasal and rectal samples, while E. faecalis was associated with ICU nasal samples.

。在那里，结果表明A.baumannii，K。肺炎链球菌，铜绿假单胞菌和屎肠球菌与ICU患者的鼻和直肠样本“显着相关”，而粪肠球菌与ICU鼻样本相关。

In the ordinary wards, E. coli was mostly associated with rectal samples. .

在普通病房，大肠杆菌主要与直肠样本有关。

To extrapolate possible hospital-associated transmissions, the researchers conducted pairwise bacterial single-nucleotide polymorphism distance analysis and concluded that HAIs were 'likely to be a significant mode of acquisition' for each of the seven pathogen species identified in the study. That is especially likely for A.

为了推断可能与医院相关的传播，研究人员进行了成对细菌单核苷酸多态性距离分析，并得出结论，HAIs“可能是研究中确定的七种病原体中每一种的重要获取方式”。这对于A来说尤其可能。

baumannii, K. pneumoniae, P. aeruginosa, and E. faecium, for which the authors believe that more than 75 percent of occurrences can be attributed to potential hospital transmission. .

鲍曼不动杆菌，肺炎克雷伯菌，铜绿假单胞菌和屎肠球菌，作者认为超过75%的发生可归因于潜在的医院传播。

Beyond transmission events, Thorpe's team also leveraged the whole-genome data to look for common antimicrobial-resistant (AMR) genes in the samples. From there, they found that the prevalence of AMR genes was 'very high' in ICU samples, with key carbapenemase and extended spectrum ß-lactamase resistance genes detected in over 40 percent of the critical care patients.

除了传播事件之外，索普的团队还利用全基因组数据在样本中寻找常见的抗微生物（AMR）基因。从那里，他们发现ICU样本中AMR基因的患病率“非常高”，超过40%的重症监护患者检测到关键的碳青霉烯酶和超广谱β-内酰胺酶耐药基因。

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'The approach described in the paper is pretty exciting if the methods can hold true and are accurate,' said Alexander Sundermann, an infectious disease professor at the University of Pittsburgh who was not involved in the study.

匹兹堡大学传染病教授亚历山大·桑德曼（AlexanderSundermann）没有参与这项研究，他说：“如果这些方法能够成立且准确，那么论文中描述的方法非常令人兴奋。”。

Echoing the study authors' points, Sundermann said one of the potential limitations of current sequencing-based pathogen surveillance methods is that they typically focus on a single colony. As such, researchers might be under-sampling the microbial diversity in the patient and, therefore, cannot fully capture all the transmission events.

与研究作者的观点相呼应，桑德曼说，目前基于测序的病原体监测方法的潜在局限性之一是它们通常集中在单个菌落上。因此，研究人员可能正在对患者的微生物多样性进行采样，因此无法完全捕获所有传播事件。

The newly described method, on the other hand, enables researchers to have a better grasp of the microorganism diversity within a patient, he added..

另一方面，新描述的方法使研究人员能够更好地掌握患者体内的微生物多样性，他补充道。。

'I would characterize this method as kind of like a Goldilocks — not too hot, not too cold, just right,' agreed Daria Van Tyne, another infectious disease professor at Pitt who was not involved in the study. In collaboration with Sundermann and others, Van Tyne has been leading a real-time WGS surveillance program at Pitt to help identify and curb HAIs.

皮特大学另一位未参与该研究的传染病教授达里亚·范泰恩（DariaVanTyne）表示同意：“我认为这种方法有点像金发姑娘——不太热，不太冷，恰到好处。”。范·泰恩（VanTyne）与桑德曼（Sundermann）等人合作，一直在皮特领导WGS实时监视计划，以帮助识别和遏制HAIs。

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While the team has been mostly doing single-isolate sequencing, Van Tyne said, she and collaborators are also exploring adopting a similar WGS strategy to capture greater microbial diversity in their studies. As such, Van Tyne said her team is interested in giving 'a test drive' of the method described in the paper.

Van Tyne说，虽然该团队主要是进行单分离测序，但她和合作者也在探索采用类似的WGS策略，以在研究中捕获更多的微生物多样性。因此，范泰恩说，她的团队有兴趣对论文中描述的方法进行“试驾”。

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Despite the method's promises, Van Tyne said one limitation of the paper is that the study authors did not go beyond the genomic analysis to investigate if there was a true epidemiologic link among the patients suspected of HAIs. 'Genomic analysis can simply generate a hypothesis,' she noted. 'That hypothesis still would need to be tested before it could be proven.'.

尽管该方法有承诺，但范泰恩说，该论文的一个局限性在于，研究作者没有超越基因组分析，以调查疑似HAIs患者之间是否存在真正的流行病学联系她指出，基因组分析可以简单地产生一个假设这一假设在得到证实之前仍需要进行检验。”。

Additionally, given that the method is a reference-based approach, it could hinder data reproducibility if people are using different reference databases. Moreover, as WGS is still not widely adopted within the healthcare setting for infection surveillance, Van Tyne said a general hurdle for all sequencing-based methods is to prove their feasibility and boost their implementation.

此外，鉴于该方法是基于参考的方法，如果人们使用不同的参考数据库，它可能会阻碍数据的可重复性。此外，由于WGS尚未在医疗保健环境中广泛用于感染监测，Van Tyne说，所有基于测序的方法的一个普遍障碍是证明其可行性并促进其实施。

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Calling the current study a 'proof of concept,' Thorpe said that while the method developed by his team demonstrated some potential utility, there is still a ways to go before it can be deployed in hospitals. As such, he noted some of the future directions for the team are to further validate the method's accuracy and to analyze the method's cost and implementation, which the current study did not explore.

索普称目前的研究是“概念验证”，他说，虽然他的团队开发的方法显示出一些潜在的效用，但在医院部署之前还有一段路要走。因此，他指出，该团队未来的一些方向是进一步验证该方法的准确性，并分析该方法的成本和实施情况，而目前的研究并未对此进行探索。

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'We're definitely not saying that this could be implemented tomorrow,' Thorpe said. 'I think the challenge is still to get institutions to adopt sequencing-based surveillance in general.'

索普说：“我们绝对不是说明天就能实施。”我认为，挑战仍然是让机构普遍采用基于测序的监测。”