Ahmadi Asal, Khezri Abdolrahman, Nørstebø Håvard, Ahmad Rafi
Department of Biotechnology, Inland Norway University of Applied Sciences, Hamar, Norway.
TINE SA, Oslo, Norway.
Front Microbiol. 2023 Jan 6;13:1104701. doi: 10.3389/fmicb.2022.1104701. eCollection 2022.
Rapid and accurate diagnosis of causative pathogens in mastitis would minimize the imprudent use of antibiotics and, therefore, reduce the spread of antimicrobial resistance. Whole genome sequencing offers a unique opportunity to study the microbial community and antimicrobial resistance (AMR) in mastitis. However, the complexity of milk samples and the presence of a high amount of host DNA in milk from infected udders often make this very challenging.
Here, we tested 24 bovine milk samples (18 mastitis and six non-mastitis) using four different commercial kits (Qiagens' DNeasy PowerFood Microbial, Norgens' Milk Bacterial DNA Isolation, and Molzyms' MolYsis™ Plus and Complete5) in combination with filtration, low-speed centrifugation, nuclease, and 10% bile extract of male bovine (Ox bile). Isolated DNA was quantified, checked for the presence/absence of host and pathogen using PCR and sequenced using MinION nanopore sequencing. Bioinformatics analysis was performed for taxonomic classification and antimicrobial resistance gene detection.
The results showed that kits designed explicitly for bacterial DNA isolation from food and dairy matrices could not deplete/minimize host DNA. Following using MolYsis™ Complete 5 + 10% Ox bile + micrococcal nuclease combination, on average, 17% and 66.5% of reads were classified as bovine and reads, respectively. This combination also effectively enriched other mastitis pathogens, including and . Furthermore, using this approach, we identified important AMR genes such as ), fosB-Saur, and blaZ. We showed that even 40 min of the MinION run was enough for bacterial identification and detecting the first AMR gene.
We implemented an effective method (sensitivity of 100% and specificity of 92.3%) for host DNA removal and bacterial DNA enrichment (both gram-negative and positive) directly from bovine mastitis milk. To the best of our knowledge, this is the first culture- and amplification-independent study using nanopore-based metagenomic sequencing for real-time detection of the pathogen (within 5 hours) and the AMR profile (within 5-9 hours), in mastitis milk samples. These results provide a promising and potential future on-farm adaptable approach for better clinical management of mastitis.
快速准确地诊断乳腺炎的致病病原体可尽量减少抗生素的不当使用，从而减少抗菌药物耐药性的传播。全基因组测序为研究乳腺炎中的微生物群落和抗菌药物耐药性（AMR）提供了独特的机会。然而，牛奶样本的复杂性以及感染乳房的牛奶中存在大量宿主DNA，常常使这项工作极具挑战性。
在这里，我们使用四种不同的商业试剂盒（Qiagens公司的DNeasy PowerFood Microbial、Norgens公司的Milk Bacterial DNA Isolation以及Molzyms公司的MolYsis™ Plus和Complete5），结合过滤、低速离心、核酸酶和雄性牛（牛胆汁）的10%胆汁提取物，对24份牛乳样本（18份乳腺炎样本和6份非乳腺炎样本）进行了检测。对分离出的DNA进行定量，使用PCR检查宿主和病原体的存在与否，并使用MinION纳米孔测序进行测序。进行生物信息学分析以进行分类学分类和抗菌药物耐药基因检测。
结果表明，专门设计用于从食品和乳制品基质中分离细菌DNA的试剂盒无法去除/最小化宿主DNA。使用MolYsis™ Complete 5 + 10%牛胆汁+微球菌核酸酶组合后，平均分别有17%和66.5%的读数被分类为牛和读数。这种组合还有效地富集了其他乳腺炎病原体，包括和。此外，使用这种方法，我们鉴定出了重要的AMR基因，如）、fosB-Saur和blaZ。我们表明，即使MinION运行40分钟也足以进行细菌鉴定和检测第一个AMR基因。
我们实施了一种有效的方法（灵敏度为100%，特异性为92.3%），可直接从牛乳腺炎牛奶中去除宿主DNA并富集细菌DNA（革兰氏阴性和阳性）。据我们所知，这是第一项不依赖培养和扩增的研究，使用基于纳米孔的宏基因组测序实时检测乳腺炎牛奶样本中的病原体（5小时内）和AMR谱（5-9小时内）。这些结果为乳腺炎的更好临床管理提供了一种有前景且潜在适用于农场的未来方法。
