Ruzickova Michaela, Palkovicova Jana, Papousek Ivo, Cummins Max L, Djordjevic Steven P, Dolejska Monika
Central European Institute of Technology, University of Veterinary Sciences Brno, Brno, South Moravian Region, Czechia.
Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, Brno, South Moravian Region, Czechia.
mSystems. 2025 May 20;10(5):e0101024. doi: 10.1128/msystems.01010-24. Epub 2025 Apr 8.
IncF plasmids are mobile genetic elements found in bacteria from the family and often carry critical antibiotic and virulence gene cargo. The classification of IncF plasmids using the plasmid Multi-Locus Sequence Typing (pMLST) tool from the Center for Genomic Epidemiology (CGE; https://www.genomicepidemiology.org/) compares the sequences of IncF alleles against a database to create a plasmid sequence type (ST). Accurate identification of plasmid STs is useful as it enables an assessment of IncF plasmid lineages associated with pandemic enterobacterial STs. Our initial observations showed discrepancies in IncF allele variants reported by pMLST in a collection of 898 ST131 genomes. To evaluate the limitations of the pMLST tool, we interrogated an in-house and public repository of 70,324 genomes of various STs and other genomes ( = 1247). All short-read assemblies and representatives selected for long-read sequencing were used to assess pMLST allele variants and to compare the output of pMLST tool versions. When multiple allele variants occurred in a single bacterial genome, the Python and web versions of the tool randomly selected one allele to report, leading to limited and inaccurate ST identification. Discrepancies were detected in 5,804 of 72,469 genomes (8.01%). Long-read sequencing of 27 genomes confirmed multiple IncF allele variants on one plasmid or two separate IncF plasmids in a single bacterial cell. The pMLST tool was unable to accurately distinguish allele variants and their location on replicons using short-read genome assemblies, or long-read genome assemblies if the same allele variant was present more than once.
Plasmid sequence type is crucial for describing IncF plasmids due to their capacity to carry important antibiotic and virulence gene cargo and consequently due to their association with disease-causing enterobacterial lineages exhibiting resistance to clinically relevant antibiotics in humans and food-producing animals. As a result, precise reporting of IncF allele variants in IncF plasmids is necessary. Comparison of the FAB formulae generated by the pMLST tool with annotated long-read genome assemblies identified inconsistencies, including examples where multiple IncF allele variants were present on the same plasmid but missing in the FAB formula, or in cases where two IncF plasmids were detected in one bacterial cell, and the pMLST output provided information only about one plasmid. Such inconsistencies may cloud interpretation of IncF plasmid replicon type in specific bacterial lineages or inaccurate assumptions of host strain clonality.
IncF质粒是在肠杆菌科细菌中发现的可移动遗传元件，通常携带关键的抗生素和毒力基因载荷。使用来自基因组流行病学中心（CGE；https://www.genomicepidemiology.org/）的质粒多位点序列分型（pMLST）工具对IncF质粒进行分类，是将IncF等位基因序列与数据库进行比较以创建质粒序列类型（ST）。准确鉴定质粒STs很有用，因为它能够评估与大流行性肠杆菌STs相关的IncF质粒谱系。我们最初的观察结果显示，在898个ST131基因组集合中，pMLST报告的IncF等位基因变体存在差异。为了评估pMLST工具的局限性，我们查阅了一个包含70324个各种STs基因组和其他基因组（n = 1247）的内部和公共数据库。所有用于长读长测序的短读长组装序列和代表性序列都用于评估pMLST等位基因变体，并比较pMLST工具不同版本的输出结果。当单个细菌基因组中出现多个等位基因变体时，该工具的Python版本和网络版本会随机选择一个等位基因进行报告，导致ST鉴定有限且不准确。在72469个基因组中的5804个（8.01%）中检测到差异。对27个基因组进行长读长测序证实，单个细菌细胞中的一个质粒或两个单独的IncF质粒上存在多个IncF等位基因变体。如果短读长基因组组装序列中存在相同的等位基因变体，或者长读长基因组组装序列中存在多个相同的等位基因变体，pMLST工具无法准确区分等位基因变体及其在复制子上的位置。
质粒序列类型对于描述IncF质粒至关重要，因为它们能够携带重要的抗生素和毒力基因载荷，因此与在人类和产食动物中对临床相关抗生素表现出抗性的致病肠杆菌谱系相关。因此，准确报告IncF质粒中的IncF等位基因变体是必要的。将pMLST工具生成的FAB公式与注释的长读长基因组组装序列进行比较，发现了不一致之处，包括同一质粒上存在多个IncF等位基因变体但在FAB公式中缺失的例子，或者在一个细菌细胞中检测到两个IncF质粒，而pMLST输出结果仅提供了关于一个质粒的信息。这种不一致可能会模糊特定细菌谱系中IncF质粒复制子类型的解释，或对宿主菌株克隆性的不准确假设。
