利用全基因组测序解析耐多药结核病传播路径

目的 应用全基因组测序技术解析耐多药结核分枝杆菌的传播路径。方法 对2009-2012年上海市一起经VNTR分型鉴定的耐多药结核病传播案例中8例簇病例进行全基因组测序,描述耐多药结核分枝杆菌的传播路径。结果 该起传播案例中源病例是1例复发耐多药结核病患者,在3年内传播耐药菌株导致另外至少7例患者发病。结论 全基因组测序技术可以弥补传统流行病学调查与基因分型技术的不足,准确地描述结核病传播的方向与细节,鉴定传染源与缺失病例,有助于准确揭示耐多药结核病的传播路径。     

全基因组测序技术在结核病流行病学调查中的应用

目的 评估全基因组测序技术在结核病分子流行病学调查中的应用。方法 对2008-2012年在上海市两家结核病定点医院发现9名耐多药患者中分离的结核分枝杆菌具有相同的可变数目串联重复序列,本研究对此进行流行学调查,并对9株结核分枝杆菌进行全基因组测序,分析其传播关系。结果 全基因组序列分析将9株结核分枝杆菌分为两个有传播关系的网络,一个为包括7株结核分枝杆菌（5例和2例患者分别来自不同的医院）的大簇,一个为只有2株结核分枝杆菌的小簇。两个簇之间相差15个单核苷酸多态性（SNP）位点,提示两个簇的遗传距离相对较远,基于菌株SNP差异构建的传播链显示了每个簇内菌株的传播方向和耐药突变积累的过程。结论 基于全基因组测序数据研究耐药结核病的传播网络,能准确判断传播路径和方向,识别传染源和传播缺失环节。     

Phylogenetic understanding of clonal populations in an era of whole genome sequencing

Phylogenetic hypotheses using whole genome sequences have the potential for unprecedented accuracy, yet a failure to understand issues associated with discovery bias, character sampling, and strain sampling can lead to highly erroneous conclusions. For microbial pathogens, phylogenies derived from whole genome sequences are becoming more common, as large numbers of characters distributed across entire genomes can yield extremely accurate phylogenies, particularly for strictly clonal populations. The availability of whole genomes is increasing as new sequencing technologies reduce the cost and time required for genome sequencing. Until entire sample collections can be fully sequenced, harnessing the phylogenetic power from whole genome sequences in more than a small subset of fully sequenced strains requires the integration of whole genome and partial genome genotyping data. Such integration involves discovering evolutionarily stable polymorphic characters by whole genome comparisons, then determining allelic states across a wide panel of isolates using high-throughput genotyping technologies. Here, we demonstrate how such an approach using single nucleotide polymorphisms (SNPs) yields highly accurate, but biased phylogenetic reconstructions and how the accuracy of the resulting tree is compromised by incomplete taxon and character sampling. Despite recent phylogenetic work detailing the strengths and biases of integrating whole genome and partial genome genotype data, these issues are relatively new and remain poorly understood by many researchers. Here, we revisit these biases and provide strategies for maximizing phylogenetic accuracy. Although we write this review with bacterial pathogens in mind, these concepts apply to any clonally reproducing population or indeed to any evolutionarily stable marker that is inherited in a strictly clonal manner. Understanding the ways in which current and emerging technologies can be used to maximize phylogenetic knowledge is advantageous only with a complete understanding of the strengths and weaknesses of these methods.     

High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis

The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 β-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe’s largest hospitals, the General Hospital Vienna, were screened for the presence of 31 β-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of β-lactamase genes was estimated using biostatistical models.The carbapenemase genes bla_KPC-2, bla_KPC-3, and bla_VIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, bla_OXA-72 in an extended-spectrum β-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired bla_DHA-1 and bla_CMY AmpC β-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all β-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated β-lactamases and other resistance genes in one of Europe’s largest hospitals.     

全基因测序法分析肺炎克雷伯菌JM45株对喹诺酮类药物耐药基因

目的分析泛耐药肺炎克雷伯菌JM45株携带的喹诺酮类药物耐药基因,研究其对喹诺酮类药物的耐药机制。方法采用Roche454高通量测序技术对肺炎克雷伯菌JM45株做全基因组测序(完成图),分析喹诺酮类耐药基因携带状况,再将gyrA与parC全长基因与其他6株肺炎克雷伯菌做分子进化分析。结果最终得到JM45株一条完整的基因组(染色体)序列及两条质粒序列;基因组(染色体)序列大小为5 273 812bp(GC含量65.8%),质粒1序列大小为317 156bp(GC含量53.0%),质粒2序列大小为12 209bp(GC含量55.3%);在基因组(染色体)序列中携带了gyrA基因(全基因组Feature ID:KPN_1614),parC基因(Feature ID:KPN_0743);与肺炎克雷伯菌喹诺酮类药物敏感株相比较,gyrA基因第83位密码子由TCC→ATC,翻译成氨基酸序列后丝氨酸(Ser)→异亮氨酸(Ile),parC基因第80位密码子由AGC→ATC,翻译成氨基酸序列后丝氨酸(Ser)→异亮氨酸(Ile)。结论肺炎克雷伯菌JM45株喹诺酮类药物耐药是gyrA基因和parC基因喹诺酮耐药决定区(QRDR)突变所致;gyrA与parC全长基因的分子进化分析提示JM45株与肺炎克雷伯菌HS11286关系最为接近(序列相同),与肺炎克雷伯菌342关系最远。     

Molecular epidemiology and drug-resistance of tuberculosis in Luodian revealed by whole genome sequencing

In this study, we aimed to investigate the molecular epidemiology and drug-resistance profiles of tuberculosis (TB) in Luodian, an area with highest TB incidence and limited healthcare resources in Guizhou, China. The passive case finding strategy was used to identify suspected pulmonary TB with symptoms, and individuals with positive Mycobacterium tuberculosis (MTB) culture were enrolled from May 22, 2018 to April 21, 2019. All the 107 cases except three came from nine towns, including 55.1% from Longping and Bianyang. The phylogeny tree showed that 53.3% of strains were Lineage 2 (Beijing genotype), while 46.7% were Lineage 4 (Euro-American genotype). Among Lineage 2 strains, 66.7% were of “modern” Beijing type. Seven clusters with genomic distance within 12 SNPs were identified. The clusters included 14 strains, accounting for a clustering rate of 13.1%. The distance separating the clustered cases was between 2.1 and 71.0 km (Km), with an average paired distance of 21.8 Km (interquartile range, 2.8–38.0 Km). Based on the gene mutations associated with drug-resistance, we predicted that 4.8% of strains were resistant to isoniazid, 3.7% to rifampicin, and 3.7% to streptomycin; only one strain (0.9%) had multidrug resistance (MDR). This study found low drug-resistance rates in Luodian, and the sub-lineage of the “modern” Beijing branch has recent expansion in Luodian. This work may also serve as a genomic baseline to assess the evolution and spread of MTB in Guizhou.     

全基因组测序技术在结核病分子流行病学中的应用进展

结核病给全球带来了严重的疾病负担,防控结核病具有重要意义.结核病分子流行病学将结核分枝杆菌分型技术与流行病学资料相整合,在研究结核病的传播和流行特点中起到了极其重要的作用.全基因组测序能够构建生物体基因组的完整DNA序列,具有高分辨率、高通量、结果精确等优势,在结核病分子流行病学研究中非常有吸引力和发展潜力.比较全基因...     

用全基因组测序追踪临床重要耐药菌院内感染的暴发流行

新一代高通量测序技术的成熟和普及使微生物基因组学获得长足发展,采用全基因组序列分析追踪和溯源临床重要耐药菌院内感染的暴发,已成为近年临床微生物研究领域的热点,并形成了一个新的学科方向——基因组流行病学。在将全基因组序列分析用于临床耐药菌流行溯源时,可利用基因组数据的强大分辨率和丰富信息,在进行菌株充分分型的基础上,更精确地判断耐药菌同源性的远近,分辨出不同的进化路线,推断耐药菌在医院环境中的演化过程,这对临床耐药菌院内感染控制工作意义重大。     

Whole genome sequencing uses for foodborne contamination and compliance: Discovery of an emerging contamination event in an ice cream facility using whole genome sequencing

We review how FDA surveillance identifies several ways that whole genome sequencing (WGS) improves actionable outcomes for public health and compliance in a case involving Listeria monocytogenes contamination in an ice cream facility. In late August 2017 FDA conducted environmental sampling inside an ice cream facility. These isolates were sequenced and deposited into the GenomeTrakr databases. In September 2018 the Centers for Disease Control and Prevention contacted the Florida Department of Health after finding that the pathogen analyses of three clinical cases of listeriosis (two in 2013, one in 2018) were highly related to the aforementioned L. monocytogenes isolates collected from the ice cream facility. in 2017. FDA returned to the ice cream facility in late September 2018 and conducted further environmental sampling and again recovered L. monocytogenes from environmental subsamples that were genetically related to the clinical cases. A voluntary recall was issued to include all ice cream manufactured from August 2017 to October 2018. Subsequently, FDA suspended this food facility's registration. WGS results for L. monocytogenes found in the facility and from clinical samples clustered together by 0–31 single nucleotide polymorphisms (SNPs). The FDA worked together with the Centers for Disease Control and Prevention, as well as the Florida Department of Health, and the Florida Department of Agriculture and Consumer Services to recall all ice cream products produced by this facility. Our data suggests that when available isolates from food facility inspections are subject to whole genome sequencing and the subsequent sequence data point to linkages between these strains and recent clinical isolates (i.e., <20 nucleotide differences), compliance officials should take regulatory actions early to prevent further potential illness. The utility of WGS for applications related to enforcement of FDA compliance programs in the context of foodborne pathogens is reviewed.     

Whole genome sequencing and phylogenetic analysis of feline anelloviruses

Torque teno felis virus (FcTTV) was detected in the cat population in the Czech Republic. A total of 110 serum samples were tested by a nested PCR technique using specific primers, situated in the highly conserved untranslated region of the virus genome. The frequency of feline TT virus in the Czech Republic was found to be 33.63%. Sequencing of PCR product from several virus strains showed that all of them are closely related and belong to the same virus species. Whole genome sequencing of three strains was performed to compare overall genetic heterogeneity of feline TT viruses. One of these three strains showed more that 10% difference at the nucleotide level. Furthermore we didn’t find any correlation between FcTTV infection and sex or health status of examined animals.     

A semiquantitative whole genome screen analysis of alcohol dependence

Two whole genome screens were applied to sibling pairs from the Collaborative Study on the Genetics of Alcoholism (COGA) family data to compare a semiquantitative method with a standard qualitative approach. The semiquantitative method used a score derived from 11 symptoms, and the qualitative approach used the COGA criteria for alcohol dependence. There was no concordance in the regions identified by the two models. Three regions of nominal significance were identified using the symptom score. In these three regions, correlated traits were also analyzed to determine whether linkage could be attributed to their intermediate effect. The evidence for linkage to one locus on chromosome 6 could be explained by linkage to the personality trait harm avoidance.     

基于全基因组测序估算结核分枝杆菌分子钟

目的 基于全基因组测序数据,通过内源性激活期间菌株基因组突变分析,开展结核分枝杆菌（MTB）分子钟研究。方法 筛选MTB内源性激活全基因组研究文献,下载全基因组测序数据,提取初治-复发配对样本单核苷酸多态性（SNP）差异和菌株分离时间,运用Poisson回归模型拟合初治-复发时间间隔与差异SNP的关系,计算MTB分子钟,估算突变率。结果 若MTB传代时间为18 h,0~2年短期内源性激活突变率为6.47×10^-10（95%CI：5.59×10^-10~7.44×10^-10）,明显高于2~14年长期内源性激活突变率（3.27×10^-10,95%CI：2.88×10^-10~3.69×10^-10）。0~、1~、2~、3~、5~、7~14年突变率分别为7.10×10^-10、6.06×10^-10、4.24×10^-10、5.34×10^-10、2.59×10^-10、1.26×10^-10。结论 内源性激活复发期间,MTB突变率随初治-复发时间间隔增加而下降,本研究从微观层面验证了临床实践观察到的初治结核病2年内易复发现象。     

A review of methods for subtyping Yersinia pestis: From phenotypes to whole genome sequencing

Numerous subtyping methods have been applied to Yersinia pestis with varying success. Here, we review the various subtyping methods that have been applied to Y. pestis and their capacity for answering questions regarding the population genetics, phylogeography, and molecular epidemiology of this important human pathogen. Methods are evaluated in terms of expense, difficulty, transferability among laboratories, discriminatory power, usefulness for different study questions, and current applicability in light of the advent of whole genome sequencing.     

A lack of genetic basis for biovar differentiation in clinically important Corynebacterium diphtheriae from whole genome sequencing

The differentiation of clinically important Corynebacterium diphtheriae into specific biovars is complex and phylogenetically unclear. Comparative genomic analyses of 17 strains indicate that the division of C. diphtheriae into different biovars does not correlate with the variation in the gene content in the relevant metabolic categories that are potentially involved in the biovar discrimination. The biochemical separation is also not supported by phylogenetic analyses, suggesting molecular methods of typing C. diphtheriae strains should be adopted much more widely.     

Rapid genome sequencing of RNA viruses

We developed a system for rapid determination of viral RNA sequences whereby genomic sequence is obtained from cultured virus isolates without subcloning into plasmid vectors. This method affords new opportunities to address the challenges of unknown or untypeable emerging viruses.     

Bioinformatics tools and databases for whole genome sequence analysis of Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease of global public health importance caused by Mycobacterium tuberculosis complex (MTC) in which M. tuberculosis (Mtb) is the major causative agent. Recent advancements in genomic technologies such as next generation sequencing have enabled high throughput cost-effective generation of whole genome sequence information from Mtb clinical isolates, providing new insights into the evolution, genomic diversity and transmission of the Mtb bacteria, including molecular mechanisms of antibiotic resistance. The large volume of sequencing data generated however necessitated effective and efficient management, storage, analysis and visualization of the data and results through development of novel and customized bioinformatics software tools and databases. In this review, we aim to provide a comprehensive survey of the current freely available bioinformatics software tools and publicly accessible databases for genomic analysis of Mtb for identifying disease transmission in molecular epidemiology and in rapid determination of the antibiotic profiles of clinical isolates for prompt and optimal patient treatment.