Application of Genomic Approaches to Bacterial Pathogenesis and Mechanisms of Antimicrobial Resistance

基因组方法在细菌发病机制和抗菌素耐药性机制中的应用

• 批准号: 10927916
• 负责人: John Dekker
• 金额: $ 222.02万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10927916
• 关键词: Acute; Amino Acids; Animals; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antimicrobial Resistance; Antimicrobial susceptibility; Area; Bacteremia; Bacteroides fragilis; Biology; Bioreactors; Bordetella; Burkholderia; Ceftazidime; Cell Line; Chromosome Deletion; Chromosomes; Citric Acid Cycle; Clinical; Collaborations; Collection; Combinatorics; Communication; Complex; Cryoelectron Microscopy; DNA; DNA Insertion Elements; DNA Methylation; DNA Polymerase III; DNA Replication Proofreading; Development; Dipeptides; Elements; Engineering; Environment; Enzymes; Episome; Event; Evolution; Experimental Designs; Gene Expression; Genes; Genetic Drift; Genetic Transcription; Genome; Genomic approach; Genomics; Gluconeogenesis; Goals; Host Defense; Human; IL12RB1 gene; Immunocompromised Host; In Vitro; Individual; Infection; Insertional Mutagenesis; Interruption; Mediating; Mediator; Metabolic; Metabolism; Methionine; Methylation; Methyltransferase; Methyltransferase Gene; Microbe; Mismatch Repair Deficiency; Modern Medicine; Multi-Drug Resistance; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Nitrogen; Organism; Pathogenesis; Pathway interactions; Patients; Pattern; Phase; Phenotype; Phenylalanine; Phylogenetic Analysis; Physiological; Physiology; Plasmids; Population Dynamics; Positioning Attribute; Pseudomonas aeruginosa; Publishing; Pump; Resistance; Structure; System; Systems Biology; Tazobactam; Testing; Texas; Treatment Protocols; United States National Institutes of Health; Work; Zoonoses; antibiotic resistant infections; attributable mortality; auxotrophy; chromosome fusion; chronic infection; clinical center; efflux pump; emerging pathogen; epigenome; experimental study; fitness; genome sequencing; homologous recombination; human microbiota; improved; in vivo; infectious disease treatment; member; metabolic phenotype; metabolomics; methylome; nanopore; neutrophil; novel; pathogen; pathogenic bacteria; promoter; protein purification; reconstruction; resistance gene; resistance mechanism; resistance mutation; screening; transcriptome; transcriptome sequencing; transcriptomics; trend; whole genome

MDR gram-negative bacterial pathogens undergo selection and evolution in the natural context of antibiotic treatment in a human host, though important features of host context are often not included in studies of AMR. Our work employs a systems biology approach to study the evolutionary mechanisms by which resistance - defined broadly to include resistance to antibiotics and to host defenses - emerges in the natural context of host infection. Current work is organized around three primary projects: Project 1: Mechanisms by which mismatch repair (MMR) deficiencies can facilitate rapid evolution of antimicrobial resistance in P. aeruginosa. We previously demonstrated that evolved MMR deficiencies may be dynamically exploited by P. aeruginosa to facilitate rapid acquisition of mutations mediating resistance to two critical broad-spectrum antibiotics, ceftazidime-avibactam (CZA), and ceftolozane-tazobactam (C/T), in the context of acute clinical infection (Khil et al, mBio, 2019). Then we characterized the detailed mutational and transcriptional events underlying the development of CZA and C/T resistance in wild type (WT) and MMR-deficient P. aeruginosa and identified a number of potential novel resistance mutations unique to the MMR deficient isolates. Work performed in FY23 included ongoing screening of a number of potential novel resistance genes identified in the study above. Specific work focused on completing experiments establishing the MexVW efflux pump as a novel mediator of resistance to CZA and C/T (published in Dulanto et al, PLoS Biology, 2022). Ongoing work seeks to characterize the structure and function of this pump. We have engineered a number of cell lines expressing MexV, MexW, and OprM proteins for purification to facilitate cryo-EM structural studies, and cryo-EM studies of these purified components is currently ongoing in collaboration with Susan Buchanan's lab in NIDDK. Other work in FY23 has involved completion of the initial phases of a large scale comprehensive genomic study of antimicrobial resistance in a 20-year collection of P. aeruginosa bacteremia isolates (320 isolates from 300 unique patients) from the NIH Clinical Center. This work is focusing particularly on mechanisms and targets that we have defined in our separate in vitro evolution work above, and for evidence of hypermutation. Potential novel targets will be cloned into the isogenic lab strain system for further study. We will also introduce MexVW deletions into clinical isolates that are have evolved resistance to CZA and C/T naturally in patients to assess the contribution of MexVW to this resistance. All isolates will undergo genomic sequencing and characterization of antimicrobial resistance mechanisms. The genomes of the first 220 of the isolates have been sequenced and assembled and sequencing of the remainder is ongoing. Project 2: In vivo evolution of an emerging zoonotic pathogen Bordetella hinzii in an immunocompromised host. This project applies system biology approaches, including genomics, transcriptomics, and metabolomics to understanding the adaptive evolution in the emerging pathogen Bordetella hinzii following presumptive zoonotic transfer from an animal reservoir to an individual with IL-12RB1 deficiency. Initial work (Launay et al, Nature Communications, 2021) demonstrated that a mutation in the DNA Pol III epsilon proofreading subunit resulted in a replicative DNA proofreading deficiency and drove genetic divergence among the isolates over the course of 45 months of persistent infection. Evidence of mutational targeting and positive selection was present in multiple sequential enzymes of the tricarboxylic acid cycle and gluconeogenesis pathways, suggesting specialized metabolic adaptation to the host environment. To study the transcriptional landscape of adaptation in these isolates, >100 transcriptomes were sequenced with Illumina short reads and a subset underwent nanopore-based direct long read RNA sequencing. Work completed during FY23 focused on continued metabolic phenotyping of the entire set of isolates. The preliminary findings from this work are that (1) the efficiency of metabolism of a variety of nitrogen containing compounds, including amino acids and dipeptides appears to have been substantially modified during the course of host adaptation; (2) subsets of isolates evolved specific amino acid auxotrophies involving methionine and phenylalanine. Current ongoing experiments are designed to quantify the fitness consequences of these adaptations in physiologically appropriate media environments using a bioreactor and test the specific hypotheses that these evolved auxotrophies confer tolerance or persistence to antibiotics and/or improved survivability to host attack within neutrophils. Presentation of this work was made at ASM Microbe in Houston, Texas, June 2023 (Ellis et al, 2023). Work completed in FY23 on a second project to study within-host evolution and adaptation of Burkholderia vietnamiensis isolates from another patient with IL-12RB1 deficiency has involved genomic analysis of 183 B. vietnamiensis isolates including both short-read and long-read sequencing. The main findings include (1) a remarkably complex and dynamic population structure that was present in the intravascular space; (2) superimposed mobile element insertional mutagenesis mediating secondary genomic plasticity, including interruption of genes and positioning of cis-acting IS promoters; and (3) homologous recombination-based events resulting in chromosomal fusions and deletions. Ongoing work aims to understand how these changes may have facilitated adaptive evolution. Presentation of this work was made at ASM Microbe in Houston, Texas, June 2023 (Moller et al, 2023) Project 3: Comprehensive whole genome sequencing and genomic analysis of a historical collection of clinical Bacteroides fragilis group (BFG) isolates spanning decades. Members of the BFG are important constituents of the human microbiota, but they can also behave as significant pathogens in certain contexts. Historically, antimicrobial susceptibility patterns in BFG isolates were largely predictable, allowing effective use of empiric treatment regimens. Alarming increases in AMR have recently necessitated reconsideration of empiric strategies. To understand the genomic basis of these AMR trends, we have initiated an effort to sequence a large group of clinical BFG isolates spanning a period of five decades. Previous involved long-read nanopore-based sequencing of 386 BFG genomes facilitating end-to-end contiguous assemblies of chromosomes, episomes, and plasmids. Detailed phylogenetic reconstruction and exhaustive annotation of AMR elements in both genome and plasmids has been performed. Work completed in FY 2023 involved using nanopore sequencing in combination with recently developed computational approaches to complete characterization the 6mA, 5mC, and 4mC methylomes of 268 BFG isolates selected on the basis of AMR phenotype, revealing that single BFG species harbor hundreds of DNA methylation motifs, with most individual motif combinations occurring uniquely in single isolates, implying immense unsampled combinatoric diversity within BFG epigenomes. Additionally, we have refined existing computational approaches to mine methylase genes from the sequenced genomes and identified more than 6000 methyltransferase genes within the genome set, explaining this profound diversity of methylation motifs. Many of the observed methylated motifs are located adjacent to, or within, the gene bodies of AMR genes. This work was published as Tisza et al, Nature Communications, 2023. Ongoing work aims to study how AMR gene expression and phenotypes are regulated by methylation.

MDR革兰氏阴性细菌病原体在人类宿主的抗生素处理自然背景下经历选择和进化，尽管宿主环境的重要特征通常不包括在AMR研究中。我们的工作采用系统生物学方法来研究抗药性（包括对抗生素的抗药性和宿主防御）的进化机制 - 在宿主感染的自然背景下出现。当前的工作是围绕三个主要项目组织的： 项目1：不匹配修复（MMR）缺陷可以促进铜绿假单胞菌中抗菌抗性的快速进化的机制。我们先前证明，铜绿假单胞菌可能会动态利用进化的MMR缺乏症，以促进对两种关键抗药性介导的突变，从而介导两种关键的宽光谱抗生素，头孢兹兹兹省 - 阿acavibactam（CZA）和ceftolozane-tazobactam（C/T）（C/T）（C/T）（C/T）（C/T）。然后，我们表征了野生型（WT）和MMR缺陷型铜绿假单胞菌中CZA和C/T抗性发展的详细突变和转录事件，并确定了MMR缺乏分离物所特有的许多潜在的新型抗药性突变。 在FY23中进行的工作包括对上述研究中确定的许多潜在的新型抗药性基因进行筛查。特定的工作着重于完成实验，以建立MEXVW外排泵作为对CZA和C/T的耐药性的新型介体（发表在Dulanto等人，PLOS Bioology，2022年）。正在进行的工作旨在表征该泵的结构和功能。我们已经设计了许多表达MEXV，MEXW和OPRM蛋白的细胞系，以促进冷冻EM结构研究，而这些纯化组件的冷冻EM研究目前正在与NIDDK的Susan Buchanan的实验室合作进行。 FY23中的其他工作涉及完成一项大规模的抗菌耐药性抗菌耐药性基因组研究的初始阶段，该研究是从NIH临床中心收集了20年的铜绿假单胞菌细菌分离株（来自300名独特患者的320个分离株）。这项工作特别关注我们在上面的单独的体外进化工作中定义的机制和目标，并提供了超称的证据。潜在的新靶标将被克隆到Isogenic Lab菌株系统中，以进行进一步研究。我们还将将MEXVW缺失引入临床分离株中，这些分离株在患者中自然而然地对CZA和C/T产生了抗性，以评估MEXVW对这种耐药性的贡献。所有分离株将接受基因组测序和抗菌耐药机制的表征。分离株的前220个基因组已经进行了测序并组装，其余的测序正在进行中。 项目2：在免疫功能低下的宿主中，新兴的人畜共患病原体hinzii的体内进化。该项目采用系统生物学方法，包括基因组学，转录组学和代谢组学来理解从动物储层到患有IL-12RB1缺乏症个体的个体的人们的人畜共患病后，新兴病原体hinzii的适应性演变。最初的工作（Launay等人，自然通信，2021）表明，DNA Polii epsilon校对亚基的突变导致复制性DNA校对缺乏症，并在45个月的持续感染过程中脱离了分离株的遗传差异。突变靶向和阳性选择的证据存在于三羧酸周期的多种顺序酶和糖异生途径中，这表明对宿主环境的专门代谢适应。为了研究这些分离株适应性的转录景观，用Illumina简短读数对> 100个转录组进行了测序，并进行了基于纳米孔的直接长读RNA测序。 在第23财年完成的工作重点是整个分离株的持续代谢表型。这项工作的初步发现是（1）在宿主适应过程中，包括氨基酸和二肽在内的各种氮的代谢效率似乎已实质性改变； （2）分离株的亚群进化出了涉及蛋氨酸和苯丙氨酸的特异性氨基酸辅助营养物。当前正在进行的实验旨在使用生物反应器在生理上适当的培养基环境中量化这些适应性的适应性后果，并测试这些发展的假设，即这些假设表明，这些假设赋予了相互作用的耐受性或持续性，并赋予抗生素和/或改善中性粒细胞中宿主攻击的生存能力。这项工作的介绍是在2023年6月在德克萨斯州休斯敦市的ASM微生物（Ellis等，2023年）进行的。 从23财年完成的第二个项目完成的工作，以研究来自另一名IL-12RB1缺乏症患者的伯克霍尔德越南人分离株的宿主进化和适应，涉及对183 B.越南B.越南b的基因组分析，包括短读和长阅读测序。 主要发现包括（1）在血管内空间中存在的非常复杂且动态的种群结构； （2）叠加的移动元件插入诱变介导次级基因组可塑性，包括基因中断和顺式作用的定位是启动子； （3）基于同源重组的事件，导致染色体融合和缺失。正在进行的工作旨在了解这些变化如何促进适应性演变。 这项工作的介绍是在2023年6月在德克萨斯州休斯敦市的ASM微生物（Moller等，2023）进行的 项目3：跨越数十年的临床细菌片（BFG）分离株的历史集合的全面基因组测序和基因组分析。 BFG的成员是人类微生物群的重要成分，但在某些情况下它们也可以作为重要的病原体。从历史上看，BFG分离株中的抗菌敏感性模式在很大程度上是可以预测的，从而有效地使用了经验治疗方案。 AMR的令人震惊的增加需要重新考虑经验策略。为了了解这些AMR趋势的基因组基础，我们开始努力对跨越五十年的大量临床BFG分离株进行测序。以前涉及386个BFG基因组的长阅读基于纳米孔的测序，促进染色体，插发体和质粒的端到端连续组件。已经进行了详细的系统发育重建和基因组和质粒中AMR元素的详尽注释。 在2023年完成完成的工作涉及使用纳米孔测序与最近开发的计算方法结合使用，以完全表征268 BFG分离株的6mA，5MC和4MC甲基甲基组的6mA，5MC和4MC甲基粒子，这是基于AMR表型选择的，这表明单个BFG物种具有数百种DNA甲基化基序，并置于单身甲基化的单身成分，是单一偶然的成分。 BFG表观基因组中的组合多样性。此外，我们从测序的基因组中完善了现有的计算方法来挖掘甲基酶基因，并确定了基因组集合中的6000多个甲基转移酶基因，从而解释了甲基化基序的多样性。许多观察到的甲基化基序位于AMR基因的基因体或内部。这项工作是随着Tisza等人的《自然通讯》（2023年）发表的。

项目成果

Association between minimum inhibitory concentration values and mortality risk in patients with Stenotrophomonas maltophilia infections: a retrospective cohort study of electronic health records from 148 US hospitals.

嗜麦芽寡养单胞菌感染患者的最低抑菌浓度值与死亡风险之间的关联：对美国 148 家医院电子健康记录的回顾性队列研究。

• DOI: 10.1093/jacamr/dlad049
• 发表时间: 2023
• 期刊: JAC-antimicrobial resistance
• 影响因子: 3.4
• 作者: Sarzynski,SadiaH;Lawandi,Alexander;Warner,Sarah;Demirkale,CumhurY;Strich,JeffreyR;Dekker,JohnP;Babiker,Ahmed;Li,Willy;Kadri,SameerS
• 通讯作者: Kadri,SameerS

Adjunctive clindamycin therapy in invasive β-haemolytic streptococcal infections - Authors' reply.

克林霉素辅助治疗侵袭性β溶血性链球菌感染-作者的回复。

• DOI: 10.1016/s1473-3099(21)00259-0
• 发表时间: 2021
• 期刊: The Lancet. Infectious diseases
• 作者: Babiker,Ahmed;Dekker,JohnP;Danner,RobertL;Kadri,SameerS
• 通讯作者: Kadri,SameerS

Protracted course of disseminated adenovirus disease with necrotizing granulomas in the liver.

播散性腺病毒病的迁延病程，伴有肝脏坏死性肉芽肿。

• DOI: 10.1016/j.diagmicrobio.2018.12.012
• 发表时间: 2019
• 期刊: Diagnostic microbiology and infectious disease
• 影响因子: 2.9
• 作者: Lerner,AndreaM;Bennett,JohnE;Pittaluga,Stefania;Khil,PavelP;Youn,JH;Fahle,GaryA;Frank,KarenM;Dekker,JohnP;Jerussi,TheresaD;Sun,Clare;Wiestner,Adrian;Gea-Banacloche,Juan
• 通讯作者: Gea-Banacloche,Juan

Disseminated Mycobacterium marinum in Human Immunodeficiency Virus Unmasked by Immune Reconstitution Inflammatory Syndrome.

免疫重建炎症综合征揭示了人类免疫缺陷病毒中传播的海分枝杆菌。

• DOI: 10.1093/infdis/jiaa769
• 发表时间: 2021
• 期刊: The Journal of infectious diseases
• 作者: Manion,Maura;DulantoChiang,Augusto;Pei,Luxin;Wong,Chun-Shu;Khil,Pavel;Hammoud,DimaA;Anderson,Megan;Laidlaw,Elizabeth;Kuriakose,Safia;Lisco,Andrea;NISCComparativeSequencingProgram;Zelazny,AdrianM;Dekker,JohnP;Sereti,Irini
• 通讯作者: Sereti,Irini

Effectiveness of adjunctive clindamycin in β-lactam antibiotic-treated patients with invasive β-haemolytic streptococcal infections in US hospitals: a retrospective multicentre cohort study.

• DOI: 10.1016/s1473-3099(20)30523-5
• 发表时间: 2021-05
• 期刊: The Lancet. Infectious diseases
• 作者: Babiker A;Li X;Lai YL;Strich JR;Warner S;Sarzynski S;Dekker JP;Danner RL;Kadri SS
• 通讯作者: Kadri SS