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Biofilm-Facilitated Dissemination of Integrative Antibiotic Resistance Elements in Streptococcus pneumoniae

肺炎链球菌中生物膜促进的综合抗生素抗性元件的传播

基本信息

批准号：
10542656
负责人：
Brenda Stephanie Antezana
金额：
$ 4.68万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10542656
关键词：
Antibiotic Resistance Antimicrobial Resistance Automobile Driving Bacteria Bioreactors Bulla Cells Centers for Disease Control and Prevention (U.S.)Cessation of life Characteristics Child Chromosomes Coculture Techniques Collection Competence Complex Conjugate Vaccines DNA DNA Insertion Elements Disease Elements Environment Frequencies Gene Expression Genes Genetic Genomic DNA Genomics Goals Human In Vitro Incidence Macrolide-resistance Macrolides Measures Membrane Microbial Biofilms Modeling Molecular Multi-Drug Resistance Nasopharynx National Institute of Allergy and Infectious Disease Operon Phylogeny Play Pneumococcal Infections Pneumococcal conjugate vaccine Population Prevalence Production Quantitative Reverse Transcriptase PCR Recombinants Reporting Research Role Scanning Electron Microscopy Serotyping Site Streptococcus pneumoniae Structure Surface System Testing Tetracycline Resistance Transference Treatment Protocols Type IV Secretion System Pathway Vaccines Vesicle Western Blotting Work World Health Organization base bioinformatics tool combat competence factor design early childhood extracellular extracellular vesicles genetic element host colonization human pathogen in silico in vivo innovation microscopic imaging molecular dynamics mutant novel novel strategies opportunistic pathogen population based priority pathogen protein expression resistance gene transmission process uptake vaccine development whole genome

项目摘要

I. PROJECT SUMMARY Streptococcus pneumoniae (Spn) colonizes the human nasopharynx during early childhood. This opportunistic pathogen causes ~15 million cases of invasive pneumococcal disease (IPD) and ~500,000 deaths in children worldwide each year. While pneumococcal conjugate vaccine (PCV) development has impacted disease incidence, the emergence and spread of antimicrobial resistance in Spn, an NIAID priority, has complicated IPD treatment regimens. Consequently, the CDC and WHO list Spn amongst the antibiotic-resistant priority pathogens. In particular, antibiotic resistance in Spn is increasingly due to acquisition of >20 kb, Tn916-like integrative and conjugative elements (ICEs), most notably Tn2009, Tn6002, and Tn2010. The specific mechanisms facilitating the wide spread of these large ICEs among Spn have not been elucidated. In other bacteria, ICEs are transferred via transposition with the formation of circular intermediates and a conjugative type 4 secretion system. Although conjugative genes are conserved within Spn ICEs, their roles in Spn ICE transference have not been established. ICEs are unusually large elements for efficient in vitro transformation of naturally competent Spn, which typically acquire ~2-6 kb DNA fragments. Accordingly, we confirmed that competent Spn strains did not uptake ICE DNA (<10-9) under in vitro conditions. Yet, when two Spn strains were co-inoculated in an ex vivo bioreactor system, forming a mixed biofilm on human nasopharyngeal cells, the transference of the entire ICE occurred at frequencies up to 10-4, which likely recapitulates the scenario during in vivo host colonization. In Aim 1, I propose to use the bioreactor system and a collection of various mutant strains to decipher the mechanism(s) responsible for this five-orders of magnitude increase in dissemination of large ICEs among naturally competent Spn strains. Specifically, we will assess the roles of ICE-encoded conjugation genes, competence factors, and the DNA uptake apparatus. Conjugative and competence gene expression will be measured via qRT-PCR and ICE circular intermediates, if present, will also be quantified using qPCR. Conjugative protein expression will be investigated by Western blots. The role of extracellular membrane vesicles (EVs) in the delivery of >20 kb ICE DNA will also be evaluated. Whether EVs carry ICE DNA will be examined by ICE-targeting qPCR and purified biofilm-derived EVs will be tested via in vitro transformations. In Aim 2, genomic characteristics of multi-drug resistance-conferring ICE elements will be defined in the global Spn population. Using innovative bioinformatics tools and approaches, publicly available whole genome sequences from global Spn isolates will be examined to identify new ICEs and novel ICE insertion sites in addition to previously defined sites from a regional isolate collection. Analyses will be based on pre- and post-vaccine periods, vaccine or non-vaccine serotypes, and clonal complexes. Prevalence, incidence rates, phylogeny, and recombinant regions will be analyzed for ICE-positive isolates. This work will provide a greater understanding of the molecular mechanism and transmission dynamics for global spread of multi-drug resistance elements in Spn.

I.项目摘要肺炎链球菌（Spn）在儿童早期在人类鼻咽定植。这个机会主义者病原体导致约1500万例侵袭性肺炎球菌病（IPD）和约50万例儿童死亡全球每年都有。虽然肺炎球菌结合疫苗（PCV）的发展影响了疾病结核病的发病率、抗生素耐药性的出现和传播（NIAID的优先事项）使IPD复杂化治疗方案。因此，疾病预防控制中心和世界卫生组织将Spn列为耐药性优先药物之一，病原体特别地，Spn中的抗生素抗性越来越多地是由于获得>20 kb的Tn 916样突变体。整合和结合元件（ICE），最著名的是Tn 2009、Tn 6002和Tn 2010。具体促进这些大ICE在Spn中广泛传播的机制尚未阐明。换句在细菌中，ICE通过转座转移，形成环状中间体和接合的 4型分泌系统。虽然接合基因在Spn ICE中是保守的，但它们在Spn ICE中的作用转移尚未建立。ICE是用于高效体外转化的不寻常的大元件。天然感受态Spn，其通常获得约2-6 kb DNA片段。因此，我们确认，感受态Spn菌株在体外条件下不摄取ICE DNA（<10-9）。然而，当两个Spn菌株被在离体生物反应器系统中共接种，在人鼻咽细胞上形成混合生物膜，整个ICE的转移发生在高达10-4的频率上，这可能概括了体内宿主定殖。在目标1中，我建议使用生物反应器系统和各种突变体的集合压力，以破译机制（S）负责这五个数量级的增加，在传播大ICE之间的自然能力的Spn菌株。具体来说，我们将评估ICE编码的接合基因、感受态因子和DNA摄取装置。接合感受态基因表达将通过qRT-PCR测量，ICE环状中间体（如果存在）也将使用 qPCR。将通过蛋白质印迹研究结合蛋白表达。细胞外膜的作用还将评估在递送>20 kb ICE DNA中的微囊泡（EV）。电动汽车是否携带ICE DNA将是将通过ICE靶向qPCR检查纯化的生物膜衍生EV，并通过体外转化测试纯化的生物膜衍生EV。在目的2，在全球Spn中定义多药耐药赋予ICE元件的基因组特征人口利用创新的生物信息学工具和方法，将对全球Spn分离株的分离物进行检查，以确定新的ICE和新的ICE插入位点，以前定义的站点从区域隔离收集。分析将基于疫苗接种前和接种后时期、疫苗或非疫苗血清型和克隆复合物。患病率、发病率、发病率和将分析重组区域的ICE阳性分离株。这项工作将提供一个更好的理解，多药耐药元件在Spn中全球传播的分子机制和传播动力学。