Impact of Antibiotics and Vaccines on the in vivo Evolution of S. pneumoniae

抗生素和疫苗对肺炎链球菌体内进化的影响

• 批准号: 7736126
• 负责人: Garth D. Ehrlich
• 金额: $ 63.94万
• 依托单位: ALLEGHENY-SINGER RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-17 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7736126
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Revised 1R01AI080935-01A1 "Impact of Antibiotics and Vaccines on the in vivo Evolution 1R01A1080935-OIAI "Impact of Antibiotics and Vaccines on the in vivo Evolution of S. pneumoniae" S. C'! Streptococcus pneumoniae has remained one of the major causative agents of pneumoniae remained the potentially life threatening human disease in our era. Nevertheless, introduction of Nevertheless, introduction potentially life threatening human disease in our era. antibiotics (penicillin) and -- more recently - the conjugate vaccine provoked major the conjugate vaccine provoked major antibiotics (penicillin) and evolutionary changes in the structure of natural populations of this pathogen. The evolutionary changes in the structure of natural populations of this pathogen. purpose of this research proposal is to combine the power of whole genome sequencing purpose this research proposal is to combine the whole (WGS) and epidemiology to obtain insights into the mechanisms by which antibiotic (WGS) and epidemiology to obtain mechanisms which resistant and non-vaccine type pneumococcal lineages emerged in the in vivo resistant and non-vaccine type pneumococcal emerged in the in environment. The studies will have three foci of concentration. environment. The studies will have three foci of concentration. Proiect I. penicillin resistant pneumococcal (PRPn) clones: ST-I, Project I. Three highly penicillin resistant pneumococcal (PRPn) clones: ST-1, ST-2 and ST-3, each with a unique sequence type (MLST) and capsular type have ST-3, with achieved pandemic spread and appear to remain stable over long times and distant achieved pandemic spread and to remain long times and geographic sites of isolation. Such genetic stability in a highly recombinogenic pathogen sites isolation. Such genetic stability in is unusual and it contrasts with the well documented diversity of penicillin susceptible and contrasts with pneumococci. The purpose of this project is to use whole genome sequencing (WGS) to pneumococci. whole sequencing (WGS) to better document and understand the nature and mechanisms of genetic stability in these and three Pen R three PenR clones. Project II. Pneumococcal strains expressing the non-vaccine type (NVT) Proiect II. Pneumococcal strains expressing capsular polysaccharides, such as 1 IA, 6A or 1 9A have been identified as minority as 11A, 6A or 19A have been components of the nasopharyngeal flora of children attending Day Care Centers -- even components of the nasopharyngeal flora of children attending Day Care Centers even before the introduction of the conjugate vaccine. WGS will be used to test if the the introduction of the conjugate vaccine. emergence of these strains from minority to majority status in the nasopharynx and from these strains from in colonizers to disease causing strains -- is accompanied by changes in genetic makeup disease causing strains is accompanied Project III. Because the pandemic penicillin-resistant strains show much less Proiect Ill. Because the pandemic penicillin-resistant strains show much less their genomic variation over time and distance than the nonclonal strains, and because their cell wall structures are profoundly different in structure from the nonclonal types we will investigate these strains to see if they are less recombinogenic and/or limited with these strains to respect to donor DNA source as opposed to being less capable or less likely to form mixed strain biofilm structures with penicillin-sensitive strains thus resulting in lower transformation and evolution. rates of inter-strain transformation and evolution. 3.0 O." (/1 ... .,, -0-0 '"; f/1 (gyp f0/1 Z E m CO) =(D +-' 0-0 O-2 53' =_i. 0-0 r-. +L-' 8)o I-a) Revised Specific Aim Section Revised Specific 1R01AI080935-01A1 "Impact of Antibiotics and Vaccines on the in vivo 1R01A1080935-OIAI"Impact of Antibiotics and Vaccines on the in vivo Evolution of S. pneumoniae" S. Below are the modified specific aims we propose to complete within the two year are the modified we propose funding period SA-1 Perform whole genome sequencing (WGS) and comparative genomic analyses SA-1 Perform whole genome sequencing (WGS) and comparative among: 1) Sp carriage isolates of the pandemic clone, SP1 (N=10); 2) Sp carriage among: 1) Sp carriage isolates of the pandemic clone, SPI (N10); carriage isolates the pandemic clones SP2, & SP3 (n's = 4 and 4, respectively), and 3) invasive respectively), isolates the clones SP2, & SP3 isolates of SP1, 2, & 3 (n = 16) as well as 8 nonclonal invasive isolates to: isolates SPI, 2, & 3 (n = 16) as to: determine the supragenome of the pandemic clones is restricted in size a: determine ififthe supragenome of the pandemic clones is restricted in size by our preliminary results compared with the nonclonal clinical Sp isolates (as suggested by our preliminary results SP-1 examining a limited number of SP-1 isolates) b: by characterizing nasopharyngeal (NP) isolates from pre-school age children (as it nasopharyngeal isolates pre-school age children (as it is generally agreed that the NP is the primary site of Sp evolutionary change) from a a constellation of sources including multiple: 1) isolates recovered from the same child at the same time; 2) isolates of the same clone recovered from multiple children attending multiple attending the same day care center (DCC) at the same time; 3) isolates of the same pandemic clone recovered from children in different DCCs within weeks of one another; 4) isolates clone of the same pandemic clone recovered from various disease sites in different countries on different continents; and 5) isolates recovered over long time periods c: Perform whole genome alignments, and bioinformatic comparative genomic and Perform whole genome alignments, and bioinformatic comparative genomic and metabolic reconstructions among the isolates' sequences identified in SA-1a and b to metabolic reconstructions among the isolates sequences identified in SA-la to build gene-sharing trees showing the evolutionary relationships among strains and build groups of strains; --(Q (s] ,-. CAN SA-2: The pathogenic potential of pneumococcal strains selected by the conjugate The pathogenic potential of pneumococcal strains selected by the conjugate These non-vaccine type (NVT) strains could be either: a) vaccine is unknown. These non-vaccine type (NVT) strains could be either: a) an pre-existing at colonization sites; or b) the expansion of NVT strains pre-existing as minority isolates at colonization sites; or b) the products of recombination in which "successful" clonal lineages acquired novel NVT which "successful" clonal capsules. The purpose of the proposed experiments are to capsules. The purpose of the proposed experiments from SA-1 invasive NVT strains and compare them a: analyze the WGS from SA-1 from the invasive NVT strains and compare them strains 1) if any of against themselves and all other sequenced pneumococcal strains to see: 1) if any of the strains are derived from the known pandemic strains; 2) if there are novel clones if which, if any of the other Sp they cluster with evolutionarily. emerging; and 3) which, if any of the other Sp they cluster with evolutionarily. b: characterize the colonizing and disease potential of these strains by using 1) in of strains by using 1) in intraperitoneal vitro tissue culture assays for adherence and invasion; and 2) the mouse intraperitoneal infection model. 3:r 00p N-.0 y.0 O7y qty 3-p O-0 Determine the pen R strains: 1) are less recombinogenic and/or limited with SA-3: Determine ififthe penR strains: 1) are less recombinogenic and/or limited with respect to donor DNA source in in vitro transformation experiments; 2) are less capable or incapable of forming mixed strain biofllm structures, and demonstrate lower biofilm lower transformation rates within in vitro biofilms (Oil -U) frail cap �-U (L) C.-.0 -�O 0)0' acs 4)> o-� L-. `,� m�> o�� `off Health

修改 1R01AI080935-01A1“抗生素和疫苗对肺炎链球菌体内进化的影响” 1R01A1080935-OIAI“抗生素和疫苗对肺炎链球菌体内进化的影响”S. C'！ 肺炎链球菌仍然是我们这个时代潜在威胁生命的人类疾病的主要病原体之一，然而，在我们这个时代引入了潜在威胁生命的人类疾病，以及最近的抗生素。结合疫苗引发了主要抗生素（青霉素）和该病原体自然种群结构的进化变化 该研究提案的目的是。为了结合全基因组测序的力量，本研究计划是将全基因组测序（WGS）和流行病学结合起来，深入了解抗生素（WGS）和流行病学的机制，以获得耐药性和非疫苗型肺炎球菌谱系出现的机制。体内环境中出现的耐药性和非疫苗型肺炎球菌将具有三个集中环境。耐青霉素肺炎球菌 (PRPn) 克隆：ST-I，项目 I。 三种高度耐青霉素肺炎球菌 (PRPn) 克隆：ST-1、ST-2 和 ST-3，每种克隆都有独特的序列类型 (MLST) 和荚膜类型ST-3，实现了大流行传播，并且似乎在很长一段时间内保持稳定，并且在很远的距离实现了大流行传播，并且在高度隔离的地理位置上保持了长时间和地理上的稳定性。重组病原体位点的分离是不寻常的，它与有据可查的青霉素敏感性多样性形成鲜明对比，并且与肺炎球菌形成鲜明对比。更好地记录和了解这些表达非疫苗型 (NVT) Proiect 的肺炎球菌菌株和三个 Pen R 克隆的遗传稳定性的性质和机制。 II. 表达荚膜多糖的肺炎球菌菌株，如 1 IA、6A 或 1 9A，已被确定为少数，而 11A、6A 或 19A 已成为日托中心儿童鼻咽菌群的组成部分，甚至是儿童鼻咽菌群的组成部分。甚至在引入结合疫苗之前就进入日托中心的儿童将被用来测试是否引入了疫苗。这些菌株在鼻咽中从少数到多数状态的出现，以及这些菌株从定殖者到致病菌株的出现，都伴随着致病菌株基因组成的变化，因为大流行的青霉素耐药菌株。由于大流行的青霉素抗性菌株随着时间和距离的变化，其基因组变异比非克隆菌株要少得多，并且因为它们的细胞壁结构在结构上与非克隆类型有很大不同，所以我们将研究这些菌株，看看它们是否具有较低的重组原性和/或在供体 DNA 来源方面受到这些菌株的限制，而不是与青霉素敏感菌株形成混合菌株生物膜结构的能力较低或不太可能，从而导致较低的转化和种系间转化和进化的速率。 3.0 哦。” (/1 ... .,, -0-0 '”； f/1 （吉卜赛人 f0/1 Z 乙 米 一氧化碳） =(D +-' 0-0 O-2 53' =_i。 0-0 r-。 +L-' 8）o I-a) 修订的具体目标部分修订的具体 1R01AI080935-01A1“抗生素和疫苗对体内的影响 1R01A1080935-OIAI”抗生素和疫苗对肺炎链球菌体内进化的影响”S. 以下是我们建议在两年内完成的修改后的具体目标，我们建议修改后的资助期限 SA-1 执行全基因组测序 (WGS) 和比较基因组分析 SA-1 执行全基因组测序 (WGS) 并比较：1 ) 大流行克隆的 Sp 携带分离株，SP1 (N=10)；2) 大流行克隆的 Sp 携带分离株，SPI (N10)；大流行克隆 SP2 和 SP3 的 Sp 携带分离株（n =分别为 4 和 4），以及 3）侵入性），分离出 SP1、2 和 3 的克隆 SP2 和 SP3 分离株（n = 16）以及 8 个非克隆侵入性分离株：分离出 SPI、2 和 3 (n = 16)：确定大流行克隆的超基因组的大小是否受到限制 a：确定大流行克隆的超基因组的大小是否受到限制b：通过我们的初步结果与非克隆临床 Sp 分离株进行比较（如我们的初步结果 SP-1 检查有限数量的 SP-1 分离株所示） b：通过表征学龄前儿童的鼻咽 (NP) 分离株（因为它是鼻咽分离株）从一系列来源中分离出学龄前儿童（因为人们普遍认为 NP 是 Sp 进化变化的主要位点），这些来源包括多个：1）在同一时间从同一儿童中回收的分离株2) 从同时就诊于同一日托中心 (DCC) 的多个儿童中分离出的同一克隆；3) 在数周内从不同 DCC 的儿童中分离出的同一克隆分离株； ) 从不同大陆不同国家的不同疾病地点回收的同一大流行克隆的分离克隆；以及 5) 长时间回收的分离株 c: 进行全基因组比对和生物信息学比较基因组和 进行全基因组比对和生物信息学SA-1a和b中鉴定的分离株序列之间的比较基因组和代谢重建与SA-1a中鉴定的分离株序列之间的代谢重建，以构建基因共享树，显示菌株之间的进化关系并构建菌株组； --(问 （s] ,-. 能 SA-2：由缀合物选择的肺炎球菌菌株的致病潜力 这些非疫苗型 (NVT) 菌株可能是： a) 疫苗未知）菌株可能是： a) 定殖位点预先存在的菌株；或b) 先前作为少数分离株存在于定殖位点的 NVT 菌株的扩增，或 b) 重组产物；其中“成功的”克隆谱系获得了新的 NVT，其中“成功的”克隆胶囊 所提议的实验的目的是从 SA-1 侵入性 NVT 菌株中进行胶囊并进行比较：分析来自 SA 的 WGS。 -1 来自侵入性 NVT 菌株，并比较它们菌株 1) 是否有任何菌株与自身和所有其他已测序的肺炎球菌菌株进行比较，以查看： 1) 是否有任何菌株源自已知的大流行菌株； 2) 是否有新的克隆，如果有的话，它们在进化上与其他 Sp 聚类；以及 3) 如果有任何其他 Sp，它们在进化上聚类 b：表征这些菌株的定植和疾病潜力。通过使用1)在菌株中通过使用1)在腹膜内体外组织培养测定中进行粘附和侵袭；以及2)小鼠腹膜内感染模型。 3：r 00p N-.0 y.0 O7y 数量 3-p O-0 确定 pen R 菌株：1) 重组原性较低和/或受 SA-3 限制：确定 penR 菌株是否：1) 在体外转化实验中重组原性较低和/或受供体 DNA 来源限制；2) 重组原性较低和/或受 SA-3 限制。能够或不能形成混合菌株生物膜结构，并表现出较低的生物膜在体外生物膜内较低的转化率 （油 -U) 脆弱 帽 �-U (大) C.-.0 -�O 0)0' ACS 4)> o-� L-。 `,� 米�> Ø `关闭 健康