PENICILLIN RESPONSE GENES IN DRUG-RESISTANT PNEUMOCOCCI

自从他们在一个偏远的村庄的临床标本中首次被鉴定以来， 1967年在巴布亚新几内亚，耐青霉素肺炎球菌已经传播 在全球肺炎球菌耐药性的发生率极高， 报告（西班牙所有侵袭性分离株的50%和所有儿科分离株的70 匈牙利的隔离物）。最近令人震惊的事态发展包括： 出现对第三代头孢菌素的耐药性; 青霉素MIC和某些能力的证明 多抗性克隆传播到很大的地理距离， 上呼吸道肺炎球菌感染比例高， 广泛地寄生在健康的孩子身上为了获得对 这些危险的社区获得性病原体的爆炸性传播， 将是强制性的，以更好地了解β-内酰胺的分子基础 抗生素耐药性;控制抗生素水平（MIC）的机制 青霉素耐药性和遗传因素的起源和性质， 定义了这些细菌复杂的多形表型。名单 这些性质是相当长的，并且基本的机制是未知的。 在高表达的大肠杆菌中，5种青霉素结合蛋白（PBP）中至少有4种 抗性菌株对青霉素的亲和力降低。抗性分离株 在其PBP中表现出高度多态性，并含有PBP基因 “马赛克”结构。绝大多数青霉素耐药菌株 仅限于83种肺炎球菌荚膜中的少数（4至6种） 类型许多耐药菌株产生细胞壁肽聚糖， 改变化学结构自溶抑制或缺陷 青霉素治疗通常与耐药性有关， 耐药菌株对杀菌剂的敏感性也降低 青霉素的作用我们建议将遗传决定因素 这些表型统称为青霉素反应基因。的目的 这项拨款提案是为了识别和分析这些基因， 分子水平的理解。具体而言：（A）我们 我将尝试识别镶嵌PBP中的最小序列改变 与抗生素亲和力降低相关的基因， 特别是PBP和增加的抗生素耐药性（MIC值）， 细胞(B)同样的方法将用于识别基因 化学成分改变的元素 在抗性细胞中的肽聚糖。这些研究将在 与苏塞克斯大学的克里斯·道森博士合作。(C)的 大多数青霉素耐药肺炎球菌也携带遗传性 与青霉素作用的不可逆性机制相关的性状， 由于耐药分离株通常显示出降低的抗生素诱导的 溶解和活力丧失。具有相似表型的细菌最近 在我们的实验室中通过插入/重复诱变分离， 一个关键的遗传决定因素控制杀菌敏感性 肺炎球菌也已被鉴定。将大力克隆 这些基因并阐明所涉及的生化（酶）机制。我们 预计这些方法将提供重要的信息， 控制抗生素耐药菌株的传播。研究 还应该允许新的见解，尚未探索的领域， 肺炎球菌细胞壁合成的分子生物学和遗传学 其他细菌。

Since their first identification in clinical specimen in a remote village in Papua New Guinea in 1967 penicillin resistant pneumococci have spread globally. Exceedingly high incidence of pneumococcal resistance has been reported (50% of all invasive isolates in Spain and 70% of all pediatric isolates in Hungary). Recent and alarming developments include: the appearance of resistance to third-generation cephalosporins; increase in the penicillin MIC and demonstration of the capacity of certain multiresistant clones to spread over large geographic distances, to cause a high proportion of upper respiratory pneumococcal infections, and to extensively colonize healthy children. In order to gain some control over the explosive spread of these dangerous community-acquired pathogens, it will be mandatory to better understand the molecular basis of beta-lactam antibiotic resistance; the mechanism(s) that control the level (MIC) of penicillin resistance and the origin and nature of genetic elements that define the complex pleiomorphic phenotypes of these bacteria. The list of these properties is quite long and the underlying mechanisms are unknown. At least 4 out of the 5 penicillin-binding proteins (PBPs) of highly resistant strains have reduced affinity for penicillin. Resistant isolates exhibit a high degree of polymorphism in their PBPs and contain PBP genes of "mosaic" structure. The great majority of penicillin resistant strains are restricted to only a few (4 to 6) of the 83 pneumococcal capsular types. Many resistant isolates produce cell wall peptidoglycans of grossly altered chemical structure. Inhibited or defective autolysis during penicillin treatment is frequently associated with resistance and some resistant isolates also show reduced sensitivity to the bactericidal effect of penicillin. We propose to refer to the genetic determinants of these phenotypes collectively as penicillin response genes. The purpose of this grant proposal is to identify and dissect these genes and to provide molecular level understanding for their functioning. Specifically: (A) We shall try to identify the minimal sequence alterations in the mosaic PBP genes that are associated with the reduced antibiotic affinity of the particular PBP and increased antibiotic resistance (MIC value) of the cells. (B) The same approach will be used to identify the genetic element(s) responsible for the altered chemical composition of peptidoglycan in resistant cells. These studies will be done in collaboration with Dr. Chris Dowson of the University of Sussex. (C) The great majority of penicillin resistant pneumococci also carry genetic traits related to the mechanism of irreversibility of penicillin action, since resistant isolates often show reduced rates of antibiotic-induced lysis and viability loss. Bacteria with similar phenotypes have recently been isolated in our laboratory by insertion/duplication mutagenesis and a key genetic determinant controlling bactericidal sensitivity of pneumococci has also been identified. A major effort will be made to clone these genes and to clarify biochemical (enzymatic) mechanisms involved. We anticipate that these approaches will provide information important for the control of the spread of antibiotic resistant strains. The studies should also allow novel insights into as yet unexplored areas of the molecular biology and genetics of cell wall synthesis in pneumococci and other bacteria.