下载文档原格式
耐药机制及特殊耐药性检测知识整理1.耐药类型1)靶位改变●MRSA：抗药性金黄色葡萄球菌●VRE：抗万古霉素肠球菌2)产酶●ESBL：超广谱β-内酰胺酶●AmpC： AmpCβ内酰胺酶●CRE：耐碳青霉烯类肠杆菌3)多重耐药●MDR-PA：多重耐药铜绿假单胞菌●MDR-AB：多重耐药鲍曼不动杆菌2.M RSA抗药性金黄色葡萄球菌1)耐药机制：MRSA携带mecA基因，可编码产生低亲和力的青霉素结合蛋白（PBP2a），阻止药物与靶位的结合而造成耐药。
2)流行现状3)检测方法●1、苯唑西林的筛选平板。
不是甲氧西林！●2、常规药敏试验(苯唑西林MIC，头孢西丁MIC或纸片法)检测。
●3、mecA基因(PCR法)及其产物PBP2a(乳胶凝集试验)阳性均提示MRSA●抑菌圈内任何生长视为耐药。
4)检测●mecA和PBP2a：确定葡萄球菌属对甲氧西林(苯唑西林)耐药性最可靠的方法，阳性应报告为该菌株对甲氧西林(苯唑西林)耐药●苯唑西林筛选平板（显色平板）●常规药敏（苯唑西林MIC、头孢西丁MIC、纸片法）●PCR●金葡或所有凝固酶阴性葡萄球菌，如对苯唑西林(或甲氧西林)耐药，则对青霉素类、头孢菌素类(头孢罗膦除外)、碳青霉烯类和含酶抑制剂的复方制剂均应报告耐药，而不考虑其体外药敏结果。
3.V RSA-vanA耐万古霉素金葡1)定义：金黄色葡萄球菌对万古霉素●MIC＞16μg/ml——耐药●MIC=4-8μug/ml——中介●MIC＜2μg/ml——敏感。
2)耐药机制：葡萄球菌细胞壁有一段由4个氨基酸组成的短肽， 4肽的末端有2个重复氨基酸是万古霉素结合的靶位，由vanA基因介导的4肽末端改变为万古霉素低亲和力靶位，与万古霉素结合的亲和力下降1000倍。
3)检测方法：不推荐纸片法做万古霉素的敏感性检测4.V RE-vanA耐万古霉素肠球菌1)流行现状：几乎无药可治2)检测方法●肉汤或平板筛查法，万古霉素琼脂平板●纸片法、E-test●琼脂稀释法VRE筛选试验※3)耐药机制：vanA基因介导的4肽末端（D-丙氨酸- D-丙氨酸）改变为D-丙氨酸-D-乳酸4)结果解释：MIC高水平耐药，根据对万古霉素和替考拉宁耐药的程度可进⼀步区分为vanA、vanB、vanC、vanD等。
耐药机制总结汇报耐药机制是指微生物对抗抗生素或其他抗微生物药物产生抵抗的能力。
随着抗生素的广泛应用，许多微生物逐渐产生了耐药性，这给人类医药卫生系统带来了巨大的挑战。
本文将总结常见的耐药机制，并分析其对公共卫生的影响。
一、突变突变是一种常见的微生物耐药机制。
通过基因突变，细菌可以改变其代谢途径或生理过程，从而使抗生素失去对其的效果。
例如，青霉素对革兰氏阳性菌起作用的机制是抑制菌体合成，而通过突变，细菌可以改变其细胞壁的结构，使其免疫于青霉素的作用。
二、抗药基因水平转移细菌之间可以通过水平基因转移的方式分享耐药基因。
细菌可以将带有抗药性的质粒转移到其他细菌中，使其也获得相应的耐受能力。
这种机制可大大加快抗药基因在细菌群体中的传播速度，从而导致耐药性的迅速蔓延。
三、药物代谢增强某些细菌能够产生酶，可以降解或转化抗生素分子，从而减少其对细菌的杀伤作用。
例如，某些细菌可以产生β-内酰胺酶，能够水解β-内酰胺类抗生素，使其失去杀菌活性。
这种机制使得细菌可以继续繁殖，而抗生素的作用受到抑制。
四、多重耐药泵细菌可以通过表面膜上的泵系统将抗生素排出细胞外。
这种多重耐药泵能够识别并主动排出多种抗生素，从而保护细菌免受药物的侵害。
由于多重耐药泵是一种主动排出机制，因此即使抗生素浓度较高，细菌仍能生存下去。
以上是常见的几种耐药机制，然而，这些机制只是细菌耐药的冰山一角。
随着科技的不断进步，越来越多的研究发现了耐药机制的新变种。
细菌在面对抗生素压力时显示出惊人的适应能力，这给人类抗感染治疗带来了前所未有的挑战。
耐药机制对公共卫生的影响是巨大的。
首先，抗药性的细菌可以引发难以治疗的感染，导致更高的病死率。
其次，治疗耐药细菌感染的药物选择变得有限，可能需要使用更昂贵、毒性更大的药物进行治疗。
这不仅增加了患者的负担，还可能导致更多的不良反应和治疗失败。
此外，耐药机制的传播也给公共卫生控制带来了困难，可能导致疫情的蔓延和传播。
病原微生物耐药机制及防控策略病原微生物的耐药性是指其对抗抗生素治疗的能力增强，从而导致常规药物对其治疗效果降低甚至失效。
随着时间的推移，病原微生物已逐渐发展出多种耐药机制，对公共卫生产生了严重的影响。
为了建立有效的防控机制，了解病原微生物的耐药机制以及相关的防控策略是至关重要的。
病原微生物的耐药机制主要包括自然耐药、获得性耐药和产生性耐药。
首先，自然耐药是指某些病原微生物在其进化过程中就具备的抗药性。
这种耐药通常是由于其细胞壁、细胞膜或细胞质中的特殊结构和物质所致，使得抗生素无法有效作用于其上。
例如，革兰氏阳性菌通常具有较厚的细胞壁，而革兰氏阴性菌则存在外膜，这些特殊的结构能够阻碍抗生素的进入，从而导致自然耐药。
其次，获得性耐药是指病原微生物在暴露于抗生素压力下，通过遗传变异或基因水平的横向转移等途径，获取抗药性基因，从而发展出对抗抗生素的能力。
这种耐药机制主要通过突变和基因水平的水平转移来实现。
突变是指病原微生物基因组中发生的随机变异，可以导致基因表达的变化。
而水平基因转移则是指病原微生物之间通过质粒等外源DNA传输抗药性基因。
最后，产生性耐药是指在病原微生物感染过程中，抗生素的过量使用导致病原微生物在宿主体内产生耐药性。
这种耐药机制是由于抗生素的选择压力导致病原微生物的种群选择，使得一些原本敏感的菌株逐渐替代为耐药菌株。
此外，产生性耐药还可能与病原微生物内部的共生菌群有关，因为这些共生菌群可能对抗生素具有天然的耐药性。
面对不断增长的病原微生物耐药问题，采取有效的防控策略显得尤为重要。
首先，强调预防为主的原则非常关键。
通过加强公众的健康教育，提倡正确使用和合理使用抗生素，消除滥用抗生素的现象。
此外，加强环境卫生和个人卫生的管理是预防耐药性传播的另一重要措施。
其次，加强监测和监控工作。
建立完善的抗菌药物耐药性监测网络，定期收集和分析相关数据，及时发现和应对新型耐药菌株的出现。
并加强病原微生物耐药性的监测和评估，提供针对性的防控措施。
抗菌药物耐药性的分子机制和预防策略在近年来，抗菌药物耐药性问题日益严峻，已成为全球公共卫生领域的重大挑战。
抗菌药物耐药性是指细菌、病毒、真菌等微生物对药物产生耐受能力的现象，使得常规治疗手段失效，增加了感染的疾病负担和医疗成本。
因此，深入了解抗菌药物耐药性的分子机制以及采取有效预防策略显得尤为重要。
一、抗菌药物耐药性分子机制1. 靶位变异：细菌可以通过基因突变改变其蛋白质靶点结构，降低与抗生素结合的亲和力，从而减少或避免了抗生素对其起效。
2. 耐药基因传递：细菌间可通过水平基因传递方式获取新产生的针对特定抗生素的耐药基因。
3. 药物外排泵：细菌可通过表面外排泵将已进入胞内的抗生素排出细胞，降低药物在细菌内部的有效浓度。
4. 酶降解：某些细菌能够产生特定酶类，例如β-内酰胺酶，能够迅速降解抗生素，使其失去活性。
二、预防抗菌药物耐药性的策略1. 合理使用抗生素：医务人员和患者应当遵循相关指南，并仅在确实需要时使用抗菌药物。
避免滥用或过度使用抗生素可以减少细菌对药物的耐受性产生。
2. 多途径干预：除了合理使用抗生素外，采取其他多种干预措施也是有效的预防策略。
例如加强手卫生和个人卫生习惯，推行消毒制度，提高环境卫生水平等。
3. 发展新型抗菌药物：目前已有许多常见病原微生物对一线治疗常用的抗生素产生了耐药性。
因此，研究和开发新型抗菌药物是投入未来防治耐药性问题的重要措施。
4. 加强监测和警戒：建立全面的抗菌药物耐药性监测系统，对医院、社区等地点进行定期监测，及早发现和预防潜在的耐药性问题。
5. 促进国际合作：由于抗菌药物耐药性是全球性问题，各国需要加强合作，共同应对。
通过分享信息、资源和技术，开展跨国合作研究，加快新型抗菌药物的研发。
总结起来，抗菌药物耐药性的分子机制主要包括靶位变异、耐药基因传递、药物外排泵以及酶降解等。
为了预防抗菌药物耐药性的发展，需要采取一系列策略，包括合理使用抗生素、多途径干预、发展新型抗菌药物、加强监测和警戒以及促进国际合作等。
药物耐药机制与对策药物耐药是全球范围内的一个严重问题，严重影响了传染性疾病的防治工作，也给临床治疗带来了极大的挑战。
药物耐药机制的研究和对策措施成为当前医学领域的热点问题之一。
本文将对药物耐药机制进行深入分析，并提出相应的对策建议。
药物耐药机制细菌药物耐药机制细菌是引起人类、动物和植物感染性疾病的重要致病因子，而细菌对抗生素产生耐药性的机制主要包括基因突变、外源基因水平转移和表型适应等。
其中，外源基因水平转移是导致耐药菌株产生最快速、最有效的途径，且易导致多重抗药菌株的出现。
此外，细菌通过产生酶类物质使抗生素失效，或者通过减少药物进入细胞内部来降低抗生素的杀菌效果。
病毒药物耐药机制与细菌不同，病毒没有自身的新陈代谢功能，它必须寄生在宿主细胞内才能进行复制。
病毒耐药性主要来自于其高度变异的特性。
病毒的遗传物质DNA或RNA在复制过程中往往发生变异，从而产生新的突变株，这些突变株可能对已有的抗病毒药物产生耐药性。
此外，在长期用药情况下，病毒亦可产生多种代谢途径抵抗或降解药物。
真菌药物耐药机制真菌对抗真菌药物产生耐药性是近年来备受关注的问题。
真菌耐药性主要包括位点靶点突变、靶点过度表达、底物外泌、多效途径高级表达、躲避免疫检测等多种机制。
这些耐药机制使得许多常用的抗真菌剂在临床治疗真菌感染时逐渐失去了活性。
对策建议加强监管，并避免滥用抗微生物剂当前，国际上普遍存在着对抗微生物剂滥用的问题，滥用不仅会降低其对微生物的杀灭能力，同时还会刺激微生物形成耐受性。
因此，对于严格限制和规范抗微生物剂的使用显得尤为重要。
开发新型抗微生物治疗手段随着科学技术水平和医学水平不断发展，人们需要不断开发新型抗微生物治疗手段以克服当前耐药现象。
例如，微生态调节治疗策略可能成为制约和减缓微生物耐受关键控制点之一。
又如蛋白质及DNA/RNA酶水平干预等新型治疗手段也是涉及今后发展趋势之一。
加大科学研究力度并鼓励科技创新在全球范围内加大对耐药机制和防控策略相关领域的科学研究力度，并推动这些领域技术创新取得跨越式发展。
肺炎克雷伯杆菌对氟喹诺酮类抗生素耐药机制探究作者：许超张春秋蒋邦栋俞蕾盛成兰来源：《健康必读·下旬刊》2019年第12期【摘要】目的：肺炎克雷伯杆菌（KP）对氟喹诺酮类药物（FQNs）的耐药机制，以更好的指导临床药物治疗。
方法：选择2018.6～2019.5期间临床分离对环丙沙星耐药的10株KP，为明确菌株对5种FQNs的MIC值，应用微量肉汤稀释法检测;用PCR法检测菌株染色体和质粒带有的FQNs基因（gyrA、parC与qnr基因）并测量序列，在试验法的支撑下检验qnr是否出现转移情况。
结果：10株KP对5种FQNs均产生了耐药性，检测扩增产物序列后发现10株KP染色体的gyrA、parC基因均有突变，有2株菌株带有gyrA，以上2菌株的接合菌对FQNs的MIC值上升了5～30倍;未检测出qnrB阳性的菌株。
结论：gyrA、parC基因突变是诱导KP对FQNs产生耐药性的住院原因，质粒上有gyrA基因吸附，也是形成耐药性的主要因素之一。
【关键词】肺炎克雷伯杆菌;氟喹诺酮类;药物耐药性;机制分析【中图分类号】R978.1 【文献标识码】A 【文章编号】1672-3783（2019）12-03--01FQNs是一类以萘啶酸为基础、人工合成的抗生素，在细菌感染类疾病临床治疗中有广泛应用。
其作用机制是通过有目的性抑制细菌DNA回旋酶与拓扑异构酶Ⅳ的形式，对细菌DNA 复制、转录与修复重组过程形成有效抑制，最后中断细菌的传代过程[1]。
伴随本药品的广泛使用，随即出现的便是耐药性问题。
KP是一种较为常见的条件致病菌，其发病率仅位居大纲杆菌与铜绿假单胞菌。
本文主要对KP的耐FQNs进行分析与研究。
1 材料与方法1.1 材料选择在2018.6～2019.5期间经临床分离的10株对环丙沙星耐药（MIC≥32μg/ml）的菌株，所有菌株均采用API系统与ATB系统鉴定到种、实验质控菌株是KP ATCC700603，结合试验受体菌是大肠埃希菌j53RifR。
细菌耐药性的分子机制与防治措施细菌耐药性是指细菌对抗生素及其他药物的抵抗力，是当今公共卫生领域的一大难题。
随着抗生素的广泛使用和滥用，细菌耐药性不断加剧。
很多病菌如金葡菌、大肠杆菌等已经产生了多重耐药，甚至无药可治。
下面我们来探讨一下细菌耐药性的分子机制和防治措施。
一、分子机制1.基因水平的变异细菌在繁殖过程中，由于DNA复制和修复过程中出现的错误或环境压力等原因，导致其基因序列发生变异，从而产生新的基因。
一些突变体表现出对抗生素的耐药性，并能通过基因水平的传递来将这种耐药性传递给后代。
这种基因水平的耐药性是细菌耐药性的一种重要机制。
2.质粒介导的传递质粒是细菌细胞外面积为常染色体的小环状DNA分子。
质粒可以携带多种抗生素耐药基因，通过与宿主菌的染色体结合，形成可移植的耐药质粒，传递耐药性。
3.核糖体保护蛋白的合成核糖体是细胞内生产蛋白质的重要组成部分。
抗生素可以通过影响细菌细胞的核糖体的正常功能，使细菌无法维持其正常生理功能并死亡。
然而，一些细菌通过合成核糖体保护蛋白来避免抗生素对核糖体的干扰，从而保持其存活和繁殖的能力。
4.多药泵的表达多药泵是一种可将化学物质从细胞内向外排出的蛋白质，它在细菌耐药性的形成中起到了重要的作用。
多药泵可以通过从细胞内排放抗生素来降低抗生素在细菌细胞内的浓度，从而减少抗生素的杀菌效果。
多药泵表达的增加是细菌对多种抗生素产生耐药的重要机制之一。
二、防治措施1.抗生素合理应用抗生素是用来治疗感染性疾病的药物，而不是预防疾病。
我们应该严格按照医师开具的用药指示，不要滥用抗生素，避免抗生素的滥用和过度使用。
2.控制感染预防是细菌耐药性最重要、最经济的策略之一。
通过控制感染，可以降低细菌耐药性的发生率。
我们应该保持手卫生，妥善贮藏食物，保持清洁卫生等措施来减少感染的风险。
3.开展监测和调查实时监测耐药菌株的变化，对于制定和调整治疗策略具有重要意义。
建立相关数据库，可及时收集和传递有关耐药性新信息，及时生产有力、有效的抗生素。
铜绿假单胞菌耐药与多重抗药性机制解析铜绿假单胞菌是一种常见的病原菌，具有广泛的耐药性和多重抗药性。
了解铜绿假单胞菌的耐药和抗药机制对于防治感染疾病具有重要意义。
本文将通过对铜绿假单胞菌的耐药和多重抗药性机制进行详细解析。
1. 铜绿假单胞菌的耐药机制铜绿假单胞菌的耐药机制主要包括药物内流、药物靶标突变、药物排出通道改变以及药物降解等多种方式。
首先，铜绿假单胞菌具有药物内流通道的改变能力。
细菌通过改变药物通道的结构和功能来限制药物进入细胞，从而减少药物的有效浓度。
比如，铜绿假单胞菌可通过调节外膜蛋白的表达量和构象来限制药物进入细胞。
其次，铜绿假单胞菌可以发生药物靶标的突变，从而降低药物对其的结合亲和力。
这种突变可能导致药物分子无法与目标蛋白结合，从而失去药物的作用。
例如，铜绿假单胞菌的GyrA蛋白具有突变位点，可导致青霉素等β-内酰胺类抗生素的失效。
此外，铜绿假单胞菌还可以改变药物排出通道，从而增加药物外流的速率。
这主要通过调节药物外排泵的表达量和功能来实现。
铜绿假单胞菌具有多种外排泵，如MexAB-OprM和MexXY-OprM等，这些泵能有效地将药物从细胞内排出，从而使得细菌对药物具有更高的耐药性。
最后，铜绿假单胞菌还可以通过药物的降解来降低药物的有效浓度。
菌体表面的酶可以降解一些抗生素，从而使得药物无法发挥作用。
此外，铜绿假单胞菌还可以产生β-内酰胺酶等药物降解酶，从而使得广谱抗生素失去活性。
2. 铜绿假单胞菌的多重抗药性机制多重抗药性是指细菌对多种抗生素产生耐药性的能力。
铜绿假单胞菌往往具有复杂的多重抗药性机制，其主要包括快速突变、基因水平的水平转移和基因调控的变化等。
首先，铜绿假单胞菌具有高度的突变率，导致其对不同抗生素产生快速适应和耐药。
这种快速突变能力使得铜绿假单胞菌能够在长期的抗生素选择压力下迅速适应新的环境，从而产生多重抗药性。
其次，铜绿假单胞菌具有基因水平的耐药基因转移能力。
细菌可以通过水平基因转移将耐药基因传递给其他菌株，从而扩大耐药菌株的范围。
药物耐药机制与对策药物耐药是指细菌、病毒或其他微生物对药物产生抗性，导致药物失去原有的疗效。
药物耐药是一个全球性的问题，严重影响了人类的健康和医疗治疗效果。
本文将探讨药物耐药的机制以及对策。
一、药物耐药机制1.基因突变：细菌、病毒等微生物通过基因突变来产生耐药性。
基因突变可以导致药物靶点的结构改变，使药物无法与其结合，从而失去疗效。
2.药物代谢：某些微生物可以通过改变药物的代谢途径来降解药物，从而减少药物的疗效。
3.药物外排：微生物可以通过增加药物外排泵的表达来将药物排出细胞外，从而减少药物在细胞内的浓度，降低药物的疗效。
4.药物靶点变化：微生物可以通过改变药物的靶点结构来降低药物的结合能力，从而减少药物的疗效。
二、药物耐药对策1.合理使用抗生素：抗生素是治疗细菌感染的重要药物，但滥用和不合理使用抗生素是导致细菌耐药的主要原因之一。
合理使用抗生素包括准确诊断感染类型、选择适当的抗生素、正确使用抗生素剂量和疗程等。
2.开发新的药物：随着微生物耐药性的不断增强，现有的药物已经失去了对某些细菌、病毒的疗效。
因此，开发新的药物是解决药物耐药问题的关键。
科学家们需要不断研究和开发新的药物，以应对不断变化的微生物耐药性。
3.联合用药：联合用药是指同时使用两种或多种药物来治疗感染。
联合用药可以减少单一药物对细菌、病毒的选择压力，降低耐药性的发生。
4.加强监测和控制：及时监测和控制药物耐药性的发生是预防和应对药物耐药的重要手段。
医疗机构和卫生部门应建立健全的监测体系，及时发现和报告耐药性的发生情况，并采取相应的控制措施。
5.加强宣传教育：药物耐药是一个复杂的问题，需要全社会的共同努力来解决。
宣传教育可以提高公众对药物耐药的认识和理解，引导公众正确使用药物，减少滥用和不合理使用药物的行为。
三、结语药物耐药是一个严重影响人类健康的问题，需要全球范围内的合作来解决。
通过合理使用药物、开发新的药物、联合用药、加强监测和控制以及加强宣传教育等对策，我们可以有效预防和应对药物耐药性的发生，保障人类的健康和医疗治疗效果。
A NTIMICROBIAL A GENTS AND C HEMOTHERAPY,Feb.2008,p.385–392Vol.52,No.2 0066-4804/08/$08.00ϩ0doi:10.1128/AAC.01617-06Copyright©2008,American Society for Microbiology.All Rights Reserved.MINIREVIEWQuinolone-Mediated Bacterial DeathᰔKarl Drlica,1*Muhammad Malik,1Robert J.Kerns,2and Xilin Zhao1 Public Health Research Institute,New Jersey Medical School,University of Medicine and Dentistry of New Jersey, 225Warren St.,Newark,New Jersey07103,1and Division of Medicinal and Natural Products Chemistry,University of Iowa,Iowa City,Iowa522422Thefluoroquinolones are broad-spectrum antibacterial agents that are becoming increasingly popular as bacterial re-sistance erodes the effectiveness of other agents(fluoroquin-olone sales accounted for18%of the antibacterial market in 2006)(41).One of the attractive features of the quinolones is their ability to kill bacteria rapidly,an ability that differs widely among the various derivatives.For example,quinolones differ in rate and extent of killing,in the need for aerobic metabolism to kill cells,and in the effect of protein synthesis inhibitors on quinolone lethality.Understanding the mechanisms underlying these differences could lead to new ways for identifying the most bactericidal quinolone derivatives.Before describing the types of damage caused by the quin-olones,it is useful to define lethal activity.Operationally,it is the ability of drug treatment to reduce the number of viable cells,usually measured as CFU on drug-free agar after treat-ment.This assay is distinct from measurements that detect inhibition of growth(e.g.,MIC),since with the latter bacteria are exposed to drug throughout the measurement.The distinc-tion between killing and blocking growth is important because it allows susceptibility determinations to be related to partic-ular biological processes.For example,inhibition of growth is typically reversed by the removal of drug,while cell death is not.Thus,biochemical events associated with blocking growth should be readily reversible,while those responsible for cell death should be difficult to reverse.Reversibility can be used to distinguish among quinolone derivatives and assign functions to particular aspects of drug structure.Moreover,protective functions,such as repair and stress responses,can be distin-guished by whether their absence affects inhibition of growth, killing,or both.The intracellular targets of the quinolones are two DNA topoisomerases:gyrase and topoisomerase IV.Gyrase tends to be the primary target in gram-negative bacteria,while topo-isomerase IV is preferentially inhibited by most quinolones in gram-positive organisms(28).Both enzymes use a double-strand DNA passage mechanism,and it is likely that quinolone biochemistry is similar for both.However,physiological differ-ences between the enzymes exist,some of which may bear on quinolone lethality.In the present minireview we consider cell death through a two-part“poison”hypothesis in which the quinolones form reversible drug-topoisomerase-DNA complexes that subse-quently lead to several types of irreversible(lethal)damage. Other consequences of quinolone treatment,such as depletion of gyrase and topoisomerase IV activity,are probably less immediate(42).To provide a framework for considering quin-olone lethality,we begin by briefly describing the drug-topo-isomerase-DNA complexes.Readers interested in a more comprehensive discussion of quinolones are referred to a pre-viously published work(28).QUINOLONE-TOPOISOMERASE-DNA COMPLEXES As a normal part of their reaction mechanism,gyrase and topoisomerase IV introduce a pair of staggered,single-strand breaks(nicks)into DNA and become covalently bound to the 5Јends of the cleaved DNA(55,57).Quinolones bind rapidly to enzyme-DNA complexes(35),probably before DNA cleav-age occurs(Fig.1,step b1);drug binding occurs with mutant gyrase(gyrA)or topoisomerase IV(parC)that fails to cleave DNA(9,54).After drug binding,a slower,DNA cleavage-associated step occurs(35)that results in drug-mediated inhi-bition of religation of the DNA ends by topoisomerases(1).In a sense,quinolones trap the bacterial type II topoisomeraseson DNA(17,23,73,75)(Fig.1,step b2).The resulting struc-tures have been termed cleavable,cleavage,and cleaved com-plexes.In the present study we refer to them as cleaved com-plexes to emphasize that phosphodiester bonds in the DNA moiety are broken(the term ternary complex is reserved for the early step in drug-enzyme-DNA complex formation in which the DNA is unbroken).A variety of quinolone-mediated phenomena follow from formation of cleaved complexes.To better understand lethal processes,we briefly describe key fea-tures of the DNA and protein components of the complexes. Since crystal structures have not been reported for cleaved complexes,tentative inferences concerning the relative posi-tions of drug,protein,and DNA are drawn from biochemical experiments,partial structures of the bacterial topoisomerases, and complete structures of eukaryotic topoisomerase II. Evidence for the DNA being cleaved derives from the re-covery of DNA fragments when cleaved complexes are treated with protein denaturants,such as sodium dodecyl sulfate (SDS)(Fig.1,step g).These DNA fragments are covalently bound to the GyrA or ParC proteins(55,57).While cleaved complexes preferentially form at particular sites on DNA(44,*Corresponding author.Mailing address:Public Health Research Institute,New Jersey Medical School,University of Medicine and Dentistry of New Jersey,225Warren St.,Newark,NJ07103.Phone: (973)854-3360.Fax:(973)854-3101.E-mail:drlica@.ᰔPublished ahead of print on27August2007.38548,60),the nucleotide sequence requirement is loose,and the fragment size is small relative to the whole chromosome (4,7,16,73).Consequently,quinolone treatment creates many DNA-break-containing complexes throughout the chromo-some.Cleaved complex formation is reversed by the removal of quinolone,by the addition of the chelating agent EDTA,or by mild thermal treatment.In each case,the topoisomerase re-seals the DNA nicks,and intact DNA is seen after treatment with SDS (17,53,64,73,75).Since chromosomal DNA super-coiling is not relaxed by bacteriostatic quinolone treatment (73)nor is plasmid DNA superhelicity altered when quino-lone-gyrase complexes are reversed by EDTA (44),the DNA ends must be constrained in the complexes.The reversible nature of the cleaved complexes emphasizes that they are not by themselves lethal.The location and conformation of DNA in the complexes is poorly defined.However,examination of quinolone binding suggests that gyrase and topoisomerase IV may perturb DNA structure (54),perhaps by forming a single-stranded bubble that facilitates quinolone binding (44).Preferential binding of quinolones to single-stranded DNA correlates with in vitro quinolone potency (i.e.,the 50%inhibitory concentration),while binding to double-stranded DNA does not (59).These data are consistent with the observation that norfloxacin bind-ing to negatively supercoiled DNA is saturable (70),since negative supercoils confer some single-stranded character.Binding of quinolone to circular,relaxed DNA occurs only when gyrase,which by itself binds quinolones poorly,is also present to form complexes (71).Then saturable binding re-quires ATP or a nonhydrolyzable analogue,and it correlates with DNA cleavage and inhibition of gyrase-mediated DNA supercoiling.Quinolone binding to protein probably involves GyrA and ParC helix-4,which is located near the DNA gate region (Fig.2).Amino acid substitutions causing the greatest decrease in susceptibility are located there (24,36,39,43,56,84).More-over,amino acid substitutions in helix-4reduce quinolone binding (3),and replacing the helix-4-containing region of ParC with that of GyrA (61)or the Escherichia coli GyrA helix-4with the comparable region from Staphylococcus aureus (74)causes quinolone sensitivity to more closely resemble that of the replacing region.The orientation of quinolone binding to helix-4can be in-ferred from interactions between particular amino acids and quinolone substituents.For example,a strain of Mycobacterium smegmatis containing a cysteine substitution at the N terminus of GyrA helix-4is less susceptible to quinolones in which the C-7ring contains an ethyl attached to the distal nitrogen (PD161144,Fig.3)than to compounds in which the ethyl is attached to the side of the C-7ring (PD161148,Fig.3)(72).Bacterial strains with other amino acid substitutions in helix-4fail to distinguish the two fluoroquinolones.These dataindi-FIG.1.Schematic representation of quinolone action with gyrase as the primary target.(Step a)Binding of gyrase to DNA.(Step b)Reversible formation of quinolone-gyrase-DNA complexes that rap-idly block DNA replication.Step b 1depicts binding of quinolone to gyrase-DNA complexes before DNA cleavage;step b 2represents bind-ing after DNA cleavage.(Step c)Inhibition of replication leads to induction of the SOS response and cell filamentation.(Step d)Lethal chromosome fragmentation that requires ongoing protein synthesis in aerobic conditions,as seen with nalidixic acid treatment of E.coli .(Step e)Lethal chromosome fragmentation that requires on-going protein synthesis but not aerobic conditions,as seen with norfloxacin treatment of E.coli .(Step f)Lethal chromosome fragmentation that requires neither ongoing protein synthesis nor aerobic conditions,as seen for PD161144with E.coli .(Step g)DNA breakage detected after treatment of cell lysates with an ionic detergent such as SDS.Not shown are effects on transcription.Question marks indicate uncer-tainty about slow death and the nature of the DNAends.FIG.2.Sketch of dimerized GyrA59fragment with DNA and pro-tein gates closed.GyrA-GyrA dimer interfaces are located along the axis of symmetry;arrows point to helix-4of each GyrA subunit and to positions of E.coli amino acid number 67.386MINIREVIEW A NTIMICROB .A GENTS C HEMOTHER .cate a negative interaction between an N-terminal cysteine of helix-4and the distal ethyl group of PD161144.Such an inter-action suggests that quinolones are oriented on the helix-4with the C-7ring pointing toward the N terminus of the helix,which would be toward the GyrA-GyrA (or ParC-ParC)dimer inter-face (Fig.4A).While additional work is required to firmly establish this orientation,it explains why a Gly81-to-Asp mu-tation near the N terminus of helix-4of E.coli GyrA confers resistance to ciprofloxacin but not to nalidixic acid (5),a quin-olone that lacks a C-7ring.The proposed orientation fits with ideas about lethal chromosome fragmentation,as discussed below.GyrB undergoes a conformational change (34)during the DNA strand-passage reaction that may bring distal regions of the protein toward the DNA gate region of GyrA (discussed in reference 22).Two amino acid substitutions in GyrB,D426N and K447E,interfere with drug binding and quinolone-medi-ated inhibition of DNA supercoiling,relaxation,and DNA cleavage (22).These two amino acid changes,which are asso-ciated with resistance (82,83),may be part of the drug-binding pocket.However,in the drug-free model of gyrase they are not located adjacent to the GyrA protein.It appears that D426and K447move to the DNA gate region during the GyrB confor-mational transition (15,22).In summary,the quinolones keep the DNA gate open in cleaved complexes,probably through binding to GyrA (ParC)helix-4,regions of GyrB (ParE),and DNA.Conformational energy associated with alterations in the structure of DNA andgyrase or topoisomerase IV may contribute to the complexes being reversible.INHIBITION OF DNA REPLICATIONOne of the consequences of cleaved complex formation is inhibition of nucleic acid biosynthesis.In the case of DNA replication,inhibition occurs within minutes of drug addition when gyrase is the target (20,73).When topoisomerase IV is the target,inhibition of replication occurs slowly (39).We begin by discussing situations involving gyrase;at the end of the section we consider topoisomerase IV-mediated effects.In cell-free systems containing gyrase,both quinolone bind-ing and DNA cleavage are required to block DNA replication (78).The replication fork movement then halts 10bp upstream from the DNA cleavage sites (78).Cleavage sites are also close to where replication fork blockage is seen in plasmid DNA extracted from norfloxacin-treated cells (64).Thus,gyrase-me-diated inhibition of DNA synthesis appears to arise from col-lision of replication forks with cleaved complexes,which ex-plains correlations between inhibition of DNA synthesis and cleaved complex formation in drug-treated cells(73).FIG.3.Quinolone structures.Positions in the core ring structure are numbered forciprofloxacin.FIG.4.Fluoroquinolone orientation and cleaved-complex destabi-lization.(A)Proposed orientation of fluoroquinolone on helix-4of GyrA dimer.Fluoroquinolones are shown as arrows,with the arrow-heads representing the C-7ring moieties.Each cylinder represents the ␣-helix-4of a GyrA subunit (N and C indicate the amino-and carboxy-terminal orientations of helix-4,respectively).The dashed line indi-cates the axis of symmetry at the GyrA dimer interface.Elements of the figure are not drawn to scale.(B)Proposed chromosome fragmen-tation arising from cleaved complex destabilization by some fluoro-quinolones.Two GyrA subunits,shown as a dimer with GyrB omitted for clarity,are attached covalently to the 5Јend of cleaved DNA.Fluoroquinolone at a moderate concentration (arrow)binds to helix-4and traps gyrase on DNA.At higher fluoroquinolone concentration,the GyrA subunits separate,thereby fragmenting the chromosome.V OL .52,2008MINIREVIEW 387Rapid inhibition of DNA synthesis is reversible(20);thus,it cannot be the immediate cause of cell death.Consistent with this conclusion,inhibition of DNA synthesis correlates with bacteriostatic drug susceptibility(i.e.,the MIC)(8,63),while it fails to correlate with rapid cell death in terms of kinetics, quinolone concentration,or interference through the inhibi-tion of protein synthesis(7,53).Moreover,situations have been found in which quinolones block DNA replication and cell growth with only some derivatives killing rapidly(52). Finally,the lethal action of nalidixic acid and ciprofloxacin does not depend on the compounds actively blocking DNA replication:both drugs kill E.coli even after replication is inhibited by shifting a temperature-sensitive dnaB mutant to high temperature(85).Quinolone action contrasts with that of camptothecin,an inhibitor of eukaryotic topoisomerase I.For camptothecin, collision of replication forks with cleaved complexes generates lethal DNA breaks(31).Work with a cell-free system showed that this double-strand DNA breakage depends on the orien-tation of the complexes relative to the replication fork(76):in one orientation the topoisomerase-generated nick appears to be released from constraint by the replication fork,while in the other orientation it is not.In contrast,collision of replication forks and replication helicases with gyrase and topoisomerase IV trapped by quinolones fails to release DNA breaks(26,67, 68,78).The two bacterial enzymes appear to prevent replica-tion forks from reaching the trapped DNA nicks from either side(discussed in reference69).When a helicase forces its way through a quinolone-containing cleaved complex,as is seen with RuvAB,DNA nicks are reversed and not released from constraint(69).Thus,rapid quinolone-mediated cell death is probably not due simply to DNA breakage associated with collision of replication forks with cleaved complexes.A context is still needed for thefinding that replication-associated breaks occur in plasmid DNA after quinolone treat-ment.Pohlhaus and Kreuzer(64)developed an intracellular system in which plasmid replication is halted by high concen-trations of norfloxacin(20-fold MIC).Within6min after drug addition,a distinct cleaved form of plasmid DNA is observed that is not reversed when cells are incubated under resealing conditions(EDTA plus incubation at65°C).This cleaved DNA form is thought to arise from replication fork breakage,per-haps by an endonuclease acting on DNA extruded by a blocked replication fork(27,64).Such cleavage could provide RecBC with access to circular DNA,as required for induction of the SOS response(6,58).Association of replication-mediated cleavage with the lethal action of norfloxacin requires addi-tional work that considers downstream events requiring ongo-ing RNA and protein synthesis(29,53).With topoisomerase IV,the rate at which replication is in-hibited is50to100times slower than with gyrase(39).The difference is generally explained by topoisomerase IV function-ing behind replication forks(38),while gyrase is probably ahead of them(13,38).Creation of additional replication forks through deletion of seqA renders topoisomerase IV-mediated inhibition rapid,as if topoisomerase IV behind one fork is ahead of another that in the mutant started prematurely(38). Thus,it is likely that cleaved complexes containing topoisomer-ase IV are capable of blocking replication forks(38,68),but inhibition is slow due to topoisomerase IV location.Neverthe-less,topoisomerase IV can be the primary quinolone target. With gram-positive bacteria such as S.aureus,in vitro reactions with norfloxacin and gyrase fail to block replication fork move-ment(25).That explains why norfloxacin inhibits DNA repli-cation largely through topoisomerase IV with S.aureus(16). While most data argue against rapid cell death arising from quinolone-mediated inhibition of DNA replication,blocked replication forks could stimulate secondary events that kill cells slowly.Indeed,when drug treatment is long(overnight with E. coli),cell death can occur at quinolone concentrations that are only twice MIC(rapid killing generally requires concentrations 5to10times the MIC).Slow killing,which is poorly under-stood mechanistically,is commonly expressed by the parame-ter called the minimal bactericidal concentration.In the fol-lowing sections we mention two types of secondary damage that might contribute to quinolone-mediated cell death.We then consider chromosome fragmentation,which has been studied only with gyrase.SOS RESPONSE AND CELL FILAMENTATIONA consequence of quinolone treatment is the induction of the SOS regulon,a set of more than30genes controlled by the lexA repressor(37).One of the genes induced is sfiA(sulA), which encodes an inhibitor of cell division that causes E.coli cells to form long,filamentous structures(12,19).Filamenta-tion has been thought to contribute to quinolone lethality, sincefilamentation is maximal at quinolone concentrations that give maximal killing(63)and since sfiA mutants that reducefilamentation are reported to the lower killing rate for several quinolones(62).However,sfiA-dependentfilamenta-tion is clearly reversible(49).Thus,filamentation is unlikely to be a direct cause of rapid quinolone-mediated lethality(12). Whetherfilamentation and other consequences of the SOS response contribute to slow death has not been studied.The availability of lexA mutations that prevent induction of the SOS response allows the contribution of the response to be examined with various quinolone derivatives.For the potent fluoroquinolones,rapid lethality is increased by a lexA IndϪallele,while under the same conditions the lexA mutation has no effect on nalidixic acid-dependent killing(30,45).More-over,nalidixic acid lethality shifts from being unaffected by the lexA IndϪmutation in a gyrAϩbackground to being10-fold enhanced in a gyrA mutant thought to have an altered GyrA-GyrA interface(53).Thus,the quinolones appear to produce at least two types of lethal damage that can be distinguished by protective elements of the SOS response.TOXIN-ANTITOXIN MODULES AND PROGRAMMEDCELL DEATHToxin-antitoxin modules may also contribute to quinolone lethality.These modules appear to be part of the bacterial stress response,acting when elimination of a short-lived anti-toxin allows the cognate toxin to interfere with the bacterial transcription-translation machinery(14,18).It has been sug-gested that activation of some toxins leads to programmed cell death,an irreversible series of events that causes the loss of colony-forming ability even after removal of the stressor(14, 21,65).Several types of stress result in toxin-mediated cell388MINIREVIEW A NTIMICROB.A GENTS C HEMOTHER.death.One is nalidixic acid treatment at500to1,000times the MIC for10min.A modest(90%)decrease in the number of CFU follows unless the chpAIK(mazEF)toxin-antitoxin mod-ule is absent(40).In wild-type cells,high-level expression of the antitoxin chp AI(mazE)reverses lethality if applied within 90min after drug treatment,but antitoxin is ineffective if its expression is induced later.Thus,a critical time window for reversal exists.A similar behavior is observed with a variety of stresses,suggesting that the response is general.Although the 10-min treatment with nalidixic acid is probably sufficient to form cleaved complexes and to block DNA replication(73),it has not been established that the very high concentrations of nalidixic acid(21,40)cause lethal effects mediated only by the topoisomerases.Additional information is needed to relate toxin-mediated cell death to other lethal effects of quinolones. Generation of hydroxyl radicals,high levels of ATP,and depletion of NADH pools may also contribute to programmed cell death after norfloxacin treatment.Defects in several genes involved in pathways leading to these events(acnB,atpC,fur, icdA,and iscS)lower the lethal activity of norfloxacin,and deletion of sodB raises it.Since the sodB effect is slow while norfloxacin kills E.coli rapidly,production of oxidative species is likely to be a secondary effect associated with a variety of lethal antimicrobials.It is not known whether these events occur when norfloxacin lethality is blocked by inhibitors of protein synthesis or when quinolones kill anaerobically(see below).CHROMOSOME FRAGMENTATIONSince cleaved complexes are distributed throughout the chromosome,the release of DNA breaks from constraint is expected to fragment the chromosome into small pieces.Initial support for this hypothesis came from a chromosome super-coiling study.When E.coli is treated with concentrations of oxolinic acid sufficient to form cleaved complexes and block DNA replication,DNA supercoils are observed in isolated nucleoids(73).However,if quinolone concentrations are raised to levels that kill cells rapidly,supercoils are absent from nucleoids,even in the presence of an intercalating dye that would otherwise introduce supercoils(7).Since nucleoid su-percoiling is partitioned into many topologically independent domains(11,81),the inability to maintain supercoiling is most easily explained by widespread chromosome fragmentation (release of DNA breaks from constraint).When inhibition of protein synthesis blocks quinolone-mediated lethality,it also blocks quinolone-mediated loss of supercoiling(7).Thus,chro-mosome fragmentation correlates with cell death.More direct assays of chromosome fragmentation now sup-port the supercoiling experiments.One involves measuring cell lysate viscosity after the treatment of bacteria with quinolone. With E.coli,gently prepared cell lysates exhibit little viscosity; subsequent heating unfolds the nucleoids and causes lysates to become very viscous.If chromosomes are fragmented by quin-olone action,lysates would fail to become viscous when heated (53).Another assay involves sedimentation of DNA into su-crose density gradients:broken DNA sediments very slowly. To minimize DNA aggregation,sucrose gradients contain SDS,which would artificially release constrained breaks(Fig. 1,step g);consequently,sedimentation experiments generally involve preincubation of lysates under resealing conditions (addition of EDTA)to identify irreversible breaks.Both viscosity and sedimentation measurements reveal that nalidixic acid causes irreversible chromosome fragmentation with kinetics similar to cell death and much slower than inhi-bition of DNA replication(53).Additional correlations are revealed by two environmental perturbations:anaerobic growth and inhibition of protein synthesis by chloramphenicol. Forfirst-generation quinolones,both perturbations block le-thal action and chromosome fragmentation(51,53).However, neither prevents cleaved complex formation,as shown by the presence of DNA breakage when cell lysates are treated with SDS.Thus,quinolone lethality correlates with chromosome fragmentation,while DNA breaks can be found in cleaved complexes even when cells are not killed.The inhibitory effect of chloramphenicol on killing by nali-dixic acid and norfloxacin suggests that a suicide factor is involved(Fig.1,steps d and e).Such a factor is likely to be short-lived,since the lethal action of nalidixic acid is rapidly blocked by chloramphenicol or by a shift to anaerobiosis(10, 51).Although mutants have been obtained that block nalidixic acid lethality without affecting bacteriostatic action(80;X. Zhao,unpublished observation),the genes involved have proven difficult to identify.Suicide factors are unlikely to be part of the SOS response because a lexA IndϪmutation can have no effect on lethality caused by nalidixic and oxolinic acids (45,53).The lethal activity of norfloxacin is also blocked by chloram-phenicol(29).However,unlike nalidixic acid,norfloxacin kills cells after anaerobic shock(51)or after suspension of cells in cold saline(29).Moreover,lethal action is increased if the SOS response is blocked(30).Thus,norfloxacin action(Fig.1,step e)is distinct from that of nalidixic acid(Fig.1,step d).The molecular basis of this difference has not been determined.DESTABILIZATION OF CLEAVED COMPLEXES The chromosome fragmentation described above,which de-pends on a suicide factor,cannot be the only way that quino-lones kill cells rapidly because manyfluoroquinolones are le-thal even in the absence of protein synthesis(7,46,47,53).As pointed out above,the lethal activity of potentfluoroquino-lones is increased by a lexA IndϪallele,unlike the activity of first-generation quinolones(30,45).Thus,at least two modes of rapid killing must exist:one that requires ongoing protein synthesis(Fig.1,steps d and e)and one that does not(Fig.1, step f).It has been suggested that step f involves destabilization of cleaved complexes,since that would not require ongoing protein synthesis(7,53).A prediction of the destabilization hypothesis is that chromosome fragmentation will be observed in cell-free systems.Such is the case when isolated nucleoids are incubated with purified gyrase plus gatifloxacin(53).A potential outcome of cleaved complex destabilization is dissociation of GyrA subunits attached to the ends of DNA (Fig.4B).Such an idea emerged from studies of quinolone-stimulated illegitimate recombination(32,33,66),a phe-nomenon that is best explained by gyrase subunit dissocia-tion-reassociation(this concept also appears to apply to interactions of eukaryotic topoisomerase II with certain anti-tumor agents[2]).If quinolones stimulate dissociation of GyrAV OL.52,2008MINIREVIEW389subunits,an amino acid substitution located on the GyrA-GyrA dimer interface might promote it.This prediction was tested by examining an E.coli mutant in which Ala-67(Fig.2) is replaced with Ser.This amino acid substitution,which con-fers a modest fourfold increase in MIC,allows nalidixic acid to kill E.coli and fragment chromosomes in the presence of chloramphenicol(53).With wild-type cells both events are blocked by chloramphenicol.Moreover,when isolated nucle-oids are incubated with mutant gyrase,nalidixic acid causes chromosome fragmentation,which is not seen with wild-type gyrase and nalidixic acid(53).Finally,a noninducible lexA mutation renders E.coli hypersusceptible to nalidixic acid in the gyrA A67S mutant,a phenomenon not observed with gyrAϩcells(53).Thus,the mutation appears to shift lethal activity from step d in Fig.1to step f,perhaps by the alanine-to-serine change weakening hydrophobic interactions between the GyrA subunits.The connection between cleaved complex destabilization and chloramphenicol-insensitive killing(Fig.1,step f)allows conclusions to be drawn about effects of quinolone structure. For example,the N-1cyclopropyl group is probably important for cleaved complex destabilization,since this moiety is the only difference between ciprofloxacin,a compound that kills in the presence of chloramphenicol,and norfloxacin,a compound that does not(29,51).A C-8-methoxy group can also be im-portant,since it increases chloramphenicol-insensitive killing offluoroquinolones with E.coli(87),S.aureus(86),M.smeg-matis(52),and M.tuberculosis(50).A third factor is the struc-ture of the C-7substituent.With mycobacteria,the large C-7 ring system of moxifloxacin(Fig.3)increases chloramphenicol-insensitive lethality relative to that of another C-8-methoxy compound,gatifloxacin(50).With E.coli,the C-7N-ethyl piperazine of PD161144(Fig.3)confers greater chloramphen-icol-insensitive lethal activity than a C-7C-ethyl derivative (PD161148[53]).We speculate that the quinolone C-7rings point toward each other across a GyrA-GyrA interface(Fig. 4A)and that particular C-7rings have a negative interaction. Such an interaction might destabilize cleaved complexes and ultimately lead to gyrase subunit dissociation(Fig.4B).CONCLUDING REMARKSIt now appears that inhibition of replication is an unlikely source of rapid killing by the quinolones,since inhibition of replication is neither necessary nor sufficient for quinolone-mediated rapid cell death and since quinolone-mediated rep-lication inhibition and cell death fail to correlate with respect to kinetics,quinolone concentration,and the effects of protein synthesis inhibitors.Nor is blockage of RNA polymerase movement by cleaved complexes(79)likely to be lethal,since blockage does not fragment DNA(79)and since inhibition of RNA synthesis is protective(46).We propose chromosome fragmentation as an alternative source of rapid death.Accord-ing to this hypothesis,quinolone lethality can be described as a two-step process in which thefirst step is reversible(bacte-riostatic)formation of cleaved complexes.This step blocks bacterial DNA replication,induces the SOS response,and leads to cellfilamentation(Fig.1,pathway c).Although these events do not appear to be rapidly lethal,their involvement in slow death has not been ruled out.In a second,lethal step that requires higher quinolone concentrations,DNA breaks are released from constraint by at least two processes.One(Fig.1, steps d and e)requires protein synthesis;the other(Fig.1,step f)does not.The relative contribution of each pathway to cell death depends on quinolone structure,with the older quino-lones requiring protein synthesis and some newfluoroquino-lones functioning without it.This dichotomy between steps d and f extends to fragmentation of nucleoids in vitro and to protective effects of the SOS response.Norfloxacin(step e) appears to represent an intermediate situation in which some properties are similar to step d and others are similar to step f. How norfloxacin-mediated replication fork breakage(64)fits into these categories is not currently understood.The chromosome fragmentation hypothesis requires much more testing:proteins involved in steps d and e of Fig.1need to be identified,the cell-free test for step f needs to be ex-tended to plasmid systems that can be readily studied,and the effects of high drug concentration,including the paradoxical loss of lethality at very high concentrations(10),need to be explained in molecular terms.Nevertheless,the scheme in Fig. 1has implications for developing new quinolones.For exam-ple,the distinction between blocking growth(Fig.1,step b) and cell death(Fig.1,steps c,d,and e)means that a compound with a low MIC is not necessarily highly lethal.Indeed,a ciprofloxacin dimer blocks growth of mycobacteria while fail-ing to kill,even at a high concentration(52).The distinction also means that quinolones can be compared for lethal activity independent of drug uptake,efflux,and the ability to form cleaved complexes:by normalizing lethality to MIC,substitu-ents that preferentially increase killing can be identified.Fi-nally,quinolones that kill via pathway f may be lethal with nongrowing bacterial cells.Such a feature could be particularly important with pathogens,such as M.tuberculosis,that enter a dormant state in which they become tolerant to many antimi-crobials(77).ACKNOWLEDGMENTSWe thank M.Gennaro,H.Hiasa,A.Maxwell,R.Pine,and anony-mous reviewers for critical comments on the manuscript.This study was supported by NIH grants AI35257,AI073491,and AI063431.REFERENCES1.Anderson,V.E.,R.P.Zaniewski,F.S.Kaczmarek,T.D.Gootz,and N.Osheroff.1999.Quinolones inhibit DNA religation mediated by Staphylo-coccus aureus topoisomerase IV.J.Biol.Chem.274:35927–35932.2.Asami,Y.,D.Jia,K.Tatebayashi,K.Yamagata,M.Tanokura,and H.Ikeda.2002.Effect of the DNA topoisomerase II inhibitor VP-16on illegitimate recombination in yeast chromosomes.Gene291:251–257.3.Barnard,F.,and A.Maxwell.2001.Interaction between DNA gyrase andquinolones:the effect of alanine mutations at A subunit residues Ser-83and Asp-87.Antimicrob.Agents Chemother.45:1994–2000.4.Bejar,S.,and J.Bouche.1984.The spacing of Escherichia coli DNA gyrasesites cleaved in vivo by treatment with oxolinic acid and sodium dodecyl sulfate.Biochimie66:693–700.5.Cambau,E.,F.Borden,E.Collatz,and L.Gutmann.1993.Novel gyrA pointmutation in a strain of Escherichia coli resistant tofluoroquinolones but not to nalidixic acid.Antimicrob.Agents Chemother.37:1247–1252.6.Chaudhury,A.M.,and G.R.Smith.1985.Role of Escherichia coli RecBCenzyme in SOS induction.Mol.Gen.Genet.201:525–528.7.Chen,C.-R.,M.Malik,M.Snyder,and K.Drlica.1996.DNA gyrase andtopoisomerase IV on the bacterial chromosome:quinolone-induced DNA cleavage.J.Mol.Biol.258:627–637.8.Chow,R.,T.Dougherty,H.Fraimow,E.Bellin,and ler.1988.Asso-ciation between early inhibition of DNA synthesis and the MICs and MBCs of carboxyquinolone antimicrobial agents for wild-type and mutant[gyrA nfxB(ompF)acrA]Escherichia coli K-12.Antimicrob.Agents Chemother.32:1113–1118.390MINIREVIEW A NTIMICROB.A GENTS C HEMOTHER.。
