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qnr介导细菌对喹诺酮类耐药机制的研究
【作者】 王明华；
【作者基本信息】 复旦大学 ， 内科学， 2010， 博士
【摘要】 全球范围内革兰阴性菌对氟喹诺酮类抗菌药耐药性的上升引起了医学界的重视。传统的理解认为细菌对喹诺酮类的耐药是由于突变所致,耐药性为垂直传递。这难以解释为何原本非常敏感的微生物迅速产生了耐药,且细菌常同时对喹诺酮类及其他类别抗菌药耐药。最近发现的质粒介导喹诺酮类耐药机制可部分解释耐药率上升迅速的原因。目前发现的质粒介导耐药机制有三种：1.五肽重复序列蛋白Qnr：保护DNA旋转酶免受喹诺酮类药物的抑制,此类蛋白很可能来源于水生细菌。2.AAC(6’)-Ib-cr:一种氨基糖肽类乙酰转移酶,可以修饰环丙沙星及诺氟沙星而使其抗菌活性下降。这是首次发现自然界存在的酶修饰完全人工合成药物。3.外排泵QepA和OqxaAB。这三种机制都可以导致细菌对喹诺酮类药物敏感性下降,进而促进细菌产生更高水平的耐药。本研究对qnr新基因、qnr基因的表达调控及其在霍乱弧菌对喹诺酮类耐药性形成中的作用进行了研究,发现了一个新的qnr基因(qnrC),发现qnrB受SOS的调控,明确了qnrVC3与染色体的靶位突变共同促进了霍乱弧菌对喹诺酮类耐药性的上升,本研究有助于我们进一步了解细菌对喹诺酮类耐药性的形成机制,指导临床合理应用喹诺酮类药物。研究背景：自1998年发现第一个质粒介导喹诺酮类耐药基因-qnrA以来,国际上又陆续报道了另外两个qnr基因-qnrB和qnrS。这三个耐药基因在世界范围内临床分离菌中广泛分布,但不同基因间的同源性低,我们推测临床株中尚存在未被发现的新的qnr基因。在此部分研究中,我们在肠杆菌科细菌临床株中进行新的喹诺酮类耐药基因筛选,以期发现新的耐药基因。材料与方法：收集复旦大学附属华山医院2005-2007年临床分离的肠杆菌科细菌。通过临床菌株与耐叠氮钠的大肠埃希菌J53接合试验,筛选对环丙沙星敏感性下降的接合子。提取接合子的质粒,并对质粒进行酶切克隆分析和测序,确定引起敏感性下降的基因。通过PCR方法检测新qnr基因在临床分离菌中的流行情况。结果：在尿路感染患者的尿标本中分离到的一株奇异变形杆菌对氨苄西林、磺胺甲基异噁唑、甲氧苄啶耐药外对大多数抗菌药物敏感。这株细菌中含有的质粒pHS10,可以通过接合转移至大肠埃希菌J53。含有pHSl0的大肠埃希菌J53对环丙沙星敏感性下降,但是通过PCR检测并未发现已知的qnr基因、aac(6’)-Ib-cr和qepA。奇异变形杆菌临床株和含pHS10 J53接合子的环丙沙星最低抑菌浓度(MIC)均为0.25μg／ml,较受体菌J53有32倍的上升。为进一步确定引起喹诺酮类敏感性下降的基因,对pHS10通过多种限制性内切酶消化后与质粒载体pUC18连接,连接后的产物转化至大肠埃希菌TOP10,通过氨苄西林和环丙沙星选择转化子。通过对转化子质粒的测序分析,发现了由HindⅢ酶切后的一个4.4kb的DNA片段可以介导喹诺酮类耐药。测序分析和进一步的克隆证明引起耐药的是其中一个含有666碱基对的基因,按照国际认可的命名原则,命名为qnrC。该基因编码221氨基酸的五肽重复序列蛋白QnrC。QnrC与QnrA1、QnrB1、QnrS1、QnrD相比,氨基酸同源性分别为64%、42%、59%、43%。QnrC基因的上游存在一个新的IS3家族的插入序列(insertion sequence, IS),命名为ISPmill,编码含有读码框移动的转座酶,推测这一结构与qnrC的摄入与传播有关。以PCR方法对2020株临床分离的肠杆菌科细菌进行检测,未发现qnrC基因。结论：在这部分研究中,在奇异变形杆菌中发现了新的质粒介导喹诺酮类耐药基因qnrC。研究背景：通过DNA结构分析发现,在qnrB基因的上游含有一个LexA结合位点。而这一位点在qnrA或qnrS周边则不存在。LexA蛋白是细菌SOS调控系统中的重要组成部分。在大肠埃希菌中,RecA刺激LexA的分解,可诱导40多种基因表达的SOS调控系统。细菌籍SOS调控系统对DNA损伤做出反应。已知喹诺酮类药物和丝裂霉素等可以损伤细菌DNA,诱导SOS反应。该部分研究旨在了解qnrB的表达是否受SOS系统的调控,此研究有利于理解Qnr蛋白的原始功能。材料与方法：大肠埃希菌GWl000,含有recA411基因,编码的RecA蛋白酶更容易被激活；大肠埃希菌AB1157,具有完整的SOS调控系统；AB1157LexA300::spec,其LexA蛋白有缺陷,不能与LexA结合位点结合；DM49,其LexA蛋白不能被蛋白酶水解。将qnrB1、qnrB2、qnrB3、qnrB4质粒和qnrA1、qnrS1质粒导入大肠埃希菌GW1000或J53,并测定不同温度下环丙沙星对这些菌株的MIC。以环丙沙星或丝裂霉素为诱导剂,用实时定量PCR方法检测qnr基因在诱导情况下的表达。在LexA蛋白有缺陷的菌株中重复诱导表达实验。使用管家基因mdh为实时定量PCR内对照。结果：含有不同亚型的qnrB质粒的GW1000菌株在培养温度从21℃升高到43℃时,对环丙沙星的敏感性降低2到8倍。含有qnrA1和qnrS1质粒的GW1000在温度升高时有2到3倍的敏感性降低。在具有野生型的SOS调控系统的大肠埃希菌J53中,温度升高时细菌对环丙沙星敏感性降低2倍。上述现象提示qnrB在SOS系统调控之下。qnrB在大肠埃希菌J53(具有完整的lexA和recA基因)中表达可受到诱导因素(环丙沙星、丝裂霉素)影响上升2.1至9.9倍,而qnrA1的表达则不受诱导。在含有野生型lexA和recA基因的大肠埃希菌AB1157内,qnrB4的表达也可被环丙沙星和丝裂霉素诱导,但是在LexA蛋白变异的ABll57LexA300::spec和DM49菌株内则不能被诱导。结论：与qnrA和qnrS不同,质粒上的qnrB基因在SOS系统调控之下,qnrB的诱导表达需要完整的SOS系统。研究背景：因为霍乱弧菌对很多抗菌药物的耐药上升,在很多地区,环丙沙星用于治疗霍乱弧菌感染。但是随着时间的推移,霍乱弧菌对环丙沙星敏感性下降,环丙沙星的临床疗效随之降低。我们对一个腹泻中心分离的霍乱弧菌耐药机制进行了研究,以明确qnr基因在耐药性形成过程中的影响。材料与方法：霍乱弧菌来源于一个腹泻研究国际中心2002至2008年的临床分离菌株。随机选择了16株对环丙沙星的敏感性水平不同的霍乱弧菌,首先用Etest测定氨苄西林、环丙沙星、庆大霉素、左氧氟沙星、萘啶酸、链霉素、四环素、甲氧苄啶和甲氧苄啶／磺胺甲基异噁唑对这些菌株MIC。应用PCR检测喹诺酮类耐药决定区突变和已知的质粒介导喹诺酮类耐药基因。以大肠埃希菌J53为受体菌进行接合试验。提取供体菌和接合子的质粒,以Southern印迹实验对水平传播耐药基因定位。使用反向PCR方法,对耐药基因周边结构进行分析。结果：根据MIC水平将16株霍乱弧菌分为3组,第一组(MIC 0.023-0.032μg／ml),第二组(MIC 0.38-0.5μg/ml),第三组(MIC>0.5μg/ml)。第一组菌株含有gyrA突变Ser83Ile,第二组菌株和第三组菌株除gyrA突变外,含有parC突变Ser85Leu,这两种突变都位于喹诺酮类耐药决定区域(QRDR)。株第二组菌株和所有的第三组菌株含有一个新的qnrVC基因亚型,其编码的蛋白与QnrVC1相差11个氨基酸,命名为qnrVC3。qnrVC3可通过接合转移至大3肠埃希菌。将qnrVC3克隆至表达载体pQE60并转化至大肠埃希菌J53可使其环丙沙星的MIC上升16-32倍。提取qnrVC3阳性的霍乱弧菌全基因组DNA对qnrVC3进行Southern印迹,对qnrVC3的周边结构进行测序,发现qnrVC3位于染色体的整合接合元件上(integrated conjugative element, ICE)。结论：霍乱弧菌对喹诺酮类药物耐药水平逐渐上升与喹诺酮类靶位突变的累积和位于可移动元件上的qnrVC3基因有关,这给霍乱的抗菌治疗带来了更大的挑战。
【Abstract】 Fluoroquinolone resistance has been increasing in Gram-negative pathogens worldwide. The traditional understanding that quinolone resistance is acquired only through mutation and transmitted only vertically does not entirely account for the relative ease with which resistance develops in exquisitely susceptible organisms, or for the very strong association between resistance to quinolones and to other agents. The recent discovery of plasmid-mediated horizontally transferable genes encoding quinolone resistance might shed light on these phenomena. The Qnr proteins, capable of protecting DNA gyrase from quinolones, have homologues in water-dwelling bacteria, and seem to have been in circulation for some time, having achieved global distribution in a variety of plasmid environments and bacterial genera. AAC(6’)-Ib-cr, a variant aminoglycoside acetyltransferase capable of modifying ciprofl oxacin and reducing its activity, seems to have emerged more recently, but might be even more prevalent than the Qnr proteins. Two plasmid-mediated quinolone transporters have now been found:OqxAB and QepA. Plasmid-medicated quinolone resistance mechanisms provide low-level quinolone resistance that facilitates the emergence of higher-level resistance in the presence of quinolones at therapeutic levels. Much remains to be understood about these genes, but their insidious promotion of substantial resistance, their horizontal spread, and their co-selection with other resistance elementsindicate that a more cautious approach to quinolone use and a reconsideration of clinical breakpoints are needed.Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6_)-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25μg/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindⅢ, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP 10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%,42%,59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.Plasmid-mediated Qnr proteins provide low-level quinolone resistance and protect bacterial DNA gyrase and topoisomeraseⅣfrom quinolone inhibition. QnrA, QnrB, and QnrS are currently known. All are pentapeptide repeat proteins differing from each other by 40% or more in amino acid sequence, while within each type minor variations in sequence define alleles such as QnrB1 and QnrB2. In addition to protecting DNA gyrase, QnrB1 (but not QnrA1) at high concentrations has been shown to inhibit the enzyme in vitro, which may explain the bacterial growth inhibition observed when the gene is maximally expressed. We have discovered that qnrB is regulated by the SOS system so that quinolone exposure augments its expression. To determine whether expression of qnrB alleles is under SOS control, plasmids were introduced into Escherichia coli GW1000 with recA441, which encodes a RecA protease that is more easily activated. GW1000 derivatives containing plasmids with qnrB alleles demonstrated two-to eightfold decreases in ciprofloxacin susceptibility as the growth temperature increased from 21℃to 43℃. A two-to threefold decrease in susceptibility was also seen in strains with plasmids carrying qnrA1 or qnrS1 alleles. In E. coli J53 with unmodified SOS regulation, temperature had only a twofold effect on the level of qnrB1-mediated ciprofloxacin resistance. While the trend observed suggested that qnrB alleles are specifically regulated by the SOS system, the MIC results were not clear-cut because of a background effect of temperature on quinolone susceptibility. To document SOS regulation directly, the expression of qnr genes was measured by real-time quantitative PCR after a 15-to 30-min exposure to agents known to trigger the SOS response. In E. coli J53 with intact lexA and recA genes, expression of qnrB alleles increased between 2.1-and 9.9-fold in response to the inducing agents while expression of qnrA1 was unchanged. Proof that this increase in qnrB expression required an intact SOS system was obtained with a set of related strains. Expression of qnrB4 increased in response to ciprofloxacin or mitomycin C in E. coli AB1157 with wild-type lexA and recA genes but not in two strains derived from it:strain AB1157 LexA300::spec, which has a defective LexA protein so that LexA-regulated genes are constitutively expressed, or strain DM49, which has a protease-resistant LexA product and consequently is defective in SOS induction. SOS regulation of QnrB could be a carryover reflecting a role for this topoisomerase-interacting protein in response to DNA damage. Alternatively, SOS regulation serves to protect the host cell from the potentially toxic effects of QnrB while allowing augmented production upon exposure to quinolone antimicrobial agents.Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of the high resistance rates to other agents, but its utility has been compromised by decreasing ciprofloxacin susceptibility of Vibrio cholerae over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in gyrA, encoding a subunit of DNA gyrase, followed by mutation in parC, encoding a subunit of DNA topoisomeraseⅣ. We found that ciprofloxacin activity was more recently further compromised in strains containing qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that qnrVC3 confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae and on the chromosome of Escherichia coli transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in V. cholerae are linked to cumulative mutations in quinolone targets and most recently to a qnr gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.