尿路致病性大肠埃希菌持留相关机制的研究
|
摘要
细菌感染治疗领域,目前面临以下两大问题。一方面,全球抗菌药物使用负荷不断增大,不合理应用甚至滥用抗菌药物现象突出,日益严峻的细菌耐药性问题成为全球关注的焦点。细菌耐药性往往由于存在某些耐药基因或固有基因发生突变而引起,细菌不仅可将耐药性遗传给子代细菌,而且可通过基因水平转移而造成耐药性的广泛播散。耐药细菌引起的感染已构成抗感染治疗的新挑战,人们正逐渐陷入“无药可用”的窘境。另一方面,临床细菌感染治疗过程中发现,某些病原菌,体外药敏试验显示对抗菌药敏感,但在体内可耐受药物的抗菌作用,并可躲避宿主的免疫防御体系,在宿主体内长期存在,难以被彻底清除,成为持续感染、慢性感染或反复发作性感染的“原凶”,比如结核病,葡萄球菌菌血症,尿路致病性大肠埃希菌(Uropathogenic Escherichia coli, UPEC)引起的反复发作性尿路感染等,这一问题又带来“有药无效”的困惑。
对抗菌药物敏感的细菌难以被清除的这一特性称之为持留(persistence)。广义而言,与细菌粘附相关的致病因子、生物膜的形成以及形成持留菌(persister)等因素,使得细菌难以被清除,均称为“持留”。狭义的“持留”则是特指持留菌能耐受高浓度抗菌药物以及各种环境应激因素而存活的能力。基因型完全相同的菌群中的很少一部分细菌亚群能耐受大剂量抗菌药物作用而存活下来,而在脱离抗菌药物的环境后又可繁殖,这一细菌亚群的子代仍保持对该抗菌药物的敏感性,这些存活下来的细菌称之为持留菌。本文题目中的“持留”取其广义含义,主要包括与粘附因子相关以及与phoU基因相关的持留。
尿路感染(Urinary tract infection, UTI)是最常见的感染性疾病之一,女性发病率远高于男性。据统计,60%女性一生至少发生一次尿路感染,而25%急性膀胱炎的患者可反复发作。大肠埃希菌是尿路感染最常见的病原菌,70-95%的社区获得性尿路感染和约50%的医院获得性尿路感染都由UPEC引起。在众多致病因子中,粘附因子被认为与UPEC难以被清除有关。UPEC的粘附因子种类繁多,主要包括1型菌毛、P型菌毛、S/F1C菌毛、Afa/Dr粘附因子家族,以及某些与生物膜形成相关的粘附因子等。其中Afa/Dr粘附因子Afa可通过与泌尿道上皮细胞表面特异性受体的相互作用,介导细菌进入宿主细胞内,躲避宿主对细菌的清除和部分抗菌药物的杀菌作用；而进入胞内的细菌可大量繁殖,随着泌尿道上皮更新脱落,胞内大量细菌释放,从而引起尿路感染再次发作,故认为携带afa基因的UPEC容易引起尿路感染的反复发作。而粘附因子PapG,由于其受体主要分布于肾脏,有研究认为其主要与急性肾盂肾炎关系密切。flu基因编码的粘附因子Ag43主要参与生物膜的形成,从而亦可使菌体难以被清除。
除了粘附因子外,UPEC持留菌的形成,亦是病原菌难以被清除的一个重要方面。关于细菌形成持留的机制,至今尚不明确,目前普遍认为是与持留菌低代谢状态有关。2007年有研究在大肠埃希菌W3110菌株中发现了与持留相关的新基因——phoU,插入突变使该基因失活后,突变株能量代谢相关基因的表达上调,细菌处于高代谢状态,而对许多抗菌药物以及各种应激状态的持留能力明显下降,突变恢复后,菌株持留能力得以恢复。本课题组比对已公布的UPEC菌株phoU基因序列与野生株W3110phoU基因序列发现：W3110phoU第236位氨基酸为甘氨酸G,而UPEC菌株,包括UTI189与CFT073、536等的phoU第236位突变为谷氨酸E。本课题第三部分探讨该氨基酸位点的突变对UPEC持留能力的影响。
基于上述研究背景,本课题收集华山医院2008年1月至2009年12月尿路感染住院患者分离培养的UPEC47株,以及从健康志愿者肛拭子标本培养分离大肠埃希菌26株,进行菌株耐药性、同源性分析、粘附因子基因以及phoU基因相关持留机制的研究,探索临床尿路感染反复发作,UPEC菌株难以清除的机制；为开发新型尿路感染治疗药物提供理论基础。本研究内容包括以下三部分。
第一部分UPEC临床株与肠道定植大肠埃希菌药物敏感性试验与同源性分析
我们收集了2008年1月至2009年12月住院尿路感染患者尿液标本中分离的47株UPEC,并从健康志愿者肛拭子标本分离培养26株肠道定植大肠埃希菌作为对照。用琼脂稀释法测定哌拉西林、头孢唑啉、头孢呋辛、头孢噻肟、头孢他啶、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、亚胺培南、美罗培南、庆大霉素、阿米卡星、环丙沙星、左氧氟沙星和呋喃妥因等15种抗菌药物对两组菌株的最低抑菌浓度(Minimal Inhibitorty Concentration, MIC)。根据2010年版CLSI (Clinical and Laboratory Standard Institute)药敏判断标准判定结果,统计两组菌株对各种抗菌药物的敏感率和耐药率。
药敏结果显示,UPEC对大多数抗菌药物的敏感性明显低于肠道定植E.coli菌株(P<0.05)。特别是对于临床常用的氟喹诺酮类药物(环丙沙星和左氧氟沙星),UPEC的敏感率仅为23%,而肠道定植菌株的敏感率为96%；而对头孢噻肟,UPEC的敏感率仅为32%,而肠道定植菌为89%。但对哌啦西林/他唑巴坦、亚胺培南、美罗培南、阿米卡星和呋喃妥因等药物,UPEC仍有较高的敏感率(敏感率均在89%-100%之间)。
根据2009年CLSI的规定,对头孢噻肟MIC≥4μm/m1,头孢他啶MIC≥16μg/ml的菌株,用CTX和CTX/CV,或CAZ和CAZ/CV双纸片进行产ESBLs菌株确证试验。结果47株UPEC中产ESBLs菌株为28株(60%),而在26株肠道定植大肠埃希菌菌株中发现1株产ESBLs菌株(4%)。产ESBLs菌株对头孢噻肟的耐药率为100%,而对头孢他啶的耐药率为50%。用PCR方法对28株UPEC菌株进行bla-CTX-M分型,其中14株(50%)属于CTX-M-1组,12株(43%)属于CTX-M-9组,CTX-M-1组菌株对头孢他啶的耐药率(86%)显著高于CTX-M-9组(17%)。
从大肠埃希菌MLST分型官方网站查询并获取七个管家基因adk、fumC、 gyrB、icd、mdh、pur A和recA的引物序列和PCR实验方案,扩增47株UPEC菌株和26株肠道定植菌株上述7个管家基因片段,比对获取等位基因编码及ST型别,用eburst软件分析菌株同源性并作图。除5株UPEC和4株肠道定植大肠埃希菌同属ST10型以及1株UPEC和1株肠道定植大肠埃希菌同属ST453外,其他UPEC菌株MLST分型与肠道定植大肠埃希菌菌株完全不同。本研究47株UPEC菌株中最多见的ST型为国际流行大肠埃希菌株ST131型(9株,19.1%),其次依次为ST405(7株,14.9%),ST10(5株,10.6%),ST393(4株,8.5%),ST69(4株,8.5%)以及ST95(3株,6.4%)。26株肠道定植大肠埃希菌菌株来自于22个不同ST型,分布更为分散,其中18个ST型在大肠埃希菌MLST官方网站资料库已有记录,另发现4个新的ST型。ST10型在肠道定植大肠埃希菌中最为常见(4株,15.4%),其次为一新发现ST型(2株,7.7%),其余20株均属于20个不同的ST分型。
第二部分UPEC与正常人肠道定植大肠埃希菌粘附因子相关基因分析
根据患者临床资料,47株UPEC菌株分为反复发作性下尿路感染组(21例)、急性肾盂肾炎组(7例)和急性单纯性膀胱炎组(19例)。用PCR方法检测三组UPEC菌株以及健康志愿者肠道定植大肠埃希菌粘附因子基因afa、dra、daa、 papG、flu、fimH、sfa和focG的携带情况。
①共有6株UPEC菌株afa基因阳性,且该6株UPEC均属于反复发作性下尿路感染UPEC菌株组(29%),在健康人肠道定植大肠埃希菌菌株未检测到afa基因携带株(P=0.004)。在所有实验菌株中均未发现携带draE或daaE基因的菌株。②47株UPEC菌株中11株(23%)papG基因阳性,其中10株为papGⅡ型,1株为papGⅢ型,另有1株肠道定植大肠埃希菌为papGⅡ型。papG基因在急性肾盂肾炎UPEC中的检出率达71%,显著高于反复发作性下尿路感染组(14%)和急性单纯性膀胱炎组(16%)(P=0.005)。③80.9%UPEC菌株与50.0%肠道定植菌株生物膜相关粘附因子flu基因阳性(P=0.006)。但3组UPEC菌株flu基因携带率无显著差异。@UPEC中fimH基因检出率为89.4%,而肠道定植菌株则为76.9%(P=0.155)。⑤反复发作性下尿路感染组、急性肾盂肾炎组和单纯性膀胱炎组UPEC菌株中各有一株为sfa基因阳性,其中有一株同时携带有focG基因。而健康中肠道定殖大肠埃希菌未检测到sfa/foc基因。
UPEC菌株携带粘附因子基因数多于肠道定植大肠埃希菌。76.6%的UPEC菌株携带2-3种粘附因子基因,而80.8%的肠道定植大肠埃希菌携带1-2种粘附因子基因。有1株(2.1%)UPEC菌株携带5种粘附因子基因,1株(2.1%)携带4种,但也有2株UPEC(4.3%)未检测到粘附因子基因。
产ESBLs UPEC菌株携带粘附因子数少于非产ESBLs菌株。28株产ESBLs UPEC菌株中有2株(7%)未检出任何粘附因子,6株(21%)检出1种粘附因子,15株(54%)携带2种粘附因子,4株(14%)携带3种粘附因子,另有1株(4%)有4个粘附因子基因为阳性。而19株不产ESBLs UPEC菌株,仅含1种粘附因子基因的菌株为1株(5%),分别有8株(42%)和9株(47%)携带2种和3种粘附因子基因,另有1株(5%)含有5种粘附因子基因。故产ESBLs UPEC菌株的大多数(80%)所含粘附因子数目不多于2种,而超过一半(52%)的不产ESBLs UPEC菌株携带至少3种粘附因子基因(P=0.012)。
第三部分UPEC临床分离株phoU基因相关持留机制研究
为研究临床分离UPEC菌株phoU基因相关持留机制,本部分研究首先用PCR方法扩增比对47株UPEC临床分离株和26株肠道定植大肠埃希菌phoU基因全长序列。47株UPEC菌株中10株(2l%)为phoU基因野生型(同W3110),37株(79%)phoU基因为突变型,其中20株为phoUG236E突变型(同CFT073、UTI89等UPEC菌株),而17株为phoUS239P+D240K+K241E突变型。而26株肠道定植大肠埃希菌均为phoU基因野生型。
UPEC菌株MLST型别与phoU基因型存在相关性。9株ST131UPEC菌株均为phoUG236E突变型,7株ST405和4株ST393UPEC菌株均为phoUS239P+D240K+K241E突变型,而5株ST10UPEC菌株则均为phoU基因野生型。另外,papG基因阳性菌株均为phoU突变型,且phoU突变型UPEC菌株所携带的粘附因子基因数多于phoU基因野生型UPEC菌株。但phoU基因型不同的UPEC菌株在引起尿路感染的类型和对主要抗菌药物的敏感性等方面无明显差异。
为进一步了解phoU基因突变型,尤其是phoUG236E突变型UPEC菌株与phoU野生型UPEC菌株持留能力的差异,本研究用杀菌试验的方法,研究phoUG236E突变型UPEC菌株与phoU野生型UPEC菌株,在三种抗菌药物头孢他啶、阿米卡星和环丙沙星较高浓度作用下的持留情况。①头孢他啶2×或4xMIC作用30小时后,phoU野生型UPEC菌落计数降至0,而phoUG236E突变型菌落计数仍有1-5×101CFU/ml；改用8×MIC头孢他啶作用,在实验观察时间(56h)内phoUG236E突变型和phoU野生型菌株均能被杀灭,但phoUG236E突变型UPEC菌株被完全杀灭所需时间(44h)明显长于phoU野生型UPEC菌株(20h)。②阿米卡星4xMIC作用20h后,完全杀灭phoU野生型UPEC菌株,而phoUG236E突变型UPEC菌落计数仍有1-7.5×101CFU/ml；增加药物浓度至8xMIC,phoUG236E突变型UPEC亦能被完全杀灭,但时间(30h)明显较phoU野生型(4h)延长。③环丙沙星8×MIC作用30h后,phoU野生型UPEC被杀灭完全,而phoUG236E突变型仍有1-3.5×101CFU/ml；而增加药物浓度于16xMIC,phoUG216E突变型可在药物作用44h后被完全杀灭,而在药物作用后4h,phoU野生型UPEC菌落计数已降为零。所以,在抗菌药物作用下,phoUG236E突变型UPEC菌株的持留能力较phoU野生型UPEC菌株持留能力增加。
为进一步探讨在抗菌药物作用下,phoUG236E突变型UPEC菌株的持留能力较phoU野生型UPEC菌株持留能力增加的机制,本研究用相对荧光定量PCR的方法测定在抗菌药物作用下phoUG236E突变型和phoU野生型UPEC菌株phoU基因相对表达量(以管家基因mdh为内参基因)。结果显示：4×MIC头孢他啶、4×MIC阿米卡星和8×MIC环丙沙星作用12h后,phoUG236E突变型UPEC菌株phoU基因相对表达量是phoU野生型UPEC菌株的6.2、20.6和6.8倍,即在上述三种抗菌药物作用下,phoU G236E突变型phoU基因表达量水平均相对更高。上述结果提示可能由于phoU G236E突变型在抗菌药物作用下phoU表达量较phoU野生型UPEC菌株高,从而增强前者的持留能力,但具体机制仍不明确。
Two major problems have arisen in the field of antimicrobial therapy. One is the emergence of heritable antibiotic resistance and their spread by horizontal gene transfer. With the growing consumption burden and the irrational use or even abuse of antimicrobials, the increasingly serious problem of antimicrobial resistance has finally caught the global attention. Some pathogens have become resistant to most of the commonly used antibiotics. Therefore, when the infectious diseases caused by these "superbugs" occur, the patients and doctors are inevitably trapped into "no drug available" dilemma. On the other hand, the persistence of bacterial infections, in which the pathogens linger in the host for long periods of time in spite of prolonged treatment, has made another big deal to the antimicrobial therapy. Most of the persistent pathogens are susceptible to antibiotics in the in-vitro test of drug susceptibility, yet leaving the difficult problem of "no drug effective" in vivo. Some well-known examples are tuberculosis, lung infections in patients with cystic fibrosis, typhoid fever and urinary tract infections.
Apart from the formation of biofilm and the virulence factors, such as adhesins as the most important ones, the persistence property of persister makes the great contribution to the bacterial long-term stay with the host, recalcitrant to therapeutic agents and host defense mechanisms. The persisters are a small fraction, of the order of10"6or less, of genetically identical bacterial cells that can survive the exposure to lethal concentrations of bactericidal antibiotics and regrow after removal of the antibiotics, with the descendants as sensitive to the same antibiotics as the original population, again leaving behind a tiny fraction of survivors.
Urinary tract infections (UTIs), including cystitis and pyelonephritis, are the most common bacterial infections in women, which account for significant morbidity and substantial medical costs on individual, family and society. In terms of urinary anatomical differences between the genders, UTIs primarily affect women. It is estimated that the overall lifetime prevalence of UTIs in women is greater than60%. Further recurrent UTIs are reported in about25%of women within6months of an acute UTI episode and pose a major problem.
Uropathogenic Escherichia coli (UPEC) is the causative pathogen in70-95%community acquired UTIs and over50%nosocomial UTIs. Many virulence factors made great contributions to the pathogenesis of UPEC. And the presence of adhesins is arguably the most important determinant of the pathogenicity for UPEC. Adhesive organelles, including type1, P and S/F1C pili along with afa/Dr adhesins, mediate bacterial attachment to host tissues within the urinary tract. E. coli expressing Dr/Afa adhesins may predispose the establishment of chronic or recurrent infections. Adhesin PapG is thought to be associated with bacterial adhesion to the kidney, so as to be related to the acute pyelonephoritis. Adhesin Ag43, encoded by flu gene, enables bacterial cells to adhere to each other and form biofilm over the host urothelial cells, leading to a hard clearance of the pathogens.
Persisters also play an important role in the hard clearance of the UPEC. The mechanisms of persistence and persister generation are still to be understood. As pointed out early in1944, the suggestion that persisters are in a state of dormancy, which enables them to tolerate antibiotic exposure, has not changed with time. In2007, Zhang and his colleges identified in E. coli a new persistence gene phoU whose inactivation caused a metabolically hyperactive status of the cells with an increased expression of energy production genes, but a defect in persister formation when exposed to a diverse range of antibiotics and different kinds of stresses. In this study, we compared the phoU gene sequences of all the known E. coli strains published in the Genebank (http://www.ncbi.nlm.nih.gov/gene), and we identified an specific difference in the236th amino acid among some UPEC strains and the wild type W3110strains. In the W3110E. coli strain, there was glycine (G) in its236th amino acid locus, while glutamate(E) shown in the same locus within the noticed UPEC strains such as CFT073, UTI89,536and APEC01strains. We wondered if this amino acid mutation in the phoU gene of the UPEC strains evoked a variant role of PhoU played in the persistence of UPEC.
In the present study, a total of47non-duplicate clinical isolates of UPEC were collected from clean midstream urine specimens of inpatients with urinary tract infections at Huashan Hospital, Shanghai from January2008to December2009, and26intestinal commensal E. coli strains, as control, were isolated from the anal swab specimens of the health volunteers. The objectives are to understand the UPEC drug suscepibility and adhesin genes pattern, and to investigate phoU related persistence mechanism of the clinical UPEC strains. The study included the following three parts.
Part One Antimicrobial susceptibility testing and homology analysis of UPEC and intestinal commensal strains
The47clinical UPEC isolates were collected according to standard laboratory protocols and isolated from urine with>105colony-forming units/ml. Together with the26intestinal commensal E. coli strains as control, the minimum inhibit concentration of15kinds of antimicrobial drugs to all of the73E.coli strains was detected using the agar dilution method according to the2010updated version of the CLSI (Clinical and Laboratory Standard Institute) files. The15antimicrobials included piperacillin, cefazolin, cefuroxime, cefotaxime, ceftazidime, cefepime, piperacillin/tazobactam, cefoperazone/sulbactam, imipenem, meropenem, gentamycin, amikacin, ciprofloxacin, levofloxacin and nitrofurantoin. E. coli ATCC25922was used as quality control strain.
UPEC isolates demonstrated a remarkably lower susceptibility rates to most of the tested antimicrobials than the intestinal commensal strains. Especially, the sensitivity rate of UPEC to cefotaxime was only32%while89%of the commensal counterparts were susceptible to cefotaxime. Less than one quarter of the UPEC were sensitive to the fluoroquinolones (including ciprofloxacin and levofloxacin), yet the susceptibility rate of intestinal isolates were96%. Fortunately, there were still a relatively high susceptibility rate of UPEC to piperacillin/tazobactam, imipenem, meropenem, amikacin and nitrofurantoin (89%-100%).
We carried out disk diffusion ESBL confirmatory test to the isolates with a MIC to cefotaxime≥4μg/ml, or a MIC to cefotazidime>16μg/ml according to the2009version of the CLSI files. We found28ESBLs-producing strains(60%) among the47UPEC stains, while1(4%) out of26commensal isolates was also proved ESBLs-producing. All the ESBLs-producing strains were cefotaxime resistant, and50%were resistant to cefotazidime. CTX-M-1,2,8,9,25/26group specific genes of28ESBLs-producing UPEC were determined by PCR with the primers reported previously. BlaCTX-M-1type genes were detected in14(50%) isolates.12(43%) isolates harbored BlaCTX-M-9type genes. The cefotazidime resistance rate (86%) of UPEC isolates carrying blaCTX-M-1type genes were significantly higher than that (17%) of the isolates with blaCTX-M-9type genes.
The7housekeeping genes (adk、fumC、gyrB、icd、mdh、pur A and recA) for multilocus sequence typing (MLST) were selected from the E. coli database at the University College Cork E. coli MLST website (http://mlst.ucc.ie/mlst/dbs/Ecoli) and sequence type determination of the47UPEC and26commensal isolates was performed using the MLST PCR scheme accordingly. The based upon related sequence types (BURST) clustering algorithm (eburst.mlst.net) was used to analyze the allelic profiles and define clonal complexes.5UPEC and4commensal E. coli isolates shared the same type ST10, and one isolate respectively from the two of the groups belonged to ST453. In addition to that, the MLST of UPEC was thoroughly different from that of the intestinal commensal counterparts. The47UPEC isolates analyzed were assigned to18distinct STs. The most common STs were ST131(n=9,19.1%of isolates), followed by ST405(7,14.9%), ST10(5,10.6%), ST393(4,8.5%), ST69(4,8.5%), and ST95(3,6.4%). The six STs accounted for68.1%(32/47) of the isolates, demonstrating the diversity of lineages. The26isolates analyzed by MLST were grouped into22different STs, more diverse than the UPEC isolates. Of the22STs,18were already included in the E. coli website database, Among which ST10(n=4,15.4%) was the most popular group. And an unknown ST included2isolates, while the other20isolates were classified into the20STs, respectively.
Part two. Comparison of adhesin genes between uropathogenic and intestinal commensal Escherichia coli strains
Among the47UPEC isolates,21were from patients with recurrent lower UTIs. The recurrent lower UTIs were defined as at least two episodes of UTI within6month. And each time the patients had:a) typical clinical symptoms as urinary urgency and pain and frequent voiding, b) pyuria, c) without fever, chill or other systematic symptoms. Seven strains were isolated from acute pyelonephritis patients by the following criteria:a) the temperature>38.5℃, b) positive percussion pain on kidney area, c) an elevated leukocytes count and percentage of neutrophils in the blood routine test. The rest19strains were from patients with acute uncomplicated cystitis.
The presence of adhesin genes afa, draE, daaE, papGI, papGⅡ, papGⅢ, flu, fimH, sfa and foc were investigated by PCR amplification using primers sets as described previously.
①afa gene was present in6UPEC isolates, all of which belonged to the recurrent lower UTIs group (28.6%). None of the isolates from the acute pyelonephritis group or the acute uncomplicated cystitis group carried afa gene. And no afa gene was found in26intestinal commensal E. coli isolates. Neither draE nor daaE gene was detected among all the strains we screened.(2)11of the47UPEC isolates were papG gene positive (23.4%) including10papG Ⅱ and1papG Ⅲ, and1of the intestinal commensal isolates (3.8%) carried papG Ⅱ gene. Further more, the prevalence of papG in acute pyelonephritis (71.4%) was significantly higher than that of recurrent lower UTI group (14.3%) and that of acute uncomplicated cystitis group (15.8%).(3)80.9%of the UPEC isolates while50.0%of the intestinal commensal isolates were found biofilm related flu gene positive. However, the prevalence difference of the three UTI groups was not significant.(4)Gene fimH was screened positive in42of47(89%) UPEC isolates and20of26(76.9%) commensal isolates.⑤Sfa gene was present in one isolate each in the three UTI groups and one of the sfa positive isolates was also detected focG positive. But none isolate in the intestinal commensal group was screened sfa/foc positive.
Most of the UPEC strains (72.3%,34/47) carried2to3kinds of adhesin genes, while80.8%(21/26) of the commensal isolates had1to2kinds of adhesin genes. The difference of the adhesin gene numbers between UPEC and commensal isolates were statistically significant.
The ESBLs-producing UPEC carried fewer adhesin genes than the non-ESBLs-producing ones, of28ESBLs-producing UPEC,2(14%) were detected no adhesion genes,6(21%) carried1adhesin gene, and15(54%) were screened2kinds of adhesin genes. On the other hand, among the19non-ESBLs-producing UPEC isolates,1(5%) carried only one adhesin gene, and8(42%) and9(47%) isolates were respectively detected2and3kinds of adhesin genes, with the left carried5kinds of the detected genes. Therefore, most of the ESBLs-producing UPEC isolates carried no more than2adhesin genes, while over half of the non-ESBLs-producing UPEC were screened no less than3kinds.
Part Three, phoU gene related persistence mechanism of the clinical UPEC isolates
With the help of PCR and gene sequencing, we amplified and compared the complete phoU gene sequence of the47clinical UPEC isolates and26intestinal commensal E. coli strains.10of47(21%) UPEC was feathered with the same wild type phoU gene sequence as the strain W3110. The phoU gene sequences of79% (37/47) UPEC had amino acid differences from the wild type, so as to be called mutant phoU gene types in this study.20of the37mutant phoU UPEC carried the phoU G236E mutation and the rest17feathered with the same phoU S239P+D240K+K24JE mutation. Interestingly, all of the26intestinal commensal E. coli were phoU gene wild type.
The MLSTs of the phoU wild type UPEC isolates were totally different from those of the phoU mutant ones.9ST131UPEC were all phoU G236mutant strains,7ST405and4ST393UPEC belonged to the phoU S239P+D240K+K241E mutation type, while the5ST10UPEC all carried wild type phoU gene. The UPEC isolates with papG gene positive were all included in the mutant phoU gene group. Furthermore, the UPEC isolates with mutant phoU gene carried more adhesin genes than those with wild type phoU gene. However, there were no significant differences between the wild type phoU UPEC and the mutant type phoU UPEC, whether referring to the urinary tract infection types or the antimicrobial susceptibilities.
So as to study whether the phoU G236E mutation provoked a variant role of PhoU played in the persistence of UPEC, we performed the in vitro bactericidal test with phoU G236E mutant UPEC and phoU wild type UPEC. We chose3kinds of antimicrobials:cefotazidime, amikacin and ciprofloxacin. In the killing curve experiment, the UPEC with phoU G236E mutation showed increased ability of persistence to cefotazidime, amikacin and ciprofloxacin. Under the relatively low dose of the three antibiotics (2,4×MIC cefotazidime,4×MIC amikacin and8×MIC ciprofloxacin), the UPEC strain of wild type phoU gene was completely killed after20-30h, while3.5-7×101CFU/ml phoU G236E mutant isolate survived at the same time and remained no less than1×101CFU/ml until the56h after the administration of the antimicrobials. When shifted to the high concentration level of drugs (8×MIC cefotazidime,8×MIC amikacin and16×MIC ciprofloxacin), the colony counts of phoU gene wild type isolate sloped to zero as early as4h (20h for8×MIC cefotazidime), but yet the phoU G236E mutant isolate were not killed until30-44h exposure to the antimicrobials. Therefore, it was probable that the phoU G236E mutant isolates left more persisters and then more tolerant to antibiotics.
In order to understand how the phoU G236E mutation might be involved in the enforcement in persister formation of the UPEC as shown by increased tolerance to various antibiotics, we performed a relatively quantitative PCR of phoU gene under the treatment of cefotazidime, amikacin and ciprofloxacin respectively (we chose the housekeeping gene mdh as the endogenous control). The result indicated that after12h exposure to cefotazidime, amikacin and ciprofloxacin, the relative phoU gene expression level of phoU G236E mutant UPEC isolate was upregulated6.2,20.6and6.8-fold higher than the phoU wild type isolate, which suggested the higher level of phoU gene expression in the phoU G236E mutant isolate leading to a more powerful formation of persisters to the threat of the various antimicrobials. However, the excact mechanisms are still to be understood.
对抗菌药物敏感的细菌难以被清除的这一特性称之为持留(persistence)。广义而言,与细菌粘附相关的致病因子、生物膜的形成以及形成持留菌(persister)等因素,使得细菌难以被清除,均称为“持留”。狭义的“持留”则是特指持留菌能耐受高浓度抗菌药物以及各种环境应激因素而存活的能力。基因型完全相同的菌群中的很少一部分细菌亚群能耐受大剂量抗菌药物作用而存活下来,而在脱离抗菌药物的环境后又可繁殖,这一细菌亚群的子代仍保持对该抗菌药物的敏感性,这些存活下来的细菌称之为持留菌。本文题目中的“持留”取其广义含义,主要包括与粘附因子相关以及与phoU基因相关的持留。
尿路感染(Urinary tract infection, UTI)是最常见的感染性疾病之一,女性发病率远高于男性。据统计,60%女性一生至少发生一次尿路感染,而25%急性膀胱炎的患者可反复发作。大肠埃希菌是尿路感染最常见的病原菌,70-95%的社区获得性尿路感染和约50%的医院获得性尿路感染都由UPEC引起。在众多致病因子中,粘附因子被认为与UPEC难以被清除有关。UPEC的粘附因子种类繁多,主要包括1型菌毛、P型菌毛、S/F1C菌毛、Afa/Dr粘附因子家族,以及某些与生物膜形成相关的粘附因子等。其中Afa/Dr粘附因子Afa可通过与泌尿道上皮细胞表面特异性受体的相互作用,介导细菌进入宿主细胞内,躲避宿主对细菌的清除和部分抗菌药物的杀菌作用；而进入胞内的细菌可大量繁殖,随着泌尿道上皮更新脱落,胞内大量细菌释放,从而引起尿路感染再次发作,故认为携带afa基因的UPEC容易引起尿路感染的反复发作。而粘附因子PapG,由于其受体主要分布于肾脏,有研究认为其主要与急性肾盂肾炎关系密切。flu基因编码的粘附因子Ag43主要参与生物膜的形成,从而亦可使菌体难以被清除。
除了粘附因子外,UPEC持留菌的形成,亦是病原菌难以被清除的一个重要方面。关于细菌形成持留的机制,至今尚不明确,目前普遍认为是与持留菌低代谢状态有关。2007年有研究在大肠埃希菌W3110菌株中发现了与持留相关的新基因——phoU,插入突变使该基因失活后,突变株能量代谢相关基因的表达上调,细菌处于高代谢状态,而对许多抗菌药物以及各种应激状态的持留能力明显下降,突变恢复后,菌株持留能力得以恢复。本课题组比对已公布的UPEC菌株phoU基因序列与野生株W3110phoU基因序列发现：W3110phoU第236位氨基酸为甘氨酸G,而UPEC菌株,包括UTI189与CFT073、536等的phoU第236位突变为谷氨酸E。本课题第三部分探讨该氨基酸位点的突变对UPEC持留能力的影响。
基于上述研究背景,本课题收集华山医院2008年1月至2009年12月尿路感染住院患者分离培养的UPEC47株,以及从健康志愿者肛拭子标本培养分离大肠埃希菌26株,进行菌株耐药性、同源性分析、粘附因子基因以及phoU基因相关持留机制的研究,探索临床尿路感染反复发作,UPEC菌株难以清除的机制；为开发新型尿路感染治疗药物提供理论基础。本研究内容包括以下三部分。
第一部分UPEC临床株与肠道定植大肠埃希菌药物敏感性试验与同源性分析
我们收集了2008年1月至2009年12月住院尿路感染患者尿液标本中分离的47株UPEC,并从健康志愿者肛拭子标本分离培养26株肠道定植大肠埃希菌作为对照。用琼脂稀释法测定哌拉西林、头孢唑啉、头孢呋辛、头孢噻肟、头孢他啶、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、亚胺培南、美罗培南、庆大霉素、阿米卡星、环丙沙星、左氧氟沙星和呋喃妥因等15种抗菌药物对两组菌株的最低抑菌浓度(Minimal Inhibitorty Concentration, MIC)。根据2010年版CLSI (Clinical and Laboratory Standard Institute)药敏判断标准判定结果,统计两组菌株对各种抗菌药物的敏感率和耐药率。
药敏结果显示,UPEC对大多数抗菌药物的敏感性明显低于肠道定植E.coli菌株(P<0.05)。特别是对于临床常用的氟喹诺酮类药物(环丙沙星和左氧氟沙星),UPEC的敏感率仅为23%,而肠道定植菌株的敏感率为96%；而对头孢噻肟,UPEC的敏感率仅为32%,而肠道定植菌为89%。但对哌啦西林/他唑巴坦、亚胺培南、美罗培南、阿米卡星和呋喃妥因等药物,UPEC仍有较高的敏感率(敏感率均在89%-100%之间)。
根据2009年CLSI的规定,对头孢噻肟MIC≥4μm/m1,头孢他啶MIC≥16μg/ml的菌株,用CTX和CTX/CV,或CAZ和CAZ/CV双纸片进行产ESBLs菌株确证试验。结果47株UPEC中产ESBLs菌株为28株(60%),而在26株肠道定植大肠埃希菌菌株中发现1株产ESBLs菌株(4%)。产ESBLs菌株对头孢噻肟的耐药率为100%,而对头孢他啶的耐药率为50%。用PCR方法对28株UPEC菌株进行bla-CTX-M分型,其中14株(50%)属于CTX-M-1组,12株(43%)属于CTX-M-9组,CTX-M-1组菌株对头孢他啶的耐药率(86%)显著高于CTX-M-9组(17%)。
从大肠埃希菌MLST分型官方网站查询并获取七个管家基因adk、fumC、 gyrB、icd、mdh、pur A和recA的引物序列和PCR实验方案,扩增47株UPEC菌株和26株肠道定植菌株上述7个管家基因片段,比对获取等位基因编码及ST型别,用eburst软件分析菌株同源性并作图。除5株UPEC和4株肠道定植大肠埃希菌同属ST10型以及1株UPEC和1株肠道定植大肠埃希菌同属ST453外,其他UPEC菌株MLST分型与肠道定植大肠埃希菌菌株完全不同。本研究47株UPEC菌株中最多见的ST型为国际流行大肠埃希菌株ST131型(9株,19.1%),其次依次为ST405(7株,14.9%),ST10(5株,10.6%),ST393(4株,8.5%),ST69(4株,8.5%)以及ST95(3株,6.4%)。26株肠道定植大肠埃希菌菌株来自于22个不同ST型,分布更为分散,其中18个ST型在大肠埃希菌MLST官方网站资料库已有记录,另发现4个新的ST型。ST10型在肠道定植大肠埃希菌中最为常见(4株,15.4%),其次为一新发现ST型(2株,7.7%),其余20株均属于20个不同的ST分型。
第二部分UPEC与正常人肠道定植大肠埃希菌粘附因子相关基因分析
根据患者临床资料,47株UPEC菌株分为反复发作性下尿路感染组(21例)、急性肾盂肾炎组(7例)和急性单纯性膀胱炎组(19例)。用PCR方法检测三组UPEC菌株以及健康志愿者肠道定植大肠埃希菌粘附因子基因afa、dra、daa、 papG、flu、fimH、sfa和focG的携带情况。
①共有6株UPEC菌株afa基因阳性,且该6株UPEC均属于反复发作性下尿路感染UPEC菌株组(29%),在健康人肠道定植大肠埃希菌菌株未检测到afa基因携带株(P=0.004)。在所有实验菌株中均未发现携带draE或daaE基因的菌株。②47株UPEC菌株中11株(23%)papG基因阳性,其中10株为papGⅡ型,1株为papGⅢ型,另有1株肠道定植大肠埃希菌为papGⅡ型。papG基因在急性肾盂肾炎UPEC中的检出率达71%,显著高于反复发作性下尿路感染组(14%)和急性单纯性膀胱炎组(16%)(P=0.005)。③80.9%UPEC菌株与50.0%肠道定植菌株生物膜相关粘附因子flu基因阳性(P=0.006)。但3组UPEC菌株flu基因携带率无显著差异。@UPEC中fimH基因检出率为89.4%,而肠道定植菌株则为76.9%(P=0.155)。⑤反复发作性下尿路感染组、急性肾盂肾炎组和单纯性膀胱炎组UPEC菌株中各有一株为sfa基因阳性,其中有一株同时携带有focG基因。而健康中肠道定殖大肠埃希菌未检测到sfa/foc基因。
UPEC菌株携带粘附因子基因数多于肠道定植大肠埃希菌。76.6%的UPEC菌株携带2-3种粘附因子基因,而80.8%的肠道定植大肠埃希菌携带1-2种粘附因子基因。有1株(2.1%)UPEC菌株携带5种粘附因子基因,1株(2.1%)携带4种,但也有2株UPEC(4.3%)未检测到粘附因子基因。
产ESBLs UPEC菌株携带粘附因子数少于非产ESBLs菌株。28株产ESBLs UPEC菌株中有2株(7%)未检出任何粘附因子,6株(21%)检出1种粘附因子,15株(54%)携带2种粘附因子,4株(14%)携带3种粘附因子,另有1株(4%)有4个粘附因子基因为阳性。而19株不产ESBLs UPEC菌株,仅含1种粘附因子基因的菌株为1株(5%),分别有8株(42%)和9株(47%)携带2种和3种粘附因子基因,另有1株(5%)含有5种粘附因子基因。故产ESBLs UPEC菌株的大多数(80%)所含粘附因子数目不多于2种,而超过一半(52%)的不产ESBLs UPEC菌株携带至少3种粘附因子基因(P=0.012)。
第三部分UPEC临床分离株phoU基因相关持留机制研究
为研究临床分离UPEC菌株phoU基因相关持留机制,本部分研究首先用PCR方法扩增比对47株UPEC临床分离株和26株肠道定植大肠埃希菌phoU基因全长序列。47株UPEC菌株中10株(2l%)为phoU基因野生型(同W3110),37株(79%)phoU基因为突变型,其中20株为phoUG236E突变型(同CFT073、UTI89等UPEC菌株),而17株为phoUS239P+D240K+K241E突变型。而26株肠道定植大肠埃希菌均为phoU基因野生型。
UPEC菌株MLST型别与phoU基因型存在相关性。9株ST131UPEC菌株均为phoUG236E突变型,7株ST405和4株ST393UPEC菌株均为phoUS239P+D240K+K241E突变型,而5株ST10UPEC菌株则均为phoU基因野生型。另外,papG基因阳性菌株均为phoU突变型,且phoU突变型UPEC菌株所携带的粘附因子基因数多于phoU基因野生型UPEC菌株。但phoU基因型不同的UPEC菌株在引起尿路感染的类型和对主要抗菌药物的敏感性等方面无明显差异。
为进一步了解phoU基因突变型,尤其是phoUG236E突变型UPEC菌株与phoU野生型UPEC菌株持留能力的差异,本研究用杀菌试验的方法,研究phoUG236E突变型UPEC菌株与phoU野生型UPEC菌株,在三种抗菌药物头孢他啶、阿米卡星和环丙沙星较高浓度作用下的持留情况。①头孢他啶2×或4xMIC作用30小时后,phoU野生型UPEC菌落计数降至0,而phoUG236E突变型菌落计数仍有1-5×101CFU/ml；改用8×MIC头孢他啶作用,在实验观察时间(56h)内phoUG236E突变型和phoU野生型菌株均能被杀灭,但phoUG236E突变型UPEC菌株被完全杀灭所需时间(44h)明显长于phoU野生型UPEC菌株(20h)。②阿米卡星4xMIC作用20h后,完全杀灭phoU野生型UPEC菌株,而phoUG236E突变型UPEC菌落计数仍有1-7.5×101CFU/ml；增加药物浓度至8xMIC,phoUG236E突变型UPEC亦能被完全杀灭,但时间(30h)明显较phoU野生型(4h)延长。③环丙沙星8×MIC作用30h后,phoU野生型UPEC被杀灭完全,而phoUG236E突变型仍有1-3.5×101CFU/ml；而增加药物浓度于16xMIC,phoUG216E突变型可在药物作用44h后被完全杀灭,而在药物作用后4h,phoU野生型UPEC菌落计数已降为零。所以,在抗菌药物作用下,phoUG236E突变型UPEC菌株的持留能力较phoU野生型UPEC菌株持留能力增加。
为进一步探讨在抗菌药物作用下,phoUG236E突变型UPEC菌株的持留能力较phoU野生型UPEC菌株持留能力增加的机制,本研究用相对荧光定量PCR的方法测定在抗菌药物作用下phoUG236E突变型和phoU野生型UPEC菌株phoU基因相对表达量(以管家基因mdh为内参基因)。结果显示：4×MIC头孢他啶、4×MIC阿米卡星和8×MIC环丙沙星作用12h后,phoUG236E突变型UPEC菌株phoU基因相对表达量是phoU野生型UPEC菌株的6.2、20.6和6.8倍,即在上述三种抗菌药物作用下,phoU G236E突变型phoU基因表达量水平均相对更高。上述结果提示可能由于phoU G236E突变型在抗菌药物作用下phoU表达量较phoU野生型UPEC菌株高,从而增强前者的持留能力,但具体机制仍不明确。
Two major problems have arisen in the field of antimicrobial therapy. One is the emergence of heritable antibiotic resistance and their spread by horizontal gene transfer. With the growing consumption burden and the irrational use or even abuse of antimicrobials, the increasingly serious problem of antimicrobial resistance has finally caught the global attention. Some pathogens have become resistant to most of the commonly used antibiotics. Therefore, when the infectious diseases caused by these "superbugs" occur, the patients and doctors are inevitably trapped into "no drug available" dilemma. On the other hand, the persistence of bacterial infections, in which the pathogens linger in the host for long periods of time in spite of prolonged treatment, has made another big deal to the antimicrobial therapy. Most of the persistent pathogens are susceptible to antibiotics in the in-vitro test of drug susceptibility, yet leaving the difficult problem of "no drug effective" in vivo. Some well-known examples are tuberculosis, lung infections in patients with cystic fibrosis, typhoid fever and urinary tract infections.
Apart from the formation of biofilm and the virulence factors, such as adhesins as the most important ones, the persistence property of persister makes the great contribution to the bacterial long-term stay with the host, recalcitrant to therapeutic agents and host defense mechanisms. The persisters are a small fraction, of the order of10"6or less, of genetically identical bacterial cells that can survive the exposure to lethal concentrations of bactericidal antibiotics and regrow after removal of the antibiotics, with the descendants as sensitive to the same antibiotics as the original population, again leaving behind a tiny fraction of survivors.
Urinary tract infections (UTIs), including cystitis and pyelonephritis, are the most common bacterial infections in women, which account for significant morbidity and substantial medical costs on individual, family and society. In terms of urinary anatomical differences between the genders, UTIs primarily affect women. It is estimated that the overall lifetime prevalence of UTIs in women is greater than60%. Further recurrent UTIs are reported in about25%of women within6months of an acute UTI episode and pose a major problem.
Uropathogenic Escherichia coli (UPEC) is the causative pathogen in70-95%community acquired UTIs and over50%nosocomial UTIs. Many virulence factors made great contributions to the pathogenesis of UPEC. And the presence of adhesins is arguably the most important determinant of the pathogenicity for UPEC. Adhesive organelles, including type1, P and S/F1C pili along with afa/Dr adhesins, mediate bacterial attachment to host tissues within the urinary tract. E. coli expressing Dr/Afa adhesins may predispose the establishment of chronic or recurrent infections. Adhesin PapG is thought to be associated with bacterial adhesion to the kidney, so as to be related to the acute pyelonephoritis. Adhesin Ag43, encoded by flu gene, enables bacterial cells to adhere to each other and form biofilm over the host urothelial cells, leading to a hard clearance of the pathogens.
Persisters also play an important role in the hard clearance of the UPEC. The mechanisms of persistence and persister generation are still to be understood. As pointed out early in1944, the suggestion that persisters are in a state of dormancy, which enables them to tolerate antibiotic exposure, has not changed with time. In2007, Zhang and his colleges identified in E. coli a new persistence gene phoU whose inactivation caused a metabolically hyperactive status of the cells with an increased expression of energy production genes, but a defect in persister formation when exposed to a diverse range of antibiotics and different kinds of stresses. In this study, we compared the phoU gene sequences of all the known E. coli strains published in the Genebank (http://www.ncbi.nlm.nih.gov/gene), and we identified an specific difference in the236th amino acid among some UPEC strains and the wild type W3110strains. In the W3110E. coli strain, there was glycine (G) in its236th amino acid locus, while glutamate(E) shown in the same locus within the noticed UPEC strains such as CFT073, UTI89,536and APEC01strains. We wondered if this amino acid mutation in the phoU gene of the UPEC strains evoked a variant role of PhoU played in the persistence of UPEC.
In the present study, a total of47non-duplicate clinical isolates of UPEC were collected from clean midstream urine specimens of inpatients with urinary tract infections at Huashan Hospital, Shanghai from January2008to December2009, and26intestinal commensal E. coli strains, as control, were isolated from the anal swab specimens of the health volunteers. The objectives are to understand the UPEC drug suscepibility and adhesin genes pattern, and to investigate phoU related persistence mechanism of the clinical UPEC strains. The study included the following three parts.
Part One Antimicrobial susceptibility testing and homology analysis of UPEC and intestinal commensal strains
The47clinical UPEC isolates were collected according to standard laboratory protocols and isolated from urine with>105colony-forming units/ml. Together with the26intestinal commensal E. coli strains as control, the minimum inhibit concentration of15kinds of antimicrobial drugs to all of the73E.coli strains was detected using the agar dilution method according to the2010updated version of the CLSI (Clinical and Laboratory Standard Institute) files. The15antimicrobials included piperacillin, cefazolin, cefuroxime, cefotaxime, ceftazidime, cefepime, piperacillin/tazobactam, cefoperazone/sulbactam, imipenem, meropenem, gentamycin, amikacin, ciprofloxacin, levofloxacin and nitrofurantoin. E. coli ATCC25922was used as quality control strain.
UPEC isolates demonstrated a remarkably lower susceptibility rates to most of the tested antimicrobials than the intestinal commensal strains. Especially, the sensitivity rate of UPEC to cefotaxime was only32%while89%of the commensal counterparts were susceptible to cefotaxime. Less than one quarter of the UPEC were sensitive to the fluoroquinolones (including ciprofloxacin and levofloxacin), yet the susceptibility rate of intestinal isolates were96%. Fortunately, there were still a relatively high susceptibility rate of UPEC to piperacillin/tazobactam, imipenem, meropenem, amikacin and nitrofurantoin (89%-100%).
We carried out disk diffusion ESBL confirmatory test to the isolates with a MIC to cefotaxime≥4μg/ml, or a MIC to cefotazidime>16μg/ml according to the2009version of the CLSI files. We found28ESBLs-producing strains(60%) among the47UPEC stains, while1(4%) out of26commensal isolates was also proved ESBLs-producing. All the ESBLs-producing strains were cefotaxime resistant, and50%were resistant to cefotazidime. CTX-M-1,2,8,9,25/26group specific genes of28ESBLs-producing UPEC were determined by PCR with the primers reported previously. BlaCTX-M-1type genes were detected in14(50%) isolates.12(43%) isolates harbored BlaCTX-M-9type genes. The cefotazidime resistance rate (86%) of UPEC isolates carrying blaCTX-M-1type genes were significantly higher than that (17%) of the isolates with blaCTX-M-9type genes.
The7housekeeping genes (adk、fumC、gyrB、icd、mdh、pur A and recA) for multilocus sequence typing (MLST) were selected from the E. coli database at the University College Cork E. coli MLST website (http://mlst.ucc.ie/mlst/dbs/Ecoli) and sequence type determination of the47UPEC and26commensal isolates was performed using the MLST PCR scheme accordingly. The based upon related sequence types (BURST) clustering algorithm (eburst.mlst.net) was used to analyze the allelic profiles and define clonal complexes.5UPEC and4commensal E. coli isolates shared the same type ST10, and one isolate respectively from the two of the groups belonged to ST453. In addition to that, the MLST of UPEC was thoroughly different from that of the intestinal commensal counterparts. The47UPEC isolates analyzed were assigned to18distinct STs. The most common STs were ST131(n=9,19.1%of isolates), followed by ST405(7,14.9%), ST10(5,10.6%), ST393(4,8.5%), ST69(4,8.5%), and ST95(3,6.4%). The six STs accounted for68.1%(32/47) of the isolates, demonstrating the diversity of lineages. The26isolates analyzed by MLST were grouped into22different STs, more diverse than the UPEC isolates. Of the22STs,18were already included in the E. coli website database, Among which ST10(n=4,15.4%) was the most popular group. And an unknown ST included2isolates, while the other20isolates were classified into the20STs, respectively.
Part two. Comparison of adhesin genes between uropathogenic and intestinal commensal Escherichia coli strains
Among the47UPEC isolates,21were from patients with recurrent lower UTIs. The recurrent lower UTIs were defined as at least two episodes of UTI within6month. And each time the patients had:a) typical clinical symptoms as urinary urgency and pain and frequent voiding, b) pyuria, c) without fever, chill or other systematic symptoms. Seven strains were isolated from acute pyelonephritis patients by the following criteria:a) the temperature>38.5℃, b) positive percussion pain on kidney area, c) an elevated leukocytes count and percentage of neutrophils in the blood routine test. The rest19strains were from patients with acute uncomplicated cystitis.
The presence of adhesin genes afa, draE, daaE, papGI, papGⅡ, papGⅢ, flu, fimH, sfa and foc were investigated by PCR amplification using primers sets as described previously.
①afa gene was present in6UPEC isolates, all of which belonged to the recurrent lower UTIs group (28.6%). None of the isolates from the acute pyelonephritis group or the acute uncomplicated cystitis group carried afa gene. And no afa gene was found in26intestinal commensal E. coli isolates. Neither draE nor daaE gene was detected among all the strains we screened.(2)11of the47UPEC isolates were papG gene positive (23.4%) including10papG Ⅱ and1papG Ⅲ, and1of the intestinal commensal isolates (3.8%) carried papG Ⅱ gene. Further more, the prevalence of papG in acute pyelonephritis (71.4%) was significantly higher than that of recurrent lower UTI group (14.3%) and that of acute uncomplicated cystitis group (15.8%).(3)80.9%of the UPEC isolates while50.0%of the intestinal commensal isolates were found biofilm related flu gene positive. However, the prevalence difference of the three UTI groups was not significant.(4)Gene fimH was screened positive in42of47(89%) UPEC isolates and20of26(76.9%) commensal isolates.⑤Sfa gene was present in one isolate each in the three UTI groups and one of the sfa positive isolates was also detected focG positive. But none isolate in the intestinal commensal group was screened sfa/foc positive.
Most of the UPEC strains (72.3%,34/47) carried2to3kinds of adhesin genes, while80.8%(21/26) of the commensal isolates had1to2kinds of adhesin genes. The difference of the adhesin gene numbers between UPEC and commensal isolates were statistically significant.
The ESBLs-producing UPEC carried fewer adhesin genes than the non-ESBLs-producing ones, of28ESBLs-producing UPEC,2(14%) were detected no adhesion genes,6(21%) carried1adhesin gene, and15(54%) were screened2kinds of adhesin genes. On the other hand, among the19non-ESBLs-producing UPEC isolates,1(5%) carried only one adhesin gene, and8(42%) and9(47%) isolates were respectively detected2and3kinds of adhesin genes, with the left carried5kinds of the detected genes. Therefore, most of the ESBLs-producing UPEC isolates carried no more than2adhesin genes, while over half of the non-ESBLs-producing UPEC were screened no less than3kinds.
Part Three, phoU gene related persistence mechanism of the clinical UPEC isolates
With the help of PCR and gene sequencing, we amplified and compared the complete phoU gene sequence of the47clinical UPEC isolates and26intestinal commensal E. coli strains.10of47(21%) UPEC was feathered with the same wild type phoU gene sequence as the strain W3110. The phoU gene sequences of79% (37/47) UPEC had amino acid differences from the wild type, so as to be called mutant phoU gene types in this study.20of the37mutant phoU UPEC carried the phoU G236E mutation and the rest17feathered with the same phoU S239P+D240K+K24JE mutation. Interestingly, all of the26intestinal commensal E. coli were phoU gene wild type.
The MLSTs of the phoU wild type UPEC isolates were totally different from those of the phoU mutant ones.9ST131UPEC were all phoU G236mutant strains,7ST405and4ST393UPEC belonged to the phoU S239P+D240K+K241E mutation type, while the5ST10UPEC all carried wild type phoU gene. The UPEC isolates with papG gene positive were all included in the mutant phoU gene group. Furthermore, the UPEC isolates with mutant phoU gene carried more adhesin genes than those with wild type phoU gene. However, there were no significant differences between the wild type phoU UPEC and the mutant type phoU UPEC, whether referring to the urinary tract infection types or the antimicrobial susceptibilities.
So as to study whether the phoU G236E mutation provoked a variant role of PhoU played in the persistence of UPEC, we performed the in vitro bactericidal test with phoU G236E mutant UPEC and phoU wild type UPEC. We chose3kinds of antimicrobials:cefotazidime, amikacin and ciprofloxacin. In the killing curve experiment, the UPEC with phoU G236E mutation showed increased ability of persistence to cefotazidime, amikacin and ciprofloxacin. Under the relatively low dose of the three antibiotics (2,4×MIC cefotazidime,4×MIC amikacin and8×MIC ciprofloxacin), the UPEC strain of wild type phoU gene was completely killed after20-30h, while3.5-7×101CFU/ml phoU G236E mutant isolate survived at the same time and remained no less than1×101CFU/ml until the56h after the administration of the antimicrobials. When shifted to the high concentration level of drugs (8×MIC cefotazidime,8×MIC amikacin and16×MIC ciprofloxacin), the colony counts of phoU gene wild type isolate sloped to zero as early as4h (20h for8×MIC cefotazidime), but yet the phoU G236E mutant isolate were not killed until30-44h exposure to the antimicrobials. Therefore, it was probable that the phoU G236E mutant isolates left more persisters and then more tolerant to antibiotics.
In order to understand how the phoU G236E mutation might be involved in the enforcement in persister formation of the UPEC as shown by increased tolerance to various antibiotics, we performed a relatively quantitative PCR of phoU gene under the treatment of cefotazidime, amikacin and ciprofloxacin respectively (we chose the housekeeping gene mdh as the endogenous control). The result indicated that after12h exposure to cefotazidime, amikacin and ciprofloxacin, the relative phoU gene expression level of phoU G236E mutant UPEC isolate was upregulated6.2,20.6and6.8-fold higher than the phoU wild type isolate, which suggested the higher level of phoU gene expression in the phoU G236E mutant isolate leading to a more powerful formation of persisters to the threat of the various antimicrobials. However, the excact mechanisms are still to be understood.
引文
[1]Jayaraman R. Bacterial persistence:some new insights into an old phenomenon. J Biosci[J]. 2008 Dec;33(5):795-805.
[2]Foxman B, Barlow R, D'Arcy H, Gillespie B, Sobel JD. Urinary tract infection:Self reported incidence and associated costs. Ann Epidemiol[J].2000;10(8):509-515.
[3]Foxman B. Epidemiology of urinary tract infections:incidence, morbidity, and economic costs. Dis Mon[J].2003 Feb;49(2):53-70.
[4]Yamamoto S. Molecular epidemiology of uropathogenic Escherichia coli. J Infect Chemother [J].2007;13(2):68-73.
[5]Mulvey MA. Adhesion and entry of uropathogenic Escherichia coli. Cell Microbiol[J]. 2002;4(5):257-271.
[6]Rosen DA, Hooton TM, Stamm WE, Humphrey PA, Hultgren SJ. Detection of intracellular bacterial communities in human urinary tract infection. PLos Med[J].2007 Dec;4(12):1949-1958.
[7]Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, Hultgren SJ. Intracellular bacterial biofilm-like pods in urinary tract infections. Science[J].2003 Jul;301(5629):105-107.
[8]Mulvey MA, Lopez-Boado YS, Wilson CL, Roth R, Parks WC, Heuser J, et al. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science[J].1998 Nov;282(5393):1494-1497.
[9]Lane MC, Mobley HLT. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney Int[J]. 2007;72(1):19-25.
[10]van der Woude MW, Henderson IR. Regulation and function of Ag43 (Flu). Annu Rev MicrobiolfJ].2008;62:153-169.
[11]Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. Bacterial persistence as a phenotypic switch. Science[J].2004 Sep 10;305(5690):1622-1625.
[12]Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett[J].2004 Jan 15;230(1):13-18.
[13]Dobrindt U, Zdziarski J, Salvador E, Hacker J. Bacterial genome plasticity and its impact on adaptation during persistent infection. Int J Med Microbiol[J].2010 Aug;300(6):363-366.
[14]Lewis K. Multidrug tolerance of biofilms and persister cells. Curr Top Microbiol Immunol[J]. 2008;322:107-131.
[15]Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol[J].2007 Jan;5(1):48-56.
[16]Li Y, Zhang Y. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob Agents Chemother[J].2007 Jun;51(6):2092-2099.
[17]Rice CD, Pollard JE, Lewis ZT, McCleary WR. Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli. Appl Environ Microbiol[J].2009 Feb;75(3):573-582.
[18]Buckles EL, Wang X, Lockatell CV, Johnson DE, Donnenberg MS. PhoU enhances the ability of extraintestinal pathogenic Escherichia coli strain CFT073 to colonize the murine urinary tract. Microbiology[J].2006 Jan; 152(Pt 1):153-160.
[19]朱德妹汪,胡付品,蒋晓飞,倪语星,孙景勇,徐英春.2010年中国CHINET细菌耐药性监测.中国感染与化疗杂志[J].2011；11(5)：321-329.
[20]Sawma-Aouad G, Hashwa F, Tokajian S. Antimicrobial resistance in relation to virulence determinants and phylogenetic background among uropathogenic Escherichia coli in Lebanon. J Chemother[J].2009 Apr;21(2):153-158.
[21]Molina-Lopez J, Aparicio-Ozores G, Ribas-Aparicio RM, Gavilanes-Parra S, Chavez-Berrocal ME, Hernandez-Castro R, et al. Drug resistance, serotypes, and phylogenetic groups among uropathogenic Escherichia coli including O25-ST131 in Mexico City. J Infect Dev Ctries[J].2011 Dec;5(12):840-849.
[22]Gundogdu A, Long YB, Vollmerhausen TL, Katouli M. Antimicrobial resistance and distribution of sul genes and integron-associated intI genes among uropathogenic Escherichia coli in Queensland, Australia. J Med Microbiol[J].2011 Nov;60(Pt 11):1633-1642.
[23]Totsika M, Beatson SA, Sarkar S, Phan MD, Petty NK, Bachmann N, et al. Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage:genome analysis and virulence mechanisms. PLoS One[J].2011;6(10):e26578.
[24]Zhao L, Chen X, Zhu X, Yang W, Dong L, Xu X, et al. Prevalence of virulence factors and antimicrobial resistance of uropathogenic Escherichia coli in Jiangsu province (China). Urology [J]. 2009 Sep;74(3):702-707.
[25]徐叶进朱,王晓东,张慧芳,卢明芹,陈永平.尿路感染病原菌分布及药物敏感趋势分析,中华医院感染学杂志[J].2007；17(5)：591-595.
[26]Gibreel TM, Dodgson AR, Cheesbrough J, Fox AJ, Bolton FJ, Upton M. Population structure, virulence potential and antibiotic susceptibility of uropathogenic Escherichia coli from Northwest England. J Antimicrob Chemother[J].2012 Feb;67(2):346-356.
[27]Croxall G, Hale J, Weston V, Manning G, Cheetham P, Achtman M, et al. Molecular epidemiology of extraintestinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospital care settings. J Antimicrob Chemother[J].2011 Nov;66(11):2501-2508.
[28]Tartof SY, Solberg OD, Manges AR, Riley LW. Analysis of a uropathogenic Escherichia coli clonal group by multilocus sequence typing. J Clin Microbiol[J].2005 Dec;43(12):5860-5864.
[29]Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J Antimicrob Chemother[J].2006 Jan;57(1):154-155.
[30]CLSI. Performance strandards for antimicrobial susceptibility testing;Twentieth informational supplement M100-S20. Twentieth informational supplement M100-S20. Wayne, PA2010.
[31]Kot B, Wicha J, Zak-Pulawska Z. Susceptibility of Escherichia coli strains isolated from persons with urinary tract infections in 2007-2008 to antimicrobial agents. Przegl Epidemiol [J]. 2010;64(2):307-312.
[32]Kawamura-Sato K, Yoshida R, Shibayama K, Ohta M. Virulence genes, quinolone and fluoroquinolone resistance, and phylogenetic background of uropathogenic Escherichia coli strains isolated in Japan. Jpn J Infect Dis[J].2010 Mar;63(2):113-115.
[33]Qi C, Pilla V, Yu JH, Reed K. Changing prevalence of Escherichia coli with CTX-M-type extended-spectrum beta-lactamases in outpatient urinary E. coli between 2003 and 2008. Diagn Microbiol Infect Dis[J].2010 May;67(1):87-91.
[34]Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis[J].2008 Mar;8(3):159-166.
[35]卢连元刘.96株大肠埃希菌耐药性及其产ESBLs、AmpC酶菌株基因型检测.山东医药[J].2009；49(47)：37-39.
[36]Wang P, Hu F, Xiong Z, Ye X, Zhu D, Wang YF, et al. Susceptibility of extended-spectrum-beta-lactamase-producing Enterobacteriaceae according to the new CLSI breakpoints. J Clin Microbiol[J].2011 Sep;49(9):3127-3131.
[37]Williamson DA, Roberts SA, Smith M, Heffernan H, Tiong A, Pope C, et al. High rates of susceptibility to ceftazidime among globally prevalent CTX-M-producing Escherichia coli: potential clinical implications of the revised CLSI interpretive criteria. Eur J Clin Microbiol Infect Dis[J].2011 Sep6.
[38]Blanco J, Mora A, Mamani R, Lopez C, Blanco M, Dahbi G, et al. National survey of Escherichia coli causing extraintestinal infections reveals the spread of drug-resistant clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 with high virulence gene content in Spain. J Antimicrob Chemother[J].2011 Sep;66(9):2011-2021.
[39]Lee MY, Choi HJ, Choi JY, Song M, Song Y, Kim SW, et al. Dissemination of ST131 and ST393 community-onset, ciprofloxacin-resistant Escherichia coli clones causing urinary tract infections in Korea. J Infect[J].2010 Feb;60(2):146-153.
[40]Servin AL. Pathogenesis of Afa/Dr diffusely adhering Escherichia coli. Clin Microbiol Rev[J]. 2005 Apr; 18(2):264-292.
[41]Nowicki B, Truong L, Moulds J, Hull R. Presence of the Dr receptor in normal human tissues and its possible role in the pathogenesis of asending urinary tract infection. Am J Pathol[J].1988 Oct; 133(1):1-4.
[42]Korotkova N, Yarova-Yarovaya Y, Tchesnokova V, Yazvenko N, Carl MA, Stapleton AE, et al. Escherichia coli DraE adhesin associated bacterial intemalization by epithelial cells is promoted independently by decay accelerating factor and carcinoembryonic antigen related cell adhesion molecule binding and does not require the DraD invasin. Infect Immun[J].2008;76(9):3869-3880.
[43]Dhakal BK, Kulesus RR, Mulvey MA. Mechanisms and consequences of bladder cell invasion by uropathogenic Escherichia coli. Eur J Clin Invest[J].2008;38:2-11.
[44]Jouve M, Garcia MI, Courcoux P, Labigne A, Gounon P, LeBouguenec C. Adhesion to and invasion of HeLa cells by pathogenic Escherichia coli carrying the afa-3 gene cluster are mediated by the AfaE and AfaD proteins respectively. Infect Immun[J].1997;65(10):4082-4089.
[45]Jones CH, Dodson K, Hultgren SJ. Structure, function, and assembly of adhesive P pili. Urinary tract infections:Molecular pathogenesis and clinical management J].1996:175-219.
[46]Johnson JR, Kaster N, Kuskowski MA, Ling GV. Identification of urovirulence traits in Escherichia coli by comparison of urinary and rectal E-coli isolates from dogs with urinary tract infection. Journal of Clinical Microbiology[J].2003 Jan;41(1):337-345.
[47]Wright KJ, Hultgren SJ. Sticky fibers and uropathogenesis:bacterial adhesins in the urinary tract. Future Microbiology[J].2006 Jun;1(1):75-87.
[48]Hanisch FG, Hacker J, Schroten H. Specificity of S-Fimbriae on Recombinant Escherichia-Coli-Preferential Binding to Gangliosides Expressing Neugc-Alpha(2-3)Gal and Neuac-Alpha(2-8)Neuac. Infection and Immunity[J].1993;61(5):2108-2115.
[49]Wang Y, Wen ZG, Kim KS. Role of S fimbriae in Eschericbia coli K1 binding to brain microvascular endothelial cells in vitro and penetration into the central nervous system in vivo. Microbial Pathogenesis[J].2004 Dec;37(6):287-293.
[50]Sakarya S, Ertem GT, Oncu S, Kocak I, Erol N, Oncu S. Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence. Fems Immunology and Medical Microbiology[J].2003;39(1):45-50.
[51]Johnson JR, Owens K, Gajewski A, Kuskowski MA. Bacterial characteristics in relation to clinical source of Escherichia coli isolates from women with acute cystitis or pyelonephritis and uninfected women. J Clin Microbiol[J].2005 Dec;43(12):6064-6072.
[52]Plos K, Connell H, Jodal U, Marklund BI, Marild S, Wettergren B, et al. Intestinal carriage of P fimbriated Escherichia Coli and the susceptibility to urinary tract infection in young children. J Infect Dis[J].1995;171(3):625-631.
[53]Blanco M, Blanco JE, Alonso MP, Mora A, Balsalobre C, Munoa F, et al. Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains:relationship with expression of adhesins and production of toxins. Res Microbiol[J].1997 Dec;148(9):745-755.
[54]Johnson JR, Russo TA, Brown JJ, Stapleton A. papG alleles of Escherichia coli strains causing first episode or recurrent acute cystitis in adult women. J Infect Dis[J]. 1998;177(1):97-101.
[55]Sokurenko EV, Feldgarden M, Trintchina E, Weissman SJ, Avagyan S, Chattopadhyay S, et al. Selection footprint in the FimH adhesin shows pathoadaptive niche differentiation in Escherichia coli. Mol Biol Evol[J].2004;21(7):1373-1383.
[56]Ulett GC, Valle J, Beloin C, Sherlock O, Ghigo JM, Schembri MA. Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in long-term persistence in the urinary tract. Infect Immun [J].2007;75(7):3233-3244.
[57]Poljakovic M, Svensson L, Persson K. The influence of uropathogenic Escherichia coli and proinflammatory cytokines on the inducible nitric oxide synthase response in human kidney epithelial cells. J Urol[J].2005;173(3):1000-1003.
[58]Johnson JR, O'Bryan TT, Delavari P, Kuskowski M, Stapleton A, Carlino U, et al. Clonal relationships and extended virulence genotypes among Escherichia coli isolates from women with a first or recurrent episode of cystitis. J Infect Dis[J].2001; 183(10):1508-1517.
[59]Soto SM, Smithson A, Horcajada JP, Martinez JA, Mensa JP, Vila J. Implication of biofilm formation in the persistence of urinary tract infection caused by uropathogenic Escherichia coli. Clin Microbiol Infect[J].2006 Oct; 12(10):1034-1036.
[60]Wang MC, Tseng CC, Wu AB, Huang JJ, Sheu BS, Wu JJ. Different roles of host and bacterial factors in Escherichia coli extra-intestinal infections. Clin Microbiol Infect[J].2009 Apr;15(4):372-379.
[61]Schlager TA, Whittam TS, Hendley JO, Bhang JL, Wobbe CL, Stapleton A. Variation in frequency of the virulence factor gene in Escherichia coli clones colonizing the stools and urinary tracts of healthy prepubertal girls. J Infect Dis[J].2003 Oct 1;188(7):1059-1064.
[62]Sokurenko EV, Chesnokova V, Doyle RJ, Hasty DL. Diversity of the Escherichia coli type 1 fimbrial lectin-Differential binding to mannosides and uroepithelial cells. J Biol Chem[J].1997 Jul;272(28):17880-17886.
[63]Sokurenko EV, Courtney HS, Maslow J, Siitonen A, Hasty DL. Quantitative differences in adhesiveness of type-1 fimbriated Escherichia coli due to structural differences in fim H genes. J Bacteriol[J].1995 Jul;177(13):3680-3686.
[64]Soto SM, Jimenez de Anta MT, Vila J. Quinolones induce partial or total loss of pathogenicity islands in uropathogenic Escherichia coli by SOS-dependent or-independent pathways, respectively. Antimicrob Agents Chemother[J].2006 Feb;50(2):649-653.
[65]Bigger JW. Treatment of staphylococcal infections with penicillin-By intermittent sterilisation. Lancet[J].1944 1944;2:497-500.
[66]Lamarche MG, Wanner BL, Crepin S, Harel J. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiol Rev[J].2008 May;32(3):461-473.
[67]Zeiler HJ, Grohe K. The in vitro and in vivo activity of ciprofloxacin. Eur J Clin Microbiol[J]. 1984 Aug;3(4):339-343.
[68]Zeiler HJ. Evaluation of the in vitro bactericidal action of ciprofloxacin on cells of Escherichia coli in the logarithmic and stationary phases of growth. Antimicrob Agents Chemother[J].1985 Oct;28(4):524-527.
[69]Wang M, Jacoby GA, Mills DM, Hooper DC. SOS regulation of qnrB expression. Antimicrob Agents Chemother[J].2009 Feb;53(2):821-823.
[1]Foxman B. Recurring Urinary-Tract Infection-Incidence and Risk-Factors. American Journal of Public Health[J].1990;80(3):331-333.
[2]Mulvey MA. Adhesion and entry of uropathogenic Escherichia coli. Cellular Microbiology[J]. 2002;4(5):257-271.
[3]Wiles TJ, Kulesus RR, Mulvey MA. Origins and virulence mechanisms of uropathogenic Escherichia coli. Experimental and molecular pathology[Jj.2008 Aug;85(1):11-19.
[4]Wright KJ, Hultgren SJ. Sticky fibers and uropathogenesis:bacterial adhesins in the urinary tract. Future Microbiology[J].2006 Jun;1(1):75-87.
[5]Wright KJ, Seed PC, Hultgren SJ. Development of intracellular bacterial communities of uropathogenic Escherichia coli depends on type 1 pili. Cellular Microbiology [J].2007 Sep;9(9):2230-2241.
[6]Bahrani-Mougeot FK, Buckles EL, Lockatell CV, Hebel JR, Johnson DE, Tang CM, et al. Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract. Mol Microbiol[J].2002 Aug;45(4):1079-1093.
[7]Mulvey MA, Lopez-Boado YS, Wilson CL, Roth R, Parks WC, Heuser J, et al. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science[J].1998 Nov;282(5393):1494-1497.
[8]Sokurenko EV, Courtney HS, Maslow J, Siitonen A, Hasty DL. Quantitative Differences in Adhesiveness of Type-1 Fimbriated Escherichia-Coli Due to Structural Differences in Fimh Genes. J Bacteriol[J].1995 Jul;177(13):3680-3686.
[9]Eto DS, Jones TA, Sundsbak JL, Mulvey MA. Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli. PLoS Pathog[J].2007 Jul;3(7):949-961.
[10]Justice SS, Hung C, Theriot JA, Fletcher DA, Anderson GG, Footer MJ, et al. Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc Natl Acad Sci U S A[J].2004 Feb;101(5):1333-1338.
[11]Mysorekar IU, Hultgren SJ. Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract. Proc Natl Acad Sci U S A[J].2006 Sep; 103(38):14170-14175.
[12]Klumpp DJ, Rycyk MT, Chen MC, Thumbikat P, Sengupta S, Schaeffer AJ. Uropathogenic Escherichia coli induces extrinsic and intrinsic cascades to initiate urothelial apoptosis. Infection and Immunity[J].2006 Sep;74(9):5106-5113.
[13]Lane MC, Mobley HLT. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney International[J].2007;72(1):19-25.
[14]Jones CH, Dodson K, Hultgren SJ. Structure, function, and assembly of adhesive P pili. Urinary tract infections:Molecular pathogenesis and clinical management[J].1996:175-219.
[15]Johnson JR, Kaster N, Kuskowski MA, Ling GV. Identification of urovirulence traits in Escherichia coli by comparison of urinary and rectal E-coli isolates from dogs with urinary tract infection. Journal of Clinical Microbiology[J].2003;41(1):337-345.
[16]Plos K, Connell H, Jodal U, Marklund BI, Marild S, Wettergren B, et al. Intestinal Carriage of P-Fimbriated Escherichia-Coli and the Susceptibility to Urinary-Tract Infection in Young-Children. Journal of Infectious Diseases[J].1995;171(3):625-631.
[17]Otto G, Sandberg T, Marklund BI, Ulleryd P, Svanborg C. Virulence Factors and Pap Genotype in Escherichia-Coli Isolates from Women with Acute Pyelonephritis, with or without Bacteremia. Clinical Infectious Diseases[J].1993;17(3):448-456.
[18]Kisielius PV, Schwan WR, Amundsen SK, Duncan JL, Schaeffer AJ. Invivo Expression and Variation of Escherichia-Coli Type-1 and P-Pili in the Urine of Adults with Acute Urinary-Tract Infections. Infection and Immunity[J].1989;57(6):1656-1662.
[19]Mobley HLT, Jarvis KG, Elwood JP, Whittle DI, Lockatell CV, Russell RG, et al. Isogenic P-Fimbrial Deletion Mutants of Pyelonephritogenic Escherichia-Coli-the Role of Alpha-Gal(1-4)Beta-Gal Binding in Virulence of a Wild-Type Strain. Mol Microbiol[J]. 1993;10(1):143-155.
[20]Hanisch FG, Hacker J, Schroten H. Specificity of S-Fimbriae on Recombinant Escherichia-Coli-Preferential Binding to Gangliosides Expressing Neugc-Alpha(2-3)Gal and Neuac-Alpha(2-8)Neuac. Infection and Immunity[J].1993;61(5):2108-2115.
[21]Wang Y, Wen ZG, Kim KS. Role of S fimbriae in Escherichia coli K1 binding to brain microvascular endothelial cells in vitro and penetration into the central nervous system in vivo. Microbial Pathogenesis [J].2004 Dec;37(6):287-293.
[22]Sakarya S, Ertem GT, Oncu S, Kocak I, Erol N, Oncu S. Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence. Ferns Immunology and Medical Microbiology[J].2003;39(1):45-50.
[23]Morin MD, Hopkins WJ. Identification of virulence genes in uropathogenic Escherichia coli by multiplex polyinerase chain reaction and their association with infectivity in mice. Urology [J]. 2002;60(3):537-541.
[24]Mitsumori K, Terai A, Yamamoto S, Yoshida O. Identification of S, F1C and three PapG fimbrial adhesins in uropathogenic Escherichia coli by polymerase chain reaction. Ferns Immunology and Medical Microbiology[J].1998;21(4):261-268.
[25]Daigle F, Harel J, Fairbrother JM, Lebel P. Expression and Detection of Pap-Encoded, Sfa-Encoded, and Afa-Encoded Fimbrial Adhesin Systems among Uropathogenic Escherichia-Coli. Canadian Journal of Microbiology[J].1994;40(4):286-291.
[26]Nowicki B, Selvarangan R, Nowicki S. Family of Escherichia coli Dr Adhesins: Decay-accelerating factor receptor recognition and invasiveness. Journal of Infectious Diseases[J]. 2001;183:S24-S27.
[27]Foxman B, Zhang LX, Tallman P, Palin K, Rode C, Bloch C, et al. Virulence Characteristics of Escherichia-Coli Causing First Urinary-Tract Infection Predict Risk of 2nd Infection. Journal of Infectious Diseases[J].1995;172(6):1536-1541.
[28]Selvarangan R, Goluszko P, Singhal J, Carnoy C, Moseley S, Hudson B, et al. Interaction of Dr adhesin with collagen type IV is a critical step in Escherichia coli renal persistence. Infect Immun[J].2004;72(8):4827-4835.
[29]Miettinen A, Westerlund B, Tarkkanen AM, Tornroth T, Ljungberg P, Renkonen OV, et al. Binding of Bacterial Adhesins to Rat Glomerular Mesangium Invivo. Kidney International [J]. 1993;43(3):592-600.
[30]Kaul AK, Khan S, Martens MG, Crosson JT, Lupo VR, Kaul R. Experimental gestational pyelonephritis induces preterm births and low birth weights in C3H/HeJ mice. Infect Immun[J]. 1999;67(11):5958-5966.
[31]Goluszko P, Selvarangan R, Popov V, Pham T, Wen JW, Singhal J. Decay-accelerating factor and cytoskeleton redistribution pattern in HeLa cells infected with recombinant Escherichia coli strains expressing Dr family of adhesins. Infect Immun[J].1999;67(8):3989-3997.
[32]Goluszko P, Niesel D, Nowicki B, Selvarangan R, Nowicki S, Hart A, et al. Dr operon-associated invasiveness of Escherichia coli from pregnant patients with pyelonephritis. Infect Immun[J].2001;69(7):4678-4680.
[33]Kansau I, Berger C, Hospital M, Amsellem R, Nicolas V, Servin AL, et al. Zipper-like internalization of Dr-positive Escherichia coli by epithelial cells is preceded by an adhesin-induced mobilization of raft-associated molecules in the initial step of adhesion. Infect Immun[J].2004;72(7):3733-3742.
[1]Jayaraman R. Bacterial persistence:some new insights into an old phenomenon. J Biosci[J]. 2008 Dec;33(5):795-805.
[2]Bigger JW. Treatment of staphylococcal infections with penicillin-By intermittent sterilisation. Lancet[J].1944 1944;2:497-500.
[3]Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol[J]. 1986 May; 132(5):1297-1304.
[4]Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett[J].2004 Jan 15;230(1):13-18.
[5]Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol[J].2007 Jan;5(1):48-56.
[6]Dobrindt U, Zdziarski J, Salvador E, Hacker J. Bacterial genome plasticity and its impact on adaptation during persistent infection. Int J Med Microbiol[J].2010 Aug;300(6):363-366.
[7]Spoering AL, Lewis K. Biofilms and pianktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol[J].2001 Dec;183(23):6746-6751.
[8]Lewis K. Multidrug tolerance of biofilms and persister cells. Curr Top Microbiol Immunol[J]. 2008;322:107-131.
[9]Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. Bacterial persistence as a phenotypic switch. Science[J].2004 Sep 10;305(5690):1622-1625.
[10]Dhar N, McKinney JD. Microbial phenotypic heterogeneity and antibiotic tolerance. Curr Opin Microbiol[J].2007 Feb;10(1):30-38.
[11]Miller C, Thomsen LE, Gaggero C, Mosseri R, Ingmer H, Cohen SN. SOS response induction by beta-lactams and bacterial defense against antibiotic lethality. Science[J].2004 Sep 10;305(5690):1629-1631.
[12]Kim JS, Heo P, Yang TJ, Lee KS, Jin YS, Kim SK, et al. Bacterial persisters tolerate antibiotics by not producing hydroxyl radicals. Biochem Biophys Res Commun[J].2011 Sep 16;413(1):105-110.
[13]Moyed HS, Bertrand KP. hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol[J].1983 Aug;155(2):768-775.
[14]Korch SB, Henderson TA, Hill TM. Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis. Mol Microbiol [J].2003 Nov;50(4):1199-1213.
[15]Keren I, Shah D, Spoering A, Kaldalu N, Lewis K. Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol[J].2004 Dec;186(24):8172-8180.
[16]Hayes F. Toxins-antitoxins:plasmid maintenance, programmed cell death, and cell cycle arrest. Science[J].2003 Sep 12;301(5639):1496-1499.
[17]Maisonneuve E, Shakespeare LJ, Jorgensen MG, Gerdes K. Bacterial persistence by RNA endonucleases. Proc Natl Acad Sci U S A[J].2011 Aug 9; 108(32):13206-13211.
[18]Gerdes K. Toxin-antitoxin modules may regulate synthesis of macromolecules during nutritional stress. J Bacteriol[J].2000 Feb;182(3):561-572.
[19]Christensen SK, Mikkelsen M, Pedersen K, Gerdes K. RelE, a global inhibitor of translation, is activated during nutritional stress. Proc Natl Acad Sci U S A[J].2001 Dec 4;98(25):14328-14333.
[20]Rodionov DG, Ishiguro EE. Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli. J Bacteriol[J].1995 Aug;177(15):4224-4229.
[21]Korch SB, Hill TM. Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli:effects on macromolecular synthesis and persister formation. J Bacteriol[J].2006 Jun;188(11):3826-3836.
[22]Correia FF, D'Onofrio A, Rejtar T, Li L, Karger BL, Makarova K, et al. Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J Bacteriol[J].2006 Dec;188(24):8360-8367.
[23]Hansen S, Lewis K, Vulic M. Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli. Antimicrob Agents Chemother[J].2008 Aug;52(8):2718-2726.
[24]Li Y, Zhang Y. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob Agents Chemother[J].2007 Jun;51(6):2092-2099.
[25]Lamarche MG, Wanner BL, Crepin S, Harel J. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiol Rev[J].2008 May;32(3):461-473.
[26]Rice CD, Pollard JE, Lewis ZT, McCleary WR. Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli. Appl Environ Microbiol[J].2009 Feb;75(3):573-582.
[27]Buckles EL, Wang X, Lockatell CV, Johnson DE, Donnenberg MS. PhoU enhances the ability of extraintestinal pathogenic Escherichia coli strain CFT073 to colonize the murine urinary tract. Microbiology [J].2006 Jan;152(Pt 1):153-160.
[28]Tiller JC, Liao CJ, Lewis K, Klibanov AM. Designing surfaces that kill bacteria on contact. Proc Natl Acad Sci U S A[J].2001 May 22;98(11):5981-5985.
[2]Foxman B, Barlow R, D'Arcy H, Gillespie B, Sobel JD. Urinary tract infection:Self reported incidence and associated costs. Ann Epidemiol[J].2000;10(8):509-515.
[3]Foxman B. Epidemiology of urinary tract infections:incidence, morbidity, and economic costs. Dis Mon[J].2003 Feb;49(2):53-70.
[4]Yamamoto S. Molecular epidemiology of uropathogenic Escherichia coli. J Infect Chemother [J].2007;13(2):68-73.
[5]Mulvey MA. Adhesion and entry of uropathogenic Escherichia coli. Cell Microbiol[J]. 2002;4(5):257-271.
[6]Rosen DA, Hooton TM, Stamm WE, Humphrey PA, Hultgren SJ. Detection of intracellular bacterial communities in human urinary tract infection. PLos Med[J].2007 Dec;4(12):1949-1958.
[7]Anderson GG, Palermo JJ, Schilling JD, Roth R, Heuser J, Hultgren SJ. Intracellular bacterial biofilm-like pods in urinary tract infections. Science[J].2003 Jul;301(5629):105-107.
[8]Mulvey MA, Lopez-Boado YS, Wilson CL, Roth R, Parks WC, Heuser J, et al. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science[J].1998 Nov;282(5393):1494-1497.
[9]Lane MC, Mobley HLT. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney Int[J]. 2007;72(1):19-25.
[10]van der Woude MW, Henderson IR. Regulation and function of Ag43 (Flu). Annu Rev MicrobiolfJ].2008;62:153-169.
[11]Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. Bacterial persistence as a phenotypic switch. Science[J].2004 Sep 10;305(5690):1622-1625.
[12]Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett[J].2004 Jan 15;230(1):13-18.
[13]Dobrindt U, Zdziarski J, Salvador E, Hacker J. Bacterial genome plasticity and its impact on adaptation during persistent infection. Int J Med Microbiol[J].2010 Aug;300(6):363-366.
[14]Lewis K. Multidrug tolerance of biofilms and persister cells. Curr Top Microbiol Immunol[J]. 2008;322:107-131.
[15]Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol[J].2007 Jan;5(1):48-56.
[16]Li Y, Zhang Y. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob Agents Chemother[J].2007 Jun;51(6):2092-2099.
[17]Rice CD, Pollard JE, Lewis ZT, McCleary WR. Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli. Appl Environ Microbiol[J].2009 Feb;75(3):573-582.
[18]Buckles EL, Wang X, Lockatell CV, Johnson DE, Donnenberg MS. PhoU enhances the ability of extraintestinal pathogenic Escherichia coli strain CFT073 to colonize the murine urinary tract. Microbiology[J].2006 Jan; 152(Pt 1):153-160.
[19]朱德妹汪,胡付品,蒋晓飞,倪语星,孙景勇,徐英春.2010年中国CHINET细菌耐药性监测.中国感染与化疗杂志[J].2011；11(5)：321-329.
[20]Sawma-Aouad G, Hashwa F, Tokajian S. Antimicrobial resistance in relation to virulence determinants and phylogenetic background among uropathogenic Escherichia coli in Lebanon. J Chemother[J].2009 Apr;21(2):153-158.
[21]Molina-Lopez J, Aparicio-Ozores G, Ribas-Aparicio RM, Gavilanes-Parra S, Chavez-Berrocal ME, Hernandez-Castro R, et al. Drug resistance, serotypes, and phylogenetic groups among uropathogenic Escherichia coli including O25-ST131 in Mexico City. J Infect Dev Ctries[J].2011 Dec;5(12):840-849.
[22]Gundogdu A, Long YB, Vollmerhausen TL, Katouli M. Antimicrobial resistance and distribution of sul genes and integron-associated intI genes among uropathogenic Escherichia coli in Queensland, Australia. J Med Microbiol[J].2011 Nov;60(Pt 11):1633-1642.
[23]Totsika M, Beatson SA, Sarkar S, Phan MD, Petty NK, Bachmann N, et al. Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage:genome analysis and virulence mechanisms. PLoS One[J].2011;6(10):e26578.
[24]Zhao L, Chen X, Zhu X, Yang W, Dong L, Xu X, et al. Prevalence of virulence factors and antimicrobial resistance of uropathogenic Escherichia coli in Jiangsu province (China). Urology [J]. 2009 Sep;74(3):702-707.
[25]徐叶进朱,王晓东,张慧芳,卢明芹,陈永平.尿路感染病原菌分布及药物敏感趋势分析,中华医院感染学杂志[J].2007；17(5)：591-595.
[26]Gibreel TM, Dodgson AR, Cheesbrough J, Fox AJ, Bolton FJ, Upton M. Population structure, virulence potential and antibiotic susceptibility of uropathogenic Escherichia coli from Northwest England. J Antimicrob Chemother[J].2012 Feb;67(2):346-356.
[27]Croxall G, Hale J, Weston V, Manning G, Cheetham P, Achtman M, et al. Molecular epidemiology of extraintestinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospital care settings. J Antimicrob Chemother[J].2011 Nov;66(11):2501-2508.
[28]Tartof SY, Solberg OD, Manges AR, Riley LW. Analysis of a uropathogenic Escherichia coli clonal group by multilocus sequence typing. J Clin Microbiol[J].2005 Dec;43(12):5860-5864.
[29]Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum (beta)-lactamases. J Antimicrob Chemother[J].2006 Jan;57(1):154-155.
[30]CLSI. Performance strandards for antimicrobial susceptibility testing;Twentieth informational supplement M100-S20. Twentieth informational supplement M100-S20. Wayne, PA2010.
[31]Kot B, Wicha J, Zak-Pulawska Z. Susceptibility of Escherichia coli strains isolated from persons with urinary tract infections in 2007-2008 to antimicrobial agents. Przegl Epidemiol [J]. 2010;64(2):307-312.
[32]Kawamura-Sato K, Yoshida R, Shibayama K, Ohta M. Virulence genes, quinolone and fluoroquinolone resistance, and phylogenetic background of uropathogenic Escherichia coli strains isolated in Japan. Jpn J Infect Dis[J].2010 Mar;63(2):113-115.
[33]Qi C, Pilla V, Yu JH, Reed K. Changing prevalence of Escherichia coli with CTX-M-type extended-spectrum beta-lactamases in outpatient urinary E. coli between 2003 and 2008. Diagn Microbiol Infect Dis[J].2010 May;67(1):87-91.
[34]Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis[J].2008 Mar;8(3):159-166.
[35]卢连元刘.96株大肠埃希菌耐药性及其产ESBLs、AmpC酶菌株基因型检测.山东医药[J].2009；49(47)：37-39.
[36]Wang P, Hu F, Xiong Z, Ye X, Zhu D, Wang YF, et al. Susceptibility of extended-spectrum-beta-lactamase-producing Enterobacteriaceae according to the new CLSI breakpoints. J Clin Microbiol[J].2011 Sep;49(9):3127-3131.
[37]Williamson DA, Roberts SA, Smith M, Heffernan H, Tiong A, Pope C, et al. High rates of susceptibility to ceftazidime among globally prevalent CTX-M-producing Escherichia coli: potential clinical implications of the revised CLSI interpretive criteria. Eur J Clin Microbiol Infect Dis[J].2011 Sep6.
[38]Blanco J, Mora A, Mamani R, Lopez C, Blanco M, Dahbi G, et al. National survey of Escherichia coli causing extraintestinal infections reveals the spread of drug-resistant clonal groups O25b:H4-B2-ST131, O15:H1-D-ST393 and CGA-D-ST69 with high virulence gene content in Spain. J Antimicrob Chemother[J].2011 Sep;66(9):2011-2021.
[39]Lee MY, Choi HJ, Choi JY, Song M, Song Y, Kim SW, et al. Dissemination of ST131 and ST393 community-onset, ciprofloxacin-resistant Escherichia coli clones causing urinary tract infections in Korea. J Infect[J].2010 Feb;60(2):146-153.
[40]Servin AL. Pathogenesis of Afa/Dr diffusely adhering Escherichia coli. Clin Microbiol Rev[J]. 2005 Apr; 18(2):264-292.
[41]Nowicki B, Truong L, Moulds J, Hull R. Presence of the Dr receptor in normal human tissues and its possible role in the pathogenesis of asending urinary tract infection. Am J Pathol[J].1988 Oct; 133(1):1-4.
[42]Korotkova N, Yarova-Yarovaya Y, Tchesnokova V, Yazvenko N, Carl MA, Stapleton AE, et al. Escherichia coli DraE adhesin associated bacterial intemalization by epithelial cells is promoted independently by decay accelerating factor and carcinoembryonic antigen related cell adhesion molecule binding and does not require the DraD invasin. Infect Immun[J].2008;76(9):3869-3880.
[43]Dhakal BK, Kulesus RR, Mulvey MA. Mechanisms and consequences of bladder cell invasion by uropathogenic Escherichia coli. Eur J Clin Invest[J].2008;38:2-11.
[44]Jouve M, Garcia MI, Courcoux P, Labigne A, Gounon P, LeBouguenec C. Adhesion to and invasion of HeLa cells by pathogenic Escherichia coli carrying the afa-3 gene cluster are mediated by the AfaE and AfaD proteins respectively. Infect Immun[J].1997;65(10):4082-4089.
[45]Jones CH, Dodson K, Hultgren SJ. Structure, function, and assembly of adhesive P pili. Urinary tract infections:Molecular pathogenesis and clinical management J].1996:175-219.
[46]Johnson JR, Kaster N, Kuskowski MA, Ling GV. Identification of urovirulence traits in Escherichia coli by comparison of urinary and rectal E-coli isolates from dogs with urinary tract infection. Journal of Clinical Microbiology[J].2003 Jan;41(1):337-345.
[47]Wright KJ, Hultgren SJ. Sticky fibers and uropathogenesis:bacterial adhesins in the urinary tract. Future Microbiology[J].2006 Jun;1(1):75-87.
[48]Hanisch FG, Hacker J, Schroten H. Specificity of S-Fimbriae on Recombinant Escherichia-Coli-Preferential Binding to Gangliosides Expressing Neugc-Alpha(2-3)Gal and Neuac-Alpha(2-8)Neuac. Infection and Immunity[J].1993;61(5):2108-2115.
[49]Wang Y, Wen ZG, Kim KS. Role of S fimbriae in Eschericbia coli K1 binding to brain microvascular endothelial cells in vitro and penetration into the central nervous system in vivo. Microbial Pathogenesis[J].2004 Dec;37(6):287-293.
[50]Sakarya S, Ertem GT, Oncu S, Kocak I, Erol N, Oncu S. Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence. Fems Immunology and Medical Microbiology[J].2003;39(1):45-50.
[51]Johnson JR, Owens K, Gajewski A, Kuskowski MA. Bacterial characteristics in relation to clinical source of Escherichia coli isolates from women with acute cystitis or pyelonephritis and uninfected women. J Clin Microbiol[J].2005 Dec;43(12):6064-6072.
[52]Plos K, Connell H, Jodal U, Marklund BI, Marild S, Wettergren B, et al. Intestinal carriage of P fimbriated Escherichia Coli and the susceptibility to urinary tract infection in young children. J Infect Dis[J].1995;171(3):625-631.
[53]Blanco M, Blanco JE, Alonso MP, Mora A, Balsalobre C, Munoa F, et al. Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains:relationship with expression of adhesins and production of toxins. Res Microbiol[J].1997 Dec;148(9):745-755.
[54]Johnson JR, Russo TA, Brown JJ, Stapleton A. papG alleles of Escherichia coli strains causing first episode or recurrent acute cystitis in adult women. J Infect Dis[J]. 1998;177(1):97-101.
[55]Sokurenko EV, Feldgarden M, Trintchina E, Weissman SJ, Avagyan S, Chattopadhyay S, et al. Selection footprint in the FimH adhesin shows pathoadaptive niche differentiation in Escherichia coli. Mol Biol Evol[J].2004;21(7):1373-1383.
[56]Ulett GC, Valle J, Beloin C, Sherlock O, Ghigo JM, Schembri MA. Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in long-term persistence in the urinary tract. Infect Immun [J].2007;75(7):3233-3244.
[57]Poljakovic M, Svensson L, Persson K. The influence of uropathogenic Escherichia coli and proinflammatory cytokines on the inducible nitric oxide synthase response in human kidney epithelial cells. J Urol[J].2005;173(3):1000-1003.
[58]Johnson JR, O'Bryan TT, Delavari P, Kuskowski M, Stapleton A, Carlino U, et al. Clonal relationships and extended virulence genotypes among Escherichia coli isolates from women with a first or recurrent episode of cystitis. J Infect Dis[J].2001; 183(10):1508-1517.
[59]Soto SM, Smithson A, Horcajada JP, Martinez JA, Mensa JP, Vila J. Implication of biofilm formation in the persistence of urinary tract infection caused by uropathogenic Escherichia coli. Clin Microbiol Infect[J].2006 Oct; 12(10):1034-1036.
[60]Wang MC, Tseng CC, Wu AB, Huang JJ, Sheu BS, Wu JJ. Different roles of host and bacterial factors in Escherichia coli extra-intestinal infections. Clin Microbiol Infect[J].2009 Apr;15(4):372-379.
[61]Schlager TA, Whittam TS, Hendley JO, Bhang JL, Wobbe CL, Stapleton A. Variation in frequency of the virulence factor gene in Escherichia coli clones colonizing the stools and urinary tracts of healthy prepubertal girls. J Infect Dis[J].2003 Oct 1;188(7):1059-1064.
[62]Sokurenko EV, Chesnokova V, Doyle RJ, Hasty DL. Diversity of the Escherichia coli type 1 fimbrial lectin-Differential binding to mannosides and uroepithelial cells. J Biol Chem[J].1997 Jul;272(28):17880-17886.
[63]Sokurenko EV, Courtney HS, Maslow J, Siitonen A, Hasty DL. Quantitative differences in adhesiveness of type-1 fimbriated Escherichia coli due to structural differences in fim H genes. J Bacteriol[J].1995 Jul;177(13):3680-3686.
[64]Soto SM, Jimenez de Anta MT, Vila J. Quinolones induce partial or total loss of pathogenicity islands in uropathogenic Escherichia coli by SOS-dependent or-independent pathways, respectively. Antimicrob Agents Chemother[J].2006 Feb;50(2):649-653.
[65]Bigger JW. Treatment of staphylococcal infections with penicillin-By intermittent sterilisation. Lancet[J].1944 1944;2:497-500.
[66]Lamarche MG, Wanner BL, Crepin S, Harel J. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiol Rev[J].2008 May;32(3):461-473.
[67]Zeiler HJ, Grohe K. The in vitro and in vivo activity of ciprofloxacin. Eur J Clin Microbiol[J]. 1984 Aug;3(4):339-343.
[68]Zeiler HJ. Evaluation of the in vitro bactericidal action of ciprofloxacin on cells of Escherichia coli in the logarithmic and stationary phases of growth. Antimicrob Agents Chemother[J].1985 Oct;28(4):524-527.
[69]Wang M, Jacoby GA, Mills DM, Hooper DC. SOS regulation of qnrB expression. Antimicrob Agents Chemother[J].2009 Feb;53(2):821-823.
[1]Foxman B. Recurring Urinary-Tract Infection-Incidence and Risk-Factors. American Journal of Public Health[J].1990;80(3):331-333.
[2]Mulvey MA. Adhesion and entry of uropathogenic Escherichia coli. Cellular Microbiology[J]. 2002;4(5):257-271.
[3]Wiles TJ, Kulesus RR, Mulvey MA. Origins and virulence mechanisms of uropathogenic Escherichia coli. Experimental and molecular pathology[Jj.2008 Aug;85(1):11-19.
[4]Wright KJ, Hultgren SJ. Sticky fibers and uropathogenesis:bacterial adhesins in the urinary tract. Future Microbiology[J].2006 Jun;1(1):75-87.
[5]Wright KJ, Seed PC, Hultgren SJ. Development of intracellular bacterial communities of uropathogenic Escherichia coli depends on type 1 pili. Cellular Microbiology [J].2007 Sep;9(9):2230-2241.
[6]Bahrani-Mougeot FK, Buckles EL, Lockatell CV, Hebel JR, Johnson DE, Tang CM, et al. Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract. Mol Microbiol[J].2002 Aug;45(4):1079-1093.
[7]Mulvey MA, Lopez-Boado YS, Wilson CL, Roth R, Parks WC, Heuser J, et al. Induction and evasion of host defenses by type 1-piliated uropathogenic Escherichia coli. Science[J].1998 Nov;282(5393):1494-1497.
[8]Sokurenko EV, Courtney HS, Maslow J, Siitonen A, Hasty DL. Quantitative Differences in Adhesiveness of Type-1 Fimbriated Escherichia-Coli Due to Structural Differences in Fimh Genes. J Bacteriol[J].1995 Jul;177(13):3680-3686.
[9]Eto DS, Jones TA, Sundsbak JL, Mulvey MA. Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli. PLoS Pathog[J].2007 Jul;3(7):949-961.
[10]Justice SS, Hung C, Theriot JA, Fletcher DA, Anderson GG, Footer MJ, et al. Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis. Proc Natl Acad Sci U S A[J].2004 Feb;101(5):1333-1338.
[11]Mysorekar IU, Hultgren SJ. Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract. Proc Natl Acad Sci U S A[J].2006 Sep; 103(38):14170-14175.
[12]Klumpp DJ, Rycyk MT, Chen MC, Thumbikat P, Sengupta S, Schaeffer AJ. Uropathogenic Escherichia coli induces extrinsic and intrinsic cascades to initiate urothelial apoptosis. Infection and Immunity[J].2006 Sep;74(9):5106-5113.
[13]Lane MC, Mobley HLT. Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney. Kidney International[J].2007;72(1):19-25.
[14]Jones CH, Dodson K, Hultgren SJ. Structure, function, and assembly of adhesive P pili. Urinary tract infections:Molecular pathogenesis and clinical management[J].1996:175-219.
[15]Johnson JR, Kaster N, Kuskowski MA, Ling GV. Identification of urovirulence traits in Escherichia coli by comparison of urinary and rectal E-coli isolates from dogs with urinary tract infection. Journal of Clinical Microbiology[J].2003;41(1):337-345.
[16]Plos K, Connell H, Jodal U, Marklund BI, Marild S, Wettergren B, et al. Intestinal Carriage of P-Fimbriated Escherichia-Coli and the Susceptibility to Urinary-Tract Infection in Young-Children. Journal of Infectious Diseases[J].1995;171(3):625-631.
[17]Otto G, Sandberg T, Marklund BI, Ulleryd P, Svanborg C. Virulence Factors and Pap Genotype in Escherichia-Coli Isolates from Women with Acute Pyelonephritis, with or without Bacteremia. Clinical Infectious Diseases[J].1993;17(3):448-456.
[18]Kisielius PV, Schwan WR, Amundsen SK, Duncan JL, Schaeffer AJ. Invivo Expression and Variation of Escherichia-Coli Type-1 and P-Pili in the Urine of Adults with Acute Urinary-Tract Infections. Infection and Immunity[J].1989;57(6):1656-1662.
[19]Mobley HLT, Jarvis KG, Elwood JP, Whittle DI, Lockatell CV, Russell RG, et al. Isogenic P-Fimbrial Deletion Mutants of Pyelonephritogenic Escherichia-Coli-the Role of Alpha-Gal(1-4)Beta-Gal Binding in Virulence of a Wild-Type Strain. Mol Microbiol[J]. 1993;10(1):143-155.
[20]Hanisch FG, Hacker J, Schroten H. Specificity of S-Fimbriae on Recombinant Escherichia-Coli-Preferential Binding to Gangliosides Expressing Neugc-Alpha(2-3)Gal and Neuac-Alpha(2-8)Neuac. Infection and Immunity[J].1993;61(5):2108-2115.
[21]Wang Y, Wen ZG, Kim KS. Role of S fimbriae in Escherichia coli K1 binding to brain microvascular endothelial cells in vitro and penetration into the central nervous system in vivo. Microbial Pathogenesis [J].2004 Dec;37(6):287-293.
[22]Sakarya S, Ertem GT, Oncu S, Kocak I, Erol N, Oncu S. Escherichia coli bind to urinary bladder epithelium through nonspecific sialic acid mediated adherence. Ferns Immunology and Medical Microbiology[J].2003;39(1):45-50.
[23]Morin MD, Hopkins WJ. Identification of virulence genes in uropathogenic Escherichia coli by multiplex polyinerase chain reaction and their association with infectivity in mice. Urology [J]. 2002;60(3):537-541.
[24]Mitsumori K, Terai A, Yamamoto S, Yoshida O. Identification of S, F1C and three PapG fimbrial adhesins in uropathogenic Escherichia coli by polymerase chain reaction. Ferns Immunology and Medical Microbiology[J].1998;21(4):261-268.
[25]Daigle F, Harel J, Fairbrother JM, Lebel P. Expression and Detection of Pap-Encoded, Sfa-Encoded, and Afa-Encoded Fimbrial Adhesin Systems among Uropathogenic Escherichia-Coli. Canadian Journal of Microbiology[J].1994;40(4):286-291.
[26]Nowicki B, Selvarangan R, Nowicki S. Family of Escherichia coli Dr Adhesins: Decay-accelerating factor receptor recognition and invasiveness. Journal of Infectious Diseases[J]. 2001;183:S24-S27.
[27]Foxman B, Zhang LX, Tallman P, Palin K, Rode C, Bloch C, et al. Virulence Characteristics of Escherichia-Coli Causing First Urinary-Tract Infection Predict Risk of 2nd Infection. Journal of Infectious Diseases[J].1995;172(6):1536-1541.
[28]Selvarangan R, Goluszko P, Singhal J, Carnoy C, Moseley S, Hudson B, et al. Interaction of Dr adhesin with collagen type IV is a critical step in Escherichia coli renal persistence. Infect Immun[J].2004;72(8):4827-4835.
[29]Miettinen A, Westerlund B, Tarkkanen AM, Tornroth T, Ljungberg P, Renkonen OV, et al. Binding of Bacterial Adhesins to Rat Glomerular Mesangium Invivo. Kidney International [J]. 1993;43(3):592-600.
[30]Kaul AK, Khan S, Martens MG, Crosson JT, Lupo VR, Kaul R. Experimental gestational pyelonephritis induces preterm births and low birth weights in C3H/HeJ mice. Infect Immun[J]. 1999;67(11):5958-5966.
[31]Goluszko P, Selvarangan R, Popov V, Pham T, Wen JW, Singhal J. Decay-accelerating factor and cytoskeleton redistribution pattern in HeLa cells infected with recombinant Escherichia coli strains expressing Dr family of adhesins. Infect Immun[J].1999;67(8):3989-3997.
[32]Goluszko P, Niesel D, Nowicki B, Selvarangan R, Nowicki S, Hart A, et al. Dr operon-associated invasiveness of Escherichia coli from pregnant patients with pyelonephritis. Infect Immun[J].2001;69(7):4678-4680.
[33]Kansau I, Berger C, Hospital M, Amsellem R, Nicolas V, Servin AL, et al. Zipper-like internalization of Dr-positive Escherichia coli by epithelial cells is preceded by an adhesin-induced mobilization of raft-associated molecules in the initial step of adhesion. Infect Immun[J].2004;72(7):3733-3742.
[1]Jayaraman R. Bacterial persistence:some new insights into an old phenomenon. J Biosci[J]. 2008 Dec;33(5):795-805.
[2]Bigger JW. Treatment of staphylococcal infections with penicillin-By intermittent sterilisation. Lancet[J].1944 1944;2:497-500.
[3]Tuomanen E, Cozens R, Tosch W, Zak O, Tomasz A. The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth. J Gen Microbiol[J]. 1986 May; 132(5):1297-1304.
[4]Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett[J].2004 Jan 15;230(1):13-18.
[5]Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol[J].2007 Jan;5(1):48-56.
[6]Dobrindt U, Zdziarski J, Salvador E, Hacker J. Bacterial genome plasticity and its impact on adaptation during persistent infection. Int J Med Microbiol[J].2010 Aug;300(6):363-366.
[7]Spoering AL, Lewis K. Biofilms and pianktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J Bacteriol[J].2001 Dec;183(23):6746-6751.
[8]Lewis K. Multidrug tolerance of biofilms and persister cells. Curr Top Microbiol Immunol[J]. 2008;322:107-131.
[9]Balaban NQ, Merrin J, Chait R, Kowalik L, Leibler S. Bacterial persistence as a phenotypic switch. Science[J].2004 Sep 10;305(5690):1622-1625.
[10]Dhar N, McKinney JD. Microbial phenotypic heterogeneity and antibiotic tolerance. Curr Opin Microbiol[J].2007 Feb;10(1):30-38.
[11]Miller C, Thomsen LE, Gaggero C, Mosseri R, Ingmer H, Cohen SN. SOS response induction by beta-lactams and bacterial defense against antibiotic lethality. Science[J].2004 Sep 10;305(5690):1629-1631.
[12]Kim JS, Heo P, Yang TJ, Lee KS, Jin YS, Kim SK, et al. Bacterial persisters tolerate antibiotics by not producing hydroxyl radicals. Biochem Biophys Res Commun[J].2011 Sep 16;413(1):105-110.
[13]Moyed HS, Bertrand KP. hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol[J].1983 Aug;155(2):768-775.
[14]Korch SB, Henderson TA, Hill TM. Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis. Mol Microbiol [J].2003 Nov;50(4):1199-1213.
[15]Keren I, Shah D, Spoering A, Kaldalu N, Lewis K. Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol[J].2004 Dec;186(24):8172-8180.
[16]Hayes F. Toxins-antitoxins:plasmid maintenance, programmed cell death, and cell cycle arrest. Science[J].2003 Sep 12;301(5639):1496-1499.
[17]Maisonneuve E, Shakespeare LJ, Jorgensen MG, Gerdes K. Bacterial persistence by RNA endonucleases. Proc Natl Acad Sci U S A[J].2011 Aug 9; 108(32):13206-13211.
[18]Gerdes K. Toxin-antitoxin modules may regulate synthesis of macromolecules during nutritional stress. J Bacteriol[J].2000 Feb;182(3):561-572.
[19]Christensen SK, Mikkelsen M, Pedersen K, Gerdes K. RelE, a global inhibitor of translation, is activated during nutritional stress. Proc Natl Acad Sci U S A[J].2001 Dec 4;98(25):14328-14333.
[20]Rodionov DG, Ishiguro EE. Direct correlation between overproduction of guanosine 3',5'-bispyrophosphate (ppGpp) and penicillin tolerance in Escherichia coli. J Bacteriol[J].1995 Aug;177(15):4224-4229.
[21]Korch SB, Hill TM. Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli:effects on macromolecular synthesis and persister formation. J Bacteriol[J].2006 Jun;188(11):3826-3836.
[22]Correia FF, D'Onofrio A, Rejtar T, Li L, Karger BL, Makarova K, et al. Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J Bacteriol[J].2006 Dec;188(24):8360-8367.
[23]Hansen S, Lewis K, Vulic M. Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli. Antimicrob Agents Chemother[J].2008 Aug;52(8):2718-2726.
[24]Li Y, Zhang Y. PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob Agents Chemother[J].2007 Jun;51(6):2092-2099.
[25]Lamarche MG, Wanner BL, Crepin S, Harel J. The phosphate regulon and bacterial virulence: a regulatory network connecting phosphate homeostasis and pathogenesis. FEMS Microbiol Rev[J].2008 May;32(3):461-473.
[26]Rice CD, Pollard JE, Lewis ZT, McCleary WR. Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli. Appl Environ Microbiol[J].2009 Feb;75(3):573-582.
[27]Buckles EL, Wang X, Lockatell CV, Johnson DE, Donnenberg MS. PhoU enhances the ability of extraintestinal pathogenic Escherichia coli strain CFT073 to colonize the murine urinary tract. Microbiology [J].2006 Jan;152(Pt 1):153-160.
[28]Tiller JC, Liao CJ, Lewis K, Klibanov AM. Designing surfaces that kill bacteria on contact. Proc Natl Acad Sci U S A[J].2001 May 22;98(11):5981-5985.