禽病原性大肠杆菌与尿道致病性大肠杆菌毒力基因相关性与体内外表达差异及其ftsk突变株的研究
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摘要
尿道感染(urinary tract infections,UTIs)是人类的常见疾病,40%-50%的妇女至少经历过一次尿道感染。迄今,尿道致病性大肠杆菌(uropathogenic Escherichia coli,UPEC)是所有尿道感染中最常见的病原体。UPEC通过携带的毒力因子黏附并侵入宿主,突破宿主防御系统,引起组织损伤和宿主炎性反应。UPEC在肠道外的生长能力使得它们能导致包括尿道感染在内的各种疾病。禽大肠杆菌病是全部或部分由禽病原性大肠杆菌(avian pathogenic Escherichia coli,APEC)引起的局部或全身性感染。与UPEC类似,APEC在禽类也有广泛的致病潜能。基于这种广泛的致病潜能,UPEC和APEC被归为新型致病性大肠杆菌——肠道外病原性大肠杆菌(extraintestinal pathogenic Escherichia coli,ExPEC)。由于APEC和UPEC在肠道外感染中可能面临宿主相似的挑战,它们可能具有某些相似的致病基因和致病能力。很多研究证明,APEC和UPEC的毒力因子之间存在着一定的相似性。本研究在收集本地区UPEC分离株的基础上,探明本地区UPEC的流行病学及其耐药性情况,比较本地区UPEC和APEC分离株的种系发生型、毒力基因型的相似性;选择代表性APEC、UPEC分离株感染实验小鼠和鸡,通过DNA芯片技术和相对定量反转录PCR测定受试毒力基因在体内的表达水平,从而揭示APEC、UPEC毒力基因在同一宿主体内的表达规律,最终为弄清APEC与UPEC是否作为对方毒力基因贮库这一关键问题提供依据。研究结果将加深我们对APEC、UPEC毒力基因的形成、分布、演化规律的理解,强化我们对APEC作为UPEC毒力基因贮库这一推断的认识,为深入研究UTIs和禽大肠杆菌病的致病机理和特异防制措施奠定基础。
1.江苏部分地区UPEC分子流行病学与耐药性研究
从江苏部分地区尿道感染临床病例中分离、鉴定出202个UPEC分离株。对分离株的血清型、种系发生型、毒力基因型、耐药性以及毒力因子与耐药性间的关系作了研究。分离株最常见的血清型为O1,其它主要血清型还有O6、O7、O15、O18、O26和O25;种系发生型中,D和B2型最为常见,分别占受试分离株的42%和38%,而A和B1型分别占13%和7%。在受检的38个毒力基因中,fimH和feoB的检出率超过90%;50%以上的分离株至少携带papA、papC、papEF、papG、kpsMTⅡ、ompT、iutA、fyuA、traT、irp-2这10个基因中的一种。在黏附素中,P菌毛相关基因检出率仅次于fimH;在保护素中,荚膜形成相关基因kpsMT检出率较高;而三种毒素基因出现的频率相当。分离株耐药性较强,多重耐药现象普遍。在供试的14种抗生素中,UPEC分离株对11种抗生素的耐药率均超过了50%,其中,对萘啶酮酸、氨苄西林和美洛西林的耐药率均达到了90%以上;对磺胺甲基异噁唑的耐药率达到73%,远高于国外报道;对头孢西定、呋喃妥因、氯霉素的敏感性相对较高,其耐药率分别为22%、23%、36%;按美国国家临床实验室标准委员会方法手册,受试的202个UPEC分离株均为多重耐药菌株。表明受检地区UPEC的耐药情况相当严重,供试的抗生素已不适合作为受检地区抗尿道感染临床使用首选药物。本研究结果还表明,头孢噻肟、头孢西定、氯霉素和呋喃妥因抗性菌株,相对于其敏感菌株毒力有下降的趋势,其中呋喃妥因抗性菌株与其毒力下降之间的关系为首次报道。
2. APEC与UPEC毒力基因及其在体内外表达差异的比较
以我国自行分离的APEC和UPEC菌株为对象,对其血清型、种系发生型、毒力基因型及其毒力基因在体内外表达规律等方面的相似性进行了研究。结果表明二者在血清型、种系发生型、毒力基因型和毒力基因在体内外的表达规律等方面都表现出很大的相似性。本研究中O1和O18血清型为UPEC分离株的优势血清型,而这两种血清型在APEC分离株中也是常见的血清型。UPEC分离株的种系发生型以D和B2型最为常见,分别占受试分离株的42%和38%,其次是A和B1型分别占13%和7%;而APEC分离株以A和B2为主,A型比例最高为42%,B2型次之,为36%,D型和B1型分别为12%和10%。在受检的38个毒力基因中,检出频率最高的两个基因是feoB和fimH,其在两类菌株中的出现频率都超过90%;APEC和UPEC分离株中都检测出许多黏附素相关的基因、铁摄取相关的基因及质粒相关的毒力基因。SPF鸡的LD50测定结果显示UPEC代表菌株U17略低于APEC代表菌株E058,二者差异不明显,且攻毒鸡出现的病变相似。通过DNA芯片,对两种来源的大肠杆菌代表菌株分别在鸡攻毒模型和小鼠尿道感染模型中毒力基因的表达规律进行了研究。在攻毒鸡体内,APEC E058株和UPEC U17株中89%的T测试基因在表达趋势上一致;APEC E058株在攻毒鸡体内表达水平明显上调的5个基因(cvaC、neuC、ompT、iutA和iucCD)中,有两个基因(cvaC和neuC)在UPEC U17株中也明显上调,其它3个基因iutA、iucCD和ompT在UPEC U17株中也表现为上调趋势;APEC E058株在攻毒鸡体内表达水平明显下调的4个基因(aec-30、aes-8、gyrB和mdh),在UPEC U17株中也出现下调趋势。在小鼠体内,APEC E058株与UPEC U17株中82%的T测试基因在表达趋势上一致;UPEC U17株中明显上调的2个基因iucCD、tir和明显下调的2个基因sta、catI在APEC E058株中也表现为相应的上调或下调趋势。应用DNA芯片技术在国际上首次揭示,APEC和UPEC分离株在同一感染模型中的转录子具有相似的表达谱。上述研究结果提示我们,APEC很可能是UPEC的来源或者是UPEC毒力基因的贮存库。同时,许多与铁摄取系统相关的基因在鸡和小鼠体内的表达都表现为上调甚至明显上调,提示我们铁摄取可能是大肠杆菌得以实现其肠道外感染的必备条件。
3.检测ompT和ftsk相对定量PCR方法的建立及其对DNA芯片检测ompT表达水平的验证和对ftsk表达水平的解析
本研究利用嵌合荧光(SYBR GreenⅠ)标记,以编码甘油醛3-磷酸脱氢酶的看家基因gapA为内参基因,建立了用于ompT和ftsk基因表达水平解析的两步法实时定量反转录PCR (real time quantitative reverse transcription polymerase chain reaction, qRT-PCR)。运用该定量PCR分别建立了内参基因gapA和目的基因ompT与ftsk的3个标准曲线,这些标准曲线的斜率和线性关系均符合要求,表明所建立的定量PCR体系的扩增效率高、定量准确。定量PCR对ompT和ftsk基因在感染鸡体内的表达水平的解析结果表明,相对于体外培养,APEC E058株ompT、ftsk在鸡体内的表达分别上调了6.69和23.57倍;UPEC U17株分别上调了6.72和9.21倍。DNA芯片的检测结果表明,在鸡体内APEC E058株ompT的表达上调了2.54倍,证明我们自行研制的DNA芯片的检测结果是可信的,qRT-PCR比DNA芯片检测更为灵敏。上述结果提示我们,ompT和ftsk可能与大肠杆菌对鸡的致病力有关,即它们可能是APEC新的致病基因。
4.禽病原性大肠杆菌ftsk基因突变株的构建及其致病性鉴定
运用基因重组方法分别将卡那霉素抗性基因(kan)连接到PCR扩增的ftsk两端区域的2个基因片段之间,并分别插入到pBluescriptⅡS K (+) (pBSK)载体的多克隆位点中,构建出带kan基因标志的载体pBSK-F2-Kan-F1,通过PCR扩增F2-Kan-F1片段,电转化入APEC分离株E058中,根据同源重组原理,筛选出APEC E058 ftsk基因缺失突变株E058(?ftsk)。生长曲线测定结果显示,E058(?ftsk)的生长速度低于亲本菌株E058株,动物实验结果表明E058(?ftsk)在攻毒后6、12和24 h时血液、肝脏、脾脏和肺中的细菌数显著少于APEC E058株。APEC E058株感染组在接种48h后,除扑杀鸡外,接种鸡全部死亡;而E058(?ftsk)组相应的死亡率为20%左右。因此,可以认定ftsk为APEC E058株重要的毒力基因。
综上所述,对江苏部分地区UPEC的流行病学调查表明,UPEC的常见血清型为O1、O6、O7、O15、O18、O26和O25, D和B2是其最常见的种系发生型,结果与国外报道基本一致。在供试的14种抗生素中,UPEC分离株对11种抗生素的耐药率均超过了50%,其中,对萘啶酮酸、氨苄西林和美洛西林的耐药率均达到了90%以上;对磺胺甲基异噁唑的耐药率达到73%;分离株均为多重耐药菌株,表明本地区UPEC的耐药情况相当严重。UPEC分离株中,头孢噻肟、头孢西定、氯霉素和呋喃妥因抗性菌株,相对于其敏感菌株毒力有下降的趋势,其中呋喃妥因抗性菌株与其毒力下降之间的关系为首次报道。UPEC与APEC毒力因子及毒力基因在体内表达差异的比较结果表明,二者无论是在种系发生型、毒力基因型还是毒力基因在体内的表达规律上都有很大的相似性,提示我们APEC很可能是UPEC的来源或者是UPEC毒力基因的贮存库。通过建立的相对定量反转录PCR,对APEC E058株的ompT和ftsk基因在感染鸡体内的表达水平的解析,验证了DNA芯片的检测结果是可靠的,ompT和ftsk可能是APEC的毒力因子。利用基因重组技术对APEC E058株的ftsk基因进行了突变,测定了突变株的生长速度,并应用动物实验来评价此基因在APEC E058株对人工感染鸡致病性中的作用。结果显示,突变株生长速度低于亲本株,突变株在攻毒鸡的血液、肝脏、脾脏和肺中的细菌数显著少于亲本株,攻毒后48 h内,除扑杀鸡外,亲本株接种组的鸡全部死亡,而突变株组相应的死亡率为20%左右,表明ftsk是APEC的重要毒力基因。
Urinary tract infections (UTIs) are among the most common human diseases in the world, causing significant morbidity and mortality. Most community-acquired UTIs are due to uropathogenic E. coli (UPEC) infections. UPEC strains possess specialized virulence factors, enabling them to colonize and invade to the host, disrupt the host defense mechanisms, injure host tissues, and/or stimulate noxious host inflammatory response. The abilities of UPEC to grow extraintestinally may enable them to cause a variety of diseases, not just urinary tract ones. Avian colibacillosis refers to any localized or systemic infection such as an acute fatal septicemia or subacute pericarditis and airsacculitis caused entirely or partly by avian pathogenic Escherichia coli (APEC). It is responsible for large financial losses for the poultry industry each year due to mortality, lost production, and condemnations.
Since APEC and UPEC may encounter similar challenges when establishing infection in extraintestinal locations, they may share a similar content of virulence genes and capacity to cause disease. It is becoming more and more apparent that the common presence of a set of virulence associated genes among APEC and UPEC strains as well as similar disease patterns and phylogenetic background indicate a genetic relationship between APEC and UPEC isolates. The potential whether APEC might serve as a reservoir of virulence genes for UPEC should be considered.
1. Prevalence of virulence factors and antimicrobial resistance of UPEC isolates in Jiangsu area
202 UPEC isolates were characterized for serogroups, virulence genes, the antimicrobial susceptibilities and the phylogenetic groups. Most of the typable strains belonged to O1, O6, O7, O15, O18, O26 and O25 serogroups, respectively. The most frequent phylogenetic groups were D and B2, which represented 42% and 38% of the isolates respectively, followed by A and B1 groups, which were 13% and 7%, respectively. Among 37 tested virulence genes, feoB and fimH were the most prevalent genes, which distributed in over 90% of the isolates. Above half of the isolates possessed one or more of the following 10 virulence genes—papA, papC, papEF, papG, kpsMTⅡ, ompT, iutA, fyuA, traT and irp-2. pap related genes were the most prevalent other than fimH among adhesin genes. kpsMT genes occurred in a high prevalence in protectin genes studied, and genes encoding toxins were at similar frequencies. All isolates showed over 50% resistance rate to all of 14 tested antimicrobials except for cefoxitin, nitrofurantoin and chloramphenicol, which were 22%, 23% and 36%, respectively. Among them, the resistance rate to nalidixic acid, mezlocillin, and ampicillin was higher than 90%. Importantly, all of the isolates were multi-resistant, and 73% of them were resistant to trimethoprim-sulfamethoxazole. The above evidence strongly suggests that the problem of antibiotic resistance for UPEC is rather serious in this area, and that empirical treatment of UTIs with all the above antibiotics is no longer appropriate in the same area. Our findings also showed that among human UPEC isolates originated from this area, cefotaxime-, cefoxitin-, chloramphenicol-, and nitrofurantoin-resistant isolates had a reduced virulence genes compared with those of susceptible strains. To our knowledge, this is the first report of a statistically significant reduction in virulence genes among nitrofurantoin-resistant isolates.
2. Comparison of virulence genes and the expression of specific genes between APEC and UPEC isolates in murine urinary tract infection model and chicken challenge model
As diversity, epidemiological sources, and evolutionary origins of ExPEC are so far only partially defined, 100 of APEC isolates and 202 of UPEC isolates were compared by their serogroups, content of virulence genes, phylogenetic groups, and the expression of specific virulence genes in the present study. The results showed that O1 and O18 were the dominant serogroups of UPEC isolates, which were also prevalent in APEC isolates. Besides the UPEC phylogenetic groups mentioned above, most of APEC isolates belong to A and B2 groups, which represented 42% and 36% of the isolates respectively, followed by D and B1 groups, which were 12% and 10%, respectively. Some virulence genes, such as those encoding of adhesins, iron-related genes, and pTJ100-related genes were often carried by APEC and UPEC isolates. Althogh LD50 of UPEC strain U17 was slightly lower than that of APEC strain E058, similar lesions were observed in birds inoculated with either UPEC U17 or APEC E058. To gain further information of the correlation and pathogenesis of UPEC and APEC isolates, the in vivo expression of specific genes in both murine urinary tract infection (UTI) model and chicken challenge model were compared to that of UPEC U17 and APEC E058 grown statically to exponential phase in rich medium by DNA chips, respectively. In chicken challenge model, 89% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. In chicken challenge model, 5 genes (cvaC, neuC, ompT, iutA and iucCD) were differentially up-regulated in APEC E058, and two (cvaC and neuC) of them were also differentially up-regulated in UPEC U17, and the left three (ompT, iutA and iucCD) of them were up-regulated in UPEC U17 also. At the same time, 4 genes (aec-30, aes-8, gyrB and mdh) differentially down-regulated in APEC E058 were also down-regulated in UPEC U17. In murine UTI model, 82% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. Two differentially up-regulated genes (iucCD and tir) in UPEC U17 were also up-regulated in APEC E058, and 2 differentially down-regulated genes (sta and catI) in UPEC U17 were also down-regulated in APEC E058. The results show that in the same model (murine UTI model or chicken challenge model), most of the T-test genes of UPEC U17 and APEC E058 show the similar tendency of expression. Several iron-related genes were up-regulated in murine UTI model and /or chicken challenge model, revealing that iron acquisition is critical for E. coli to survive in blood or urinary tract. Based on these results, the potential for APEC to act as human UPEC or as a reservoir of virulence genes for UPEC should be considered. Further, this study represents the first comparison of APEC and UPEC transcriptome in vivo and provides specific insights into the mechanisms necessary for extraintestinal pathogenesis.
3. Development and application of relative real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the analysis of the expression of ompT and ftsk genes
To relatively quantify the expression of ompT and ftsk genes of APEC strain E058 and UPEC strain U17, two-step real time quantitative RT-PCRs (qRT-PCR) were developed based on SYBR Green I, respectively. ompT and ftsk were as target genes and gapA as internal reference, respectively. Three standard curves were established using a series dilution of cDNA synthesized from the RNA of APEC E058 grown statically to exponential phase in rich medium. In chicken challenge model, the expression of ompT and ftsk in APEC E058 were up-regulated 6.69- and 23.57-fold compared to that of APEC E058 grown statically to exponential phase in rich medium, respectively. Meanwhile, the expression of ompT and ftsk were up-regulated 6.72- and 9.21-fold in UPEC U17 compared to that of UPEC U17 grown statically to exponential phase in rich medium, respectively. Previous study showed that the expression of ompT in APEC E058 was up-regulated 2.54-fold in chicken challenged model by using DNA chips, when compared to that of APEC E058 grown statically to exponential phase in rich medium. These data verified the results of DNA chips, and qRT-PCR analysis demonstrated a greater transcription level sensitivity than that of microarray analysis. The result also suggests that ompT and ftsk may be the important virulence genes of APEC.
4. Construcion of ftsk knock-out mutant of APEC strain E058 and evaluation of its pathogenesis in chickens
The 709-bp fragment of ftsk1, 900-bp fragment of the ftsk2 and the gene encoding kanamycin resistance were generated by PCR respectively. All of these fragments were cloned into pBluescriptⅡS K (+) (pBSK) vector, termed pBSK-F2-Kan-F1. Fragment of F2-Kan-F1 amplified by PCR were transformed into APEC strain E058, and then the ftsk knock-out mutant of APEC strain E058 were generated by allelic replacement and named E058(?ftsk). The growth rate of E058(?ftsk) was slower than that of APEC E058. In chicken challenge model, the mutant was tested to determine the individual role of this gene for virulence and persistence in 1-day old SPF chicks. At 6, 12 and 24 hours after inoculation, the number of colony forming unit (CFU) recovered from the liver, spleen, lung and blood of chickens inoculated with E058(?ftsk) were significantly less than that of chickens inoculated with the parent strain APEC E058 (p<0.05), respectively. All birds were died during 48 hours post-inoculation with E058, however, only 20% of the birds were died within the same period post-inoculation with E058(?ftsk). All data showed that E058(?ftsk) had a reduced virulence in 1-day old chickens. It was hypothesized that ftsk may be an important virulence gene of APEC E058. The actual function of ftsk gene needs further studying.
In summary, the primary conclusions were drawn as following:
1) 202 UPEC isolates were collected from patients with UTIs in Jiangsu Province. Among those isolates, O1, O6, O7, O15, O18, O25 and O26 were the prevalent serogroups, and D and B2 were the most frequent phylogenetic groups.
2) Among 37 tested virulence genes, feoB and fimH were the most prevalent genes, which distributed in over 90% of the isolates. Above half of the isolates possessed one or more of the following 10 virulence genes, papA, papC, papEF, papG, kpsMTⅡ, ompT, iutA, fyuA, traT and irp-2.
3) All UPEC strains showed over 50% resistance rate to all of 14 tested antimicrobials except for cefoxitin, nitrofurantoin and chloramphenicol, which were 22%, 23% and 36%, respectively. Among them, the resistance rate to nalidixic acid, mezlocillin, and ampicillin was higher than 90%. Importantly, all of the isolates were multi-resistant, and 73% of them were resistant to trimethoprim-sulfamethoxazole.
4) Of the UPEC isolates originated from Jiangsu area, cefotaxime-, cefoxitin-, chloramphenicol-, and nitrofurantoin-resistant isolates have a reduced virulence genes compared with those of susceptible strains. And this is the first report of a statistically significant reduction in virulence genes among nitrofurantoin-resistant isolates.
5) UPEC and APEC isolates showed substantial overlap in terms of their serogroups, phylogenetic groups, and virulence genotypes including their possession of certain genes associated with large transmissible plasmids of APEC.
6) No host specificity could be observed in both APEC and UPEC isolates, since both avian- and human-originted isolates were highly pathogenic to chickens as well as caused similar lesions in the birds inoculated.
7) In chicken challenge model, 89% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. 5 genes (cvaC, neuC, ompT, iutA and iucCD) were differentially up-regulated in APEC E058, and two (cvaC and neuC) of them were also differentially up-regulated in UPEC U17, and the left three (ompT, iutA and iucCD) of them were up-regulated in UPEC U17 also. 4 genes (aec-30, aes-8, gyrB and mdh) differentially down-regulated in APEC E058 were also down-regulated in UPEC U17.
8) In murine UTI model, 82% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. Two differentially up-regulated genes (iucCD and tir) in UPEC U17 were also up-regulated in APEC E058, and 2 differentially down-regulated genes (sta and catI) in UPEC U17 were also down-regulated in APEC E058.
9) Several iron-related genes were up-regulated in UTI model and/or chicken challenge model, revealing that iron acquisition is critical for E. coli to survive in blood or urinary tract.
10) Two-step qRT-PCRs were developed based on SYBR Green I for relatively quantifying the expression level of ompT and ftsk genes of APEC strain E058 and UPEC strain U17. Three standard curves were established using a series dilution of cDNA synthesized from the RNA of APEC E058 grown in vitro.
11) The expression of ompT and ftsk of UPEC U17 and APEC E058 were up-regulated in chicken challenge model compared to that of APEC E058 and UPEC U17 grown statically to exponential phase in rich medium, respectively. These data verify the results of DNA chips, and qRT-PCR analysis demonstrate a greater transcription level sensitivity than that of microarray analysis, and suggest that ompT and ftsk may be the important virulence genes of APEC.
12) All data show that E058(?ftsk) has a reduced virulence in 1-day old chickens. It is reasonable that ftsk gene screened in SSH and confirmed in qRT-PCR might be an important virulence gene of APEC E058.
1.江苏部分地区UPEC分子流行病学与耐药性研究
从江苏部分地区尿道感染临床病例中分离、鉴定出202个UPEC分离株。对分离株的血清型、种系发生型、毒力基因型、耐药性以及毒力因子与耐药性间的关系作了研究。分离株最常见的血清型为O1,其它主要血清型还有O6、O7、O15、O18、O26和O25;种系发生型中,D和B2型最为常见,分别占受试分离株的42%和38%,而A和B1型分别占13%和7%。在受检的38个毒力基因中,fimH和feoB的检出率超过90%;50%以上的分离株至少携带papA、papC、papEF、papG、kpsMTⅡ、ompT、iutA、fyuA、traT、irp-2这10个基因中的一种。在黏附素中,P菌毛相关基因检出率仅次于fimH;在保护素中,荚膜形成相关基因kpsMT检出率较高;而三种毒素基因出现的频率相当。分离株耐药性较强,多重耐药现象普遍。在供试的14种抗生素中,UPEC分离株对11种抗生素的耐药率均超过了50%,其中,对萘啶酮酸、氨苄西林和美洛西林的耐药率均达到了90%以上;对磺胺甲基异噁唑的耐药率达到73%,远高于国外报道;对头孢西定、呋喃妥因、氯霉素的敏感性相对较高,其耐药率分别为22%、23%、36%;按美国国家临床实验室标准委员会方法手册,受试的202个UPEC分离株均为多重耐药菌株。表明受检地区UPEC的耐药情况相当严重,供试的抗生素已不适合作为受检地区抗尿道感染临床使用首选药物。本研究结果还表明,头孢噻肟、头孢西定、氯霉素和呋喃妥因抗性菌株,相对于其敏感菌株毒力有下降的趋势,其中呋喃妥因抗性菌株与其毒力下降之间的关系为首次报道。
2. APEC与UPEC毒力基因及其在体内外表达差异的比较
以我国自行分离的APEC和UPEC菌株为对象,对其血清型、种系发生型、毒力基因型及其毒力基因在体内外表达规律等方面的相似性进行了研究。结果表明二者在血清型、种系发生型、毒力基因型和毒力基因在体内外的表达规律等方面都表现出很大的相似性。本研究中O1和O18血清型为UPEC分离株的优势血清型,而这两种血清型在APEC分离株中也是常见的血清型。UPEC分离株的种系发生型以D和B2型最为常见,分别占受试分离株的42%和38%,其次是A和B1型分别占13%和7%;而APEC分离株以A和B2为主,A型比例最高为42%,B2型次之,为36%,D型和B1型分别为12%和10%。在受检的38个毒力基因中,检出频率最高的两个基因是feoB和fimH,其在两类菌株中的出现频率都超过90%;APEC和UPEC分离株中都检测出许多黏附素相关的基因、铁摄取相关的基因及质粒相关的毒力基因。SPF鸡的LD50测定结果显示UPEC代表菌株U17略低于APEC代表菌株E058,二者差异不明显,且攻毒鸡出现的病变相似。通过DNA芯片,对两种来源的大肠杆菌代表菌株分别在鸡攻毒模型和小鼠尿道感染模型中毒力基因的表达规律进行了研究。在攻毒鸡体内,APEC E058株和UPEC U17株中89%的T测试基因在表达趋势上一致;APEC E058株在攻毒鸡体内表达水平明显上调的5个基因(cvaC、neuC、ompT、iutA和iucCD)中,有两个基因(cvaC和neuC)在UPEC U17株中也明显上调,其它3个基因iutA、iucCD和ompT在UPEC U17株中也表现为上调趋势;APEC E058株在攻毒鸡体内表达水平明显下调的4个基因(aec-30、aes-8、gyrB和mdh),在UPEC U17株中也出现下调趋势。在小鼠体内,APEC E058株与UPEC U17株中82%的T测试基因在表达趋势上一致;UPEC U17株中明显上调的2个基因iucCD、tir和明显下调的2个基因sta、catI在APEC E058株中也表现为相应的上调或下调趋势。应用DNA芯片技术在国际上首次揭示,APEC和UPEC分离株在同一感染模型中的转录子具有相似的表达谱。上述研究结果提示我们,APEC很可能是UPEC的来源或者是UPEC毒力基因的贮存库。同时,许多与铁摄取系统相关的基因在鸡和小鼠体内的表达都表现为上调甚至明显上调,提示我们铁摄取可能是大肠杆菌得以实现其肠道外感染的必备条件。
3.检测ompT和ftsk相对定量PCR方法的建立及其对DNA芯片检测ompT表达水平的验证和对ftsk表达水平的解析
本研究利用嵌合荧光(SYBR GreenⅠ)标记,以编码甘油醛3-磷酸脱氢酶的看家基因gapA为内参基因,建立了用于ompT和ftsk基因表达水平解析的两步法实时定量反转录PCR (real time quantitative reverse transcription polymerase chain reaction, qRT-PCR)。运用该定量PCR分别建立了内参基因gapA和目的基因ompT与ftsk的3个标准曲线,这些标准曲线的斜率和线性关系均符合要求,表明所建立的定量PCR体系的扩增效率高、定量准确。定量PCR对ompT和ftsk基因在感染鸡体内的表达水平的解析结果表明,相对于体外培养,APEC E058株ompT、ftsk在鸡体内的表达分别上调了6.69和23.57倍;UPEC U17株分别上调了6.72和9.21倍。DNA芯片的检测结果表明,在鸡体内APEC E058株ompT的表达上调了2.54倍,证明我们自行研制的DNA芯片的检测结果是可信的,qRT-PCR比DNA芯片检测更为灵敏。上述结果提示我们,ompT和ftsk可能与大肠杆菌对鸡的致病力有关,即它们可能是APEC新的致病基因。
4.禽病原性大肠杆菌ftsk基因突变株的构建及其致病性鉴定
运用基因重组方法分别将卡那霉素抗性基因(kan)连接到PCR扩增的ftsk两端区域的2个基因片段之间,并分别插入到pBluescriptⅡS K (+) (pBSK)载体的多克隆位点中,构建出带kan基因标志的载体pBSK-F2-Kan-F1,通过PCR扩增F2-Kan-F1片段,电转化入APEC分离株E058中,根据同源重组原理,筛选出APEC E058 ftsk基因缺失突变株E058(?ftsk)。生长曲线测定结果显示,E058(?ftsk)的生长速度低于亲本菌株E058株,动物实验结果表明E058(?ftsk)在攻毒后6、12和24 h时血液、肝脏、脾脏和肺中的细菌数显著少于APEC E058株。APEC E058株感染组在接种48h后,除扑杀鸡外,接种鸡全部死亡;而E058(?ftsk)组相应的死亡率为20%左右。因此,可以认定ftsk为APEC E058株重要的毒力基因。
综上所述,对江苏部分地区UPEC的流行病学调查表明,UPEC的常见血清型为O1、O6、O7、O15、O18、O26和O25, D和B2是其最常见的种系发生型,结果与国外报道基本一致。在供试的14种抗生素中,UPEC分离株对11种抗生素的耐药率均超过了50%,其中,对萘啶酮酸、氨苄西林和美洛西林的耐药率均达到了90%以上;对磺胺甲基异噁唑的耐药率达到73%;分离株均为多重耐药菌株,表明本地区UPEC的耐药情况相当严重。UPEC分离株中,头孢噻肟、头孢西定、氯霉素和呋喃妥因抗性菌株,相对于其敏感菌株毒力有下降的趋势,其中呋喃妥因抗性菌株与其毒力下降之间的关系为首次报道。UPEC与APEC毒力因子及毒力基因在体内表达差异的比较结果表明,二者无论是在种系发生型、毒力基因型还是毒力基因在体内的表达规律上都有很大的相似性,提示我们APEC很可能是UPEC的来源或者是UPEC毒力基因的贮存库。通过建立的相对定量反转录PCR,对APEC E058株的ompT和ftsk基因在感染鸡体内的表达水平的解析,验证了DNA芯片的检测结果是可靠的,ompT和ftsk可能是APEC的毒力因子。利用基因重组技术对APEC E058株的ftsk基因进行了突变,测定了突变株的生长速度,并应用动物实验来评价此基因在APEC E058株对人工感染鸡致病性中的作用。结果显示,突变株生长速度低于亲本株,突变株在攻毒鸡的血液、肝脏、脾脏和肺中的细菌数显著少于亲本株,攻毒后48 h内,除扑杀鸡外,亲本株接种组的鸡全部死亡,而突变株组相应的死亡率为20%左右,表明ftsk是APEC的重要毒力基因。
Urinary tract infections (UTIs) are among the most common human diseases in the world, causing significant morbidity and mortality. Most community-acquired UTIs are due to uropathogenic E. coli (UPEC) infections. UPEC strains possess specialized virulence factors, enabling them to colonize and invade to the host, disrupt the host defense mechanisms, injure host tissues, and/or stimulate noxious host inflammatory response. The abilities of UPEC to grow extraintestinally may enable them to cause a variety of diseases, not just urinary tract ones. Avian colibacillosis refers to any localized or systemic infection such as an acute fatal septicemia or subacute pericarditis and airsacculitis caused entirely or partly by avian pathogenic Escherichia coli (APEC). It is responsible for large financial losses for the poultry industry each year due to mortality, lost production, and condemnations.
Since APEC and UPEC may encounter similar challenges when establishing infection in extraintestinal locations, they may share a similar content of virulence genes and capacity to cause disease. It is becoming more and more apparent that the common presence of a set of virulence associated genes among APEC and UPEC strains as well as similar disease patterns and phylogenetic background indicate a genetic relationship between APEC and UPEC isolates. The potential whether APEC might serve as a reservoir of virulence genes for UPEC should be considered.
1. Prevalence of virulence factors and antimicrobial resistance of UPEC isolates in Jiangsu area
202 UPEC isolates were characterized for serogroups, virulence genes, the antimicrobial susceptibilities and the phylogenetic groups. Most of the typable strains belonged to O1, O6, O7, O15, O18, O26 and O25 serogroups, respectively. The most frequent phylogenetic groups were D and B2, which represented 42% and 38% of the isolates respectively, followed by A and B1 groups, which were 13% and 7%, respectively. Among 37 tested virulence genes, feoB and fimH were the most prevalent genes, which distributed in over 90% of the isolates. Above half of the isolates possessed one or more of the following 10 virulence genes—papA, papC, papEF, papG, kpsMTⅡ, ompT, iutA, fyuA, traT and irp-2. pap related genes were the most prevalent other than fimH among adhesin genes. kpsMT genes occurred in a high prevalence in protectin genes studied, and genes encoding toxins were at similar frequencies. All isolates showed over 50% resistance rate to all of 14 tested antimicrobials except for cefoxitin, nitrofurantoin and chloramphenicol, which were 22%, 23% and 36%, respectively. Among them, the resistance rate to nalidixic acid, mezlocillin, and ampicillin was higher than 90%. Importantly, all of the isolates were multi-resistant, and 73% of them were resistant to trimethoprim-sulfamethoxazole. The above evidence strongly suggests that the problem of antibiotic resistance for UPEC is rather serious in this area, and that empirical treatment of UTIs with all the above antibiotics is no longer appropriate in the same area. Our findings also showed that among human UPEC isolates originated from this area, cefotaxime-, cefoxitin-, chloramphenicol-, and nitrofurantoin-resistant isolates had a reduced virulence genes compared with those of susceptible strains. To our knowledge, this is the first report of a statistically significant reduction in virulence genes among nitrofurantoin-resistant isolates.
2. Comparison of virulence genes and the expression of specific genes between APEC and UPEC isolates in murine urinary tract infection model and chicken challenge model
As diversity, epidemiological sources, and evolutionary origins of ExPEC are so far only partially defined, 100 of APEC isolates and 202 of UPEC isolates were compared by their serogroups, content of virulence genes, phylogenetic groups, and the expression of specific virulence genes in the present study. The results showed that O1 and O18 were the dominant serogroups of UPEC isolates, which were also prevalent in APEC isolates. Besides the UPEC phylogenetic groups mentioned above, most of APEC isolates belong to A and B2 groups, which represented 42% and 36% of the isolates respectively, followed by D and B1 groups, which were 12% and 10%, respectively. Some virulence genes, such as those encoding of adhesins, iron-related genes, and pTJ100-related genes were often carried by APEC and UPEC isolates. Althogh LD50 of UPEC strain U17 was slightly lower than that of APEC strain E058, similar lesions were observed in birds inoculated with either UPEC U17 or APEC E058. To gain further information of the correlation and pathogenesis of UPEC and APEC isolates, the in vivo expression of specific genes in both murine urinary tract infection (UTI) model and chicken challenge model were compared to that of UPEC U17 and APEC E058 grown statically to exponential phase in rich medium by DNA chips, respectively. In chicken challenge model, 89% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. In chicken challenge model, 5 genes (cvaC, neuC, ompT, iutA and iucCD) were differentially up-regulated in APEC E058, and two (cvaC and neuC) of them were also differentially up-regulated in UPEC U17, and the left three (ompT, iutA and iucCD) of them were up-regulated in UPEC U17 also. At the same time, 4 genes (aec-30, aes-8, gyrB and mdh) differentially down-regulated in APEC E058 were also down-regulated in UPEC U17. In murine UTI model, 82% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. Two differentially up-regulated genes (iucCD and tir) in UPEC U17 were also up-regulated in APEC E058, and 2 differentially down-regulated genes (sta and catI) in UPEC U17 were also down-regulated in APEC E058. The results show that in the same model (murine UTI model or chicken challenge model), most of the T-test genes of UPEC U17 and APEC E058 show the similar tendency of expression. Several iron-related genes were up-regulated in murine UTI model and /or chicken challenge model, revealing that iron acquisition is critical for E. coli to survive in blood or urinary tract. Based on these results, the potential for APEC to act as human UPEC or as a reservoir of virulence genes for UPEC should be considered. Further, this study represents the first comparison of APEC and UPEC transcriptome in vivo and provides specific insights into the mechanisms necessary for extraintestinal pathogenesis.
3. Development and application of relative real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the analysis of the expression of ompT and ftsk genes
To relatively quantify the expression of ompT and ftsk genes of APEC strain E058 and UPEC strain U17, two-step real time quantitative RT-PCRs (qRT-PCR) were developed based on SYBR Green I, respectively. ompT and ftsk were as target genes and gapA as internal reference, respectively. Three standard curves were established using a series dilution of cDNA synthesized from the RNA of APEC E058 grown statically to exponential phase in rich medium. In chicken challenge model, the expression of ompT and ftsk in APEC E058 were up-regulated 6.69- and 23.57-fold compared to that of APEC E058 grown statically to exponential phase in rich medium, respectively. Meanwhile, the expression of ompT and ftsk were up-regulated 6.72- and 9.21-fold in UPEC U17 compared to that of UPEC U17 grown statically to exponential phase in rich medium, respectively. Previous study showed that the expression of ompT in APEC E058 was up-regulated 2.54-fold in chicken challenged model by using DNA chips, when compared to that of APEC E058 grown statically to exponential phase in rich medium. These data verified the results of DNA chips, and qRT-PCR analysis demonstrated a greater transcription level sensitivity than that of microarray analysis. The result also suggests that ompT and ftsk may be the important virulence genes of APEC.
4. Construcion of ftsk knock-out mutant of APEC strain E058 and evaluation of its pathogenesis in chickens
The 709-bp fragment of ftsk1, 900-bp fragment of the ftsk2 and the gene encoding kanamycin resistance were generated by PCR respectively. All of these fragments were cloned into pBluescriptⅡS K (+) (pBSK) vector, termed pBSK-F2-Kan-F1. Fragment of F2-Kan-F1 amplified by PCR were transformed into APEC strain E058, and then the ftsk knock-out mutant of APEC strain E058 were generated by allelic replacement and named E058(?ftsk). The growth rate of E058(?ftsk) was slower than that of APEC E058. In chicken challenge model, the mutant was tested to determine the individual role of this gene for virulence and persistence in 1-day old SPF chicks. At 6, 12 and 24 hours after inoculation, the number of colony forming unit (CFU) recovered from the liver, spleen, lung and blood of chickens inoculated with E058(?ftsk) were significantly less than that of chickens inoculated with the parent strain APEC E058 (p<0.05), respectively. All birds were died during 48 hours post-inoculation with E058, however, only 20% of the birds were died within the same period post-inoculation with E058(?ftsk). All data showed that E058(?ftsk) had a reduced virulence in 1-day old chickens. It was hypothesized that ftsk may be an important virulence gene of APEC E058. The actual function of ftsk gene needs further studying.
In summary, the primary conclusions were drawn as following:
1) 202 UPEC isolates were collected from patients with UTIs in Jiangsu Province. Among those isolates, O1, O6, O7, O15, O18, O25 and O26 were the prevalent serogroups, and D and B2 were the most frequent phylogenetic groups.
2) Among 37 tested virulence genes, feoB and fimH were the most prevalent genes, which distributed in over 90% of the isolates. Above half of the isolates possessed one or more of the following 10 virulence genes, papA, papC, papEF, papG, kpsMTⅡ, ompT, iutA, fyuA, traT and irp-2.
3) All UPEC strains showed over 50% resistance rate to all of 14 tested antimicrobials except for cefoxitin, nitrofurantoin and chloramphenicol, which were 22%, 23% and 36%, respectively. Among them, the resistance rate to nalidixic acid, mezlocillin, and ampicillin was higher than 90%. Importantly, all of the isolates were multi-resistant, and 73% of them were resistant to trimethoprim-sulfamethoxazole.
4) Of the UPEC isolates originated from Jiangsu area, cefotaxime-, cefoxitin-, chloramphenicol-, and nitrofurantoin-resistant isolates have a reduced virulence genes compared with those of susceptible strains. And this is the first report of a statistically significant reduction in virulence genes among nitrofurantoin-resistant isolates.
5) UPEC and APEC isolates showed substantial overlap in terms of their serogroups, phylogenetic groups, and virulence genotypes including their possession of certain genes associated with large transmissible plasmids of APEC.
6) No host specificity could be observed in both APEC and UPEC isolates, since both avian- and human-originted isolates were highly pathogenic to chickens as well as caused similar lesions in the birds inoculated.
7) In chicken challenge model, 89% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. 5 genes (cvaC, neuC, ompT, iutA and iucCD) were differentially up-regulated in APEC E058, and two (cvaC and neuC) of them were also differentially up-regulated in UPEC U17, and the left three (ompT, iutA and iucCD) of them were up-regulated in UPEC U17 also. 4 genes (aec-30, aes-8, gyrB and mdh) differentially down-regulated in APEC E058 were also down-regulated in UPEC U17.
8) In murine UTI model, 82% of the T-test genes showed the similar tendency of expression in both APEC E058 and UPEC U17. Two differentially up-regulated genes (iucCD and tir) in UPEC U17 were also up-regulated in APEC E058, and 2 differentially down-regulated genes (sta and catI) in UPEC U17 were also down-regulated in APEC E058.
9) Several iron-related genes were up-regulated in UTI model and/or chicken challenge model, revealing that iron acquisition is critical for E. coli to survive in blood or urinary tract.
10) Two-step qRT-PCRs were developed based on SYBR Green I for relatively quantifying the expression level of ompT and ftsk genes of APEC strain E058 and UPEC strain U17. Three standard curves were established using a series dilution of cDNA synthesized from the RNA of APEC E058 grown in vitro.
11) The expression of ompT and ftsk of UPEC U17 and APEC E058 were up-regulated in chicken challenge model compared to that of APEC E058 and UPEC U17 grown statically to exponential phase in rich medium, respectively. These data verify the results of DNA chips, and qRT-PCR analysis demonstrate a greater transcription level sensitivity than that of microarray analysis, and suggest that ompT and ftsk may be the important virulence genes of APEC.
12) All data show that E058(?ftsk) has a reduced virulence in 1-day old chickens. It is reasonable that ftsk gene screened in SSH and confirmed in qRT-PCR might be an important virulence gene of APEC E058.
引文
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