福氏志贺氏菌毒力基因ipaC在大肠杆菌中表达的初步研究

目的：构建福氏志贺氏菌毒力基因ipaC与质粒pET32a的重组质粒，转入大肠杆菌中表达。并检测表达产物的活性。方法：PCR扩增福氏志贺氏菌毒力质粒中ipaC基因，克隆到质粒pET32a T7启动子下游，转化感受态大肠杆菌BL21(λDE3)，IPTG诱导表达，SDS-PAGE以及Western-Blot分析。结果：获得pET32a-ipaC重组表达质粒。SDS-PAGE以及Western-Blot显示重组质粒表达产物的分子量约为63kDa。并具有一定的免疫活性。结论：福氏志贺氏菌毒力基因ipaC可在大肠杆菌中表达。且表达产物具有一定的免疫活性。     

Immunoproteome analysis of soluble and membrane proteins of Shigella flexneri 2457T

INTRODUCTION Shigellosis is a public health concern in developing countries, particularly for young children who make up 69% of all cases. The majority of shigellosis cases in the Asian, African and Central American regions are caused by Shigella flexneri…     

福氏志贺菌5型M90T株O抗原多糖细胞毒性作用的研究

本文提取并纯化了福氏志贺菌 5型M 90T的脂多糖 (lipopolysaccharide ,LPS)亚单位分子———O抗原多糖 (O anti genpolysaccharide ,OPS)进行体内外试验 ,并与该菌LPS作对比 ,揭示其亚单位OPS的毒性作用不同于完整LPS分子。OPS可引起HeLa细胞病变 ,而LPS不能 ;OPS可引起兔回肠襻肠粘膜出血 ,但不引起肠腔积液 ,而LPS引起肠腔积液 ,并严重损伤肠粘膜。说明LPS的亚单位———OPS有其特异的致病作用     

福氏志贺菌中质粒介导喹诺酮类耐药qnr基因的检测

目的 了解福氏志贺菌中qnr基因的分布.方法 采用PCR法检测26株福氏志贺菌的qnr基因并对阳性扩增产物进行测序分析,采用琼脂稀释法测定qnr基因阳性菌株对多种抗菌药物的敏感性,采用随机引物PCR法(ERIC-PCR)进行qnr基因阳性菌株同源性检测.结果 26株福氏志贺菌中,qnrAl、qnrS1和qnrS2基因的阳性率分别为30.8%、11.5%和3.8%;qnr基因阳性菌株对氯霉素、奈啶酸耐药.对诺氟沙星、哌拉西林耐药或中介;部分菌株对头孢唑啉、阿米卡星、第二、三代头孢菌素耐药.ERIC-PCR电泳图谱型显示.2株qnrAl基因阳性菌株属于同一谱型.结论 福氏志贺菌中存在qnr基因阳性菌株,部分菌株间存在克隆传播现象,临床应加强检测和监测.     

志贺菌属肠毒素ShET1/ShET2的基因型PCR分析

目的 对福氏2a志贺菌肠毒素ShET1和肠毒素ShET2进行基因分型，提高福氏2a志贺菌同源克隆鉴定系统的分析水平。方法 对93株分离自不同地区、不同时间的福氏2a志贺菌用PCR法检测志贺菌肠毒素ShET1／ShET2基因并进行基因分型。结果 93株福氏2a志贺菌按志贺菌肠毒素ShET1／ShET2基因分为4种基因型，即12株ShET1(-)／ShET2( )，14株ShET1( )／ShET2(-)，59株ShET1( )／ShET2( )，8株ShET1(-)／ShET2(-)。结论 福氏2a志贺菌肠毒素ShET1／ShET2基因PCR检测可用于福氏2a志贺菌的初步筛检。     

马鞍山市志贺菌福氏4c亚型毒力基因检测及分子分型研究

目的了解马鞍山市志贺菌福氏4 c(ShigellaF4c)分离株的毒力基因的流行模式及分子分型的特点,掌握Shi-gellaF4c在该市的流行状况。方法使用常规法进行菌株分离培养、使用全自动微生物鉴定系统进行生化鉴定,鉴定为志贺菌的阳性菌株使用PCR检测ShigellaF4c的ipa H、ial、set、sen四种毒力基因及应用PFGE进行分子分型。结果 ipa H、ial、set、sen四种毒力基因在76株ShigellaF4c的携带率分别为100%、97.4%、96.1%、90.8%,其中67株ShigellaF4c四种毒力基因全为阳性,占88.2%;本地区ShigellaF4c携带毒力基因模式分成五大类型,Ⅰ型是主要毒力基因模式。PFGE电泳条带图谱显示,ShigellaF4c中100%相同的菌株数较少;100%相同的都为同一年代,91%相同的菌株出现跨年度;不同年代分离株无完全相同基因型,地域方面没有明显联系。结论马鞍山地区S.F4c分离株携带ipa H、ial、set、sen四种毒力基因普遍存在,毒力强是本地区ShigellaF4c主要特征之一;ShigellaF4c在本地区无显著的流行迹象,属于散在发病。     

PCR快速检测食品中志贺氏菌方法的建立

目的本研究根据福氏志贺菌(Shigella lexneri)侵袭性质粒抗原H基因(invasion plasmid antigen H,ipaH),设计了一对特异性引物,预计PCR扩增的目的基因片段为435bp。对PCR的特异性、敏感性分析以及建立L16(43)正交试验对PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速检测福氏志贺菌的稳定的PCR方法。该方法检测的灵敏度为可以检测到食品中7 ng/ml福氏志贺菌的基因组DNA。并且模拟检测食品中的细菌,结果很稳定。该方法操作简单、检验周期短、特异性和灵敏度高,能够快速地实现对食品中的志贺菌的诊检和监控。     

志贺菌基因转移耐多药相关蛋白初步分析

目的:对志贺菌敏感株与基因转移耐多药株全菌蛋白进行蛋白质组学比较,寻找细菌耐多药相关蛋白.方法:采用接合基因转移实验对临床分离鉴定志贺菌敏感株进行基因转移耐多药试验,并对志贺菌敏感株及基因转移耐多药株全菌蛋白进行双向电泳;电泳图谱采用Image Master 2D Platinum软件分析,并对差异表达蛋白进行MOLDI TOF-TOF质谱分析.结果:成功获得志贺菌基因转移耐多药株,在志贺菌敏感株与耐多药株全菌蛋白质图谱中分别检测出946±37个和1013±157个蛋白质斑点;经分析共发现43个差异表达的蛋白点,初步对其中5个表达量增加的差异蛋白进行质谱鉴定,基因转移耐多药株中发现两个新出现的耐多药相关蛋白分别为CRISPR相关蛋白及Hsp60的分子伴侣蛋白(Groel-Groes-Adp7);ABC转运蛋白、胱氨酸合成酶、预测的胞浆脂蛋白表达量上调.结论:通过对鉴定的5个耐多药相关蛋白分析初步发现供体菌中一些耐多药相关基因通过基因转移方式插入志贺菌敏感株中并大量表达,同时一些在细胞代谢中起重要作用的酶类表达量上调,ABC转运蛋白在志贺菌基因转移耐多药机制中也起重要作用.     

福氏志贺菌中超广谱β-内酰胺酶的基因型分析

目的检测福氏志贺菌中超广谱G内酰胺酶（ESBLs）的基因型别。方法琼脂稀释法测定5株福氏志贺菌对多种抗菌药物的敏感性，并进行接合试验；改良三维试验检测产ESBLs菌株，同时对这些菌株进行脉冲场电泳（PFGE）检测；采用TEM、SHV、CTX-M-1组、CTX-M-2组、CTX-M-9组β-内酰胺酶通用引物以及TEM、CTX-M-9组全编码基因引物进行PCR检测，并对全编码基因PCR产物进行DNA序列分析。结果三维试验结果显示，5株福氏志贺菌均为产ESBLs菌株，对青霉素类、第一代、第二代头孢菌素以及四环素、复方磺胺甲嗯唑显著耐药，对第三代头孢菌素中头孢曲松、头孢噻肟耐药或中度敏感，对亚胺培南、头孢美唑、氟喹诺酮类、头孢噻肟克拉维酸、头孢他啶、头孢他啶一克拉维酸显示敏感。对于β-内酰胺类的耐药性可以通过接合方式发生水平转移；ESBLs基因型别为CTX-M-14，5株菌株的PFGE谱型可分为A、B两种谱型。结论5株福氏志贺菌产生CTX-M-14型超广谱β-内酰胺酶，导致对多种β-内酰胺类抗生素耐药，并存在克隆传播，需加强监控。     

Correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri and the sul genes

The aim of this study was to discuss the correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri (S. flexneri) and the antibiotic resistance genes sul1, sul2, and sul3 and SXT element.From May 2013 to October 2018, 102 isolates of S. flexneri were collected from the clinical samples in Jinan. The Kirby–Bauer (K-B) test was employed to determine the antibiotic susceptibility of the S. flexneri isolates. The antibiotic resistance rate was analyzed with the WHONET5.4 software. The isolates were subject to the PCR amplification of the sul genes (sul1, sul2, and sul3) and the SXT element. On the basis of the sequencing results, the correlation between the sulfamethoxazole-trimethoprim resistance of the S. flexneri isolates and the sul genes was analyzed.The antibiotic resistance rates of the 102 S. flexneri isolates to ampicillin, streptomycin, chloramphenicol, tetracycline, and sulfamethoxazole-trimethoprim were 90.2%, 90.2%, 88.2%, 88.2%, and 62.7%, respectively. The antibiotic resistance rates of these isolates to cefotaxime, ceftazidime, and ciprofloxacin varied between 20% and 35%. However, these isolates were 100% susceptible to cefoxitin. Positive fragments were amplified from 59.8% (61/102) of the 102 S. flexneri isolates, the sizes of the sul1 and sul2 genes being 338 bp and 286 bp, respectively. The sequence alignment revealed the presence of the sul1 and sul2 genes encoding for dihydrofolate synthase. The carrying rate of the sul1 gene was 13.7% (14/102), and that of the sul2 gene was 48.0% (49/102). No target gene fragments were amplified from the 3 isolates resistant to sulfamethoxazole-trimethoprim. The sul3 gene and SXT element were not amplified from any of the isolates. The testing and statistical analysis showed that the resistance of the S. flexneri isolates to sulfamethoxazole-trimethoprim correlated to the sul1 and sul2 genes.The acquired antibiotic resistance genes sul1 and sul2 were closely associated with the resistance of the 102 S. flexneri isolates to sulfamethoxazole-trimethoprim.     

天津地区福氏志贺菌整合子携带及耐药性研究

目的了解天津地区不同年份福氏志贺菌血清型分布,整合子携带情况,耐药性及其变化趋势。方法采用血清学方法对天津地区不同年份分离的56株福氏志贺菌进行分型;PCR扩增整合子整合酶及可变区,并测序;采用K-B法测定其对16种抗菌药物的敏感性。结果 1981~1983年分离株福氏志贺菌1类整合酶阳性率为87.50%(28/32),其中27株可变区含氨基糖苷类药物耐药基因aadA;2009~2011年分离株福氏志贺菌1类整合酶阳性率为91.67%(22/24),可变区及3’末端扩增均阴性。1981~1983年分离株福氏志贺菌2类整合酶均阴性;2009~2011年分离株福氏志贺菌2类整合酶阳性率79.17%(19/24),可变区含dfrA1、sat1、aadA1,介导对甲氧苄啶、链丝菌素和链霉素耐药。有17株福氏志贺菌1、2类整合酶均阳性。1981~1983年分离株福氏志贺菌对四环素、链霉素、氯霉素及甲氧苄啶/磺胺甲噁唑耐药率高,多重耐药率为65.63%;2009~2011年分离株福氏志贺菌对氨苄西林、链霉素、甲氧苄啶/磺胺甲噁唑、哌拉西林和四环素耐药率高,多重耐药率为83.33%。结论 1、2类整合子广泛存在于天津地区福氏志贺菌中,其中1类整合子3’保守末端可能缺失或存在变异。2009~2011年分离株福氏志贺菌对抗菌药物耐药性较1981~1983年分离株增强,多重耐药率增高。福氏志贺菌优势血清型发生转变,血清型分布呈现多样化。     

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM: To screen the immunogenic membrane proteins of Shigella flexneri 2a 2457T. METHODS: The routine two-dimensional polyacrylamide gel electrophoresis (2-DE) and Western blotting were combined to screen immunogenic proteins of S. flexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was performed to determine the molecular weight of peptides. Electrospray ionization (ESI-MS/MS) was performed to determine the sequences of the interesting peptides. RESULTS: A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein (YaeT) was detected, which might be a candidate target of vaccine. CONCLUSION: Membrane proteins of 5. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.     

福氏志贺菌主动外排基因acrA的克隆及原核表达

目的对福氏志贺菌外排基因acrA进行克隆和原核表达,为进一步研究其在志贺菌外排机制中的作用奠定基础。方法参考福氏志贺菌2aacrA基因序列设计一对特异引物,在引物的5′和3′端分别加入含有BamHⅠ和SalⅠ限制性酶切位点序列。以福氏志贺菌2a菌株基因组DNA为模板,通过PCR扩增acrA基因并与pMD18-T载体连接,然后转化DH5α。提取重组质粒pMD18-acrA,经BamHI/SalI双酶切并与载体pET30a连接后转化宿主菌BL21(DE3)pLys,通过IPTG诱导表达目的蛋白。结果克隆的acrA基因长度为1122bp,核苷酸序列与GenBank上公布的序列完全相同。原核表达经SDS-PAGE及WesternBlotting检测和鉴定,结果表明重组载体pET-30a-acrA可成功地在大肠杆菌中表达AcrA蛋白。结论成功构建了福氏志贺菌acrA基因的原核表达质粒,并在大肠杆菌中得到有效表达,该研究为了解AcrA蛋白的特性、功能以及对福氏志贺菌多药耐药机理的深入研究奠定了基础。     

HYPOXIC STRESS,HEPATOCYTES AND CACO-2 VIABILITY AND SUSCEPTIBILITY TO Shigella flexneri INVASION

SUMMARY

Inflammation due to Shigella flexneri can cause damage to the colonic mucosa and cell death by necrosis and apoptosis. This bacteria can reach the bloodstream in this way, and the liver through portal veins. Hypoxia is a condition present in many human diseases, and it may induce bacterial translocation from intestinal lumen. We studied the ability of S. flexneri to invade rat hepatocytes and Caco-2 cells both in normoxic and hypoxic microenvironments, as well as morphological and physiological alterations in these cells after infection under hypoxia. We used the primary culture of rat hepatocytes as a model of study. We analyzed the following parameters in normoxic and hypoxic conditions: morphology, cell viability, bacterial recovery and lactate dehydrogenase (LDH) released. The results showed that there were fewer bacteria within the Caco-2 cells than in hepatocytes in normoxic and hypoxic conditions. We observed that the higher the multiplicity of infection (MOI) the greater the bacterial recovery in hepatocytes. The hypoxic condition decreased the bacterial recovery in hepatocytes. The cytotoxicity evaluated by LDH released by cells was significantly higher in cells submitted to hypoxia than normoxia. Caco-2 cells in normoxia released 63% more LDH than hepatocytes. LDH increased 164% when hepatocytes were submitted to hypoxia and just 21% when Caco-2 cells were in the same condition. The apoptosis evaluated by Tunel was significantly higher in cells submitted to hypoxia than normoxia. When comparing hypoxic cells, we obtained more apoptotic hepatocytes than apoptotic Caco-2 cells. Concluding our results contribute to a better knowledge of interactions between studied cells and Shigella flexneri. These data may be useful in the future to define strategies to combat this virulent pathogen.     

福氏2a志贺菌肠毒素ShET1/ShET2基因分型在菌痢快速诊断中的应用

目的对福氏2a志贺菌肠毒素ShET1(Shigellaenterotoxin1)和肠毒素ShET2(Shigellaenterotoxin2)进行基因分型,提高菌痢爆发流行时同源克隆的鉴定分析水平。方法对93株分离自不同地区,不同时间的福氏2a志贺菌用PCR法检测志贺菌肠毒素ShET1/ShET2基因,进行基因分型和同源克隆鉴定。结果93株福氏2a志贺菌按志贺菌肠毒素ShET1/ShET2基因可分为4种基因型,即12株ShET1(-)/ShET2(+),14株ShET1(+)/ShET2(-),59株ShET1(+)/ShET2(+),8株ShET1(-)/ShET2(-)。93株福氏2a志贺菌ShET1检出率为89.24%(83/93),ShET2为65.59%(61/93)。二者至少有一种基因被检出的检出率为91.39%(85/93)。结论福氏2a志贺菌的快速诊断可应用ShET1、ShET2双基因PCR检测,具有较高的敏感性与特异性。在应用多生物学标志进行福氏2a志贺菌同源克隆鉴定系统研究时,肠毒素ShET1/ShET2基因PCR分析是不可或缺的分析指标。     

Tyrosine kinases,drugs, and Shigella flexneri dissemination

Shigella flexneri is an enteropathogenic bacterium responsible for approximately 100 million cases of severe dysentery each year. S. flexneri colonization of the human colonic epithelium is supported by direct spread from cell to cell, which relies on actin-based motility. We have recently uncovered that, in intestinal epithelial cells, S. flexneri actin-based motility is regulated by the Bruton’s tyrosine kinase (Btk). Consequently, treatment with Ibrutinib, a specific Btk inhibitor currently used in the treatment of B-cell malignancies, effectively impaired S. flexneri spread from cell to cell. Thus, therapeutic intervention capitalizing on drugs interfering with host factors supporting the infection process may represent an effective alternative to treatments with antimicrobial compounds.     

The synthesis of OspD3 (ShET2) in Shigella flexneri is independent of OspC1

Shigella flexneri is a Gram-negative pathogen that invades the colonic epithelium and causes millions of cases of watery diarrhea or bacillary dysentery predominately in children under the age of 5 years in developing countries. The effector Shigella enterotoxin 2 (ShET2), or OspD3, is encoded by the sen or ospD3 gene on the virulence plasmid. Previous literature has suggested that ospD3 is in an operon downstream of the ospC1 gene, and expression of both genes is controlled by a promoter upstream of ospC1. Since the intergenic region is 328 bases in length and contains several putative promoter regions, we hypothesized the genes are independently expressed. Here we provide data that ospD3 and ospC1 are not co-transcribed and that OspC1 is not required for OspD3/ShET2 function. Most importantly, we identified strong promoter activity in the intergenic region and demonstrate that OspD3/ShET2 can be expressed and secreted independently of OspC1. This work increases our understanding of the synthesis of a unique virulence factor and provides further insights into Shigella pathogenesis.     

Influence of Physicochemical Factors on Adsorption of Ten Shigella flexneri Phages

Bacterial viruses known as bacteriophages have been demonstrated to be effective in killing foodborne pathogens such as Shigella flexneri. Adsorption is the first step in the phage–host interaction. In the present work, 10 Shigella phages were used to characterize the adsorption process on Shigella flexneri ATCC12022 in several physicochemical conditions related to food and in a food matrix. One-step growth curves were drawn for all the Shigella-phages evaluated. Furthermore, the adsorption rate for each of the 10 phages was determined. In addition, the influence of temperature, Na⁺, Mg²⁺, pH, sucrose and glycerol on phage adsorption was investigated. Two phages (Shi22 and Shi30) showed higher burst sizes values (67 and 64 PFU cell⁻¹, respectively) and burst times of 25 min to 30 min, while the other eight phages exhibited burst sizes ranging from 14 to 17 PFU cell⁻¹ with slower burst times. Furthermore, most phages achieved a high adsorption rate, and the adsorption constants (k) ranged from ~10⁻⁹ to 10⁻¹⁰ mL min⁻¹. Regarding the influence of temperature, cations and pH, a high or moderate percentage of adsorption was observed for most of the phages evaluated. The adsorption decreased at increasing concentrations of Na⁺, sucrose and glycerol, although at different levels, since adsorption was more affected by sucrose than by glycerol and Na⁺ for most phages. The adsorption obtained in Triptein soy broth (TSB) for most of the phages/strain systems evaluated was moderate or high, as well as those observed in a food matrix. Thus, our phages could potentially be used to improve food safety under a wide range of environmental conditions against foodborne pathogens.     

HeLa细胞感染痢疾杆菌前后差异表达的新EST序列的电子延伸及验证

目的:研究痢疾杆菌福氏2a入侵前和入侵后3h的人上皮细胞差异表达的新基因.方法:采用增毒的痢疾杆菌福氏2a 2457T侵袭HeLa细胞,检查HeLa细胞中细菌的数量并用RT-PCR方法从HeLa细胞中提取总RNA,制备探针.采用含有约3000个代表新基因的芯片进行芯片杂交,分析杂交结果.将所获得的新的156条EST序列为种子序列用cDNA微阵列实验,以人类EST数据库为基础库,应用siClone软件进行EST拼接、校对并尽可能延长获得大片段或全长cDNA.选取基因组未注释的编码区序列作为新基因进行RT-PCR实验验证.结果:共鉴定到≥3倍或≤0.33的差异表达的代表新基因的EST序列45条,其中25个延伸序列鉴定为与重要的肠道功能相关的已知基因,并有3个在痢疾杆菌侵袭期间高表达的存在显著差异的新EST序列,他们分别名为:NPCCKH12、ADBCSB02和HTBAMG05,这3个基因是新的人基因或假基因.结论:鉴定出了在HeLa细胞感染痢疾杆菌前后差异表达的新EST序列,为进一步研究痢疾杆菌与寄主相互作用奠定了基础.