The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology

Avian pathogenic Escherichia coli(APEC)belonging to extraintestinal pathogenic E.coli(ExPEC)can cause severe infections in extraintestinal tissues in birds and humans,such as the lungs and blood.MprA(microcin production regulation,locus A,herein renamed AbsR,a blood survival regulator),a member of the MarR(multiple antibiotic resistance regulator)transcriptional regulator family,governs the expression of capsule biosynthetic genes in human ExPEC and represents a promising druggable target for antimicrobials.However,a deep understanding of the AbsR regulatory mechanism as well as its regulon is lacking.In this study,we present a systems-level analysis of the APEC AbsR regulon using ChIP-Seq(chromatin immunoprecipitation sequencing)and RNA-Seq(RNA sequencing)methods.We found that AbsR directly regulates 99 genes and indirectly regulates 667 genes.Furthermore,we showed that:1)AbsR contributes to antiphagocytotic effects by macrophages and virulence in a mouse model for systemic infection by directly activating the capsular gene cluster;2)AbsR positively impacts biofilm formation via direct regulation of the T2SS(type II secretion system)but plays a marginal role in virulence;and 3)AbsR directly upregulates the acid tolerance signaling system EvgAS to withstand acid stress but is dispensable in ExPEC virulence.Finally,our data indicate that the role of AbsR in virulence gene regulation is relatively conserved in ExPEC strains.Altogether,this study provides a comprehensive analysis of the AbsR regulon and regulatory mechanism,and our data suggest that AbsR likely influences virulence primarily through the control of capsule production.Interestingly,we found that AbsR severely represses the expression of the type I-F CRISPR(clustered regularly interspaced short palindromic repeats)-Cas(CRISPR associated)systems,which could have implications in CRISPR biology and application.

Distribution of Virulence-Associated Genes of Avian Pathogenic Escherichia coli Isolates in China

216 avian pathogenic Escherichia coli （APEC） isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as 078, 088, and 093. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD （70.8%）, iss （58.8%）, and tsh （51.4%） in APEC isolates. The detection rate of high pathogenicity islands （HPI）-associatedfyuA and irp2 genes were both 44.9%, with no LEE （the locus of enterocyte effacement） island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained onlyfimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% 078 isolates had a gene distribution patterns of fimC^＋iucD^＋irp2^＋fyuA^＋iss^＋colV^＋tsh^＋.

Prevalence of shiga toxins(stx_1,stx_2),eaeA and hly genes of Escherichia coli O157:H7 strains among children with acute gastroenteritis in southern of Iran

Objective:To survey the prevalence severe diarrhea arising from these bacteria in children under 5 years old in Marvdasht.Methods:In this study faecal sample from 615 children aged <5years old who were hospitalized lor gastroenteritis in Fars hospitals in Iran were collected and then enriched in Escherichia coli(E.coli) broth and modified tryplone soy broth with novobiocin media,fermentation of sorbitol,lactose and β— glucoronidase activity of isolated strains was examined by CT—SMAC,VRBA and chromogenic media respectively.Then isolation of E.coli O157:H7 have been confirmed with the use of specific antisera and with multiplex PCR method presence of virulence genes including:xtx_1.stx_2,eae.A.hly has been analyzed.Results:E.coli O157:H7 was detected in 7(1.14%) stool specimens.A significanl difference was seen between detection rale of isolated bacteria from age groups 18-23 months and other age groups(P=0.004).Out of considered virulence genes.only 1 of the isolated strains(0.16%)he stx,and eaeA genes were seen and also all isolated hacleria had resistance to penicillin,ampicillin and erythromycin antibiotics.Conclusions:We found thai children < 2 years of age were at highest risk of infection with E.coli O157:H7.Regarding severity of E.coli O157:H7 pathogenesis,low infectious dose and lack of routine assay for detection ol these bacleria in clinical laboratory,further and completed studies on diagnosis and genolyping of this E.coli O157:H7 strain has been recommended.

Probiotic bacteria change Escherichia coli-induced gene expression in cultured colonocytes:Implications in intestinal pathophysiology

AIM: To investigate the change in eukaryotic gene expression profile in Caco-2 cells after infection with strains of Escherichia coli and commensal probiotic bacteria. METHODS: A 19 200 gene/expressed sequence tag gene chip was used to examine expression of genes after infection of Caco-2 cells with strains of normal flora E. coli,Lactobacillus plantarum,and a combination of the two. RESULTS: The cDNA microarray revealed up-regulation of 155 and down-regulation of 177 genes by E. coli . L. plantarum up-regulated 45 and down-regulated 36 genes. During mixed infection,27 genes were up-regulated and 59 were down-regulated,with nullification of stimulatory/inhibitory effects on most of the genes. Expression of several new genes was noted in this group. CONCLUSION: The commensal bacterial strains used in this study induced the expression of a large number of genes in colonocyte-like cultured cells and changed the expression of several genes involved in important cellular processes such as regulation of transcription,protein biosynthesis,metabolism,cell adhesion,ubiquitination,and apoptosis. Such changes induced by the presence of probiotic bacteria may shape the physiologic and pathologic responses they trigger in the host.

The Pathogenicity of Chicken Pathogenic <i>Escherichia coli</i>Is Associated with the Numbers and Combination Patterns of Virulence-Associated Genes

Various virulence-associated genes or pathogenicity island are responsible for determining the pathogenicity of Escherichia coli strains. However, the correlation of the number and combination patterns of virulence-associated genes in Escherichia coli strains with their pathogenicity remains largely unknown. In this work, 581 chicken Escherichia coli strains were isolated from 1045 liver samples of dead chickens from 50 chicken farms at four provinces in China during 2007-2012. Based on the pathogenic test of SPF chickens, 320 chickens pathogenic Escherichia coli isolates were identified as highly (n = 193), intermediate (n = 98) and low pathogenic (n = 29) strains, respectively. Furthermore, the number of virulence genes in the 320 chicken pathogenic and 50 non-pathogenic Escherichia coli strains was examined. Our results reveal that thirteen virulence genes in Escherichia coli strains were detected, and all strains carried at least two or more than two virulence-associated genes. This study also suggests that highly pathogenic E. coli strains simultaneously carried at least 8 to13 virulence genes while intermediate pathogenic strains carried at least 5 to 8 virulence genes. The number of virulence-associated genes detected in highly pathogenic strains showed there were more significant differences than that in low pathogenic strains (P irp2, fyuA, and colV in high pathogenic strains was significantly higher than that in low and non-pathogenic strains (P irp2, fyuA, iucA, iucD, iutA, papC, iss, tsh, and colV were more often detected in highly and intermediate pathogenic E. coli strains. Taken together, our results provide evidences demonstrating that the pathogenicity of Escherichia coli strains is closely associated with the number and combination patterns of virulence-associated genes.

Regulation of fim genes in uropathogenic Escherichia coli

Uropathogenic Escherichia coli(UPEC)is the leading cause of urinary tract infections in women,causing significant morbidity and mortality in this population.Adherence to host epithelial cells is a pivotal step in the pathogenesis of UPEC.One of the most important virulence factors involved in mediating this attachment is the type 1 pilus(type 1 fimbria)encoded by a set of fim genes arranged in an operon.The expression of type 1 pili is controlled by a phenomenon known as phase variation,which reversibly switches between the expression of type 1 pili(Phase-ON)and loss of expression(Phase-OFF).Phase-ON cells have the promoter for the fimA structural gene on an invertible DNA element called fimS,which lines up to allow transcription,whereas transcription of the structural gene is silenced in Phase-OFF cells.The orientation of the fimS invertible element is controlled by two site-specific recombinases,FimB and FimE.Environmental conditions cause transcriptional and post-transcriptional changes in UPEC cells that affect the level of regulatory proteins,which in turn play vital roles in modulating this phase switching ability.The role of fim gene regulation in UPEC pathogenesis will be discussed.

Antibiotic susceptibility and molecular characterization of resistance genes among Escherichia coli and among Salmonella subsp. in chicken food chains

Objective: To investigate the occurrence of resistance genes among Escherichia coli(E. coli) and Salmonella subsp. isolated in chicken food chains in Phnom Penh, 2012–2013.Methods: Six hundred eighty two E. coli and 181 Salmonella Albany, Corvallis, and Kentucky strains were examined for susceptibilities to eight antimicrobials and following resistance genes were identified by PCR: blaTem, Str A, aad A, sul1, sul2, gyr A, Tet(A), and Tet(B).Results: E. coli presented high resistances to tetracycline, amoxicillin, and sulfamethoxazole(63.1%–76.1%). Salmonella Albany and Salmonella Kentucky traduced high resistance percentages to amoxicillin, tetracycline, sulfamethoxazole, and nalidixic acid(84.6%–100%). Among amoxicillin-resistant isolates, blaTemgenes were observed for 62% of E. coli isolates and 20% of 65 Salmonella Kentucky. The Str A gene was prevalent in 36% of 331 aminoglycoside-resistant E. coli and 90% of 40 aminoglycoside-resistant Salmonella Corvallis. The sul2 gene was predominant among sulfamethoxazole-resistant isolates, for 56% of 431 E. coli and 53% of 66 Salmonella Corvallis; the sul1 gene was observed in 54% of Salmonella Albany. The Tet(A) resistance gene was prevalent in E.coli(86%), Salmonella Corvallis(82%), Salmonella Kentucky(84%). High percentages of gyr A genes observed among nalidixic-acid resistant E. coli(91%), Salmonella Albany(92%), Salmonella Corvallis(75%) and Salmonella Kentucky(85%).Conclusions: Important occurrences of resistance gene were observed among E. coli and Salmonella in chicken food chains in Cambodia.

Antibiotic Sensitivity Test and Detection of Sulfonamide Resistance Gene Sul2 in Escherichia coli Isolates from Swine

[ Objective] This study aimed to analyze antibiotic sensitivity and detect sulfonamide resistance gene Sul2 in 17 Escherichia coli isolates from swine. [ Method] The antibiotic sensitivity of 17 E. coil isolates from swine was analyzed with Kirby-Bauer disk diffusion method. Sulfonamide resistance gene Sul2 was detected by PCR amplification with the extracted genomic DNA as a template. [ Result] The 17 swine-derived E. coli isolates from different regions were resistant to various antibiotics and exhibited different multi-antibiotic resistance phenotypes, including nine isolates resistant to sulfamethoxazole, seven isolates resistant to oxacillin, ten isolates resistant to cefazolin and eight isolates resistant to erythromycin. Sulfonamide resistance gene Sul2 was successfully amplified from genomic DNA of E. coli isolates. The PCR results were basically consistent with antibiotic resistance phenotypes of these strains. [ Conclusion] Sul2 gene was widespread in swine-derived E. coli, which was closely associated with sulfonamide resistance phenotvpes of E. coli.

Performance Parameters:Demobilization Antibiotic Resistant Bacteria(ARB)and Carrying Genes(ARG)in Wastewater Disinfection

The UV irradiation is used for removing Antibiotic Resistant Bacteria(ARB)and Antibiotic Resistance Genes(ARG)from wastewater treatment.Bacteriophages are viruses that infect within bacteria,are recognized for bacterial control.The influence of some parameters in quantification and performance influencing of pathogen demobilization could be considered in disinfection of wastewater.The comparison of Polyvalent phage(NE1)versus Coliphage(NE4)in suppressing a bacterium Escherichia coli(NDM-1:b-lactam-resistant)with UV irradiation was observed the efficacy in reduction of cells in the disinfection and parameter process.The results with the effect of UV-C irradiation on NDM-1 infected with 1%of NE4 showed a decrease of cells from 8×10^(6)to 2×10^(5)in 60 min with UV-C dose.The NDM1(E.coli)was infected with 1%of NE4(Polyvalent Phage)under magnetic stirring for 1 h,the cells count was 8×10^(6).After 1 h in UV-C e×posure,the cells number reached 3×10^(5).The NDM1 that was e×posed in 1 h of UV-C irradiation and then was infected with 1%of NE4.Cells counting were done 24 h after this procedure.These cells were e×posed in UV-C and showed a reduction in the number of cells from 1×10^(8)to 4×10^(5)after 60 min.The results indicate that bacteriophages can mitigate bacteria species,and combined the conventional water disinfection technologies that can support the microbial safety control strategies.

Metabolic regulation of <i>Escherichia coli</i>cultivated under anaerobic and aerobic conditions in response to the specific pathway gene knockouts

Effect of the specific gene knockout on the main metabolism in Escherichia coli was reviewed, and the regulation mechanisms were clarified based on different levels of information such as gene expressions, enzyme activities, intracellular metabolite concentrations, and metabolic fluxes together with fermentation data. The effects of the knockout of such genes as pflA, pta, ppc, pykF, adhE, and ldhA on the metabolic changes were analyzed for the case under anaerobic condition. The effects of the knockout of such genes as pgi, zwf, gnd, ppc pck, pyk, and lpdA on the metabolic changes were also analyzed for the case under aerobic condition. The metabolic regulation analysis was made focusing on the roles of transcription factors.

Reconstruction of tyrosol synthetic pathways in Escherichia coli

Tyrosol is a pharmacologically active phenolic compound widely used in the medicine and chemical industries.Traditional methods of plant extraction are complicated and chemical synthesis of tyrosol is not commercially viable. In this study, a recombinant Escherichia coli strain was constructed by overexpressing the phenylpyruvate decarboxylase ARO10 from Saccharomyces cerevisiae, which could produce tyrosol from glucose. Furthermore,genes encoding key enzymes from the competing phenylalanine and tyrosine synthesis pathways and the repression protein TyrR were eliminated, and the resulting engineered strain generated 3.57 mmol·L^(-1) tyrosol from glucose. More significantly, codon optimization of ARO10 increased expression and tyrosol titer. Using the novel engineered strain expressing codon-optimized AR10 in shake-flask culture, 8.72 mmol·L^(-1) tyrosol was obtained after 48 h. Optimization of the induction conditions improved tyrosol production to 9.53 mmol·L^(-1)(1316.3 mg·L^(-1)). A higher titer of tyrosol was achieved by reconstruction of tyrosol synthetic pathway in E. coli.

Escherichia coli tetracycline efflux determinants in relation to tetracycline residues in chicken

Objective:To screen for Escherichia coli(E.coli)resistant to tetracycline,followed by identification of tet efflux genes by polymerase chain reaction(PCR).In addition,detection of tetracycline residues in chicken livers and kidneys were conducted using high performance liquid chromatography-tandem quadrupole mass spectrometry(HPLC-MS-MS).Methods:Strains of E.coli were isolated from samples of chicken colon and screened for tetracycline resistance.Tetracycline genes conferring resistance(Tc^r)were detected by polymerase chain reaction(PCR).Most of the isolates were resistant to tetracycline(97.9%).Results:PCR analysis indicated that Tc^r E.coli R-plasmids contained tet(A),tet(B)and a combination of both efflux genes.None of the isolates contained other efflux tet genes tet(C,D,E and Y).High performance liquid chromatography-tandem quadrupole mass spectrometry(HPLC-MS-MS),a sensitive technique,was used to detect residues of chlortetracycline(CTC),oxytetracyeline(OTC),doxveycline(DC)in chicken livers and kidneys.The samples containing tetracycline residues were at 0.13-0.65pg/μL levels.Conclusions:Tetracycline and other antibiotics are commonly used in the poultry and meat production industry for prevention of microbial infections.Multiple antibiotic resistant bacteria in Oman have increased to alarming levels,threatening public health,domestic and may have adverse effect on environment.

Comparison of extended spectrum β-lactamasesproducing Escherichia coli with non-ESBLsproducing E.coli:drug-resistance and virulence

BACKGROUND:The virulent factors of Escherichia coli(E.coli) play an important role in the process of pathopoiesis.The study aimed to compare drug-resistant genes and virulence genes between extended spectrum β-lactamases(ESBLs)-producing E.coli and non-ESBLs-producing E.coli to provide a reference for physicians in management of hospital infection.METHODS:From October 2010 to August 2011,96 drug-resistant strains of E.coli isolated were collected from the specimens in Qingdao Municipal Hospital,Qingdao,China.These bacteria strains were divided into a ESBLs-producing group and a non-ESBLs-producing group.Drug sensitivity tests were performed using the Kirby-Bauer(K-B) method.Disinfectant gene,qacEA1-sull and 8 virulence genes(CNF2,hlyA,eaeA,VT1,est,bfpA,elt,and CNF1) were tested by polymerase chain reaction(PCR).RESULTS:Among the 96 E.coli isolates,the ESBLs-producing E.coli comprised 46(47.9%)strains and the non-ESBLs-producing E.coli consisted of 50(52.1%) strains.The detection rates of multiple drug-resistant strain,qacEA1-sull,CNF2,hlyA,eaeA,VT1,est,bfpA,elt,and CNF1 in 46ESBLs-producing E.coli isolates were 89.1%,76.1%,6.5%,69.6%,69.6%,89.1%,10.9%,26.1%,8.7%,and 19.6%,respectively.In the non-ESBLs-producing E.coli strains,the positive rates of multiple drug-resistant strain,qacEA1-sull,CNF2,hlyA,eaeA,VT1,est,bfpA,elt,and CNF1 were 62.0%,80.0%,16.0%,28.0%,64.0%,38.0%,6.0%,34.0%,10.0%,and 24.0%,respectively.The difference in the detection rates of multiple drug-resistant strain,hlyA and VT1 between the ESBLs-producing E.coli strains and the non-ESBLs-producing E.coli strains was statistically significant(P<0.05).CONCLUSION:The positive rate of multiple drug-resistant strains is higher in the ESBLsproducing strains than in the non-ESBLs-producing strains.The expression of some virulence genes hlyA and VT1 varies between the ESBLs-producing strains and the non-ESBLs-producing strains.Increased awareness of clinicians and enhanced testing by laboratories are required to reduce treatment failures and prevent the spread of multiple drug-resistant strains.

代谢工程改造Escherichia coli生产3-羟基丙酸

以提高3-羟基丙酸的产量为目标,对实验室构建的基因工程大肠杆菌进行改造,敲除形成副产物1,3-丙二醇的主要酶基因——乙醛脱氢酶基因yqh D,得到E.coli W3110Δyqh D(p CDFDuet-tac-gpd1-TUkgsadh/p ACYCDuet-tac-dha B1-4),该工程菌摇瓶发酵产量达到2.53 g/L,相比未敲除yqh D基因的菌株,产量提高了5.8倍。另外,敲除了甘油代谢途径中的抑制因子glpR基因,得到E.coli W3110ΔglpR(p CDFDuet-tac-gpd1-TUkgsadh/p ACYCDuet-tac-dha B1-4),该工程菌摇瓶发酵产量达到2.86 g/L,相比未敲除glpR基因的菌株,产量提高了6.7倍。后经5 L罐发酵培养后,3-羟基丙酸的产量提升到15.4 g/L。该实验为进一步利用大肠杆菌工程菌发酵生产3-羟基丙酸提供了研究基础。

Multiple Strategies for Metabolic Engineering of Escherichia coli for Efficient Production of Coenzyme Q_（10）

Escherichia coli BW25113 被从 Gluconobacter suboxydans 用 ddsA 代替 ispB 新陈代谢地为 CoQ10 生产设计。先锋平衡和减少的菸碱腺嘌 dinucleotide 磷酸盐(NADPH ) 的效果在 E 的 CoQ10 生产上的可获得性。coli 被调查。与 pck overexpression 一起的 pykFA 的大美人能维持在 glyceraldehyde 3 磷酸盐和 pyruvate 之间的平衡，增加 CoQ10 生产。和 gapC 的 overexpression，有从 Clostridium acetobutylicum 的 NADP 依赖的 gapC 的本国的 NAD 依赖的 gapA 的代替能增加 NADPH 可获得性然后提高 CoQ10 生产。在 E 的 CoQ10 生产上的三效果，在 CoQ 生合成和中央新陈代谢的各种各样的基因的 overexpressions，不同向量和文化条件。coli，都被调查。不同向量的调查显示那低拷贝数字向量可能为在 E 的 CoQ10 生产是更有益的。coli。recombinant E。 coli ( ispB::ddsA ， pykFA 和 gapA::gapC )，怀有在 pQE30 上在 PT5 的控制下面编码 pck ， dxs ， idi 和 ubiCA 基因的二 plasmids ， ispA ，从 Gluconobacter suboxydans 的 ddsA 和从在 pBAD33 上的 PBAD 的控制下面的 Clostridium acetobutylicum 的 gapC ，能生产 CoQ10 直到 3.24 mg 吗？

Microarray Analysis of Transcriptomic Response of <i>Escherichia coli</i>to Nonthermal Plasma-Treated PBS Solution

We developed a technique of generating nonthermal atmospheric plasma-activated solution that had broad-spectrum antibacterial properties. Plasma-activated phosphate-buffered saline (PBS) causes rapid inactivation of bacteria following generation of oxidative stress. However, dose optimization requires understanding of cellular mechanisms. The objective of this study was to explore genome-wise response to develop gene expression profile of Escherichia coli using DNA microarray following exposure to plasma-activated PBS solution. Upon exposure to plasma-treated PBS solution, E. coli cells had differentially expressed genes involved in oxidative stress, and cell envelope and membrane associated porin and transporters. The genes involved in house-keeping and metabolism, energy generation, motility and virulence were conversely downregulated. This is the first report which demonstrates a severe oxidative stress induced in E. coli cells in response to an exposure to nonequilibrium nonthermal dielectric-barrier discharge plasma-activated PBS solution, and the genes that are responsive to reactive oxygen species appeared to play a role in cellular stress. Such studies are important to identify targets of inactivation, and to understand plasma-treated solution and bacterial cell interactions.

Characterization of Multidrug Resistant Escherichia coli Isolates Recovered from Humans and Chickens, Trinidad and Tobago

To characterize extended-spectrum beta-lactamase (ESBL) and extra-intestinal pathogenic Escherichia coli (ExPEC) associated virulence genes in E. coli isolates from chickens and humans in Trinidad and Tobago. This cross sectional study was conducted over a three-month period. A total of 471 E. coli isolates;160 from humans treated at a regional tertiary hospital and 311 from chicken caecal samples from “pluck shops” in Trinidad & Tobago were identified using both conventional and molecular microbiological methods. Phenotypic confirmation of ESBL producing E. coli isolates from humans was by Microscan system (Siemens, USA) while the double disk diffusion method was used for the chicken isolates. Polymerase chain reaction (PCR) analysis was used to determine the ESBL and ExPEC-associated virulence genes in representative human isolates and all chicken isolates. From the 311 chicken E. coli isolates, 49.2% (153/311) produced ESBL, while 56.3% (90/160) from humans were ESBL positive. All human and chicken ESBL isolates were 100% susceptible to carbapenems and aminoglycosides antimicrobials. PCR detected 21.1% blaCTX-M, 13.3% blaTEM and 7.8% blaSHV genes among E coli isolates from humans compared to 0.6% blaCTX-M and 48.6% blaTEM genes in chickens. PCR analysis revealed diverse virulence profiles among the isolates. There was a high occurrence rate of ExPEC-asso- ciated virulence genes in E. coli isolates from both humans and chickens. However, the CTX-M-1 genes were most predominant in humans while TEM occurred in chic- ken isolates. The diverse ESBL and virulence associated gene profiles encountered in E. coli isolates from humans and chickens on the surface depicts no similarity or relationships despite occurrence in both cohort groups. Therefore E. coli strains from chickens and humans require further investigation to determine their clonal relatedness or transmission in the country.

Cloning of polyadenylated mRNA fragments of Escherichia coli with restriction display-polymerase chain reaction

Objective:To investigate the polyadenylation of mRNA in E. Coli. Methods: The mRNA of E. Coli was enriched from the total RNA with oligo(dT)-cellulose, prior to reverse transcription using oligo(dT)18as the primer. Double-stranded cDNA was subsequently synthesized, which was subjected to digestion with Sau3A I to produce multiple gene fragments for ligation with the adapters. PCR was carried out in 10 groups according to 10 different pairs of the selective primers, and the PCR products were then cloned into T-vectors. Results: More than 100 gene fragments had been cloned, 30 of which were sequenced. Conclusion:Polyadenylation of E. Coli mRNA may not be a biochemical curiosity but a general attribute of bacterial mRNA.

北京某医院感染性腹泻患者致泻性大肠埃希菌毒力和耐药特征分析

目的明确本院感染性腹泻患者致泻性大肠埃希菌毒力和耐药特征。方法采用VITEK MS微生物质谱检测系统初步鉴定,多重实时荧光PCR检测毒力基因,对本院感染性腹泻患者临床分离的致泻性大肠埃希菌(Diarrheagenic Escherichia coli,DEC)进行5种型别鉴定。微量肉汤稀释法和E-test法药敏试验检测DEC菌株的耐药表型特征。二代测序及生物信息学分析其耐药分子特征。采用Fisher确切概率法进行统计学分析。结果本院DEC检出率为11.9%,其中EAEC占比37.5%,非典型EPEC占比34.38%,ETEC占比25.0%,EIEC占比3.12%,未检出EHEC菌株。32株DEC对氨苄西林、四环素、甲氨苄啶/磺胺异恶唑耐药率最高,分别为53.12%、43.75%和37.5%。ESBLs(+)株占比18.75%,其多重耐药菌株检出率为83.83%,显著高于ESBLs(-)菌株,差异有统计学意义(P=0.042)。32株DEC共有25个ST型,优势基因型为ST10共4株(12.5%),ST28、ST31和ST3153各2株(各占比6.25%),其他21个型别各1株菌(各占比3.13%)。EAEC中检出1株携带bla_(NDM-1)的耐碳青霉烯类菌株。结论我院感染性腹泻患者的DEC以aggR、pic、astA、eae 4种毒力基因较为常见,以EAEC、EPEC为主要型别,基因型别呈高度多态性,检出多重耐药菌株。

rLTB/rCTB-rOmpL1/1融合基因及其原核表达系统的构建和鉴定

目的 :构建 lt B/ ct B- omp L1/ 1融合基因及其原核表达系统 ,鉴定表达产物的免疫和佐剂活性 ,检测问号钩端螺旋体 (简称钩体 )野生株 omp L 1基因的携带和表达情况及钩体患者血清特异性抗体水平。方法 :采用连接引物 PCR构建 lt B- omp L 1/ 1和 ct B- omp L 1/ 1融合基因 ,常规方法构建其原核表达系统。采用 SDS- PAGE、Westernblot和 GM1- ELISA分别检测目的重组蛋白 r LTB- r Omp L 1/ 1和 r CTB- r Omp L 1/ 1表达量、免疫反应性及与 GM1结合的活性。采用 PCR和 MAT分别检测 97株问号钩体野生株 omp L1基因及其表达情况。采用 ELISA检测 2 2 8例钩体患者血清 omp L1基因产物的抗体。结果 :与报道的相关序列比较 ,lt B- omp L1/ 1和 ct B- omp L1/ 1融合基因核苷酸和氨基酸序列相似性 ,分别为 99.7%～ 99.9%和 99.5 %～ 10 0 %。r LTB- r Omp L1/ 1和 r CTB- r Omp L1/ 1表达产量均约为细菌总蛋白的 10 % ,主要以包涵体形式存在。 r LTB- r Omp L 1/ 1和 r CTB- r Omp L1/ 1均分别能与r Omp L 1/ 1兔抗血清和牛 GM1结合。 89.7%问号钩体野生株含有 omp L1基因 ,87.6 %问号钩体野生株分别与r Omp L 1/ 1和 r Omp L1/ 2兔抗血清出现效价 ,为 1∶ 4～ 1∶ 2 5 6的 MAT阳性结果。 86 .8%和 88.6 %的患?