EXPRESSION OF TYPE 1 FIMBRIAE ENHANCES VIRULENCE OF UROPATHOGENIC E. COLI BY BIOFILM FORMATION

BACKGROUND: Many bacterial infections are associated with biofilm formation. It is one of the important virulent factors of E. coli in urinary tract, causing recurrent and drug resistant infections. Fecal E. coli colonize the urethra and spread up the urinary tract to the bladder and kidney. Type 1 fimbriae are surface located adhesion organelles of E. coli that are directly associated with adherence to the urinary tract. The present study was aimed to study biofilm production in E. coli isolated from urinary tract infection and to correlate it with expression of fimH gene and compare its sequences. METHOD: Total 150 urine samples were processed for isolation and identification of uropathogens. E. coli isolates were further processed for detection of biofilm by TCP method and screened for the presence of fimH gene by PCR using specific primers. The PCR products were purified and sequenced bidirectionally by Sanger dideoxy sequencing system using ABI 3500 Genetic analyzer. RESULTS: From the total 98 urine samples with significant bacteriuria, 77 E. coli were isolated out of which, 40 were positive for in vitro biofilm production. Among them11 were classified as strong biofilm producers and 29 as moderate. The fimH gene from E. coli isolates was amplified using specific primers and appeared as a band of about 508bp on agarose gel. It was noted that the fimH gene was detected in moderate and strong biofilm forming E. coli while absent in non biofilm isolates.The sequences showed 99% similarity with fim H gene of E coli. CONCLUSION: The high binding ability of fimH could result in increased bacterial binding to target cells and increased pathogenicity of E. Coli.

Frequency of Adhesive Virulence Factor fimH among the Clinical Isolates of Acinetobacter baumannii in India

The objective of the study was to detect the presence of fimH geneamong the drug resistanst strains of Acinetobacter baumannii.fimH gene was found to be associated with a catch bond mechanism which led to better evolution of biofilm formation. Since there are not many studies done with this gene it would be a timely investigation and this study mainly aims in molecular characterizationof fimHgene among clinical isolates of A.baumannii. Semi quantitative bio adherent assay was done by the multidrug resistant strains of A.baumannii to find the formation of biofilm. The DNA was extracted with the help of kit and PCR was performed for amplification. Pearson correlation analysis was done to find the existing correlation between the fimHgene and MDR strains of A.baumanniiwith significant p-value of (<0.05). From the screened 73 genomes of MDR A.baumannii 6.8% showed positive amplicons for the fimH gene which were related to biofilm and porin formation (Fig. 1). Correlation of its existence was high in beta lactamase (100%), cephems (100%), folate (100%) resistant strains, followed by aminoglycosides (80%), carbapenems (60%) and fluoroquinolones (60%) and efflux pumps (20%). In Spite of various measures undertaken to prevent the disease, the prevalence of the pathogen is multiplying. The current study recorded the presence of fimHgene (6.8%) among the clinical isolates of A.baumannii. This gene can be used as a target to develop new drugs and vaccines to combat the menace of A.baumannii infection

Virulence factors detection and single nucleotide polymorphism assay of extraintestinal pathogenic E.coli in elderly nosocomial infection / 天津医药

Objective To examine the detection rate of 30 known virulence factors (VFs) of extraintestinal pathogenic Escherichia coli(ExPEC), and to investigates the epidemiology of ExPEC in elderly nosocomial infection. Methods A to?tal of 140 ExPEC clinical isolates from elderly nosocomial patients in hospitals in Tianjin were investigated. Multiplex PCR was performed to detect the 30 virulence factors among the E.coli strains and the detection rate of virulence factors for Ex?PEC were compared between isolates from different sites of infection.Fifty E. coli strains were shown to carry fimH gene that was amplified and sequenced. These sequences were used besides3 references strains (CFT037、UTI89 and K-12 ) to detect SNPs of fimH gene using DNAMAN Version 6.0.3.93 these 53 fimH sequences were used for genotyping and building dendrogram by MEGA4 software. Results In ExPEC, the following virulence factor genes, fimH, traT, fyuA, iutA and kpsMT II, had a higher detection rate than those of the rest . The following virulence factor genes, kpsMT II, K5, papC, pa?pEF ,papG allele II (Internal), papA, cnf1 (CNF), sfa/focDE and rfc had a a higher detectionrate from non-urine origin sam?ples than those from urine origin samples. fimH SNPs analysis of the 50 clinical isolated samples and 3 references samples showed 60 SNPs at 57 polymorphic sites. The fimH SNPs analysis classified the 53 strains into 25 genotype. The genetic fin?gerprintings of 11 isolates were exactly the same. Conclusion Many kinds of virulence factors can be found in ExPEC of el?derly nosocomial infection. The ExPEC strain isolated from non-urine origin had a stronger pathogenicity than those from urine-origin specimens. fimH SNPs analysis is suitable for molecular epidemiological investigation of ExPEC in hospital.

Molecular typing of uropathogenic Escherichia coli isolated from Korean children with urinary tract infection / 소아과

PURPOSE: We investigated the molecular types of uropathogenic Escherichia coli (UPEC) by using conventional phylogrouping, multilocus sequence typing (MLST), and fimH genotyping. METHODS: Samples of patients younger than 18 years of age were collected from the Chung-Ang University Hospital over 2 years. Conventional phylogenetic grouping for UPEC strains was performed by polymerase chain reaction (PCR). Bacterial strain sequence types (STs) were classified on the basis of the results of partial sequencing of seven housekeeping genes. In addition, we analyzed nucleotide variations in a 424-base pair fragment of fimH, a major virulence factor in UPEC. RESULTS: Sixty-four UPEC isolates were analyzed in this study. Phylogenetic grouping revealed that group B2 was the most common type (n=54, 84%). We identified 16 distinctive STs using MLST. The most common STs were ST95 (35.9%), ST73 (15.6%), ST131 (12.5%), ST69 (7.8%), and ST14 (6.3%). Fourteen fimH allele types were identified, of which 11 had been previously reported, and the remaining three were identified in this study. f1 (n=28, 45.2%) was found to be the most common allele type, followed by f6 and f9 (n=7, 11.3% each). Comparative analysis of the results from the three different molecular typing techniques revealed that both MLST and fimH typing generated more discriminatory UPEC types than did PCR-based phylogrouping. CONCLUSION: We characterized UPEC molecular types isolated from Korean children by MLST and fimH genotyping. fimH genotyping might serve as a useful molecular test for large epidemiologic studies of UPEC isolates.

Construction,prokaryotic expression and purification of FimH1-156 fusion protein / 重庆医学

Objective To construct and express a prokaryotic expression vector carrying the gene of FimH 1-156 that comprises human lysosome membrane protein 2 P41-49 gene ,and to express and purify the fusion protein .Methods FimH1-156 gene was cloned from plasmid pPKL241 by PCR ,and inserted into vector pET-28a(+ ) to obtain prokaryotic expression plasmid pET-28a-FimH . After transforming Escherichia coli BL21(DE3) with pET-28a-FimH ,fusion protein FimH1-156 was expressed under induction .The target fusion protein was purified ,and its antigenicity was detected through Western blot .Results The expressed recombinant pro-tein was purified ,the expression of protein was the highest when IPTG was 1 mmol/L and 4h after induction ,it was expressed as include body form ,and the expressed protein was identified to react with monoclonal antibodies 6 × His by Western blotting .Conclu-sion We cloned FimH1-156 fusion protein expressed genes successfully ,constructed prokaryotic expression vector ,and won the in-clusion body purification of FimH1-156 fusion protein .

Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

Effect of fimH gene on type 1 fimbriae adhesion of uropathogenic Escherichia coli: a preliminary study / 中华临床感染病杂志

Objective To investigate the effect of fimH gene on type 1 fimbriae adhesion of uropathogenic Escherichia coli (UPEC) and the gene variations between type 1 fimbriae adhesion positive and negative bacteria.Methods A total of 171 UPEC strains (not catheter associated) were collected from the Second Hospital of Tianjin Medical University,Tianjin First Center Hospital,and Tianjin Children's Hospital during January 2012 and January 2013.fimH gene was detected by PCR technique,and type 1 fimbriae adhesion was detected by yeast agglutination test.RT-PCR was used to exterminate gene transcript factor impacting on adhesion.Chi-square and Yates' chi-squared tests were performed to comparefimH gene sequences between the adhesion positive and negative bacteria.Results Among 171 UPEC strains,142 (83％) werefimH gene positive,and type 1 fimbriae was expressed in 98 strains (57％).All adhesion positive strains carriedfimH gene.Among 44 strains with positivefimH gene and negative adhesion RT-PCR revealed thatfimH gene did not transcript in 8 strains (18％).The sequencing results showed that gene mutation on the 51 st amino acid site was more prevalent in the adhesion positive group compared with the negative group (x2 =6.64,P =0.010).In adhesion,mutations on the 190th and 219th amino acid sites were observed in 6 strains and 7 strains of negative group,respectively; while not observed in the adhesion positive group (x2 =4.69 and 5.87,P ＜ 0.05).Negative adhesion in other 23 strains was not attributed to single nucleotide polymorphism.Conclusion Adhesion function of type 1 fimbriae mignt be affected by mutation and deletion offimH gene.Three key site mutations may also affect the adhesion function of type 1 fimbriae.Besides fimH gene,there may be other genes that can affect the adhesion function of type 1 fimbriae.

Comparison among the immune effects of DNA-or protein-FimH of UPEC type 1 pilus / 中国免疫学杂志

Objective:To observe cellular,humoral and mucosal immune responses induced by DNA-or protein-based of FimH of UPEC type 1.Methods:After mice were immunized respectively with recombinant plasmid pcDNA3.1/fimH or pcDNA3.1/fimC,and the combinant FimH and FimC protein,the anti-FimH protein IgG of sera and SIgA in bladders were detected by ELISA.The lymphocyte phenotypes of CD3,CD4 and CD8 were analyzed by FCM.Results:The titers of IgG in sera and SIgA in the bladders were all low in the group immunized by recombinant FimH plamid,but the percentage of CD4+T cells in spleen was high,which revealed that recombinant FimH plamid was able to trigger better cellular immune response.The titers of IgG were very high in the group immunized by FimH protein,which suggested that the FimH protein was able to trigger better humoral immune response,but SIgA in the bladders was not detectable.Conclusion:The DNA for FimH can induce humoral,mucosal and cellular immune response.FimH protein can only induce humoral immune response.FimC protein is able to enhance the immunogenicity of FimH protein.