Effect of thymol on antimicrobial susceptibility, and adhesion genes expression of uropathogenic Escherichia coli isolated from pediatric urinary tract infection.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a common cause of urinary tract infections (UTI) in children and currently is one of the leading medical problems. Due to the increase in antibiotic resistance rate, herbal medicines with lower side effects were considered. OBJECTIVE: This study aimed to identify the afa, fimH, and sfa genes of UPEC bacteria isolated from pediatric UTI to investigate the effect of the thyme on the expression of fimH gene. STUDY DESIGN: In this cross-sectional study, 160 UPEC were isolated from pediatric UTIs. An antibiotic susceptibility test was performed on six families of antibiotics, including beta-lactams, quinolones, aminoglycosides, carbapenems, sulfonamides, and nitrofurantoin. The micro-broth dilution method was used to determine MIC of thymol. The biofilm production ability of isolated strains was quantified by the microtiter plate method. The PCR technique was used to detectfimH, afa, and sfa adhesion genes, and real-time PCR was used to measure the fimHgene expression. RESULTS: The results of the antibiogram showed that the lowest and highest resistance related to meropenem and imipenem (zero), and 72.5% for cephalothin. MIC showed 80.7% of the isolates were sensitive to thymol. The biofilm production results showed that 3.12%, 53.75%, and 43.12% of the isolates were strong, weak, and no-biofilm (Zero) producers, respectively. After thymol treatment, 26.25% and 73.75% of isolates were weak and no-producer (Zero) biofilms, respectively and there was a significant correlation (P-value = 0.042) compared to the control group. The frequency of fimH, sfa, and afa genes was 53.1%, 49.4%, and 29.4%, respectively. The expression of fimHgene after 48 h thymol treatment decreased significantly (P-value< 0.05). CONCLUSION: Due to the significant effects of thymol in preventing the expression of the adhesion gene (fimH) of UPEC bacteria, our study is a proof-of-concept study evaluating bacterial sensitivity to Thymol and its effect on biofilm production in vitro. Given the demonstrated promising results of Thymol's effectiveness and the increase in bacterial antibiotic resistance, further studies should be undertaken to determine the safety and effectiveness of Thymol use in the clinical treatment of urinary tract infection. We believe that Thymol may prove to be an effective adjunct to the treatment of bacterial urinary tract infections.

Efficient Biofilm-Based Fermentation Strategies for L-Threonine Production by Escherichia coli.

Biofilms provide cells favorable growth conditions, which have been exploited in industrial biotechnological processes. However, industrial application of the biofilm has not yet been reported in Escherichia coli, one of the most important platform strains, though the biofilm has been extensively studied for pathogenic reasons. Here, we engineered E. coli by overexpressing the fimH gene, which successfully enhanced its biofilm formation under industrial aerobic cultivation conditions. Subsequently, a biofilm-based immobilized fermentation strategy was developed. L-threonine production was increased from 10.5 to 14.1 g/L during batch fermentations and further to 17.5 g/L during continuous (repeated-batch) fermentations with enhanced productivities. Molecular basis for the enhanced biofilm formation and L-threonine biosynthesis was also studied by transcriptome analysis. This study goes beyond the conventional research focusing on pathogenic aspects of E. coli biofilm and represents a successful application case of engineered E. coli biofilm to industrial processes.

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

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.

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.