Effect of Niaogan Decoction on expulsion of uropathogenic Escheriehia coli from human bladder epithelial cells / 中成药

AIM To observe the effects of Niaogan Decoction (Portulacae Herba,Taraxaci Herba) on expulsion of uropathogenic Escheriehia coli (UPEC) from human bladder epithelial cells and to explore its mechanism.METHODS After HTB-9 was infected with UPEC,drug urine exocytosed the UPEC invaded into urinary bladder epithelial cell were observed.With molecular biology technology,the mechanism of key factors to TLR4/cAMP signal pathway such as Toll-like receptor 4 (TLR4),adeuylate cyclase 3 (AC3),cyclic adenosine monophosphate (cAMP),protien kinase A (PKA),Myosin Ⅶ A and Rab interacting protein (MyRIP),Rab27b and Caveolin-1 were analyzed.RESULTS Treatment with Niaogan Decoction resulted in a significant increase in UPEC exocytosis compared with the blank control high dose group.Niaogan Decoction group improved TLR4,AC3,MyRIP,Rab27b and Caveolin-1 protein expression,increased intracellular cAMP content and promoted PKA activation.CONCLUSION Niaogan Decoction has the capacity to exocytose UPEC,its mechanism may relate to TLR 4/cAMP signal pathway.

Curli fimbria: an Escherichia coli adhesin associated with human cystitis

Abstract Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-D-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.

Pentacyclic triterpenes combined with ciprofloxacin help to eradicate the biofilm formed in vitro by Escherichia coli.

Background & objectives: Ciprofloxacin is commonly used in clinical practice for the treatment of recurrent urinary tract infections caused by Escherichia coli. However, very often these recurrent infections are due to a failure in a complete eradication of the microorganisms colonizing the urinary tract, especially in catheterized patients. To enhance the bactericidal activity of ciprofloxacin against biofilm-forming uropathogenic E. coli (UPECs), we examined its effect in combination with two pentacyclic triterpenes – asiatic and ursolic acids. Methods: The anti-biofilm activity of ciprofloxacin and pentacyclic triterpenes - asiatic acid (AA) and ursolic acid (UA), as well as their synergistic effect were tested on two types of surfaces - polystyrene microtiter plates and silicone catheters. It was investigated using the time-killing and biofilm assays. Results: anti-biofilm activity of ciprofloxacin was not observed on microtiter plates or on the catheters. Ciprofloxacin combined with ursolic acid inhibited the biofilm formation on microtitre plates. This mixture, however, did not express such a strong activity against the synthesis of biofilm on the surface of catheters. Ciprofloxacin combined with asiatic acid had very weak inhibiting effect on the synthesis of biofilm mass on microtitre plates as well as on the catheters. Despite this, both mixtures – ciprofloxacin and asiatic acid, as well as ciprofloxacin and ursolic acid, exhibited strong and significant impact on the eradication of mature biofilm (P < 0.05). Interpretation & conclusions: Although ciprofloxacin is recommended in the treatment of urinary tract infections caused by UPECs, but its efficacy is arguable. Subinhibitory concentrations of ciprofloxacin did not inhibit the formation of biofilm. Pentacyclic triterpenes used in combination with ciprofloxacin enhanced its anti-biofilm effectiveness. However, this anti-biofilm activity was found to depend on the type of surface on which biofilm was formed.

Prevalence of adhesive genes among uropathogenic Escherichia coli strains isolated from patients with urinary tract infection in Mangalore.

The study was carried out to detect the adhesive genes pap (pyelonephritis associated pili), sfa (S fimbrial adhesin) and afa (afimbrial adhesin) from Escherichia coli strains isolated in patients diagnosed with urinary tract infection (UTI). A total of 23% of the isolates were positive for pap, sfa and afa genes with a prevalence of 60.87% (14/23), 39.1% (9/23) and 39.1% (9/23), respectively. Prevalence of multiple adhesive genes was 8.7% (2/23) for pap and afa, 30.43% (7/23) for pap and sfa. Significant numbers of isolates were positive for Congo red binding (80%) and haemolysin production 60%. The prevalence of multiple adhesive genes indicate the potential to adhere and subsequently cause a systemic infection among UTI patients.

The detection of adherence factorsby Escherichia coli cause of urinary tract infectionsin Ulaanbaatar, Mongolia / Монголын Анагаах Ухаан

INTRODUCTION: Urinary tract infections among the most common bacterial infectious diseases encountered at all ages. Escherichia coli are being the etiologic agent in 50–80%. Therefore, it is an important public health problem. E.coli causing urinary tract infections express pilli, fimbriae and others adherence virulence factors. GOAL: To detect the some adherence virulence factors of Uropathogenic Escherichia coli (UPEC) in Ulaanbaatar, Mongolia MATERIALS AND METHODS: A total of 76E.colisampleswere collected. These samples were positive bacteriological examination of urine, performed at the bacteriological laboratory of the State Central Third Hospital and State Central First Hospital, Ulaanbaatar, Mongolia. The biofilm formation was evaluated by the growth rate of E.coli on plastic surface.The detection of the virulence factors type 1 fimbriae (fimA gene) and P-fimbriae (papC) was performed by multiplex PCR using gene specific primers.Curli expression was determined by using congo red agar. RESULTS: The evaluation of bacterial biofilm formation using 96 well plates showed 40 negative (52.6%), 32 weak biofilm (42.1%) and 4 moderate biofilm (5.3%) formation for E.coli and no strong biofilm forming strain was detected. The cell surface protein (curli) was detected by Congo red agar. The result was 71% positive for studied E.coli strains. The detection result of pili genes by multiplex PCR showed that fimH gene detected for 73 (96.1%) and papC gene detected for 18 (23.7%) E.coli cultures. CONCLUSION: Almost half of surveyed Uropathogenic E.coli isolated in Ulaanbaatar, Mongolia had ability of biofilm formation and it has been determined by the bacterial surface protein (curli), which is one of bacterial adherence factors, may cause biofilm formation.

Phenotypical characterization and adhesin identification in Escherichia coli strains isolated from dogs with urinary tract infections

Pathogenic strains of Escherichia coli are the most common bacteria associated with urinary tract infections in both humans and companion animals. Standard biochemical tests may be useful in demonstrating detailed phenotypical characteristics of these strains. Thirteen strains of E. coli isolated from dogs with UTIs were submitted to biochemical tests, serotyping for O and H antigens and antimicrobial resistance testing. Furthermore, the presence of papC, sfa, and afa genes was evaluated by PCR, and genetic relationships were established using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The antimicrobial that showed the highest resistance rate among the isolates was nalidixic acid (76.9%), followed by cephalotin (69.2%), sulfamethoxazole + trimethoprim (61.5%), tetracycline (61.5%), streptomycin (53.8%), ciprofloxacin (53.8%), ampicillin (46.2%), gentamicin (30.8%) and chloramphenicol (23.1%). No isolate was resistant either to meropenem or nitrofurantoin. Among the five clusters that were identified using ERIC-PCR, one cluster (A) had only one strain, which belonged to a serotype with zoonotic potential (O6:H31) and showed the genes papC+, sfa+, afa-. Strains with the genes papC-, sfa+, afa- were found in two other clusters (C and D), whereas all strains in clusters B and E possessed papC-, sfa-, afa- genes. Sucrose and raffinose phenotypic tests showed some ability in discriminating clusters A, B and C from clusters D and E.

Genotypic characterization of virulence factors in Escherichia coli strains from patients with cystitis / Caracterização genotípica dos fatores de virulência em amostras de Escherichia coli isoladas de pacientes com cistite

Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5 percent), 86 kpsMTII (53.1 percent), 53 papC/papEF/papG (32.7 percent), 45 sfa (27.8 percent), 42 iucD (25.9 percent), 41 hly (25.3 percent), 36 usp (22.2 percent), 30 cnf-1(18.5 percent) and 10 afa (6.2 percent) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.

Adesinas (Fímbria P, fímbria S, fímbria do tipo 1 e a adesina afimbrial), toxinas (α-hemolisina e o fator necrosante citotóxico do tipo 1), sistemas de captação de ferro (aerobactina), e mecanismos de defesa do hospedeiro (cápsula ou lipopolissacarídeo) são prevalentes em amostras de Escherichia coli associadas a infecções do trato urinário. O objetivo deste trabalho foi caracterizar genotipicamente 162 amostras de Escherichia coli uropatogênica (UPEC) de pacientes com cistite através do ensaio da reação em cadeia da polimerase. Foram realizados três ensaios de PCR multiplex para os seguintes fatores de virulência: papC, papE/F, alelos de papG, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, e kpsMTII. Os resultados da PCR identificaram, 158 amostras fimH (97,5 por cento), 86 amostras kpsMTII (53,1 por cento), 53 amostras papC/papEF/papG (32,7 por cento), 45 amostras sfa (27,8 por cento), 42 amostras iucD (25,9 por cento), 41 amostras hly (25,3 por cento), 36 amostras usp (22,2 por cento), 30 amostras cnf-1 (18,5 por cento) e 10 amostras afa (6,2 por cento). Nenhuma amostra foi positiva para o gene cdtB. Neste trabalho, demonstramos que podemos encontrar múltiplas adesinas em uma única amostra e que diferentes genes de fatores de virulência podem ser encontrados em associação.

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.