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Purpose: the aim of the present study was to determine the role of some virulence determinants and biofilm production in bacteraemia of urinary tract origin by phenotypic defection of these virulence factors. In addition, this research was done to characterize and compare, using genetic techniques, bacterial genes that encode virulence determinants
Methods: a total of 111 strains of Enterobacteriaceae isolated from urine of patients with clinically diagnosed urinary tract infection in Urology and Nephrology Center, Mansoura University, were included in this study. The isolated strains were phenotypically screened for virulence factors, namely mannose-resistant, mannose-sensitive haemagglutination [MRHA, MSHA], hemolysin production, biofilm formation, serum resistance, and lipase, protease and lecithinase production. They were also genotypically examined by PCR for the presence of genes for the virulence factors MRHA [papC], MSHA [fimH], serum resistance [iss] and biofilm,formation [biofilm]
Results: among 111 urinary isolates of Enterobacteriaceae, 40 isolates of E. coli, 39 isolates of K. pneumonia and 32 isolates of Enterobacter were identified. The antibiotic sensitivity test showed that amikacin and imipinem were the most active antibiotic against all isolates[90%-100%]. While most isolates were resistant to ampicillin [95%- 100]. The phenotypic detection results revealed that 29[72%] of E.coli, 32[82%] of K.pneumoniae and 22[69%] of Enterobacter isolates showed MRHA. MSHA was defected in 11[28%] isolates of E.coli. 7[18%] of K.pneumoniae and in 10[31%] of Enterobacter isolates. Serum resistance was seen in 15[37%] o f E.coli, 13[33%] of K.pneumoniae and 7[21%] of Enterobacter isolates. Biofilm formation was observed in 27[67%] of both E.coli and K. pneumoniae and in 30[94%] of Enterobacter isolates. All E.coli isolates were negative lipase and protease producers, while 16[33%] of K.pneumoniae isolates showed lipase production and one isolate showed protease production. For Enterobacter, none of isolates produce lipase, while 4[12%] of isolates were protease producers. All isolates showed no lecithinase production. For genotypic detection, it was observed that among E.coli isolates, 69% were positive for papC gene, 31% were positive for fimH gene, 30% were positive for iss gene and 77% were positive for biofilm gene. For K.pneumoniae, papC gene was detected in 80% of isolates, 20% were positive, for fimH gene, 8% were positive for iss gene and 87% contained the biofilm gene. Results of Enterobacter isolates were 89%, 11 %, 5% and 95% positive for genes of papC, fimH, iss and biofilm respectively
Conclusion: the present study shows the significance role of virulence factors in urinary tract infections caused by Enterobacteriaceae and the genotypic characterization was more prominent compared to the phenotypic detection of these virulence factors
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Purpose: P. aeruginosa is the epitome of an opportunistic pathogen of humans. It causes a variety of serious illness and represents a major risk among patients with cystic fibrosis, hospital-acquired infections, especially in burn, and otherwise immunocompromised patients, including transplantation and cancer patients. This microbe has a high level of innate antimicrobial resistance and a large armamentarium of virulence factors that have been the subjects of vaccine development. Flagellum is one Of these virulence factors that play a major role in the recognition of bacteria by the host and in the induction of immune responses by Toll-like receptor 5[TLR5]. These data support Flagellin as an antigen candidate to design an anti -Flagellin vaccine. Isolation, cloning, expression and purification of Flagellin were aimed at in this study
Methods: flagellin gene sub type b fliC [B] was amplified by PCR from genomic DNA isolated from P. aeruginosa PA01 and then cloned into the cloning vector pCEMT Easy. Sub cloning of the amplified gene into expression vector pRSET-B. The recombinant Flagellin was expressed after transformation into E. coli BL-21 [DE3] pLysS and finally purified using Ni+2 affinity chromatography
Results: a 1460 bp fragment of the fliC [B] gene from P. aeruginosa strain PAO1 was amplified from genomic DNA by PCR. This fragment was cloned into the TA cloning vector pGEMT-Easy and after transformation into E. coli DH5 alpha, selection of the clones was carried by blue-white screening. Confirmed positive plasmid extracted from some white colonies was digested, gel purified and ligated to pRSET- B expression vector that has also been digested and gel purified. This recombinant vector was transformed into E. coli DH5 alpha and some white colonies were picked and miniprepped to screen for the presence of the insert by restriction digests. Plasmids with the fliC [B] insert were used for expression of fliC [B] by transformation into E. coli BL-21 [DE3] pLysS. SDS-PAGE electrophoresis confirmed the expression of target protein having molecular weight of 53 KDa
Conclusion: our findings confirmed that a prokaryotic expression system of recombinant fliC [B] was successfully constructed. The results of SDS-PAGE showed that our constructed prokaryotic expression system pRSET-B /fliC [B] / BL-21 [DE3] efficiently produced target recombinant protein in the form of soluble protein. Therefore we can suggest that this purified protein can effectively be a vaccine candidate
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Aim: the aim of this study is to estimate the phenotypic characters of some virulence factors in Escherichia coli and Klebsiella pneumoniae isolated from urinary tract infections and their relative gene expression level
Methods: Escherichia coli and Klebsiella pneumoniae clinical isolates were collected, identified. Their antimicrobial sensitivity pattern was determined. The presence of different virulence factors were evaluated. Relative expression level of different genes responsible for the appearance of the tested virulence factors was evaluated using QRT-PCR and PCR
Results: in this study, when testing the expression level of some genes like BssS and its genes in E. coli, we found that quinolone sensitive isolates of E, coli had shown higher expression level of the tested genes than that o f quinolone resistant E. coli isolates. These results agreed with the phenotypic characters of the tested virulence factors for the same isolates. On the other hand, in K. pneumoniae tested isolates, the expression level of BssS and fimH genes was evaluated and they showed higher expression level in quinolone resistant isolates than sensitive ones. These results agreed with the phenotypic characters of the tested virulence factors for the same isolates. PCR detection of iss gene on both plasmid and chromosomal DNA in K. pneumonia isolates has demonstrated that isolates that exhibit iss gene on both chromosomal and plasmid DNA were those that had shown higher expression at the genetic and phenotypic levels
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Aim: the present study aims to determine the efflux pump resistance by phenotypic and genotypic Methods
Methods: a total of 40 isolates of Pseudomonas aeruginosa were studied for antibiotic susceptibility pattern, , Effect of efflux pump inhibitors [reserpine, carbonyl cyanide mchloropheylhydorazone [CCCP] and omperazole] on the MIC of antibacterial agents, ethidium bromide [EtBr] efflux assay, outer membrane proteins, polymerase chain reaction for the amplification of resistance genes
Results: nearly most of the isolates were multiple resistant as they were resistant to most antimicrobial classes used in this study. CCCP was found to have the highest effect on decreasing the MIC of different antibiotics comparable with reserpine and omperazole. It was found that all tested Pseudomonas aeruginosa isolates extrude EtBr resulting in decrease in fluorescence over the time of assay. In Pseudomonas aeruginosa isolate No.3 and 14 only the control cells extrude EtBr resulting in significant loss in fluorescence. In presence of each of reserpine, CCCP and omperazole/e, an insignificant loss in fluorescence was observed, reflecting blockage of EtBr by these compounds at different levels. All the isolates produced high amount of outer membrane proteins with apparent molecular mass of 54 KDa and 50 KDa. These proteins may be designated as OprJ and OprM or OprN. MexA and MexB genes was detected and amplified in most of the tested isolates of Pseudomonas aeruginosa genomic and plasmid DNA. The amp/icons were visualized at 500 bp and 1000 hp respectively. While OprM gene was amplified successfully in the twenty tested isolates and the amp/icons were visualized at size of 900 bp
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Background With an annual tuberculosis (TB) incidence of about 350 cases per 100;000 of the population; Uganda is a high burden country. Moreover; it is evident that some TB patients have been treated for a previous episode of the disease. Objective To highlight the burden of re-treatment pulmonary TB and examine patient factors associated with re-treatment among adults at two teaching and referral hospitals; Mbarara and Mulago Methods A descriptive cross sectional study with data collection between September 2004 and March 2005; we calculated the prevalence and used logistic regression to explore factors associated with re-treatment. Results The prevalence of re-treatment pulmo-naryTB at Mbarara based on medical records was 30.0(95CI: 21.2 to 40.0); and 21.3(95CI: 12.9 to 31.8) from exit interviews.The corresponding estimates at Mulago hospital were 12.0(95CI: 6.4 to 20.0) and 43.9(33.0 to 55.3). Compared to the 18-26 year age category; the prevalence odds ratio (POR) for a seven-year increase in age was 1.54 (95CI: 1.04-2.28; p = 0.027); while female patients were 0.39 (95CI: 0.17-0.90; p = 0.025) times less likely to report re-treatment disease than males; in this facility-based study. Conclusions Re-treatment pulmonary TB is frequent at the two teaching and referral hospitals.A contribution to re-treatment prevention should entail more rigorous management of new TB cases; particularly at lower levels of care