Molecular characterization and functional analysis of the PilQ380-705: a novel secretin domain in Pseudomonas aeruginosa

Background: Type 4 pili [T4P] is an important virulence factor of Pseudomonas aeruginosa [P. aeruginosa]. T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ [PilQ380-706] was produced as a recombinant protein

Methods: A 981 bp fragment of C-terminal of the pilQ secretin [pilQ1138-2118] from was designed into the prokaryotic expression vector pET28a. The presence of the pilQ1138-2118 gene in the recombinant construct [pET28a/pilQ] was assessed by double digestion and PCR. After transformation, expression of the recombinant PilQ was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced PilQ380-706 confirmed by Western blot analysis and twitching inhibition assay

Results: The PCR and enzymatic digestion results showed the presence of the pilQ1138-2118 gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ380-706 is approximately 37 kDa. The Western blot analysis confirmed the specificity of specific IgG against the PilQ380-706 protein. The PilQ380-706 protein showed high biological activity in all of these standard assays

Conclusion: Since, the PilQ380-706 protein plays an important role in the biogenesis of pili; and thus, the primary establishment of P. aeruginosa; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies

Synthesis, characterization and antibacterial activity of some novel thiosemicarbazides, 1,2,4-triazol-3-thiols and their S-substituted derivatives

The thiosemicarbazides 3a-c were appeared by reaction of the corresponding substituted hydrazides 1a-c with allylisothiocyanate 2. Synthesis of some novel 1,2,4-triazole-thiols 4a-c bearing a pyridyl unit using 1-[x-picolinoyl]-4-allyl-thiosemicarbazides [x = 2,3,4] in an alkaline solution, is reported. Also, the S-alkylation of triazole derivatives 5-7a-c is described. The structure of the synthesized compounds resulted from the IR, [1]H and -[13]C NMR spectroscopy and elemental analysis data. The antibacterial studies to all of the synthesized compounds against B. cereus, E. coli, P. aeroginosa, S. aureus and E. faecalis as MIC values are reported. Some of these compounds such as 7a, 4a and 3a exhibited a good to significant antibacterial activity

Antibiotic resistance and frequency of class 1 integrons among Pseudomonas aeruginosa isolated from burn patients in Guilan Iran

Antibiotic resistance of Pseudomonas aeruginosa remains a major problem in bum patients. The main objective of this study was to determine the antibiotic resistance pattern and frequency of class 1 integrons among P. aeruginosa strains isolated from patients with burn wound infections in a new Burn Centre in Guilan, Iran. The bacterial isolates were collected from 182 patients with burn wound infections and P. aeruginosa species were identified by standard bacteriological methods. The drug susceptibility test, using 11 antimicrobial agents, was performed for all the isolates via agar disk diffusion method. PCR was carried out for the detection of integrons. Out of a total of 182 hospitalized patients in the burn center assessed, 86 [47%] found to have P. aeruginosa in their isolates. Resistance rates to various antibiotics were as follows: cloxacillin [91.8%], cotrimoxazole [86%], cephazolin [83.7%], carbenicillin [74.4%], piperacillin [69.9%], ceftazidime [68.8%], ciprofloxacin [66.3%], tobramycin [58.2%], amikacin [48.8%] and gentamicin [37.2%], while the most effective antibiotic was imipenem with a resistance rate of 23.3%. Thirty nine [45.3%] isolates were detected as multi-drug resistant. The PCR results showed that 37 [43%] P. aeruginosa isolates and 27 [69.2%] multi-drug resistant strains harbored class 1 integrons. A significant correlation was obtained between the presence of integrons and resistance against imipenem, ceftazidime, piperacillin and ciprofloxacin [P < 0.001]. Optimization of using antimicrobial agents and control of infection is recommended to prevent the increasing population of drug resistant organisms in the new burn centre setting in this study. Furthermore, the high frequency of class 1 integrons among multi-drug resistant strains might be responsible for dissemination of antibiotic resistance gene

Effect of hexanic and alcoholic extracts of fenugreek seed in male diabetic rats

Diabetes mellitus is recognized with severe complications. Many herbal medicines have been recommended for treatment of diabetes problem. In this study, the effect of hexanic and alcoholic extracts of fenugreek [Trigonella-foenum graecum] on serum parameters was investigated in normal and streptozotocin-induced diabetic rats. This experimental study was carried out in 2011 at paramedical school of Guilan University of Medical Sciences, 48 male Sprague Dawley rats [230- 300 gram] were divided into six groups: control, type 1 diabetic, and 2 diabetic groups that receive alcoholic extract and 2 groups receive hexanic extract of fenugreek [100, 200 mg/kg body weight] intraperitonealy for 28 days. For diabetes induction, streptozotocin [60 mg/kg/ intraperitonealy] was used. Blood glucose, total cholesterol, triglyceride [TG], urea, creatinine, uric acid, AST and ALT level was measured. Data were analyzed with spss software 16 and One Way ANOVAs and Tukey test. p<0.05 was statistically significant. Fenugreek extract inhibit weight loss especially in diabetic groups that receive hexanic extract [p=0.006]. blood glucose, total cholesterol, TG, urea, creatinin, uric acid, AST and ALT level was reduced significantly in diabetics groups that receive fenugreek extract [p=0.001]. This effect was stronger in groups that receive Hexanic extract. Fenugreek is a good candidate for reduction of diabetic complications

Evaluation of a PCR assay to detect Enterococcus faecalis in blood and determine glycopeptides resistance genes: van A and van B

Bacteremia due to Enterococcus faecalis is usually caused by strains resistant to most antibiotics. Effective management of the disease is dependent on rapid detection and characterization of the bacteria, and determination its sensitivity pattern to antimicrobial drugs. The aim of this study was to investigate a more rapid and reliable assay for simultaneous diagnosis of enterococcal bacteremia and its sensitivity pattern to antimicrobial drugs. Several bacterial suspensions with different content of two standard strains of Enterococcus faecalis resistant to vancomycin were used for inoculation to defibrinated sheep blood samples. PCR and routine assay was performed on all blood samples with different bacterial content. Routine assay and PCR for all inoculated blood samples with >/= 5 cfu/ml was positive. Mean time for PCR and routine assays was 10 hours and 5 days, respectively. PCR is a more rapid and sensitive assay for simultaneous detection and characterization for Enterococcus faecalis, and determination of its sensitivity pattern to vancomycin

comparison of two-step tuberculin skin test between health-care workers and nonhospital employees

The tuberculin test is widely used to identify tuberculosis infection. Some individuals infected with Mycobacte-rium tuberculosis may have an initial negative skin test reaction to tuberculin. The two step purified protein derivative skin test can decrease misinterpretation of tuberculin test .This study was aimed at comparing the two- step tuberculin skin test and booster phenomenon in health care workers and non hospital employees. One hundred and eighty five health service employees from Razi University Hospital and 181 non hospital employees were subjected to an initial tuberculin skin test. Those who were negative on the first test underwent a second one 2 weeks later. The reactions to the tests were measured 72 hours later. Tests with an induration of > 10 mm was considered positive. Moreover, second tests with an induration of at least 6 mm increase relative to the relevant first tests were considered positive. Compared to non hospital employees [n=79, 43.6%], a significantly higher number of health care workers [n=113, 61.1%] were positive on the first tuberculin test. 18.5% of health care workers and 31.5% of non hospital employees demonstrated a boosted reaction after the second tuberculin test. The occurrence of boosted reaction was significantly associated with age both groups. There was no association between the presence of BCG scars and the occurrence of boosted reaction. the present study shows that the prevalence of TB was higher among health care worker than non hospital employees. Moreover, it demonstrated that non hospital employees had a higher age-associated booster reaction to the second tuberculin skin test than health care workers. Therefore, to avoid missing false negative cases, it would be necessary to do a second tuberculin test for subjects with a negative reaction to the initial test, especially in subjects older than 40 years

Evaluation of cellular immune response against purified antigen 85 in patients with tuberculosis

Tuberculosis [TB] remains an important health problem throughout the world. Despite its significance in public health, mechanisms of protective immunity against Mycobacyerium Tuberculosis in humans have not yet been understood. To evaluate cell mediated immune response against purified Ag 85, PPD and Phytohemagglutinin [PHA] in patients with tuberculosis and healthy tuberculin positive and negative individuals. Thirty patients with tuberculosis and 60 healthy tuberculin skin test positive and negative volunteers were participated in this study. Cell mediated immunity was assessed by measuring [[3]H]-thymidine uptake and detection of IFN-gamma in the culture supernatant using commercial ELISA test. In the present study, we showed that IFN-gamma production and cell proliferation response to Ag 85 were significantly higher in tuberculin positive than tuberculin negative individuals [P<0.01]. Among tuberculous patients, IFN-gamma production and cell proliferative responses to Ag 85 was significantly lower in contrast to healthy tuberculin positive individuals [P<0.01]. In addition, IFN- gamma response in patients with cavitary tuberculosis was lower than patients without cavitation [P<0.05]. Based on the higher cell mediated immune responses to Ag 85 in healthy tuberculin positive volunteers compared to patients [especially with advanced disease], purified Ag 85 can be used as a sensitive marker for analysis of immune responses in tuberculosis