Synthesis and evaluation of monoclonal antibody against Plasmodium falciparum merozoite surface antigen 2

ObjectiveTo assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.MethodsMice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum (P. falciparum) MSP-2. B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation. Fusion of NS-1 and spleen cells was performed. The positive hybrids were cloned and ELISA was applied against different dilutions. The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains. The positive clones were expanded to large (75 cm²) flask and cultured under the same conditions, checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.ResultsA total number of 7 fusions including 26 plates (2 496 wells) were performed, of which 1 336 hybrids were produced and the overall efficiency (1 336/2496 × 100) was about 53%. ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3 (66 out of 315 hybrids). The supernatant of both B5 and F1 hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test. Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P. falciparum parasite were recognized by some of the positive clones and also immune sera.ConclusionsBringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.     

High Prevalence of AmpC β-Lactamases in Clinical Isolates of Escherichia coli in Ilam,Iran

Objectives

Widespread use of β-lactam antibiotics could cause resistance to this group of antibiotics in pathogenic bacteria through the production of the enzyme β-lactamases. The aim of this study is to determine the molecular detection of AmpC β-lactamases among clinical Escherichia coli isolated from Ilam hospitals in Ilam, Iran.

Methods

One hundred and twelve clinical isolates of E. coli were collected from hospitalized patients and were identified by biochemical tests. They were evaluated for extended spectrum beta-lactamases (ESBLs) production, and the positive strains were subjected to AmpC enzymes; for detection of AmpC cluster genes, multiplex polymerase chain reaction was applied.

Results

The analysis showed 62.5% of isolates were ESBLs positive and that five strains revealed the AmpC cluster genes. This is the first report of FOXM cluster genes in E. coli in Iran.

Conclusion

Based on our results, the prevalence of AmpC β-lactamases is increasing in Iran, which caused failure in antibiotic therapy. So, the current study recommended the revision of antibiotic policy in Iranian hospitals.     

Cloning,Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

Objectives

In an attempt α-amylase gene from Pyrococcus woesei was amplified and cloned into a pTYB2 vector to generate the recombinant plasmid pTY- α-amylase.

Methods

Escherichia coli BL21 used as a host and protein expression was applied using IPTG. SDS-PAGE assay demonstrated the 100 kDa protein. Amylolytic activity of proteins produced by transformed E. coli cells was detected by zymography, and the rate of active α-amylase with and without the intein tag in both soluble conditions and as inclusion bodies solubilized by 4M urea were measured.

Results

Amylolytic activity of ∼185,000 U/L of bacterial culture was observed from the soluble form of the protein using this system.

Conclusion

These results indicate that this expression system was appropriate for the production of thermostable α-amylase.     

Is the mazEF toxin-antitoxin system responsible for vancomycin resistance in clinical isolates of Enterococcus faecalis?

The current study was conducted to investigate the relationship between vancomycin-resistant Enterococcus faecalis (VRE) and the presence of mazEF toxin-antitoxin (TA) system, which may be useful as target for novel antimicrobial therapy concepts. The susceptibility of E. faecalis was determined by MIC, and the presence of the mazEF TA system was evaluated by PCR. Among 200 E. faecalis isolates 39.5% showed resistance to vancomycin (VRE), while 60.5% were susceptible strains (VSE). The mazEF TA system was positive in all VRE isolates (100%), but less prevalent (38/121, 31.4%) among the 121 VSE strains. In conclusion, our study demonstrated a positive relationship between the presence of vancomycin resistance and mazEF TA system. This observation may introduce therapeutic options against a novel antimicrobial target in enterococci.     

Genetic relatedness among isolates of <Emphasis Type="Italic">Shigella sonnei</Emphasis> carrying class 2 integrons in Tehran,Iran, 2002–2003

Background  

Shigella spp. are major cause of diarrhoeal disease in both developing and developed countries. Shigella sonnei is the serogroup of Shigella most frequently responsible for sporadic and epidemic enteritis in developed countries. In recent years the emergence and spread of S. sonnei biotype g carrying class 2 integron have been frequently reported in many countries. Recently, S. sonnei has been reported as the prevalent serogroup of Shigella in Iran.     

Antimicrobial resistance of nosocomial strain of Acinetobacter baumannii in Children's Medical Center of Tehran: a 6-year prospective study

There are increasing reports of emergence of multiple drug resistant (MDR) Acinetobacter spp in the world; however there are a few reports in our country. 145 A. baumannii isolates from distinct wards and Children's Medical Center (CMC) in Tehran were studied in order to find the profile of antibiotic resistance among them. 40.6% (59/145) of A. baumannii isolates were identified as MDR. Overall susceptibility rates to cotrimoxazole, chloramphenicole and ciprofloxacin were 23.4%, 16.9% and 20.1%, respectively. Frequency susceptibility rates to amikacin, kanamycin, gentamycin and tobramycin decreased gradually from 81.2%, 50%, 50% and 62.5% in 2002 to 25%, 15.6%, 28.1% and 25% in 2007 respectively. Overall susceptibility rates to cephalosporines cephalotin, ceftazidime, cefteriaxon, ceftizoxime and cefixime were 9.3%, 14.7%, 16.2%, 15.9% and 18%, respectively. Susceptibility to carbapenems was assessed only in 2007. The susceptibility rates of Imipenem and meropenem were shown to be 50% and 46.8%, respectively. Our data indicates that MDR A. baumannii strains are spreading and carbapenem resistance is becoming more common in Iran. Our findings also highlight the importance of clinicians' access to updated susceptibility data regarding A. baumannii in developing countries such as Iran.     

The incidence of nosocomial toxigenic clostridium difficile associated diarrhea in Tehran tertiary medical centers

Clostridium difficile is the most common cause of nosocomial diarrhea. It is usually a consequence of antibiotic treatment, But sporadic cases can occur. This study was aimed to determine the frequency of the nosocomial Clostridium difficile (C. difficile) associated diarrhea in Tehran University of Medical Sciences hospitals and study of antibacterial susceptibility of isolates. In this study a total of 942 stool samples from patients with nosocomial diarrhea that were hospitalized in Imam Khomeini hospital, Shariati hospital and Children clinical center were collected. The samples were cultured on a selective cycloserine cefoxitin fructose agar (CCFA) and incubated in anaerobic conditions, at 37°C for 5 days. Isolates were characterized to species level by conventional biochemical tests. Bacterial cytotoxicity was assayed on tissue culture (vero). Antimicrobial sensitivity of isolated toxigenic C. difficile were investigated by kirby Beuer method (disk diffusion). Our findings show that, of the total patients, 57 toxigenic C. difficile (6.1%) were isolated. Results of statistical analysis show significant differences between the rate of isolated toxigenic C. difficile and age group of patients (P<0.05). Among the wards of selected hospitals, in gastroenterology of Children clinical center, Toxigenic C. difficile was isolated from patients most frequently. The sensitivity of isolates to vancomycin, Chloramphenicol and ceftriaxone were higher than other antibiotics. Toxigenic C. difficile is a common hospital-acquired infection. The organism was found in 6.1% hospitalized patients. Further studies to evaluate the rate and role of toxigenic C. difficile in nosocomial diarrheal processes, ecological and pathogenic terms are suggested.     

Antimicrobial susceptibility patterns and distribution of blaOXA genes among Acinetobacter spp. Isolated from patients at Tehran hospitals

Multiple drug-resistant strains of Acinetobacter have created therapeutic problems worldwide. This study was conducted to determine the antimicrobial susceptibility patterns and prevalence of bla(OXA-type) carbapenemases among isolates of Acinetobacter spp. obtained from Iranian patients. Here, 128 Acinetobacter isolates were identified at the species level, and their susceptibilities to different antibiotics were determined using disk agar diffusion testing. Isolates were then subjected to multiplex-PCR targeting bla(OXA) genes. More than 50% of the isolates showed multidrug resistance to different antibiotics. The rates of susceptibility to imipenem, meropenem, piperacillin-tazobactam, and amikacin were 50.7, 50, 42.1, and 38.2%, respectively. The MICs of carbapenems for the resistant isolates ranged from 64 to > or = 256 microg/ml. All strains of Acinetobacter baumannii possessed a bla(OXA-51-like) gene. The co-existence of bla(OXA-51-like)/bla(OXA-23-like) and bla(OXA-51-like)/bla(OXA-24-like) was detected in 25% (n=32) and 17.9% (n=23) of the isolates, respectively. Over 70% of carbapenem-resistant strains contained at least two genes encoding OXA-type carbapenemase. Resistance to carbapenems in the population of Acinetobacter strains in Iran is high, with the majority of isolates showing multidrug resistance. A wide diversity of OXA genes exists among the strains of A. baumannii in Iran. Detection of bla(OXA-51-like) can be used as a simple and reliable method to differentiate A. baumannii strains from other species.