Iranian Journal of Microbiology
Volume:3 Issue: 4, Dec 2011

Pneumonia with Acinetobacter baumannii has a major therapeutic problem in health care settings. Decision to initiate correct antibiotic therapy requires rapid identification and quantification of organism. The aim of this study was to develop a rapid and sensitive method for direct detection of A. baumannii from respiratory specimens. A Taqman real time PCR based on the sequence of blaoxa-51 was designed and used for direct detection of A. baumannii from 361 respiratory specimens of patients with pneumonia. All specimens were checked by conventional bacteriology in parallel. The new real time PCR could detect less than 200 cfu per ml of bacteria in specimens. There was agreement between the results of real time PCR and culture (Kappa value 1.0, p value < 0.001). The sensitivity, specificity and predictive values of real time PCR were 100%. The prevalence of A. baumannii in pneumonia patients was 10.53 % (n = 38). Poly-microbial infections were detected in 65.71% of specimens. Acinetobacter baumannii is the third causative agent in nosocomial pneumonia after Pseudomonas aeroginosa (16%) and Staphylococcus aureus (13%) at Tehran hospitals. We recommend that 104 CFU be the threshold for definition of infection with A. baumannii using real time PCR.

The aim of study was to develop a rapid assay, dye labelled monoclonal antibody assay (DLMAA), using non-radioactive organic synthetic dyes for identification of Toxic Shock Syndrome Toxin-1 (TSST-1) producing strains of Staphylococcus aureus. The assay protocol required only two simple steps; addition of TSST-1 antigen to a nitrocellulose membrane and then adding a colloidal dye labelled antibody (D/A) suspension detection reagent. The sensitivity and specificity of the assay was determined relative to positive and negative strains compared to an ELISA assay. Overall 100% agreement was found between both assays. The sensitivity for detection of TSST-1 was 30 ng. The DLMAA did not require handling and disposal of radioactive materials. It is a rapid qualitative technique for detection of TSST-1 toxin at room temperature within a short time.

Human Papillomavirus (HPV) infection is a major risk factor for adenocarcinoma of the cervix. The high-risk types of the virus such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, are responsible for approximately 50% of all cervical cancers. A rapid, sensitive and specific test has been proposed for detection of HPV to improve cervical cancer screening programs. The aim of this study was to develop a fast PCR-ELISA assay designated as DIAPOPS (Detection of Immobilized Amplified Products in a One Phase System)for detection of HPV16 and HPV18 types in SCC samples and Pap smears. The type specific primers and probes were designed for PCR and PCR-ELISA. The amplified products were hybridized with a specific biotin-labeled probe for HPV18 inner amplicons. The hybrids were detected with peroxidase conjugated avidin. The test was performed on the paraffin block and Pap smear samples from the cervical cancer patients, and the results of DIAPOPS were compared with conventional PCR assay. The 70 samples (SCC and Pap smear samples) were collected from Imam Khomeini and Mirzakoochak Khan Hospitals in Tehran. The PCR-based method detected six HPV16 positive, three HPV18 positive and Two HPV33 positive samples. DIAPOPS results were compared with the conventional PCR results and they showed an increase in sensitivity of the DIAPOPS test. Not only all of them were confirmed by PCR-ELISA but also three samples that conventional PCR showed negative for HPV18, were demonstrated positive by the PCR-ELISA method. The results of the study show that modified PCR-ELISA assay is more sensitive to detect HPV types and can be used for diagnostic purposes.

The incidence of fungal infections in immunocompromised patients, especially by Candida species, has increased in recent years. This study was designed to identify Candida species and determine antifungal susceptibility patterns of 595 yeast strains isolated from various clinical specimens. Identification of the isolates were determined by the API 20 C AUX kit and antifungal susceptibilities of the species to fluconazole, amphotericin B, ketoconazole, itraconazole, voriconazole, and caspofungin were determined by the agar-based E-test method. Candida albicans (48%) was the most frequently isolated species, followed by Candida kruzei (16.1%), Candida glabrata (13.5%), Candida kefyr (7.4%), Candida parapsilosis (4.8%), Candida tropicalis (1.7%) and other species (8.5%). Resistance varies depending on the species and the respective antifungal agents. Comparing the MIC90 for all the strains, the lower MIC90 was observed for caspofungin (0.5 μg/ml). The MIC90 for all Candida species were 64 μg/ml for fluconazole, 0.75 μg/ml for amphotericin B, 4 μg/ml for ketoconazole, 4 μg/ml for itraconazole, and 2 μg/ml for voriconazole. Species definition and determination of antifungal susceptibility patterns are advised for the proper management and treatment of patients at risk for systemic candidiasis. Resistance to antifungal agents is an alarming sign for the emerging common nosocomial fungal infections.

Resistance among bacterial isolates is the leading cause of increased mortality and morbidity worldwide. Carbapenems once thought to be effective are becoming ineffective mostly due to the emergence of carbapenemase. This study was designed to determine in vitro efficacy of Modified Hodge test for detection of carbapenemase production in Gram negative rods. The study was done in the Department of Microbiology, Armed Forces Institute of Pathology Rawalpindi Pakistan from January 2010 to December 2010. A total of 200 Gram negative rods from different clinical samples were taken. Those isolates which showed intermediate or susceptible zones i.e 16mm-21mm on disc diffusion were included in the study. These isolates were then subjected to Modified Hodge test. Out of 200 isolates, 138 (69%) were positive for carbapenemase production by Modified Hodge test. Out of 138 MHT positive organisms, the frequency of E. coli was 38%, followed by Pseudomonas aeruginosa (30%), Klebsiella pneumoniae (17%), Acinetobacter baumannii (12%), Citrobacter diversus (2%) and Enterobacter agglomerans (1.4%). Modified Hodge test is a simple test which can be performed in the routine lab for detection of carbapenemases in isolates showing intermediate or sensitive zone diameter on disc diffusion.

The aim of this study was to evaluate the chemical composition and antimicrobial activity of Satureja hortensis and Trachyspermum copticum essential oils against different kinds of microorganisms in vitro. The antimicrobial activity was evaluated by micro broth dilution assay and analysis of essential oils chemical composition by GC and GC/MS analysis. Thymol, p-cymene, γ-terpinene and carvacrol were the main components of S. hortensis oil while thymol, γ-terpinene, and o-cymene were the major components of T. copticum oil. Two essential oils exhibited strong antimicrobial activity but the antimicrobial activity of T. copticum oil was higher than that of S. hortensis oil. Thymol as a main component of oils plays an important role in antimicrobial activity.

Proline dehydrogenase (ProDH; 1.5.99.8) plays an important role in specific determination of plasma proline level in biosensor and diagnostic kits. The goal of this research was to isolate and characterize ProDH enzyme from Iranian soil microorganisms. Screening of L-proline degradative enzymes from soil samples was carried out employing enrichment culture techniques. The isolate was characterized by biochemical reactions and specific PCR amplification. The target ProDH was purified and the effects of pH and temperature on the activity and stability were also tested. Among the 250 isolates recovered from 40 soil samples, only one strain characterized as Pseudomonas entomophila displayed the highest enzyme activity toward L-proline (350 U/l) than others. The enzyme of interest was identified as a ProDH and had Km value of 32 mM for L-proline. ProDH exhibited its best activity at temperature range of 25 to 35°C and its highest activity was achieved at 30°C. It was almost stable at temperatures between 25-30°C for 2 hours. The optimum pH activity of ProDH reaction was 8.5 and its activity was stable in pH range of 8.0-9.0 upto 24 hours. The enzyme was purified with a yield of 8.5% and a purification factor of 37.7. The molecular mass of the purified ProDH was about 40 kDa, and determined to be a monomeric protein. This is the first report concerning the ProDH production by a P. entomophila bacterium isolated from soil sample.

The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media.

Phaffia rhodozyma is a red yeast which produces astaxanthin as the major carotenoid pigment. Astaxanthin is thought to reduce the incidence of cancer and degenerative diseases in man. It also enhances the immune response and acts as a free-radical quencher, a precursor of vitamin A, or a pigment involved in the visual attraction of animals as mating partners. The impact of gamma irradiation was studied on the Phaffia rhodozyma genome. Ten mutant strains, designated Gam1-Gam10, were obtained using gamma irradiation. Ten decamer random amplified polymorphic DNA (RAPD) primers were employed to assess genetic changes. Nine primers revealed scorable polymorphisms and a total of 95 band positions were scored; amongst which 38 bands (37.5%) were polymorphic. Primer F with 3 bands and primer J20 with 13 bands produced the lowest and the highest number of bands, respectively. Primer A16 produced the highest number of polymorphic bands (70% polymorphism) and primer F showed the lowest number of polymorphic bands (0% polymorphism). Genetic distances were calculated using Jaccard''s coefficient and the UPGMA method. A dendrogram was created using SPSS (version 11.5) and the strains were clustered into four groups. RAPD markers could distinguish between the parental and the mutant strains of P. rhodozyma. RAPD technique showed that some changes had occurred in the genome of the mutated strains. This technique demonstrated the capability to differentiate between the parental and the mutant strains.