فصلنامه دنیای میکروب ها
سال ششم شماره 3 (پیاپی 16، پاییز 1392)

Bacillus subtilis is considered as a suitable host for gene cloning and protein expression due to non-pathogenicity and ability to secrete high amounts of proteins. However, transformation efficiency of ligated plasmid DNA into competent cells of B. subtilis is low, as compared to that into CaCl2 shocked cells of Escherichia coli. Therefore, cloning the target in E. coli using a shuttle vector before transfer into B. subtilis by a high amount of hybrid vector is more efficient. The aim of this study is construction a shuttle vector using pW980 and pET-16b plasmids for gene cloning and expression in B. subtilis. pWB980 plasmid was isolated from its host using alkaline lysis method. It was undergone a double digestion to obtain the segment of interest using EcoRI  and SnaBI enzymes. Then a special fragment of pET-16b plasmid was also amplified by PCR and was double digested. Ligation reaction was performed between these two segments and then it was transferred to E. coli TOP10. Following screening the cells containing shuttle vector, plasmid was extracted and was transferred to B. subtilis WB600 by an effective method. The resulting shuttle vector replicated easily in both hosts. It showed good stability in B. subtilis, which is helpful for its maintenance in its host. In this study, the resulting shuttle vector - pMR12 - had 8 unique cloning site in its polylinker and a suitable size (5.4 kb), which make it possible to simply gene cloning into it. This is the first report of construction of an expression shuttle vector for Escherichia coli and Bacillus subtilis in Iran.

In addition to safety, nanoparticles produced via biological methods show antimicrobial activity against human pathogens. This study was aimed to isolation and identification of intracellular and extracellular gold nanoparticle producing strains from soil and to investigate the antimicrobial effects of the produced nanoparticles on 10 common bacterial pathogens. In this study, after isolation of bacteria from the soil and production of the gold nanaoparticles by their supernatants and their pure cells, the bio-production was confirmed through visible spectrophotometry, transmission electron microscopy (TEM) and X-ray diffraction analysis (XRD). The nanoparticle producing stains were identified based on gene amplification by PCR and gene sequencing. Finally, the antibacterial properties of the produced gold nanoaprticles against 10 pathogenic bacteria was assessed by well diffusion method. Results showed that 38 strains had ability to produce extracellular gold nanoparticles. Among them 16 strains had ability to produce intracellular gold nanoparticles as well. TEM images of the gold nanoparticles showed sizes less than 100 nm and the XRD patterns confirmed the crystalline structure of the gold nanoparticles. Three bacterial strains, Bacillus mojavensis, Bacillus subtilis and Bacillus vallismortis, showed higher productivity. Antibacterial tests showed that the extracellular gold nanoaprticles had better activity against pathogenic bacterial strains than the intracellular produced ones. Production of gold nanoparticles was performed by biological method. This nanoparticles showed a same antibacterial activity against both tested gram positive and negative bacteria.

Trichophyton violaceum is one of the dermatophyte fungi which invades the skin, nail and hair of humans. Few studies were carried out in the field of molecular biology of this fungus. This study aimed to evaluate the presence of the squalene epoxidase encoding gene in Trichophyton violaceum. This sectional study was performed on the Trichophyton violaceum isolated from the patients who were visited at department of Mycology, Medical division of Tehran University. Following DNA extraction, the squalene epoxidase encoding gene was amplified by PCR preformed and its specified primers. The amplified gene was finally sequenced and its aminoacid sequences were identified. Based on electrophoresis of PCR products, a fragment with 600bp nt was observed, which confirmed the amplification of squalene epoxidase gene. This gene was recorded in the national the NCBI gene bank as JX869101. This gene encodes a polypeptide with 220 aminoacids. PCR Nucleotide sequence comparison of this new gene with other existing genes in the gene bank revealed a significant homology with squalene epoxidase genes in other members of the fungi and other eukaryotes, as well. The aminoacid sequence of the encoded protein was about 78% identical to the sequence of squalene epoxidase from other fungi.

Acinetobacter baumannii is one of the major causes of nosocomial infections resistant to most of available antibiotics, which is known as an important reason of nosocomial death. Although the aminoglycosides are still used as drugs of choice for treatment of Acinetobacter infections, resistance to aminoglycosides has been increasing in this bacterium. The present study investigated the prevalence of the encoding genes of aminoglycoside modifying enzymes, aphA6 and aadB, in Acinetobacter baumannii strains isolated from patients in Emam Reza hospital, in Tabriz city. Material & This cross- sectional study was carried out on 103 Acinetobacter baumannii isolated from hospitalized patient in Imam Reza hospital located at Tabriz. Identification of the strains was performed based on differential and biochemical tests. Antimicrobial susceptibility patterns of the isolates to different aminoglycoside antibiotics including gentamicin, amikacin, tobramycin and kanamycin were evaluated by disc diffusion method. Then the PCR technique used to evaluate the presence of aphA6 and aadB genes. A total of studied isolates, 52 (50.48%) and 16 (15.53%) cases were positive for aphA6 and aadB genes, respectively. Also, the highest resistance was recorded for kanamycin (94%), gentamicin (86%), amikacin (81%) and tobramycin (63%) antibiotics. The results of this study showed the remarkable prevalence of the encoding genes of aminoglycoside modifying enzymes in the A. baumannii isolates. Therefore, a widespread surveillance of resistance to antibiotics and prevention of distribution of these antibiotic-resistant genes is necessary.

Polycyclic aromatic hydrocarbons (PAHs) are a group of non-polar compounds that are categorized as one of main environmental concerns due to their highly toxicity and persistent in the environment. Biodegradation of such pollutants is a safe inexpensive clean up method. This study was aimed to isolation and molecular identification of a Nocardia strain with the ability to degrade anthracene in vitro condition.
Material & Petroleum contaminated soil sample was collected from Isfahan refinery and some chemical properties of sample were measured. Anthracene degrading bacteria were isolated by the enrichment culture technique in basal salt medium with 50 mg/l anthracene. Only one isolate with similar morphology to Nocardia sp was selected. Identification of isolate was done based on biochemical tests and 16S rRNA gene analyses. Then, the biodegradation rate of anthracene was measured after 9 days by Gas chromatography (GC).
Anthracene concentration in soil sample was 18.48 mg/kg that is more than allowable concentration. Isolate was recorded as Nocardia cyriacigeorgica ATAI20 in the NCBI database under accession number KF113844. Biodegradation of anthracene (50 mg/l) was 36.60% after 9 days.
Based on this study, Nocardia cyriacigeorgica ATAI20 was actively able to degrade aromatic hydrocarbons. Therefore, further studies would be useful for commercial application of this strain in bioremediation of antheracene from polluted wastewaters.

Listeria is a gram-positive facultative intracellular bacteria. The actA is one of the most important genes in this bacterium, which involves in bacterial movement in the host cell and so in its pathogenesis. The purpose of this study is to evaluate the actA gene in Listeria monocytogenes strains isolated from dairy products. This cross sectional study was performed on 70 samples of dairy products collected from Tehran and Babolsar, Iran from June to August 1391. The samples were grown onto BHI agar and Mueller agar. A PCR approach was used to detect the presence of the actA gene in the isolated Listeria species. Also, the isolated were grown into TSA containing 0.0015% Congo red in order to determine the invasive properties of L. monocytogenes. This study showed contamination of the milk, cheese and soft cheese samples with L. monocytogenes (10 cases), L. innocua (4 cases), L. welshimeri (2 cases) and L. seeligeri (1 case). Furthermore, no yogurt and butter samples were contaminated with Listeria. Although all of these isolates contained actA gene in their genome, only 14% of the strains isolated from vegetables were positive for this gene. A total of 10 cases of isolated L. monocytogenes, 100% of the clinical strains, 70% of the strain food and 100% of standard strains purchased from Razi Institute were positive for Congo red phenotype. According to the results obtained in this study, detection of actA gene based on PCR can be used as an alternative approach for identification of pathogenic L. monocytogenes in samples without culture method. Also, due to the widespread use of dairy by individuals, it seems necessary to reduce bacterial contamination monitoring the production process, transport and distribute this material.

Tristeza is one of the most important viral diseases in citrus products worldwide. Fars province is one the most important citrus growing regions in Iran. Infection to citrus tristeza virus (CTV) has been reported from many citrus orchards in this region. The objectives of this research were to determine CTV distribution, evaluation of citrus species infection to CTV and to compare the infection prevalence rate to CTV in different regions of Fars province.
This cross sectional study was performed on 230 samples collected from different citrus species grown in Kazeroun, Firouzabad, Ghir-o-Karzin, Jahrom, Darab and Fasa regions. First existence of infection to Tristeza virus was detected using ELISA. The prevalence of infection was calculated based on the rate of infection in the total numbers of samples. Next, a PCR amplification was performed based on the primers specific for coat protein and p23 genes.
Based on the ELISA test, all citrus species were infected to CTV. The virus infection was detected in all the regions of interest. Totally, 24.35% out of 230 collected samples were infected to CTV. The highest infection prevalence was found in Jahrom, Ghir-o-Karzin, Darab, Kazeroun, Firouzabad and Fasa, respectively. Furthermore, mandarin was found the most infected fruit between all citrus family, followed by lemon Lesbon, lime, orange and sweet lime, respectively.
The results of this research showed that CTV infection has increased during the past years. As a result, control of the disease in this region is necessary.