فصلنامه دنیای میکروب ها
سال هفتم شماره 3 (پیاپی 20، پاییز 1393)

Growth of ostrich depends to employment of growth hormone and proper nutrition. The ostrich growth hormone is a polypeptide that stimulates growth and cell reproduction. The aim of this study was to clone and generation of two new gene constructs for expression of ostrich growth hormone gene in prokaryotic and eukaryotic systems.
Materials &

In this study, RNA was extracted from ostrich pituitary gland and cDNAs synthesized using RT-PCR method. These cDNAs were cloned into TOPO vector based on T/A method. Then, cDNA sub-clones of ostrich growth hormone were prepared on pcDNA3.1 and pET32 vectors.

Cloning of ostrich growth hormone cDNA in TOPO vector was confirmed by PCR and endonuclease restriction digest. After sub-cloning of this cDNA, pcDNA3.1-gh and pET32-gh recombinant vectors were generated and the presence of genes were confirmed using restriction digestion.

According to the results, the new recombinant vectors generated in this study can serve as an effective approach for production of the ostrich growth hormone in commercial levels.

Background & α-amylase, is an endoamylase to hydrolyses amylase, amylopectin and glycogen by randomly cleavage of internal alpha 1, 4 linkages. Genus Bacillus is one of the most common microbial sources of α-amylase production. The aim of the present study is identification, overexpression and activity analysis of Bacillus licheniformis α-amylase (amyS). Materials & A polymerase chain reaction was used to identify and isolate α-amylase gene in Bacillus sp. The fragments were introduced into expression vector pET-26 (+). After sequencing, the recombinant vectors were introduced into E. coli BL21 (DE3) for expression. The recombinant enzyme were purified using amicon filter and dialysis bags. α-amylase activity was measured using di-nitro salicylic acid technique. Based on the results, the α-amylase gene was over-expressed in an expression system beyond the native host (Bacillus sp.). Based on the SDS-PAGE electrophoresis, the molecular weight of this enzyme was predicted 54 KDa. The α-amylase showed an enzyme activity about 4.77 U/ml. These results indicated a high expression of this enzyme in an expression system beyond the host, which was due to existence of a strong promoter used in this study, T7promoter. As a result, employment of this system in an industrial scale is recommended due to the secretion of the target enzyme, a proper folding of the enzyme and maintenance of its activity.

Background & Respiratory system infections is a common infectious disease and is an acute inflammation of the upper respiratory system caused by several bacterial infections including Staphylococcus aureus. The aim of this study was to investigate the coagulase gene polymorphism of S. aureus isolated from clinical respiratory system infections samples in Shahrekord of Iran.
Material & This study was conducted by a sectional-descriptive study on 200 persons suspected to the upper respiratory system infections who referred to Imam Ali clinic in Shahrekord. After growth of microorganisms on blood agar and manitol salt agar, the suspected colonies were identified by microbiological testing. Next, DNA samples were prepared and the products of PCR reactions were enzymatically digested and genes were genotyped using RFLP.
Overall, 60 patients (30%) were infected to S. aureus. Among them 42 isolates showed a 970 bp fragments and 18 isolates showed a 730bp fragments. After enzymatic digestion with AluI, 42 specimens contained three bands: 320, 490, and 160 bp (genotype I), while 16 specimens contained two bands: 490 and 240 bp (genotype VIII) and 2 specimens contained two bands: 410 and 320 bp (genotype IX).
The results obtained from present study showed that coagulase-positive S. aureus strains isolated from respiratory system infections in Shahrekord belonged mostly to genotype type I, which can be considered as a potential source for the release of the genotypes in the population.

Background & Listeria monocytogenes is one of the etiologic causes of asymptomatic infection that can cause urogenital tract infection in infants and fetal death. Given the challenges posed by growing the bacteria, this study aimed to detect Listeria in urine using polymerase chain reaction and fluorescent probes.
Materials & This cross-sectional study was carried out on 100 urine samples obtained from the patients who were referred to a specialized center for women in Jahrom and Larestan. The sensitivity of the technique was evaluated using a standard strain. Then hlyA gene probes were designed using the polymerase chain reaction, and fluorescence level was determined by fluorescent probes.
Based on FLASH-PCR assay between 5 to 10 genes were detected in each reaction. Using fluorescent probes for L. monocytogenes hlyA gene overall, 30 out of 100 clinical samples (30%) were infected to this bacterium.
The results showed that fluorescent probe increase speed, sensitivity and specificity of the molecular identification of Listeria and therefore it can be a suitable alternative to electrophoresis. Thus, with respect to the accuracy of this non-invasive method it is possible to monitor larger amounts of samples suspected to L. monocytogenes infections.

Background & Several studies have shown that fungi can cause allergenic asthma in the susceptible persons. Among these fungi, Alternaria, Penicillium and Aspergillus species are more common in this cases. This study was performed to detect and to compare allergenic bands in A.alternata, P.citrinum and A.fumigatus. Materials & This cross-sectional study was performed on 48 patients afflicted to asthma and 22 healthy controls. The fungi were grown and their extract crude were obtained using the liquid nitrogen. The protein fractions were isolated by SDS-PAGE and after electrotransfering and transfer of the bands into the nitrocellulose membrane, the proteins were used for immunoblotting against the sera obtained from patients and controls. Based on the immunobloting test, 6 protein allergenic bands for A. alternata (49-115 kDa), 4 protein bands for A. fumigatus (57-112 kDa) and 5 protein bands for P. citrinum (37-127 kDa) are detected in these samples. The highest amount of allergic band belonged to A. alternate. The bands with higher molecular weights were more effective in stimulation of IgE.

Background & Mycoplasmas are one of the causative aetiologies of infertility in human beings. Genital Mycoplasmal infections can damage reproductive systems and lead to infertility infant mortalities. The aim of this study was molecular identification of Mycoplasma hominis isolated from genital system of infertile men and women in Kerman.
Materials & This descriptive study was performed on 100 infertile men and 100 infertile women with a six month purposive sampling from the patients who referred to Infertility Center of Kerman. Semen and vaginal swab were tested by PCR assay for the presence of M. hominis. PCR product of positive samples were selected for sequencing. Sequence alignment was performed using MEGA 5 software and Neighbor-joining method.
In this study 45% of the samples taken from men were infected to Mycoplasma. Among them, 33% of the isolates belonged to M. hominis. Furthermore, 43% samples taken from women were infected to Mycoplasma, among them 41.8% belonged to M. hominis. Based on sequencing analysis, these M. hominis isolated from the patients were categorized into 5 different strains.
In this study, M. hominis was indicated as the main microbial reason of infertility in this group. Sequence analysis of different M. hominis showed significant variability among the isolates. Therefore, these isolates can be reported as the local strains of Iran.

Background & Microbial biomass show high capacity for the remediation of heavy metals in contaminated solutions. This study aimed to isolate and identify the Cd and Ni resistant bacteria from the soils polluted to heavy metals and to evaluate the biosorption & bioaccumulation of these metals in competitive solution.
Materials & In this descriptive study, samples were taken from the soils polluted to waste water of farms nearby the water refinery in west of Ahvaz. The Ca and Ni resistant bacteria were isolated and their identity were clarified using biochemical tests. Next, minimum inhibitory concentration (MIC) of Cd and Ni determined for these bacteria. Following preparation of alive and deactivated bacteria and solutions containing equal amounts of N and Ca, the levels of Ni and Ca were determined using atomic adsorption system to evaluate the levels of biosorption and bioaccumulation.
The microorganisms isolated in this study belonged to Bacillus sp., Staphylococcus sp. and Actinomycete sp. Among them, Actinomycete sp. showed the highest absorption activity for these elements. The bioaccumulation was higher than biosorption at low concentrations of the metals but the biosorption was dominant at high pollution levels. In both systems, the bacteria showed higher ability for remediation of Cd in comparison to Ni.
The bacteria in the soils polluted to heavy metals showed intensive resistance activity to high concentrations of the elements. Both bioaccumulation and biosorption methods were suitable enough to remediate these metals in aquatic environments. However, the bioaccumulation was more powerful than the second method at low concentrations of the metals whereas biosorption showed more ability at high concentrations of the metals.

Background &

Approximately 98% of total potassium in soil is in unavailable mineral forms for plants. Potassium-solubilizing bacteria are able to dissolve potassium bearing silicate minerals and release available form of potassium to the plants. The present study was intended to isolate potassium-solubilizing bacteria from rhizosphere soil of crop plants and to evaluate the ability of isolates in solubilisation of absorbable potassium. Material &

Potassium-solubilizing bacteria were isolated from the rhizosphere of different crop plants. The isolates were grown on optimized Aleksandrov agar and were assayed based on the diameter of zone of potassium solubilization. The selected isolates were identified using macroscopic, microscopic, biochemical, and molecular methods. The Flame photometry was used to quantify potassium released by isolates in Aleksandrov broth and soil.

Totally, 5 out of 30 isolates with ability to release potassium showed high activity in potassium solubilisation. The biochemical and molecular studies indicated that these isolates belonged to the genera Bacillus, Paenibacillus, and Pseudomonas. The flame photometry results showed that the amount of potassium released by the isolates ranged from 950 to 1250 mg/l in broth media and 525 to 550 mg/kg in soil.

The potassium-solubilizing bacteria were isolated and identified from rhizosphere samples and identified. These isolates showed high ability for solubilisation of silicate minerals and release of absorbable potassium and therefore they can be used in biofertilizers to enhance the availability of potassium in the soils and to improve the growth and yield of crop plants.