فصلنامه دنیای میکروب ها
سال ششم شماره 2 (پیاپی 15، تابستان 1392)

Due to rapid resistance of Acinetobacter baumannii to broad-spectrum antibiotics and to cause nosocomial infections with high mortality rates, this bacterium has associated with many therapeutic problems. This study aimed to investigate epidemiologic strains pf Acinetobacter isolates in Sahid Namazi hospital, Shiraz- Iran, based on AFLP method and to determine their antibiotic resistance levels in order to design surveillance program and effective treatment.
This descriptive study were performed on the Acinetobacter baumannii strains isolated from seven different intensive Care Units of Sahid Namazi hospital. All isolates were identified with the API20NE kits. Drug resistances of the strains were determined based on broth microdilution methods. For purpose of the phenotypic typing by obtained results, the antibiodendrogram was designed with SPSS statistical software. Next, AFLP analysis was performed with DNA digestion by MboI and MseI restriction enzymes, ligation of synthetic adapters, Pre-amplification and sensitive amplification by specific primers.
In present research, 54% (46 cases) of Acinetobacter baumannii strains were MDR, while 43% (36 cases) belonged to XDR and only 2% (2 cases) was identified as PDR. The antibiotic susceptibility pattern show prominent presence of imipenem resistance (51%) and meropenem resistance (76%) strains. In addition, the AFLP results revealed that three main clusters (1, 3, and 4) have been associated with several outbreaks of nosocomial infections.
Based on the results, rapid growth of Acinetobacter baumannii and their cross-transmission among different wards of Sahid Namazi hospital have led to increase of antibiotic resistance rate and their prevalence in the hospital. Therefore, results of this study emphasizes surveillance programs for infection control and to eradication therapy.

Microbial lipid composition is similar to the oil obtained from plants and animals. Although vegetable oils were originally used for producing biodiesel, high costs of the process encouraged industries to use  microbial lipids as biodiesel sources. This study was conducted to isolate yeast strains with high lipid productivity, to optimize the extraction process of produced lipid and to convert the lipids to biodiesel.
In this study, after isolation of the yeast Rhodotorula, the productivity of microbial lipid in nitrogen limited condition, rice straw and wheat bran hydrolyzate was evaluated. The products were analyzed based on Gas Chromatography-Mass Spectrometry technique (GC-MS). The lipid production was optimized by two techniques (one factorial and Taguchi method) and the results were compared. At the end, the yeast strain was identified using polymerase chain reaction (PCR) techniques.
The strain isolated from this study was identified as Rhodotorula musilaginosa. The strain had high lipid production and dry biomass of 10.97 g/l and 18.84 g/l in optimized conditions, respectively. The highest fatty acids were Palmitic acid (18.51%) and Oleic acid (67.29%).
The results obtained in this study indicate that there are valuable native strains in our country that they can be used in different industries , especially biodiesel production.

 Nowadays RFLP test from IGS region is a used for determination of interspecies diversities. The aim of this study was to determine pathogenic species of Alternaria from leaf spots of citrus in Mamasani region based on morphological and ITS1 sequencing and to determine genetic variation of isolates based on RFLP-IGS.
This descriptive study was performed on 70 leaf spotted samples collected from different citruses species and cultivars of Mamasany orchards including orange (Valencia, Navel and leaf spoon Cv.), lime and sweet lemon (Wikova and local Cv.) during In autumn 2011. The isolates were cultured on PDA medium and purified by using single spore method and their species were identified based on morphological characters. The pathogenicity of the isolates was evaluated by based on Koch’s rules through exposure of isolates to leaf. DNA was extracted from mycelia by CTAB and the ITS1 and IGS regions were amplified by using specific primers. The ITS1 region was sequenced and the IGS region was digested by HinfI, BsnI, RsaI, BssmI and AluI restricted enzymes.
All isolates were identified as Alternaria alternata based on morphological characters and sequencing of ITS1 region. Constructed dendrogram based on RFLP patterns showed existence of three divergent groups in 51% similarity. Orange, lime and sweet lemon isolates grouped in divergent groups. Also Wikova isolates grouped in different clade.
The obtained results clearly showed that the enzyme patterns of IGS can reveal the classification defects of Alternaria alternata form citrus hosts.

Neospora caninum is one of main etiologic factors of abortion in  in cattle. The mass production of protozoa in laboratory conditions is performed based on growing in different cell lines. Since using suspension cell lines are very cost effective for mass production of biological products, this study aimed to evaluate the suspension cell culturing for production of this protozoan.

This study was experimentally performed bases on growth of N. caninum tachyzoites on Ka6 cell line (a cell line obtained from cattle infected with Theileria lestoquardi, Razi Institute, Shiraz, Iran. Next, based on MTT assay, ability of this cell culture for production of  N. caninum was compared with  Vero cell as the best current cell line for this purpose.

The results showed that N. caninum tachyzoites could enter into K6a cell lines and after replication released from the cells successfully. Replication of the tachyzoites was significant in both Vero and K6a cell lines in compare to the control.

To our knowledge, this is the first and successful report of suspension culture of N. caninum tachyzoites. Although, the rate of N. caninum proliferation on Ka6 cell line did not show any significant difference in compare to Vero cell line, since Ka6 cell line is a transformed lymphocytic cell and it is possible to grow massively this cell line as suspension bioreactors, it is preferred to Vero cell line.

Pseudomonas aeruginosa is one of the most important infectious agent in medicine. These bacteria cause several infections especially in the patient with high degrees of burns and in immunodeficiency cases. The purpose of this study was providing conjugated antigen component that can induct production of antibody and memory immune in mouse model against Pseudomonas aeruginosa. 
After culturing P. aeruginosa strain PAO1 and extraction of alginate, Diphteria toxoid, ADH and EDAC were added as protein carrier, spacer and linker, respectively to the alginate. After passing the Alginate diphtheria toxoid conjugated (ALG-DT) through chromatography column (CL-2B), its quality was checked to get the quality control label. The prepared antigens were intraperitoneally injected to BALB/c mice (10µg/ml/mouse) for three times once each two weeks. Blood sampling was performed after two weeks of each injection and the levels of serum IgG, IgA and IgM were measured by ELISA.
The titer of serum antibodies of vaccinated group with ALG-DT increased significantly after each injection. The levels of serum IgG, IgM and IgA against alginate in vaccinated group was significantly more than the control groups, and after third injection reached to 3.5, 1.7 and 1.2 times increases, respectively.
The increases in the levels of  antibodies in vaccinated groups are an indicator of activation of T-cells and memory cells. As a result, the conjugated alginate-diphtheria toxoid can be appropriate candidates for production of vaccine.

Proteases are a useful family of enzymes in microbiology with applications in leather industry, food industry, pharmacy, cleaners, and many other industries. Due to rapid replication, fast growing and facility in their genetic manipulations, microorganisms are used as the main resources of production of proteases. This paper aimed to isolate the Serratia marcescens strains from the natural environments at western Mazandaran, with the ability to produce proteolytic enzymes.

In the present study, 150 natural samples were taken from different depth (0-10 cm) of soils and waters located at western part of Mazandaran. In order to isolate and to identify the microorganisms the samples were grown on Skim milk agar, and the isolates were underwent different biochemical tests. Next, Serratia marcescens strains were screened by molecular tests and 16SrRNA. After isolation of the strains, several time and temperature optimization steps were performed on the most potent enzyme producing strains. As well, approximate molecular weight of the enzymes were measured abased on protein deposition and SDS-PAGE.

Only two isolates were identified as Serratia marcescens. One of the two isolates belongs to a novel strain nominated as Serratia marcescens 424, which recorded in Genbank as KC790390. These two isolates could produce high levels of proteolytic enzyme in 24 hours under 37oC. The highest amount of enzyme belonged to a 52 Dalton molecular weight isolated from Serratia marcescens 424.

Our studies show a high potential of the isolated Serratia marcescens strains in production of protease and Serratiopeptidase. As a result these strains can be useful in various industries.

Polycyclic aromatic hydrocarbons are a group of organic compounds with two or more aromatic rings, and approximately 90 percent of these compounds are carcinogen. Although there are different methods to clear such contaminants from environment, microorganisms are more effective and more cost friendly. This study was designed to isolate indigenous microorganisms which are able to biodegradation fluoranthene from sediments of mangrove forests in Persian Gulf and to evaluate their biodegrading ability on fluoranthene.
This sectional study was performed on the sediment samples collected from Persian Gulf mangrove. The bacteria were counted in two series of media; one containing fluoranthene and another one without any contaminants. The degrading bacteria were isolated on two basic mineral media (MSM and MSM Agar). The degradation ability were assayed based on High pressure liquid chromatography (HPLC).
The total number of bacteria grown on the medium without Fluoranthene was significantly more than those contaminated with Fluoranthene (cfu/g). Among the isolated bacteria Bacillus circulance, Alcaligens fecalis, Entrobacter, Listeria and Staphylococcus showed the highest ability to degrade Fluoranthene. Bacillus circulance and Alcaligens fecalis showed the most biodegrading activity and growth at the presence of fluoranthene (73.4% and 71%, respectively).
Results of this study indicate that there are many fluoranthene degrading bacteria in Persian Gulf mangrove sediments.

Rhizosphere bacteria assist growth of plants via dissolving phosphate, production of special compounds such as phytohormones and release of hydrolytic enzymes such as a-amylase and kitinase. This study aimed to isolate and to identify a a-amylase producing bacillus in rhizosphere of orange orchards and to investigate microbial activity to enzyme production in presence of different source of carbon ranges.
This study was conducted in order to isolation of a-amylase bacilli from rhizosphere of orange orchards located at Kerman province, Iran. After sample collection, bacteria were isolated by growing on starch agar. Several molecular (based on 16SrRNA) and biochemical methods were used to identify bacterial species. The starch rapid hydrolysis test was used to investigate amylase activity of the isolates. Additionally, the enzyme production level was studied in the presence of several carbon sources include glucose, fructose and starch ranges.
Molecular and biochemical analysis showed that isolated bacterium is a strain of Bacillus cereus named as Bacillus cereus MR-R3 and recorded in GeneBank under accession number of KC306945.1. Moreover, results showed that starch and glucose have the highest positive effects on alpha amylase production in the presence of 0.5 g/l and fructose have the highest effect in the presence of 0.25 g/l.
Isolation and identification of orange orchards rhizospher-derived bacillus species considering their ability to produce a-amylase and phosphate solvation showed that the presence of this species in this region is very important. Moreover, increase in production of the enzyme after treatment with different carbon sources can be related to their gene expression induction effects.