فصلنامه زیست فناوری میکروبی
پیاپی 11 (زمستان 1390)

Bacillus thuringiensis is a Gram-positive and endospore-forming bacterium. Soil is main habitat of this bacteria and it have capable of producing a wide variety of crystalline proteins with insecticide properties. The purpose of this study was isolation of Bacillus thuringiensis strains which carriers Cry gene through soil around the West Mazandaran province.Methods of research: 12 strains of Bacillus thuringiensis were isolated in minimal medium from 35 regions of west Mazandaran province, after soil screening by selective method of sodium acetate and L-Serine. After extracting DNA from all strains, confirming the strains including Cry genes as Bacillus thuringiensis, has been done through using 16S rDNA PCR Sequencing. Also relative molecular weight of crystal protein of the strain containing Cry gene were assessed by SDS-PAGE. Among 12 isolated strains, Cry gene in 1Aa class was recognized only just in one strain and through molecular method. Investigating rDNA sequence of isolated strain indicated a 99% homology with a Bacillus thuringiensis strain IBL 200(Accession No: ACNK01000211.1). Also, the approximate weight of it crystal protein was measured between 90-100 kDa. due to variety of crystal protein genes and lack of accurate phenotypic methods in detecting crystalline proteins, using semi-conserve PCR method identifies Bacillus species containing the crystal gene, through low cost and in quick form.

The oral probiotic bacteria with ability to change the intestinal microbial flora play an important role as health supplement in body. After ingestion they are residing in intestinal tract and exhibit beneficial effect. Also probiotic bacteria are suitable alternative for (natural) antibiotics. The purpose of this study was screening of Arak native Bacillus isolated from poultry farms and evaluation of their probiotic properties.Methods of research: In this study 41 samples of fecal material were collected from 8 different poultry farms. After enriching and heat treatment, spore-forming bacteria were screened. The probiotic properties of the isolates (Acid, bile, salt, pepsin, and chicken gizzard extract resistance and antimicrobial compound production) were determined. In this study 140 isolates of Bacillus were screened 14 strains were subjected to more research base on negative haemolysis. Investigation of probiotic properties of isolates showed that most of them could growth in the presence of 10% NaCl, starin 7 was resistance to HCl (up 91.7%) while starins 9 and 10 were completely resistance to bile salts (cholate and deoxycholate sodium). The selected strains were tolerated to chicken gizzard extract and pepsin (up to 80%) and their adhesion ability to intestinal cells in under evaluation. All the strains showed antimicrobial activity against the common poultry pathogens such as Salmonella sp and E.coli. This study showed that the isolates are potential probiotics and after complementary and filed experiment they can be used as poultry probiotics with the ability of inhibition of poultry common disease.

In recent years, prevalence of Extended Spectrum β- Lactamase (ESBLs) Producing bacteria were increased. These bacteria can hydrolyze the most of β-lactam antibiotics and are known as one of the main problems in Drug resistances and bacterial infection control programs and its illness treatment. TEM-1 β-Lactamase gene product is one of the most important ESBLs β-Lactamase in Enterobactriacea, such as E. coli, Which is the most important factor in E. coli resistant strains to β-lactam drugs and cause Nosocomial infections. In this study, The frequency of TEM-1 gene in Damghan Clinical isolated of Extended Spectrum β-Lactamase (ESBLs) Producing E. coli was surveyed by PCR method. In this project, information of patients were collected through special forms and then, transfer 70 bacterial isolated to laboratory, The specimens were tested and bacterial type were Identified, examined for β-Lactamase producing by combined disk method (according to CLSI) and then Extended Spectrum β-Lactamase Producing bacteria were surveyed by using PCR method to determine the TEM-1 gene frequency. From 70 E. coli isolate, 27 specimens were β-Lactamase Producer. 10 of 27 specimens (%37/04), had TEM-1 β-Lactamase resistance gene. These results indicated the importance of TEM-1 gene in β-lactam antibiotics hydrolysis between E. coli strains isolated in Damghan. The identification of this gene frequency in clinical isolates such as E. coli strains can speed up the process of diagnosis and treating of patients.

Infection with hepatitis B is one of the most common infectious diseases worldwide. The presence of HBeAg correlates with high infectivity in acute HBV infection. The presences of anti-HBe indicate suppression of viral replication and low infectivity of disease. But sometimes, the appearance of anti-HBe is followed by disappearing of HBeAg in the presence of detectable HBV-DNA in serum indicates active viral replication. This situation may be due to the presence of certain precore and core mutations in the HBV genome that alter the expression of HBeAg. The aim of this study was to detect HBV-DNA and viral load in HBeAb positive patients. We studied 50 sera of hepatitis B infected patients. Hepatitis B serological markers including HBsAg, HBeAg, HBeAb and HBcAb were measured by Enzyme-Linked Immunosorbent Assay (ELISA). HBV-DNA was extracted from serum samples. Then, PCR was carried out on the extracted HBV-DNA using specific primer for C gene. HBV viral load in serum was detected by Real-Time PCR. In this study both methods PCR and Real-Time PCR were performed. Liver enzymes levels ALT& AST were measured in patients by using Pars Azmoon kit and based on kit instruction. Of 50 HBV infected patients, all patients (100%) were positive HBsAg and anti-HBc and only 92 % of them were HBeAb positive. HBV-DNA was detected in 100% of patients. Of HBeAb positive patients, 36.7% of them had high viral load. The results of this study show that there is a spectrum of HBeAb positive patients had high viral load. Therefore, it is necessary that the possibility of the presence of precore and core mutations in these patients should be studied.

Study of nontuberculous Mycobacteria is pivotal due to their undisputable impact on human health and environment. Among ecosystem factors Surface water plays an important role in the circulation of mycobacteria throughout the environment and transmission of some pathogens to a human. In addition, there are increasing evidence showing the correlation of mycobacteria isolated from water resources and nosocomial infections. Therefore, the main aim of the current study was to isolate and identify the nontuberculous mycobacteria from surface water resources. A total of 70 water samples were collected from the surface water resources in Isfahan, transferred to the research laboratory and subjected to filtration and decontamination. They were then subcultured onto Lowenstein Jensen medium and incubated at various temperatures and inspected weekly for the appearance of Mycobacterium-like colonies. A battery of biochemical and molecular tests including hsp65 based PCR were then applied to identify the isolates up to genus level. Out of 70 water samples a total of 24 (36%) mycobacteria were isolated. Among these, 5 stains isolated from water resources in local parks, 6 from urban water resources, 1 and 2 from the countryside springs and wells respectively, 1 from household tap water, 3 from agriculture and farms, 3 from rivers and 1 from water resources in hospitals. The results of the current study showed that a noticeable rate of water resources are contaminated with mycobacteria which could be considered as a potential hazardous source of infection for the people exposed to them, in particular those with immunocompromised immune systems such as the aged, the children and the patients with immunodeficiency conditions.

Escherichia coli is the most common aerobic organisms in human gut and some other warm-blood animals. It is Indicator of fecal contamination of food and water. According to available reports E.coli can be transmitted to humans through the water, meat, milk and its products. Considering that the milk is a valuable food in the food basket families, bacteriological quality control of raw and pasteurized milk is critical in production facilities, milk collection and processing. Successive cultivation methods for detection of E.coli was also very time consuming and have low accuracy and may cause false positive results. Polymerase Chain Reaction (PCR) is sensitive, accurate, inexpensive and fast method that can substitute for common testing methods. In this study grown on medium trips through standard and PCR molecular techniques were compared. For this purpose, 50 samples of raw and pasteurized milk were collected. DNA were done extracted by boiling method then evaluated by optimized PCR and Results of PCR reactions were evaluated on agarose gel. For cultivation 9 tubes MPN used at first then the tube containing the gas cultured on the EMB medium by linear method. Among the 50 samples of raw milk, PCR test result evaluated positive in 24 samples but the microbial culture was positive in 20 samples and among the 50 samples of pasteurized milk, PCR test result evaluated positive in 29 samples but microbial culture results in no instance was positive. This study showed that the PCR method compared with the successive cultures methods is more sensitive, and able to detect E.coli in milk with high specificity and less time.

K. pneumonia subsp.rhinoscleromatisis a non-motile gram negative coccobacillus, in the Enterobacteriaceae family. This organism is found in the environment and is considered to be part of the normal flora of the skin, respiratory and gastrointestinal tracts of humans and animals. Milk and its products might represent important sources of pathogenic Klebsiella species. The goal of this investigation was isolation and determination of antimicrobial susceptibility pattern of K. pneumonia subsp.rhinoscleromatis strains isolated from consumed powdered infant formula milk in NICU ward. In this cross-sectional study, total 125 consumed powdered infant formula milk in NICU ward were surveyed for microbial contamination. FDA protocol was used for Isolation and Identification of K. pneumoniaesubsp.Rhinoscleromatis from samples. Antimicrobial susceptibility test was carried out based on CLSI (2011) recommendations by using the standard disc diffusion method. In this study, K. pneumonia subsp.rhinoscleromatis was isolated from 3 (2.4%) of 125 powdered infant formula milk samples. Antimicrobial susceptibility results showed that all isolated strains are sensitive (100%) to amikacin, cefotaxime, nalidixic acid, chloramphenicol, ciprofloxacin, ticarcillin and minocycline. awareness of drug resistant Klebsiella spp. is important for the food industry. In the absence of control measures, drug resistant organisms might be transmitted to humans by the consumption of milk, powdered infant formula milk and milk products.

Single Cell Protein (SCP) is a term coined in the 1960´s to define microbial biomass products which were produced by fermentation. SCP production technologies are applied to solve the problem of worldwide protein shortage. They evolved as bioconversion processes which turned low value substrate, often wastes, into products with added nutritional value. In this research, Taguchi experimental design was applied to optimize the conditions for Single Cell Protein (SCP) production by Methylobacteriumsp. NO. 69. The objective of this research was to determine the significant parameters in the production of SCP in submerged fermentation with methanol as sole Carbone source. Four factors viz., Methanol concentration, Aeration rate, Agitation speed and pH, each at three levels, were selected and an orthogonal array layout of L9 performed. The experiment result indicated that Methanol (18 g/L), Aeration (3 vvm), agitation (800 rpm) and pH (8) were the important factors for SCP production by selected methylotrophic strain in submerged fermentation at the optimum levels.Under the optimal culture condition, the maximum biomass concentration was 16 g/L, which is about two times higher than that at the batch culture in erlenmeyer flask. The total and true protein content of the product was up to 74 and 62% respectively. The protein content, biomass concentration and amino acid profileof the microbial biomass showed that it was enriched with all essential amino acids and could be employed in protein enrichment of animal feed supplements. But Additional laboratory and field experiments are necessary to evaluate the nutritional value of this product.