فصلنامه دنیای میکروب ها
سال دهم شماره 1 (پیاپی 30، بهار 1396)

Inorganic mercury entrance into the environment through industry and agriculture is one of the most serious environmental hazards in the country. Microbial bioremediation is considered as one of the practical solutions to clean up pollutant ions from the environment. The present study was conducted to construct a recombinant vector including merA gene, and also to investigate its expression in order to clean up mercury.
Material & merA gene from mercury resistance bacterial genome was isolated, and subsequently cloned into the pET21a() expression vector. Confirmation of cloning target gene was achieved by PCR and restriction enzyme digestion. Then pET21a()-merA recombinant vector was cloned into E.coli strain BL21. In order to assess increased resistance to mercury in recombinant bacteria, and the functionality of the enzyme produced by the recombinant vector, the growth rate of recombinant BL21 E. coli strains to contain merA gene, and without it was measured in a medium containing mercury for 48 hours.
The results showed that the growth of E. coli strains without merA gene was strongly affected after introducing mercury into the media till 12 hours, and bacteria would not be able to grow at 10 ppm and 20 ppm mercury levels. However, transformed bacteria with pET21a()-merA vector showed suitable growth in mercury-containing media. SDS-PAGE analysis of recombinant bacterial proteins on acrylamide gel showed the highest MerA (62KDa) expression following 16 hours induction with 1mM IPTG at 37ºC.
Overall, the growth ability of merA- containing recombinant bacteria reflects the action of MerA protein in transformed bacteria. Furthermore, increasing the resistance of recombinant bacteria to the mercury indicates that environmental heavy metal pollutants can be cleaned up properly through the construction of recombinant vectors.

Klebsiella pneumoniae is one of the important clinical pathogens and responsible for some nosocomial infections; especially pneumonia, septicemia, and urinary tract infection (UTI). Study of the genotypic and phenotypic characteristics of virulence factors associated with pathogenicity of K. pneumoniae clinical isolates is of great importance. The aim of the present study was the molecular identification of serotypes and capsular virulence genes in cps gene group of K. pneumonia isolates.
Material & This cross-sectional study was carried out on 50 K. pneumonia clinical isolates collected from patients admitted to the Payambaran and Baqiyatallah hospitals located in Tehran. Multiplex PCR was used for identification of K1, K2, K5, K54, K57 capsular types, and detection of rmpA and wcaG virulence factors from capsular polysaccharide genes group.
In this study, K54 (68%) was the most frequent capsular serotype, while K1 (8%) had the lowest frequency. The highest frequency of virulence gene rmpA was observed in capsular serotype K5 (60%), and the highest frequency of wcaG gene was observed in serotype K54 (17.64%).
Multiplex PCR method can be used as a rapid tool to characterize and type the bacterial isolates causing nosocomial infections.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, and one of the mortality causes of nosocomial infections, specifically in patients with severe burns. One of the drug resistance mechanisms in P. aeruginosa is mutation or down-regulation of oprD gene. In this study, we investigated oprD mutations in imipenem-resistant P. aeruginosa isolates in Gilan province.
In this sectional study, 45 P. aeruginosa strains were isolated from different clinical samples of Rasht and Lahijan hospitals and laboratories. The antibiotic resistance and susceptibility of the strains were determined by Kirby Bauer and minimum inhibitory concentration (MIC) methods. Then, PCR and sequencing were performed to evaluate oprD
mutation in imipenem-resistant isolates.
44.4% isolates were imipenem- resistant. The highest resistance was shown to 512 µg/ml of imipenem. Sequencing analysis showed that 5 isolates have T103S and K115T missense mutations in the oprD gene. Furthermore, silencing mutations including L92L were detected in 7 samples, S100S in 7 samples, P116P in 5 samples, and G151G in 3 samples.
Since oprD plays, a major role in imipenem entry to the cell, oprD mutation can be the cause of imipenem resistance in P. aeruginosa isolates. Furthermore, the oprD mutation might have changed the affinity to imipenem in these isolates.

L-asparaginase enzyme is known as one of the best antineoplastic drugs. Since the available commercial asparaginases are from bacterial sources, the aim of this study was to identify and isolate the L-asparaginase- producing bacteria from natural sources.
Asparaginase- producing bacterial strains were isolated from various natural sources. The primary isolation was performed on nutrient agar and asparagine dextrose salts (ADS) agar medium, supplemented with phenol red. Asparaginase- producing colonies with pink color zone were selected for enzyme activity study, and identification using biochemical and molecular tests. Enzyme activity was determined using Nessler’s method. Some factors such as carbon, nitrogen source, and pH were examined to optimize enzyme production process.
Among 133 isolates, the strain isolated from chicken fat exhibited high glutaminase-free L-asparaginase enzyme activity (8.92 U/mg). The selected isolate was identified as Serratia marcescens strain 100 and was registered with accession number KX821734 in NCBI GenBank. The optimal enzyme-producing conditions were as follows: 1.5% (w/v) maltose as a carbon source, 1 % (w/v) ammonium sulfate as the nitrogen source, and initial pH of 6.8.
Considering the use of L-asparaginase enzyme in various pharmaceutical fields, this strain could be a suitable option for industrial production of the enzyme after further clinical studies.

L-asparaginase enzyme is known as one of the best antineoplastic drugs. Since the available commercial asparaginases are from bacterial sources, the aim of this study was to identify and isolate the L-asparaginase- producing bacteria from natural sources.
Asparaginase- producing bacterial strains were isolated from various natural sources. The primary isolation was performed on nutrient agar and asparagine dextrose salts (ADS) agar medium, supplemented with phenol red. Asparaginase- producing colonies with pink color zone were selected for enzyme activity study, and identification using biochemical and molecular tests. Enzyme activity was determined using Nessler’s method. Some factors such as carbon, nitrogen source, and pH were examined to optimize enzyme production process.
Among 133 isolates, the strain isolated from chicken fat exhibited high glutaminase-free L-asparaginase enzyme activity (8.92 U/mg). The selected isolate was identified as Serratia marcescens strain 100 and was registered with accession number KX821734 in NCBI GenBank. The optimal enzyme-producing conditions were as follows: 1.5% (w/v) maltose as a carbon source, 1 % (w/v) ammonium sulfate as the nitrogen source, and initial pH of 6.8.
Considering the use of L-asparaginase enzyme in various pharmaceutical fields, this strain could be a suitable option for industrial production of the enzyme after further clinical studies.

Due to indiscriminate use of chemical pesticides and its impact on the environment, other methods including the use of epiphytic and endophytic bacteria to control pathogens is highly regarded. Since Medicago sativa is the most important forage crop in Iran and many parts of the world, this study was aimed to evaluate the inhibitory effect of alfalfa epiphytic bacteria on the causal agent of wilt disease in in-vitro.
Undiseased alfalfa leaf samples were collected from different parts of Hamedan province, before its flowering. After isolating epiphyte bacteria from alfalfa fields, the antagonistic effect of isolates was investigated by measuring the diameter of the inhibition zone against alfalfa bacterial wilt. Protein extraction was performed to assess the pattern of protein bands and the variety of the strains. Biochemical tests and PCR were used to identify the isolates.
A total of 30 epiphyte bacteria were isolated from alfalfa shoots. Bacterial isolates were identified as Bacillus, Pseudomonas, and Xanthomonas, based on biochemical, physiological and morphological tests. The highest bactericidal effect was related to isolating No. 16 and the lowest to isolates No. 13, and No. 6, respectively. Using molecular and dendrogram methods, isolate No. 16 was most closely related to Pseudomonas monteilii.
Epiphytic bacteria isolated in this study showed a good inhibitory effect against alfalfa wilt in vitro. This can be promising for the biocontrol of this disease. However, further studies are needed to prove the efficacy of the isolates in field conditions.

Citrus bacterial canker (CBC) disease is one of the main citrus diseases that is caused by Xanthomonas citri subsp. citri. The disease has been reported from several citrus- growing regions of Iran. Due to the high loss incurred by the disease, its control is very much needed. One of the new methods to control the disease is the use of plant essential oils. This study was aimed to evaluate the effect of Marjoram essential oil on the activity of bacteria causing citrus canker.
In this experimental study, CBC- causing bacteria were isolated from infected Poldokhtar citrus gardens, and subsequently identified by phenotypic and biochemical tests, along with specific primers. The essential oil was extracted using a Clevenger extraction device. Antimicrobial effect of the essential oil was evaluated using disc diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the essential oil were investigated, as well. Then, the effect of different concentrations of marigold essential oil was studied. Following Marjoram essential oil preparation, it was injected into a Gas-Chromatography Mass-Spectrometry (GC-MS) to determine the composition.
MIC and MBC of essential oil were observed as 3.5 µl/ml, and 4 µl/ml, respectively. The most effective concentration for disease prevention on cut leaves were 4 µl/ml , and 4.5 µl/ml, which reduced disease incidence by 62.5% and 53.12 % on water agar test, and trays method.
It is concluded that marjoram essential oil can be used as a potential compound to control citrus bacterial canker disease.

Fusarium and Verticillium wilts are two major fungal wilt diseases of tomato that have restricted its production worldwide. Culturing resistant tomato cultivars is the best way to control such diseases. Molecular markers linked to resistance genes would be useful for improving tomato breeding programs. In this study allele, specific markers and cleaved amplified polymorphic sequences (CAPS) markers were used to identify tomato genotypes that are resistant to Verticillium wilt and Fusarium wilt, respectively.
This cross-sectional study was carried out on 32 tomato hybrids and commercial varieties provided from Falat company. DNA was extracted using cetyl-trimethyl ammonium bromide (CTAB) method. Then, polymerase chain reaction (PCR) was performed using molecular markers. To detect Fusarium wilt resistant varieties, PCR-RFLP was down using RsaI and FokI restriction enzymes. The results were confirmed by pathogenicity test.
Out of 35 tomato genotypes, 83% were resistant to Verticillium wilt, while 17% were sensitive. Also, 46% of genotypes were resistant to Fusarium wilt, while 54% were sensitive. Genotypes that showed resistance to Verticillium and Fusarium wilts possessed Ve1 and I-2 genes, respectively.
Planting resistant genotypes in infected areas can control fungal diseases such as Verticillium and Fusarium wilts, without using any fungicides.