فصلنامه دنیای میکروب ها
سال یازدهم شماره 3 (پیاپی 36، پاییز 1397)

  Glucose oxidase has a wide range of application in medical, pharmaceutical, food, textile, and environmental industries. Heterologous expression of the enzyme has been investigated in yeast hosts due to several industrial-scale limitations in enzyme production by Aspergillus niger. The present study aimed to investigate bioinformatics characteristics of recombinant glucose oxidase in Yarrowia lipolytica Po1g-GOX and to optimize the production conditions of the enzyme by Taguchi experimental design method.

Protein sequence, primary, secondary and tertiary structures and glycosylation rate of recombinant glucose oxidase enzyme were studied via bioinformatics tools. Optimization of recombinant glucose oxidase for glucose, peptone, yeast extract and pH at four levels in YPD media with thiamine were defined to Qualitek-4 software. The level of enzyme production was measured and analyzed by the software. Recombinant glucose oxidase had 605 amino acids, with 29% α-helix, 16% β sheet and 54% coil in its secondary structure showing similarity to that of native glucose oxidase of A. niger. Eight glycosylation sites have been located in this recombinant protein, and the tertiary structure had one domain showing high similarity to native glucose oxidase. The highest level of recombinant glucose oxidase production was obtained in medium containing 30 g/L glucose, 5 g/L yeast extract, 15 g/L peptone, and pH 7. Recombinant glucose oxidase produced in Yarrowia has a suitable potential to be used in pharmaceutical and food industries due to high similarity to native glucose oxidase.

Mercury due to stability and the high cost of conventional refinement methods is a major environmental problem in the world. Biological methods such as the use of bacterial-based bio-reactors or their enzymes are one of the bioremediation methods. MerA and MerB bacterial enzymes are used to decompose organic and inorganic compounds of mercury. This study was designed to clone merA and merB genes into the pET28a (+) expression vector for the production of MerA and MerB active enzymes. Material & At first merA and merB genes were isolated from mercury- resistant bacterial genome and subsequently cloned into pET28a(+) expression vector. Confirmation of cloning the target gene was achieved by PCR and restriction enzymes. Then pET28a(+)-merA-merB recombinant vector was transformed into E.coli strain BL21. To assess resistance to inorganic and organic mercury by transformed bacteria and the functionality of the enzyme
produced by a recombinant vector, the growth of E.coli strain BL21 containing the recombinant vector and without it were measured by adding mercury into the environment during 48 h. Recombinant bacterial growth in medium containing different levels of inorganic and organic mercury was measured at different times. The result showed that the growth of E. coli containing no target gene in the vector was affected after introducing mercury into the medium till 12 hours so that bacteria would not be able to grow at 10 and 20ppm mercury concentrations. However, transformed bacteria with pET28a(+)-merA-merB vector showed suitable growth in a mercury-containing medium. The SDS-PAGE analysis of extracted proteins from transformed bacteria with pET28a(+)-merA-merB vector on 12.5% acrylamide gel showed the highest MerA (62kDa) and MerB enzymes (23kDa) expression following 16 hours induction with 1mM IPTG at 37ºC. Growth ability of transformed E.coli with recombinant vector indicates MerA and MerB proteins function in transformed bacteria. Furthermore, increasing resistance of recombinant bacteria to inorganic and organic mercury indicates that heavy metal pollution in the environment can be cleaned up with proper management through the construction of a recombinant vector.

  Copper nanoparticles due to unique catalytic properties and electrical and optical conductivity are of great importance. This study was aimed to use the potential of aquatic bacteria as biocatalysts to reduce copper sulfate into copper sulfide nanoparticles (CuSNPs) and to assess its antimicrobial properties. The CuSNPs produced via bioconversion reaction have been characterized by spectroscopy analysis, electro-micrographs prepared by scanning electron microscopy (SEM) and particle size distribution histogram. The antimicrobial activity of CuSNPs against some bacteria and pathogenic fungi was also investigated by disc diffusion test. 105 Cu-resistant bacterial strains have been isolated according to selective enrichment technique. Based on the results, the only culture supernatant of strain Cu25 was able to reduce copper sulfate into copper sulfide nanoparticles (CuSNPs), extracellular. The cu25 strain was identified as Bacillus licheniformis based on phenotypic and molecular analysis. Subsequently, the extracellular synthesis of CuSNPs was investigated. The results showed that the supernatant of B. licheniformis Cu25 following exposure to 7.5 mM copper sulfate solution and 24 h of
incubation can produce spherical CuSNPs with the average diameter of 21.5 nm as extracellular. The current study is the first report on the extracellular synthesis of CuSNPs using B. licheniformis. Also, the produced biological nanoparticles have growth inhibitory effect against some pathogenic bacteria and fungi.

Nowadays, widespread use of fluconazole has resulted in resistance in Candida albicans strains. The conformational changes of Erg11p due to mutations in the ERG11 gene is one of the mechanisms resulting in azole resistance. The aim of our study was to investigate ERG11 gene mutations in fluconazole-resistant isolates of C. albicans in the west of Mazandaran province. In this descriptive cross-sectional study, clinical specimens were obtained from vaginal mucosa of 120 women in hospitals of the west of Mazandaran province. C. albicans isolates were identified by standard methods such as germ tubes and CHROMEagar medium culture. The fluconazole resistance and susceptibility of the isolates were evaluated by Kirby Bauer and broth micro-dilution methods. Then, ERG11 gene mutations in resistant isolates were determined by PCR-sequencing method as compared with PTCC5027 (ATCC10231) reference strain. Out of 45 C. albicans isolates, 40 isolates were resistant and 5 isolates were susceptible to fluconazole. The MIC of fluconazole was determined as ≥ 64 µg/ml. PCR-sequencing analysis revealed that 18 fluconazole-resistant isolates have six missense mutations (Y257H, E266D, V404I, D421N, V488I, and D504V) in the ERG11 gene. The identified mutations in this study may play role in developing fluconazole resistance in C. albicans isolates in the west of Mazandaran province by decreasing fluconazole affinity to ERG11p.

  Pseudomonas aeruginosa is a major nosocomial infection in immunocompromised patients. Silybin as an herbal drug has anti-inflammatory and anti-cancer effects and recently further studies have been focused on its antibacterial functions. The aim of this study was to investigate the antibacterial effect of silybin on mexY gene expression in ciprofloxacin (CP)-resistant isolates of P. aeruginosa. In this study, six ciprofloxacin- resistant isolates of P. aeruginosa were both treated by ciprofloxacin (1/2 MIC) only as a control sample and in combination with silybin-encapsulated micelles (nanoparticles) as the test sample. MBC test was performed 24 h after culturein Mueller-Hinton agar. After RNA extraction and cDNA synthesis, mexY expression was quantitatively investigated in silybin- treated and untreated cells. Our findings showed that a combined silybin-encapsulated micelles and ciprofloxacin (1/2 MIC) treatment reduces the bacterial growth up to 50% after 24h. Also Q-RT-PCR analysis revealed that silybin-encapsulated micelles decrease mexY expression. Our results suggest that silybin could inhibit bacterial growth by decreasing mexXY-oprM efflux pump count on the cell surface.

  Scientists are of the conviction that probiotic bacteria can ameliorate urinary tract infection by inhibiting colonization, attachment, and growth of uropathogenic Escherichia coli. The aim of this research was to investigate the anti-adhesive effect of two probiotic Lactobacilli including Lactobacillus plantarum and Lactobacillus casei on uropathogenic E. coli (UPEC). In research in 1395, 35 urinary tract infection (UTI) samples were collected from one hospital in Tehran and based on biochemical analysis 27 UPEC samples were identified. The ability of biofilm-formation and the presence of genes (papG and fimH) involved in biofilm- formation was investigated by microtiter plate and PCR methods, respectively. Co-aggregation and anti-adhesive effects of two probiotic lactobacilli including L. plantarum and L. casei against UPEC were studied by co-aggregation and microtiter plate method, respectively. Among 27 isolates, 77%, 15%, and 5% showed strong, mediate and the weak ability of biofilm-formation, respectively, and 3% had no ability. Among 15 UPEC which had a strong biofilm-formation ability, 13 (86%) and 15 (100%) had papG and FimH genes, respectively. The average of co-aggregation between L. planetarium and L. casei with UPEC was gained 49.13% and 46.25%, respectively. The mean anti-adhesive effect of L. plantarium and L. casei against pathogenic bacterium was 62% and 58%, respectively. Further studies on the anti-adhesive effect of probiotic lactobacilli are suggested to prevent UPEC prevalence.

Today plant sources as substitutes for chemicals in food and drugs have received increasing attention. The use of essential oils and plant extracts can certainly be a good alternative as antimicrobial effects of some plants oils has proven. This study was aimed to determine the antifungal effects of essential oils of thyme, cumin, savory and cloves in different concentrations on Penicillium fungus. The antifungal effects of the medicinal plant (thyme, cumin, savory and cloves) essential oils at concentrations of 250, 500 and 750 µl /L were investigated by pour plate method in potato dextrose agar medium. Fungal growth zone diameter was measured after 8 days at 25 ºC and data were analyzed by MSTAT-C software. The essential oils of all plants at all concentrations had significant antifungal effects as compared to the control. But it decreased with increasing the experiment time. Clove oil at all concentrations and thyme oil in 500 and 750 µl /L concentration had the highest anti-fungal effects. The result showed that thyme and clove essential oils have high inhibition effects against Penicillium sp.

  Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus bacterial canker. In Iran, for the first time, Alizadeh and Rahimian reported the presence of Xcc on Mexican lime (C. aurantifolia), in 1990. Early characterization data suggested the presence of both wide (pathotype A) and narrow (pathotype A* and Aw) host range Xcc strains in Iran. However, more recently a discriminant analysis of MLVA-31 and MLVA-14 data in 2014 showed that Iranian strains of Xcc have genetically belonged to the host-restricted pathotype A* but not to the pathotype A. This study was aimed for genomic characterization and phylogenetic analysis of a narrow-host-rang Iranian strain of X. citri sub. citri, NIGEB-88. To determine genetic characteristics and phylogenetic relationships of Iranian strains, Illumina sequencing method was used to obtain a draft genome sequence of Xcc strain NIGEB-88. General features of Iranian strain such as genome size, number of plasmids, average genomic GC content, number of CDS and structural RNA were similar to those of other Xcc pathotypes. Furthermore, studies of various potential virulence and host range determinants factors such as type III secretion system effectors, type IV secretion system and surface Lipopolysaccharides revealed that this pathogen is very close to XccA and XccAw strains. Phylogenetic and assessment of genes related to virulence and host specificity showed that XccA*NIGEB-88 is more closely related to other Iranian strain, XccA*NIGEB-386, and narrow-host-rang pathotype A*.