فصلنامه دنیای میکروب ها
سال دوازدهم شماره 2 (پیاپی 39، تابستان 1398)

Pasteurella multocida is a principal pathogen of domestic animals causing agents of pneumonia and hemorrhagic septicemia in cattle, sheep, and goats, fowl cholera in chickens, and progressive atrophic rhinitis in swine and as well as an opportunistic pathogen of humans. In this study, we investigated the humoral and cellular immune responses and protective immunity conferred by an iron-inactivated vaccine with two different bacterial DNA as an adjuvant.

P. multocida was grown in BHI broth, inactivated with FeCl3, adjuvanted with alum and P. multocida A (homologous DNA) and P. multocida B (heterologous DNA) bacterial DNAs. BALB/c mice were immunized with two whole-cell inactivated vaccine doses at 2 weeks apart. The animals were challenged 4 weeks after booster immunization. The serum antibodies titer was tested by ELISA. At 28 days post-immunization, cell mediates immunity responses were measured by assay of DTH and IL-6 and IL-12 in the serum samples.

Our results showed the levels of antibodies in bDNA adjuvant groups were higher than the alum adjuvant vaccine group. Peak antibody titers of 0.372 were obtained in the IIA+AbDNA group. The protection rate of the AbDNA adjuvant vaccine was better than of other adjuvant vaccines and they protected 100% of mice challenge groups. Peak serum IL-6 and IL-12 titers were achieved in the IIA+AbDNA groups.

These studies indicate that bDNA is effective as immune adjuvants and because of its stimulating properties it can be used as an inducer of humoral and cellular immune responses for vaccination applications. The findings also showed a better ability of homologous bDNA to induce immune responses.

Fungal is one of the most important causes of food spoilage. The use of antimicrobial active packaging increases food safety and shelf life. Hence, this study aimed to produce antifungal compounds (Ergoconin) and its application in the polylactic acid film to reduce the fungal contamination of Aspergillus niger and Penicillium notatum. 

To produce the Ergoconin recombinant protein, the target gene was entered into the pET21 expression vector, and the recombinant vector was transferred to Escherichia coli BL21 (DE3) as host of expression. Gene expression was evaluated by SDS-PAGE, western blot and dot blot. To purify the recombinant protein, a chromatography column was used. Poly-lactic acid bioactive films were prepared by casting method and adding different amounts of Ergoconin (0, 0.25, 0.5, and 0.75%) to 4% wt Poly-lactic acid solution. To investigate the antifungal effect of the poly-lactic acid film, Disc diffusion was used.  

Ergokonin gene was replicated by specific primers. The expression of recombinant protein was performed by SDS-PAGE and confirmed by dot and western blot. Ergoconin compound had an antifungal effect on both fungi. The inhibition zone diameter for both fungi increased with increasing Ergoconin concentration. The antimicrobial film had a more antifungal effect on Penicillium notatum.  

Ergoconin destroys fungi by preventing the synthesis of glucan in the wall of fungi. Therefore, Poly-lactic acid film containing Ergoconin can be used as antimicrobial active packaging to increase food safety.

Background & Fusarium wilt disease is one of the limiting factors of tomato production in most countries, especially in Iran. Considering the malicious effect of chemicals on health, the study on the alternatives for disease controlling is important. This study aimed to evaluate the ability of Trichoderma and Bacillus isolates on biological control of Fusariumoxysporum f. sp. lycopersici.
Materials & The inhibitory effects of  Trichoderma and Bacillus isolates against Fusarium were evaluated on PDA. The ability of the colonization of these antagonists was also examined. The effect of separate and combined application of the antagonists isolates against Fusarium vascular wilt disease severity index and some growth factors of tomato were determined. Also, the potential of these isolates on the induction of phenylalanine ammonlyase was measured in seedlings infected with Fusarium.
The biocontrol agents showed different levels of inhibition of Fusarium growth. The Trichoderma isolates were more successful with a high inhibitory effect (72.06% and 69.16%). These isolates were also powerful colonizers of the pathogen. The disease severity was affected by the antagonists at different levels. Separate and combined treatments of Trichoderma isolates were more effective in the reduction of the disease severity and improving the growth factors. The activity of phenylalanine ammonialyas increased in the plants treated with Trichoderma isolates compared to control treatment (Fusarium). The combined application of these isolates was more effective than the separate using of them on the induction of the enzyme.
The investigated Trichoderma isolates were successful in controlling the pathogen. Therefore, the use of these isolates as agents with multiplier effects can be effective in biocontrol of tomato Fusarium wilt.

Background & A portion of biodiversity and variability of life on earth belongs to the yeasts. The caroptenoid-producing yeasts are economically important and their distinct natural habitats are of interest. In this study, isolation and molecular identification of carotenoid-producing yeasts of the exudates of endangered birch trees (Betula pendula Roth.) at Marmisho in Northwest Iran were performed. Materials & This cross-sectional study carried out by sampling from exudates of birch trees (Betula pendula Roth.) at a natural habitat located in West Azerbaijan Province in the Northwest of Iran. Using selective media, the isolation of the yeasts was done. The screening was initially based on the color and shape of the colonies during one-month incubation along with simple rapid tests to check the existence of carotenoid pigments. Finally, the D1/D2 region rDNA sequencing was accomplished for 19 isolates. 19 out of 45 isolates with pink-red colonies were selected for ribosomal gene sequencing. Phylogenetic results showed that the carotenoid-producing yeasts are in the Basidiomycota division including Rhodotorula, Cystofilobasidium, Cystobasidium, Rhodosporidiobolus, Ustilentyloma, and Xanthophyllomyces.  The unique pattern of carotenogenic yeasts community at this ecological niche and placing in 6 genera with the capability to produce a variety of carotenoid structures indicate that the exudates of birch trees (Betula pendula Roth.) include a wide diversity for native carotenoid-producing yeasts. This study is the first report presenting the diversity of carotenoid-producing yeasts in the Northwest of Iran with a distinct consortium of native yeasts at this habitat.

Background & Paclitaxel is an effective anti-microtubule agent against lung, ovarian and breast cancers. The low production of taxol in the yew, as well as its resistance to chemotherapy, is limiting the use of paclitaxel. This study aimed to create a screening system for new sources of taxol using the taxol sensitive mutant strain of Saccharomyces cerevisiae.   Materials & To mutate the yeast chromosome TUB2 gene, primers containing a change in codon 26 from glycine to aspartic acid (GGT to GAT) were designed and TUB2-Asp26 gene amplified by three-step PCR method. The TUB2 gene was then cloned from the haploid strain into the yeast expression vector in the galactose promoter. After transplantation of haploid yeast with Gal-TUB2 construct (pZF58), transgenic screening was carried out for the second time by PCR product of the TUB2-Assp26 gene and in the medium containing galactose and against 40 μM taxol. Of the 84 transgenes, the yeast strain YNAH1 was selected from the first screening and the integration of mutation in the chromosome was confirmed by the MBC value of 50 μM taxol. The formerly generated membrane-defective yeast strain with amino acid sequences similar to the brain beta-tubulin with an exception for amino acid 26, tolerated 25 µM taxol. However, in the present study, tolerance increased by up to 2-fold due to the use of yeast hosts that had no defects in membrane transporters. The mutant yeast strain can be used in screening to find new sources of taxol producers with production capacity up to 50 µM.

Background & Alginate is one of the most important virulence factors of Pseudomonas aeruginosa. This study aimed to evaluate the synergistic effect of recombinant alginate lyase and common antibiotics in the treatment of Pseudomonas infections on the growth of planktonic cells of P. aeruginosa.
 
Materials & In this study, P. aeruginosa TAG48 was isolated and identified from the clinical sample. To purify alginate lyase and its effect on planktonic cells, the alginate lyase gene (algL) was isolated, cloned, sequenced and expressed. The resultant enzyme was purified by affinity chromatography. The ciprofloxacin, tobramycin, and cefixime were also used to test the effectiveness of these antibiotics on planktonic cells of P. aeruginosa by carrying out MIC, MBC. The synergistic effects of these antibiotics and the recombinant alginate lyase enzyme on planktonic cells were evaluated.
 
Our results indicated that the antibiotics and the enzyme have shown MIC for ciprofloxacin, tobramycin, cefixime and enzyme in the following concentrations: 4, 16,128 and 9.37 μg/ml, respectively. MBC was also calculated equal to MIC. Also, alginate lyase exhibited synergy with tobramycin and cefixime on the growth of planktonic cells of P. aeruginosa TAG48 but not with ciprofloxacin.
 
Regarding the importance of alginate in the pathogenicity of P. aeruginosa, its degradation could reduce this characteristic of the bacteria. Detection of novel algL genes in bacterial communities can also lead the way for the study of alginate lyases with specific activities against alginates from pathogenic bacteria in microbial samples.

Background & The biological method of nanoparticles synthesis by microorganisms has a special place due to its high environmental compatibility and reduced energy consumption and costs. The aim of this study was to determine the effect of bismuth oxide nanoparticles produced by green methods on bacteria causing hospital infections. Materials & In this study, 160 samples were collected from burn wound infections in patients hospitalized in Motahari Burn Rescue Hospital. Resistant strains were identified phenotypically and genotypically. Synthesis of bismuth oxide nanoparticles by Bacillus subtilis wild type strain. The synthesis of bismuth oxide nanoparticles was confirmed by infrared spectroscopy, X-ray diffraction, and scanning electron microscopy. Finally, the antibacterial activity of nanoparticles against isolated strains was investigated with the standard disk diffusion test. Biosynthesis of nanoparticles showed an average size of 44 nm using Bacillus subtilis. All 44% (71 strains) of Pseudomonas aeruginosa formed a biofilm. Bismuth oxide nanoparticles at a concentration of 2000 ppm were inhibited 5% of Pseudomonas aeruginosa. By increasing the concentration of bismuth oxide nanoparticles, its inhibitory effect increased. The results showed that there is a significant difference between the groups exposed nanoparticle and ciprofloxacin.

Rice straw is a promising agricultural residue for the production of biobutanol due to its plentitude and low commercial value. Rice straw has high potential sugars that can be converted into fermentable sugars. However, biobutanol production from rice straw-based on the current technology is not economically viable due to the challenges and restrictions throughout the overall process of rice straw-biobutanol conversion. For the process to be economically viable, the use of an appropriate pretreatment technique is of great significance since an effective pretreatment can result in efficient hydrolysis. In this article, a total number of 66 articles from 1980-2018 were first searched on databases using keywords and then were reviewed. This paper reviews the current approaches and advances available for the production of biobutanol from rice straw. The challenges encountered throughout the overall process of bioconversion of rice straw into biobutanol are discussed as well. Even though much attempt has been made to develop a proper technique for pretreatment of rice straw, efforts are still required to make the process more efficient and economically feasible. The use of genetically modified butanol tolerant strains has resulted in butanol production at higher concentrations. Therefore, in situ product-recovery systems have become more economical as substrate inhibition and butanol toxicity to the culture are drastically reduced.