University of Isfahan Journal of Microbial Biology 3060-7647 13 49 2024 03 20 The Effect of Amoxicillin, Cefixime, and Metronidazole Antibiotics on the Percentage of Resistant bacteria in Soils Contaminated with Heavy Metals The Effect of Amoxicillin, Cefixime, and Metronidazole Antibiotics on the Percentage of Resistant bacteria in Soils Contaminated with Heavy Metals 1 20 27581 10.22108/bjm.2023.135724.1508 FA Ziba Najafzadeh Nobar Department of Soil Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran. Ali Akbar Safari Sinegani Department of Soil Science, Faculty of Agriculture, Bu-Ali Sina University, Hamadan, Iran Journal Article 2022 11 14 The purpose of the present study was to investigate the antibiotic resistance of culturable bacteria following the increase of three commonly used antibiotics in three agricultural, rangeland, and mine soils with different amounts of heavy metals. Antibiotics amoxicillin, cefixime, and metronidazole were used in amounts of 100 and 200 mg per kilogram of agricultural, rangeland, and mine soils. Then, in a short time incubation (zero, 1, 3, and 7 days), the abundance of bacteria in nutrient agar medium and the percentage of resistant bacteria in nutrient agar medium with antibiotics (16 µg of amoxicillin, 2 µg of cefixime, 8 µg of gentamicin, 16 µg of metronidazole and 8 µg of tetracycline per milliliter of nutrient agar) were counted and estimated. The percentage of bacteria resistant to cefixime, gentamicin, and tetracycline in mine soil, especially rangeland soil, which had high contamination with heavy metals, was higher than in agricultural soil, and only the percentage of bacteria resistant to amoxicillin and metronidazole was higher in agricultural soil than mine soil. In rangeland soil, 100% of bacteria were resistant to the tested antibiotics except for tetracycline. Treatment of soils with amoxicillin caused an increase in the number of resistant bacteria, which was especially evident in agricultural soils at a concentration of 200 mg.kg-1. The same finding was also seen in the application of cefixime, but in mine soil, the high concentration of this antibiotic showed a decrease, and the percentage of resistant bacteria in both rangeland and agricultural soils was close to 100%, but in mine soil, it reached less than 20%. There was a significant increase in the number of resistant bacteria in the soils treated with metronidazole compared to control soils. But, in this treatment, the response of agricultural soil bacteria to a concentration of 200 mg.kg-1 was decreased, and mine soil did not show such a response. In all treatments, the bactericidal power of tetracycline and then gentamicin in the concentrations used in the culture medium was higher compared to the three antibiotics of amoxicillin, cefixime, and metronidazole. Antibiotic resistance of soil bacteria is dependent on soil contamination with heavy metals and diversity of soil bacteria, genetic pool, and ability to move resistance genes between them. The increase of antibiotics in the soil, depending on the characteristics of the soil and the antibiotic causes the dominance of special and resistant species, which increases the transfer of resistance genes and their abundance in the environment. Introduction Antibiotics are widely used in medicine, agriculture, and animal husbandry. The continuous increase of antibiotics in dry and wet ecosystems causes chemical pollution, and the emergence and spread of antibiotic stability genes and antibiotic-resistant bacteria. The spread of antibiotic resistance is a global threat to human health. Bacteria with various mechanisms can remain stable against antibiotics and at the same time against heavy metals. Although antibiotic resistance can occur naturally, biocontaminants, such as metals (and metalloids), can increase the development of antibiotic resistance by stressing bacteria through the co-selection of genes and traits that protect bacteria from both antibiotics and metals. Many studies have shown that metal pollution has a positive correlation with antibiotic stability genes in polluted habitats. In most of the studies, it has been stated that heavy metals can affect the abundance of antibiotic stability genes in the habitat. This research was conducted with the aim of investigating the transformation of the percentage of stable soil bacteria in the face of different concentrations of antibiotics in soils with different sizes of heavy metals. Materials and Methods Three commonly used antibiotics, amoxicillin (betalactam), cefixime (cephalosporin), and metronidazole (nitroimidazole) were purchased from Hamedan pharmacy. In the beginning, the tablet and the contents inside the capsule (amoxicillin capsule (500 mg), cefixime tablet (400 mg) and metronidazole tablet (500 mg)) were weighed for all three antibiotics, and the equivalent weight of the pure substance of the antibiotic was obtained. After that, the required weight was used to prepare solutions with a concentration of 100 and 200 mg of the antibiotic per kg of dry soil. Aqueous solutions of three antibiotics, amoxicillin, cefixime, and metronidazole, were added separately to the equivalent weight of dry soil and mixed well. Then sterilized distilled water was added to the soil samples to make the soil moisture close to the agricultural capacity. The treated soil samples were kept in the dark and at laboratory temperature (about 25°C). Then, at four heating times of zero (without heating), 1, 3, and 7 days, the abundance of bacteria in nutrient agar cultures without antibiotics and nutrient agar with 5 antibiotics amoxicillin, cefixime, gentamicin, metronidazole, and tetracycline were counted as below. The average number of bacteria in treated and untreated soils was estimated separately at zero, 1, 3, and 7 days for short-term heating (7 days), and the percentage of stable bacteria was statistically analyzed. This research was conducted as four separate tests of treated soil and treated soil with three antibiotics amoxicillin, cefixime, and metronidazole. Each experiment is in a factorial form with three factors: three types of soil (uncontaminated agricultural, mine-contaminated, and pasture near the mine), two antibiotic concentrations of 100 and 200 mg per kg of dry soil, and five types of antibiotics used in nutrient agar culture in short-term heating (7 Fasting) was done with a randomized complete design in three replications. Sampling was done at four times (zero, 1, 3, and 7 days) and then their average was processed and analyzed for each soil. Excel 2010 software was used to process the data of each experiment and draw graphs, and SPSS 20 software was used for statistical tests. The normality of data distribution was checked by the Shapiro-Wilk test. After standardization with Z-score, the effect of each treatment and their interaction was evaluated by analysis of variance. The mean test of each of the mentioned characteristics in the used treatments was performed with Tukey's method at the base of five percent. Research Findings Abundance and percentage of stable bacteria in treated soils: The variance analysis of the logarithm of the abundance of bacteria counted in the soils showed that the simple effect of soil type on the number of bacteria was significant (P<0.01). The test of the average logarithm of abundance of bacteria in soils is shown in Figure 1. As can be seen, agricultural soil has the highest logarithm of abundance, which is not significantly different from pasture soil; But in both soils, it was significantly more than mine soil. It should be remembered that the characteristics of these soils were very close to each other. But their heavy metal concentrations were very different. Iron in agricultural, pasture, and mine soil is 22,691, 20,708, and 73,110 mg/kg respectively, lead in them is 33/20, 67/79, and 9749/66 mg/kg respectively, zinc is 58/33, 89/16, and 3839/20 respectively. mg/kg, cadmium respectively 0.75, 1.54, and 37.53 mg/kg, copper respectively 16.45, 18.70 and 89.58 mg/kg, manganese respectively 387.50, 837.50 and 66/ 9816 mg/kg and magnesium was 6052.08, 11166.67 and 4697.32 mg/kg, respectively; Therefore, except for magnesium, the highest amount of metals was found in the soil sampled from the mine. The order of contamination of the soils was according to the type of mine soil more or closer to the pasture soil and more than the agricultural soil. The high amount of heavy metals in the pasture soil can be related to the structure of the parent material of the soil, which is near the mine. However, the abundance of bacteria in it is not significantly different from agricultural soil. Discussion of Results and Conclusions Abundance and percentage of antimicrobial stability of microorganisms in test soils: Among the three test soils, mine soil had the lowest abundance of microorganisms. This could be related to more severe mine soil contamination or unsuitable soil habitat for bacterial growth. Indication of amoxicillin use in soil: In mine soil, the percentage of stable bacteria was lower; But the addition of amoxicillin, especially at a lower concentration (100 mg/kg soil), increased the percentage of stable bacteria in the soil, and nearly 100% of the bacteria in both agricultural and pasture soils by adding both concentrations of 100 and 200 amoxicillin compared to amoxicillin, cefixime, and metronidazole. They showed stability. Indication of the use of Cefixime in soil: By adding Cefixime (both 100 and 200 concentrations) to agricultural soil, the least stability of bacteria against tetracycline was seen. The stability of agricultural soil bacteria against the other four antibiotics added to the culture was not significantly different. Also, by increasing the concentration of cefixime in the soil to 200 mg, the resistance against four antibiotics, amoxicillin, cefixime, gentamicin, and metronidazole, reached nearly 100%. In the mine soil treated with cefixime (concentration of 100 mg/kg), the resistance against five antibiotics added to the culture was not significant. But by increasing the concentration of cefixime in the soil to 200 mg/kg, the stability of bacteria decreased, which was significant for three antibiotics in the culture (amoxicillin, cefixime, and metronidazole). In other words, increasing the concentration of cefixime in the soil decreased the stability of mine soil bacteria. Indication of metronidazole application in soil: In the agricultural soil treated with metronidazole, the lowest stability against tetracycline was seen, and by increasing the concentration of metronidazole from 100 to 200 mg/kg of soil, this stability against the antibiotics added to the culture decreased significantly. The bacteria of this soil had 100% stability against four antibiotics: amoxicillin, cefixime, gentamicin, and metronidazole at a concentration of 100 metronidazole. In contrast, mine soil bacteria treated with metronidazole at a concentration of 100 mg/kg were resistant to all five antibiotics; however, by increasing the concentration of metronidazole to 200 mg/kg, the response of bacteria to four antibiotics, amoxicillin, cefixime, gentamicin, and metronidazole was still stable; but they had a significant decrease against tetracycline. In pasture soil treated with metronidazole (both 100 and 200 concentrations), the lowest stability against tetracycline was seen. In this study, the percentage of stable bacteria in agricultural soil against amoxicillin and metronidazole was higher than in mine soil; but it never reached their size in pasture soil, and in line with previous studies, the percentage of stable bacteria in soils contaminated with metals, especially in pasture soil, was higher than in agricultural soil. Adding amoxicillin, cefixime, and metronidazole antibiotics to each of the soils, especially at a concentration of 100 mg/kg, caused a significant increase in the percentage of resistant bacteria against the five antibiotics used in the farm. This stability was significantly reduced especially in cefixime and metronidazole treatments by increasing the concentration of antibiotics to 200 mg/kg of soil, and mine soil bacteria had a more specific response in cefixime soil treatment. Pasture soil bacteria, like the treated soil, in both concentrations of the three soil treatments of amoxicillin, cefixime, and metronidazole, except for tetracycline, had high stability against the antibiotics added to the culture. In general, the pattern of stability of soil bacteria against antibiotics was Amoxicillin > Cefixime > Metronidazole > Gentamicin, and 100% stability of bacteria was not seen against tetracycline; Therefore, among the five antibiotics added to the slaughterhouse, tetracycline was the most lethal, followed by gentamicin. Only mine soil bacteria treated with 100 mg/kg metronidazole had significant resistance to tetracycline. Therefore, the response of bacteria to the antibiotics added to the soil and their stability in the studied soils are not the same and depend on the characteristics of the antibiotic, the soil, and their bacteria. Therefore, the percentage of stable bacteria in pasture soil was high; However, the percentage of bacteria resistant to tetracycline in the mine soil was higher than in the other two soils, especially at zero and 100 concentrations of the used antibiotics. The purpose of the present study was to investigate the antibiotic resistance of culturable bacteria following the increase of three commonly used antibiotics in three agricultural, rangeland, and mine soils with different amounts of heavy metals. Antibiotics amoxicillin, cefixime, and metronidazole were used in amounts of 100 and 200 mg per kilogram of agricultural, rangeland, and mine soils. Then, in a short time incubation (zero, 1, 3, and 7 days), the abundance of bacteria in nutrient agar medium and the percentage of resistant bacteria in nutrient agar medium with antibiotics (16 µg of amoxicillin, 2 µg of cefixime, 8 µg of gentamicin, 16 µg of metronidazole and 8 µg of tetracycline per milliliter of nutrient agar) were counted and estimated. The percentage of bacteria resistant to cefixime, gentamicin, and tetracycline in mine soil, especially rangeland soil, which had high contamination with heavy metals, was higher than in agricultural soil, and only the percentage of bacteria resistant to amoxicillin and metronidazole was higher in agricultural soil than mine soil. In rangeland soil, 100% of bacteria were resistant to the tested antibiotics except for tetracycline. Treatment of soils with amoxicillin caused an increase in the number of resistant bacteria, which was especially evident in agricultural soils at a concentration of 200 mg.kg-1. The same finding was also seen in the application of cefixime, but in mine soil, the high concentration of this antibiotic showed a decrease, and the percentage of resistant bacteria in both rangeland and agricultural soils was close to 100%, but in mine soil, it reached less than 20%. There was a significant increase in the number of resistant bacteria in the soils treated with metronidazole compared to control soils. But, in this treatment, the response of agricultural soil bacteria to a concentration of 200 mg.kg-1 was decreased, and mine soil did not show such a response. In all treatments, the bactericidal power of tetracycline and then gentamicin in the concentrations used in the culture medium was higher compared to the three antibiotics of amoxicillin, cefixime, and metronidazole. Antibiotic resistance of soil bacteria is dependent on soil contamination with heavy metals and diversity of soil bacteria, genetic pool, and ability to move resistance genes between them. The increase of antibiotics in the soil, depending on the characteristics of the soil and the antibiotic causes the dominance of special and resistant species, which increases the transfer of resistance genes and their abundance in the environment. Introduction Antibiotics are widely used in medicine, agriculture, and animal husbandry. The continuous increase of antibiotics in dry and wet ecosystems causes chemical pollution, and the emergence and spread of antibiotic stability genes and antibiotic-resistant bacteria. The spread of antibiotic resistance is a global threat to human health. Bacteria with various mechanisms can remain stable against antibiotics and at the same time against heavy metals. Although antibiotic resistance can occur naturally, biocontaminants, such as metals (and metalloids), can increase the development of antibiotic resistance by stressing bacteria through the co-selection of genes and traits that protect bacteria from both antibiotics and metals. Many studies have shown that metal pollution has a positive correlation with antibiotic stability genes in polluted habitats. In most of the studies, it has been stated that heavy metals can affect the abundance of antibiotic stability genes in the habitat. This research was conducted with the aim of investigating the transformation of the percentage of stable soil bacteria in the face of different concentrations of antibiotics in soils with different sizes of heavy metals. Materials and Methods Three commonly used antibiotics, amoxicillin (betalactam), cefixime (cephalosporin), and metronidazole (nitroimidazole) were purchased from Hamedan pharmacy. In the beginning, the tablet and the contents inside the capsule (amoxicillin capsule (500 mg), cefixime tablet (400 mg) and metronidazole tablet (500 mg)) were weighed for all three antibiotics, and the equivalent weight of the pure substance of the antibiotic was obtained. After that, the required weight was used to prepare solutions with a concentration of 100 and 200 mg of the antibiotic per kg of dry soil. Aqueous solutions of three antibiotics, amoxicillin, cefixime, and metronidazole, were added separately to the equivalent weight of dry soil and mixed well. Then sterilized distilled water was added to the soil samples to make the soil moisture close to the agricultural capacity. The treated soil samples were kept in the dark and at laboratory temperature (about 25°C). Then, at four heating times of zero (without heating), 1, 3, and 7 days, the abundance of bacteria in nutrient agar cultures without antibiotics and nutrient agar with 5 antibiotics amoxicillin, cefixime, gentamicin, metronidazole, and tetracycline were counted as below. The average number of bacteria in treated and untreated soils was estimated separately at zero, 1, 3, and 7 days for short-term heating (7 days), and the percentage of stable bacteria was statistically analyzed. This research was conducted as four separate tests of treated soil and treated soil with three antibiotics amoxicillin, cefixime, and metronidazole. Each experiment is in a factorial form with three factors: three types of soil (uncontaminated agricultural, mine-contaminated, and pasture near the mine), two antibiotic concentrations of 100 and 200 mg per kg of dry soil, and five types of antibiotics used in nutrient agar culture in short-term heating (7 Fasting) was done with a randomized complete design in three replications. Sampling was done at four times (zero, 1, 3, and 7 days) and then their average was processed and analyzed for each soil. Excel 2010 software was used to process the data of each experiment and draw graphs, and SPSS 20 software was used for statistical tests. The normality of data distribution was checked by the Shapiro-Wilk test. After standardization with Z-score, the effect of each treatment and their interaction was evaluated by analysis of variance. The mean test of each of the mentioned characteristics in the used treatments was performed with Tukey's method at the base of five percent. Research Findings Abundance and percentage of stable bacteria in treated soils: The variance analysis of the logarithm of the abundance of bacteria counted in the soils showed that the simple effect of soil type on the number of bacteria was significant (P<0.01). The test of the average logarithm of abundance of bacteria in soils is shown in Figure 1. As can be seen, agricultural soil has the highest logarithm of abundance, which is not significantly different from pasture soil; But in both soils, it was significantly more than mine soil. It should be remembered that the characteristics of these soils were very close to each other. But their heavy metal concentrations were very different. Iron in agricultural, pasture, and mine soil is 22,691, 20,708, and 73,110 mg/kg respectively, lead in them is 33/20, 67/79, and 9749/66 mg/kg respectively, zinc is 58/33, 89/16, and 3839/20 respectively. mg/kg, cadmium respectively 0.75, 1.54, and 37.53 mg/kg, copper respectively 16.45, 18.70 and 89.58 mg/kg, manganese respectively 387.50, 837.50 and 66/ 9816 mg/kg and magnesium was 6052.08, 11166.67 and 4697.32 mg/kg, respectively; Therefore, except for magnesium, the highest amount of metals was found in the soil sampled from the mine. The order of contamination of the soils was according to the type of mine soil more or closer to the pasture soil and more than the agricultural soil. The high amount of heavy metals in the pasture soil can be related to the structure of the parent material of the soil, which is near the mine. However, the abundance of bacteria in it is not significantly different from agricultural soil. Discussion of Results and Conclusions Abundance and percentage of antimicrobial stability of microorganisms in test soils: Among the three test soils, mine soil had the lowest abundance of microorganisms. This could be related to more severe mine soil contamination or unsuitable soil habitat for bacterial growth. Indication of amoxicillin use in soil: In mine soil, the percentage of stable bacteria was lower; But the addition of amoxicillin, especially at a lower concentration (100 mg/kg soil), increased the percentage of stable bacteria in the soil, and nearly 100% of the bacteria in both agricultural and pasture soils by adding both concentrations of 100 and 200 amoxicillin compared to amoxicillin, cefixime, and metronidazole. They showed stability. Indication of the use of Cefixime in soil: By adding Cefixime (both 100 and 200 concentrations) to agricultural soil, the least stability of bacteria against tetracycline was seen. The stability of agricultural soil bacteria against the other four antibiotics added to the culture was not significantly different. Also, by increasing the concentration of cefixime in the soil to 200 mg, the resistance against four antibiotics, amoxicillin, cefixime, gentamicin, and metronidazole, reached nearly 100%. In the mine soil treated with cefixime (concentration of 100 mg/kg), the resistance against five antibiotics added to the culture was not significant. But by increasing the concentration of cefixime in the soil to 200 mg/kg, the stability of bacteria decreased, which was significant for three antibiotics in the culture (amoxicillin, cefixime, and metronidazole). In other words, increasing the concentration of cefixime in the soil decreased the stability of mine soil bacteria. Indication of metronidazole application in soil: In the agricultural soil treated with metronidazole, the lowest stability against tetracycline was seen, and by increasing the concentration of metronidazole from 100 to 200 mg/kg of soil, this stability against the antibiotics added to the culture decreased significantly. The bacteria of this soil had 100% stability against four antibiotics: amoxicillin, cefixime, gentamicin, and metronidazole at a concentration of 100 metronidazole. In contrast, mine soil bacteria treated with metronidazole at a concentration of 100 mg/kg were resistant to all five antibiotics; however, by increasing the concentration of metronidazole to 200 mg/kg, the response of bacteria to four antibiotics, amoxicillin, cefixime, gentamicin, and metronidazole was still stable; but they had a significant decrease against tetracycline. In pasture soil treated with metronidazole (both 100 and 200 concentrations), the lowest stability against tetracycline was seen. In this study, the percentage of stable bacteria in agricultural soil against amoxicillin and metronidazole was higher than in mine soil; but it never reached their size in pasture soil, and in line with previous studies, the percentage of stable bacteria in soils contaminated with metals, especially in pasture soil, was higher than in agricultural soil. Adding amoxicillin, cefixime, and metronidazole antibiotics to each of the soils, especially at a concentration of 100 mg/kg, caused a significant increase in the percentage of resistant bacteria against the five antibiotics used in the farm. This stability was significantly reduced especially in cefixime and metronidazole treatments by increasing the concentration of antibiotics to 200 mg/kg of soil, and mine soil bacteria had a more specific response in cefixime soil treatment. Pasture soil bacteria, like the treated soil, in both concentrations of the three soil treatments of amoxicillin, cefixime, and metronidazole, except for tetracycline, had high stability against the antibiotics added to the culture. In general, the pattern of stability of soil bacteria against antibiotics was Amoxicillin > Cefixime > Metronidazole > Gentamicin, and 100% stability of bacteria was not seen against tetracycline; Therefore, among the five antibiotics added to the slaughterhouse, tetracycline was the most lethal, followed by gentamicin. Only mine soil bacteria treated with 100 mg/kg metronidazole had significant resistance to tetracycline. Therefore, the response of bacteria to the antibiotics added to the soil and their stability in the studied soils are not the same and depend on the characteristics of the antibiotic, the soil, and their bacteria. Therefore, the percentage of stable bacteria in pasture soil was high; However, the percentage of bacteria resistant to tetracycline in the mine soil was higher than in the other two soils, especially at zero and 100 concentrations of the used antibiotics.

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University of Isfahan Journal of Microbial Biology 3060-7647 13 49 2024 05 07 Identification and Isolation of L-asparaginase-producing Yeast from Soil Samples Identification and Isolation of L-asparaginase-producing Yeast from Soil Samples 21 52 27853 10.22108/bjm.2023.138189.1549 FA Mansooreh Sadeghi Department of Microbiology, Faculty of Biological Sciences, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Keivan Beheshti-Maal Department of Microbiology, Faculty of Biological Sciences, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Fereshteh Ghandehari Department of Microbiology, Faculty of Biological Sciences, Falavarjan Branch, Islamic Azad University, Isfahan, Iran Journal Article 2023 07 05 In recent years, the need for L-asparaginase in different industries has increased. L-asparaginase obtained from microorganisms is cost-effective, has no harmful effects on the environment, and improves the quality of products. In the present study, L-asparaginase-producing microorganisms were isolated and optimized from different sources, and selected strain enzymes were produced. L-asparaginase-producing yeasts were randomly isolated and purified from different parts of Isfahan. Strains were screened for L-asparaginase production. Then, the superior yeast strain with the highest enzymatic activity was selected for optimization. Under suitable pH, temperature, time, carbon source, and nitrogen source, multi-factor optimization was performed using response-level statistical software to investigate the simultaneous effect of several factors on production and the highest enzyme activity. In the present study, 5S2 yeast isolate led to the formation of the highest halo of L-asparaginase. This strain was first introduced as the L-asparaginase-producing strain and was registered in the NCBI database under Diutina mesorugosa strain SBG-IAUF-2 with access number MZ901211. Yeast 5S2 with enzyme activity equivalent to 1722.8 u / ml was selected as the selected strain for optimization. The results of multivariate optimization of enzyme activity showed that at pH 7, a concentration of 2% glucose as carbon source, the attention of 10% L-asparagine, a temperature of 35°C, and a time of 120 hours, there is the highest enzyme production activity and curve RSM is located in the optimization area. In the present study, after recording 5S2 yeast isolate for the first time as an L-asparaginase-producing strain in the NCBI database, extracellular L-asparaginase produced by the 5S2 yeast strain was introduced due to its high enzymatic activity as a potent strain in the production of L-asparaginase enzyme. Introduction L-asparaginase as a food processing aid, is capable of significantly reducing acrylamide formation in starchy foods by over 90% without altering their taste or appearance. L-asparaginase enzymes are produced by various microorganisms, including bacteria like Streptomyces goulbarjensis, Enterobacter cloacae, and Serratia marcescens, as well as fungi and yeasts. Different isoforms of L-asparaginase originating from plants, eukaryotes, and prokaryotes have been identified, with those isolated from microorganisms, especially bacteria, proving more effective. Microorganisms producing L-asparaginase are found across diverse ecological areas, showcasing desirable enzymatic activity. The most effective isoforms of L-asparaginase currently available as anticancer drugs and produced commercially are sourced from microbial origins like Escherichia coli, Erwinia carotovora, and various Bacillus strains. Reports also mention the enzyme's production by filamentous fungi and yeasts such as Saccharomyces cerevisiae and Candida. Like Saccharomyces cerevisiae, Yeast is highlighted for its single-cell nature, rapid reproduction in cultivation environments, and its significance in various ecosystems like soil, swamps, and rivers due to its cellular processes post-translation and lack of endotoxins. Saccharomyces cerevisiae intricately regulates L-asparagine metabolism through complex pathways involving unrelated citrate products. The current study focuses on isolating, purifying, and identifying yeast strains producing L-asparaginase, optimizing its production under various environmental conditions to find an optimal method for generating these enzymes with reduced side effects and increased therapeutic potential. Materials and Methods Sampling and isolation of L-asparaginase-producing yeast: Soil samples were collected randomly from various points in Isfahan, particularly from agricultural fields, and diverse yeasts were isolated from these samples. For initial enrichment of the yeasts, 1 gram of collected soil samples was mixed in 100 milliliters of L-asparagine culture medium. This mixture was placed on a shaker for 3 days at 31 degrees Celsius and a speed of 130 rotations per minute, repeated thrice. Subsequently, 1 milliliter of the culture medium was taken and transferred to YPG agar medium. Yeast screening based on L-asparagine enzyme production: A rapid assay method was employed using a minimum asparagine agar medium. Colonies were placed on this medium and incubated at 30 degrees Celsius for 24 to 72 hours. Changes in color and halo diameter were examined every 24 hours. Yeasts capable of producing the enzyme hydrolyze asparagine in the culture medium, releasing ammonia and alkalinizing the medium. As a result, a shift in pH from acidic to alkaline changed the color of the medium from yellow to pink. Yeasts producing the enzyme caused an alkalinity shift, changing the medium color to a pinkish hue. Yeasts that did not produce this pink halo were eliminated. Molecular Identification of Superior Yeast Species Producing L-Asparaginase Enzyme: For molecular identification, the selected strains were incubated in YPG culture medium for 72 hours at 37 degrees Celsius. The colony-PCR method was employed to identify the genus and species of the yeasts. The DNA of each sample was added to the respective vials, and after adding the required reaction components and the designed primers, they were placed inside the thermocycler. Consequently, a 655 base pair fragment resulting from amplifying the gene encoding 18S-rRNA (ITS4, ITS1) using the forward and reverse primer was amplified via a polymerase chain reaction. Optimization of Enzyme Production via RSM Statistical Design: Multiple factors, including pH, carbon source, nitrogen source, temperature, time, and their various concentrations of carbon and nitrogen sources were examined in enzyme production. Based on the available sources in the culture medium, enzyme activity of Sizopek's Dax was subjected to various concentrations of glucose (from 1.5% to 2.5%) as a carbon source, a pH range from 5 to 9, L-asparagine as a nitrogen source (from 6% to 14%), temperature between 25 to 45 degrees Celsius, and time from 80 to 160 hours as factors in the optimization process using the Response Surface Methodology (RSM). Research Findings In this study, yeast 5S2 was examined for the first time to produce the L-asparaginase enzyme. After sequencing and obtaining the relevant file, the sequence underwent evaluation and editing using the Choromas software. Sequence comparison via the BLASTN server in the NCBI database revealed that the 18S-rDNA sequence of the 5S2 yeast strain bears the highest similarity and coverage with a strain of Diutina mesorugosa. Following assessments, this isolate was registered as a new strain for L-asparaginase production under the name Diutina mesorugosa strain SBG-IAUF-2 with accession number MZ901181 in the NCBI database. The highest enzyme activity during the optimization stage was achieved in test number 6, with 2% glucose, pH at 7, a duration of 120 hours, a temperature of 35 degrees Celsius, and 10% L-asparagine. This resulted in an enzyme activity of 1722.8 U/mL, indicating the maximum level of enzyme activity. In the optimization of L-asparaginase enzyme from the 5S2 strain, the highest activity was observed at pH 7, showing sensitivity to both acidic and alkaline conditions. Additionally, the enzyme was temperature-sensitive, exhibiting the highest activity at 35 degrees Celsius but decreasing notably at 45 degrees Celsius. Discussion of Results and Conclusions Glucose and L-asparagine levels influenced enzyme production, achieving optimal enzyme yields at their moderate levels. Time was also a significant factor affecting enzyme production. This research successfully produced L-asparaginase using the isolated yeast strain 5S2 from the soil, optimizing its activity through the response surface method. The increased enzyme activity suggests the potential application of these results in various industries, including food processing and nanotechnology. The enzyme production levels in the optimization process suggest the potential for enhanced enzyme yields through strain optimization. In recent years, the need for L-asparaginase in different industries has increased. L-asparaginase obtained from microorganisms is cost-effective, has no harmful effects on the environment, and improves the quality of products. In the present study, L-asparaginase-producing microorganisms were isolated and optimized from different sources, and selected strain enzymes were produced. L-asparaginase-producing yeasts were randomly isolated and purified from different parts of Isfahan. Strains were screened for L-asparaginase production. Then, the superior yeast strain with the highest enzymatic activity was selected for optimization. Under suitable pH, temperature, time, carbon source, and nitrogen source, multi-factor optimization was performed using response-level statistical software to investigate the simultaneous effect of several factors on production and the highest enzyme activity. In the present study, 5S2 yeast isolate led to the formation of the highest halo of L-asparaginase. This strain was first introduced as the L-asparaginase-producing strain and was registered in the NCBI database under Diutina mesorugosa strain SBG-IAUF-2 with access number MZ901211. Yeast 5S2 with enzyme activity equivalent to 1722.8 u / ml was selected as the selected strain for optimization. The results of multivariate optimization of enzyme activity showed that at pH 7, a concentration of 2% glucose as carbon source, the attention of 10% L-asparagine, a temperature of 35°C, and a time of 120 hours, there is the highest enzyme production activity and curve RSM is located in the optimization area. In the present study, after recording 5S2 yeast isolate for the first time as an L-asparaginase-producing strain in the NCBI database, extracellular L-asparaginase produced by the 5S2 yeast strain was introduced due to its high enzymatic activity as a potent strain in the production of L-asparaginase enzyme. Introduction L-asparaginase as a food processing aid, is capable of significantly reducing acrylamide formation in starchy foods by over 90% without altering their taste or appearance. L-asparaginase enzymes are produced by various microorganisms, including bacteria like Streptomyces goulbarjensis, Enterobacter cloacae, and Serratia marcescens, as well as fungi and yeasts. Different isoforms of L-asparaginase originating from plants, eukaryotes, and prokaryotes have been identified, with those isolated from microorganisms, especially bacteria, proving more effective. Microorganisms producing L-asparaginase are found across diverse ecological areas, showcasing desirable enzymatic activity. The most effective isoforms of L-asparaginase currently available as anticancer drugs and produced commercially are sourced from microbial origins like Escherichia coli, Erwinia carotovora, and various Bacillus strains. Reports also mention the enzyme's production by filamentous fungi and yeasts such as Saccharomyces cerevisiae and Candida. Like Saccharomyces cerevisiae, Yeast is highlighted for its single-cell nature, rapid reproduction in cultivation environments, and its significance in various ecosystems like soil, swamps, and rivers due to its cellular processes post-translation and lack of endotoxins. Saccharomyces cerevisiae intricately regulates L-asparagine metabolism through complex pathways involving unrelated citrate products. The current study focuses on isolating, purifying, and identifying yeast strains producing L-asparaginase, optimizing its production under various environmental conditions to find an optimal method for generating these enzymes with reduced side effects and increased therapeutic potential. Materials and Methods Sampling and isolation of L-asparaginase-producing yeast: Soil samples were collected randomly from various points in Isfahan, particularly from agricultural fields, and diverse yeasts were isolated from these samples. For initial enrichment of the yeasts, 1 gram of collected soil samples was mixed in 100 milliliters of L-asparagine culture medium. This mixture was placed on a shaker for 3 days at 31 degrees Celsius and a speed of 130 rotations per minute, repeated thrice. Subsequently, 1 milliliter of the culture medium was taken and transferred to YPG agar medium. Yeast screening based on L-asparagine enzyme production: A rapid assay method was employed using a minimum asparagine agar medium. Colonies were placed on this medium and incubated at 30 degrees Celsius for 24 to 72 hours. Changes in color and halo diameter were examined every 24 hours. Yeasts capable of producing the enzyme hydrolyze asparagine in the culture medium, releasing ammonia and alkalinizing the medium. As a result, a shift in pH from acidic to alkaline changed the color of the medium from yellow to pink. Yeasts producing the enzyme caused an alkalinity shift, changing the medium color to a pinkish hue. Yeasts that did not produce this pink halo were eliminated. Molecular Identification of Superior Yeast Species Producing L-Asparaginase Enzyme: For molecular identification, the selected strains were incubated in YPG culture medium for 72 hours at 37 degrees Celsius. The colony-PCR method was employed to identify the genus and species of the yeasts. The DNA of each sample was added to the respective vials, and after adding the required reaction components and the designed primers, they were placed inside the thermocycler. Consequently, a 655 base pair fragment resulting from amplifying the gene encoding 18S-rRNA (ITS4, ITS1) using the forward and reverse primer was amplified via a polymerase chain reaction. Optimization of Enzyme Production via RSM Statistical Design: Multiple factors, including pH, carbon source, nitrogen source, temperature, time, and their various concentrations of carbon and nitrogen sources were examined in enzyme production. Based on the available sources in the culture medium, enzyme activity of Sizopek's Dax was subjected to various concentrations of glucose (from 1.5% to 2.5%) as a carbon source, a pH range from 5 to 9, L-asparagine as a nitrogen source (from 6% to 14%), temperature between 25 to 45 degrees Celsius, and time from 80 to 160 hours as factors in the optimization process using the Response Surface Methodology (RSM). Research Findings In this study, yeast 5S2 was examined for the first time to produce the L-asparaginase enzyme. After sequencing and obtaining the relevant file, the sequence underwent evaluation and editing using the Choromas software. Sequence comparison via the BLASTN server in the NCBI database revealed that the 18S-rDNA sequence of the 5S2 yeast strain bears the highest similarity and coverage with a strain of Diutina mesorugosa. Following assessments, this isolate was registered as a new strain for L-asparaginase production under the name Diutina mesorugosa strain SBG-IAUF-2 with accession number MZ901181 in the NCBI database. The highest enzyme activity during the optimization stage was achieved in test number 6, with 2% glucose, pH at 7, a duration of 120 hours, a temperature of 35 degrees Celsius, and 10% L-asparagine. This resulted in an enzyme activity of 1722.8 U/mL, indicating the maximum level of enzyme activity. In the optimization of L-asparaginase enzyme from the 5S2 strain, the highest activity was observed at pH 7, showing sensitivity to both acidic and alkaline conditions. Additionally, the enzyme was temperature-sensitive, exhibiting the highest activity at 35 degrees Celsius but decreasing notably at 45 degrees Celsius. Discussion of Results and Conclusions Glucose and L-asparagine levels influenced enzyme production, achieving optimal enzyme yields at their moderate levels. Time was also a significant factor affecting enzyme production. This research successfully produced L-asparaginase using the isolated yeast strain 5S2 from the soil, optimizing its activity through the response surface method. The increased enzyme activity suggests the potential application of these results in various industries, including food processing and nanotechnology. The enzyme production levels in the optimization process suggest the potential for enhanced enzyme yields through strain optimization.

https://bjm.ui.ac.ir/article_27853_c35b9338b0f9903e639ae5f7cd92077f.pdf

University of Isfahan Journal of Microbial Biology 3060-7647 13 49 2024 03 20 Screening of effective bacteria in the biological removal of selenium from sarcheshme copper tailing dam effluent Screening of effective bacteria in the biological removal of selenium from sarcheshme copper tailing dam effluent 53 75 28326 10.22108/bjm.2023.138438.1553 FA Seyed Mansour Meybodi Microbiology, Department of Microbiology, Faculty of Biological Sciences, Islamic Azad University, Tonekabon, Iran Amir Sarabandi Haghighi Department of Microbiology, Islamic Azad University, Sirjan, Kerman Journal Article 2023 07 18 Introduction: One of the concerns of today's world is environmental pollution caused by heavy metals, and in this regard, the element selenium is very important due to its low amount in the environment. Bioremoval of metals is one of the cleanest and cheapest methods of biological absorption. The purpose of this research is to isolate and evaluate selenium-resistant strains for the bioremoval of this metal from aquatic environments. Materials and Methods: By isolating selenium-resistant strains, the bioabsorption rate of the selected types was investigated in different conditions of acidity, temperature, and biomass amount at different times. In between, two isolates were resistant to the concentration of 400 mM sodium selenite, which were identified after performing biochemical and genetic tests. Results: The highest absorption rate in terms of inoculated biomass was related to Thalassospira permensis and Bacillus thuringiensis at 4% in 20 and 60 minutes, respectively. The highest amount of absorption was obtained by Bacillus at an acidity of 5 during 40 minutes and by Thalassospira at an acidity of 7 and a time of 60 minutes. The optimal temperature for the two strains was 25 °C and it was found that Thalassospira permensis had the best effect in 20 minutes and Bacillus thuringiensis had the best effect in 40 minutes. In addition, Thalassospira permensis was the best choice for selenium bioabsorption by absorbing 95.1% of total selenium with 4% of the biological mass in a period of 20 minutes, at acidity equal to 6 and 25 °C. Discussion and Conclusion: Thalassospira permensis absorbed 95.1 mgL-1 of 100 mgL-1 of sodium selenite in the culture medium within 20 minutes by biomass 4%. Thus, it had a high bioabsorption rate and became a suitable candidate for further studies to remove selenium from the relevant wastewater. Introduction: One of the concerns of today's world is environmental pollution caused by heavy metals, and in this regard, the element selenium is very important due to its low amount in the environment. Bioremoval of metals is one of the cleanest and cheapest methods of biological absorption. The purpose of this research is to isolate and evaluate selenium-resistant strains for the bioremoval of this metal from aquatic environments. Materials and Methods: By isolating selenium-resistant strains, the bioabsorption rate of the selected types was investigated in different conditions of acidity, temperature, and biomass amount at different times. In between, two isolates were resistant to the concentration of 400 mM sodium selenite, which were identified after performing biochemical and genetic tests. Results: The highest absorption rate in terms of inoculated biomass was related to Thalassospira permensis and Bacillus thuringiensis at 4% in 20 and 60 minutes, respectively. The highest amount of absorption was obtained by Bacillus at an acidity of 5 during 40 minutes and by Thalassospira at an acidity of 7 and a time of 60 minutes. The optimal temperature for the two strains was 25 °C and it was found that Thalassospira permensis had the best effect in 20 minutes and Bacillus thuringiensis had the best effect in 40 minutes. In addition, Thalassospira permensis was the best choice for selenium bioabsorption by absorbing 95.1% of total selenium with 4% of the biological mass in a period of 20 minutes, at acidity equal to 6 and 25 °C. Discussion and Conclusion: Thalassospira permensis absorbed 95.1 mgL-1 of 100 mgL-1 of sodium selenite in the culture medium within 20 minutes by biomass 4%. Thus, it had a high bioabsorption rate and became a suitable candidate for further studies to remove selenium from the relevant wastewater.

https://bjm.ui.ac.ir/article_28326_f2f6903578407d0063e5b8d21500c5e9.pdf

University of Isfahan Journal of Microbial Biology 3060-7647 13 49 2024 06 16 Prevalence of Campylobacter Isolates from Native Animals Based on the Presence of Certain Virulence Genes Prevalence of Campylobacter Isolates from Native Animals Based on the Presence of Certain Virulence Genes 77 94 28386 10.22108/bjm.2024.140567.1583 FA Seyedeh Ommolbanin Ghasemian Department of Veterinary, Behbahan Branch, Islamic Azad University, Behbahan, Iran 0000-0002-4805-3553 Hamid Mahmoodipour Department of Nursing and Midwifery, Behbahan Branch, Islamic Azad University, Behbahan, Iran Journal Article 2024 02 01 This study aimed to evaluate the prevalence of Campylobacter isolates based on the presence of certain invasive genes. The experimental study tested 392 samples from Behbahan city. After culturing the samples, microbial identification was performed using Gram staining. Molecular identification of Campylobacter isolates was then conducted using PCR and suitable primers for invasive genes cdtA, B, C, cadF, pldA, ciaB, and 16S rRNA. According to the Gram staining results, 50 samples were infected with Campylobacter. Of these, 37 (74%) were chicken samples, 8 (16%) were cow samples, 2 (4%) were sheep and goat samples, and 3 (6%) were water samples. Analysis of the 16S rRNA gene sequence showed that 72% of the contamination was related to Campylobacter jejuni and 28% was related to Campylobacter coli. Overall, the prevalence of Campylobacter jejuni in isolated samples was significantly higher than that of general Campylobacter (p=0.012). Among the Campylobacter jejuni isolates, the highest prevalence was related to the CdtC toxin production gene (76.2%), and among the Campylobacter coli isolates, it was related to the CdtB toxin production gene (30.2%). Although chickens are considered the main sources of Campylobacter species, it is necessary to investigate the significance of other contamination sources, especially cattle and sheep, to assess their role in human infections. Therefore, necessary precautions must be taken for human consumption, and hygiene standards must be maintained in storage areas, along the slaughter line, and in stores. This study aimed to evaluate the prevalence of Campylobacter isolates based on the presence of certain invasive genes. The experimental study tested 392 samples from Behbahan city. After culturing the samples, microbial identification was performed using Gram staining. Molecular identification of Campylobacter isolates was then conducted using PCR and suitable primers for invasive genes cdtA, B, C, cadF, pldA, ciaB, and 16S rRNA. According to the Gram staining results, 50 samples were infected with Campylobacter. Of these, 37 (74%) were chicken samples, 8 (16%) were cow samples, 2 (4%) were sheep and goat samples, and 3 (6%) were water samples. Analysis of the 16S rRNA gene sequence showed that 72% of the contamination was related to Campylobacter jejuni and 28% was related to Campylobacter coli. Overall, the prevalence of Campylobacter jejuni in isolated samples was significantly higher than that of general Campylobacter (p=0.012). Among the Campylobacter jejuni isolates, the highest prevalence was related to the CdtC toxin production gene (76.2%), and among the Campylobacter coli isolates, it was related to the CdtB toxin production gene (30.2%). Although chickens are considered the main sources of Campylobacter species, it is necessary to investigate the significance of other contamination sources, especially cattle and sheep, to assess their role in human infections. Therefore, necessary precautions must be taken for human consumption, and hygiene standards must be maintained in storage areas, along the slaughter line, and in stores.

https://bjm.ui.ac.ir/article_28386_618ae5bb6fd357a00f8c5d9dcc8af106.pdf

University of Isfahan Journal of Microbial Biology 3060-7647 13 49 2024 03 20 Expression changes of vanA and vanB genes of Staphylococcus saprophyticus treated with secondary metabolites of Cyanobacterium Anabaena Expression changes of vanA and vanB genes of Staphylococcus saprophyticus treated with secondary metabolites of Cyanobacterium Anabaena 95 109 28404 10.22108/bjm.2024.140026.1572 FA Zahra Sadat Shobeiri Department of biology, Faculty of Basic Sciences, East Tehran Branch, Islamic Azad University, Tehran, Iran Elahe Asgari Department of biology, Faculty of Basic Sciences, East Tehran Branch, Islamic Azad University, Tehran, Iran Kumarss Amini Department of Microbiology, School of Basic Sciences, Saveh Branch, Islamic Azad University, Saveh, Iran. 0000-0002-6419-3417 Journal Article 2023 12 08 Herein we evaluate the effect of secondary metabolites from the cyanobacteria Anabaena sp. on the expression of vancomycin resistance genes in Staphylococcus Saprophyticus.. This study is motivated by the growing concerns of multi-drug resistant S. saprophyticus strains and the need for alternative antimicrobial agents. of During a two-month period, 120 clinical samples were collected from elderly patients referred to Tehran hospitals. S. saprophyticus isolates were identified using both phenotypic and chemical tests. Subsequently, molecular methods were employed to detect the presence of van genes. The microbroth dilution method used to determine the sensitivity of the bacterial isolates to the methanolic extract of Anabaena sp. . Finally, real-time PCR was employed to measure the expression level of van genes compared to the 16S rRNA gene in S. saprophytic isolates treated with the cyanobacterial secondary metabolite. Among the 83 bacterial isolates obtained from the clinical samples, only eight were identified as S. saprophyticus. One isolate harbored the vanA gene, while two isolates possessed the vanB gene. The minimum inhibitory concentration (MIC) of the cyanobacterial secondary metabolite against S. saprophyticus was 750 μg/mL, with a sub-MIC of 325 μg/mL. Importantly, the expression level of vanA and vanB genes in the strains treated with the cyanobacterial secondary metabolite down-regulated compared to the reference gene (16S rRNA). The findings suggest that the secondary metabolites from Anabaena sp. possess a potent antimicrobial effect and can potentially reduce the expression of antibiotic resistance genes in S. saprophyticus. This paves the way for the development of a new generation of anti-staphylococcal drugs. Herein we evaluate the effect of secondary metabolites from the cyanobacteria Anabaena sp. on the expression of vancomycin resistance genes in Staphylococcus Saprophyticus.. This study is motivated by the growing concerns of multi-drug resistant S. saprophyticus strains and the need for alternative antimicrobial agents. of During a two-month period, 120 clinical samples were collected from elderly patients referred to Tehran hospitals. S. saprophyticus isolates were identified using both phenotypic and chemical tests. Subsequently, molecular methods were employed to detect the presence of van genes. The microbroth dilution method used to determine the sensitivity of the bacterial isolates to the methanolic extract of Anabaena sp. . Finally, real-time PCR was employed to measure the expression level of van genes compared to the 16S rRNA gene in S. saprophytic isolates treated with the cyanobacterial secondary metabolite. Among the 83 bacterial isolates obtained from the clinical samples, only eight were identified as S. saprophyticus. One isolate harbored the vanA gene, while two isolates possessed the vanB gene. The minimum inhibitory concentration (MIC) of the cyanobacterial secondary metabolite against S. saprophyticus was 750 μg/mL, with a sub-MIC of 325 μg/mL. Importantly, the expression level of vanA and vanB genes in the strains treated with the cyanobacterial secondary metabolite down-regulated compared to the reference gene (16S rRNA). The findings suggest that the secondary metabolites from Anabaena sp. possess a potent antimicrobial effect and can potentially reduce the expression of antibiotic resistance genes in S. saprophyticus. This paves the way for the development of a new generation of anti-staphylococcal drugs.

https://bjm.ui.ac.ir/article_28404_1155f185dbcadbebe59518328c9cdd85.pdf

University of Isfahan Journal of Microbial Biology 3060-7647 13 49 2024 03 20 Isolation of a Moderately Halophilic Bacillus sp. G362 from Garab HotSpring in Northeastern Iran and Characterization of Thermostable, Broad Range, and pH-Stable of Its Endoglucanase Isolation of a Moderately Halophilic Bacillus sp. G362 from Garab HotSpring in Northeastern Iran and Characterization of Thermostable, Broad Range, and pH-Stable of Its Endoglucanase 111 129 27632 10.22108/bjm.2023.137002.1528 FA Nazanin Gholampour-faroji Biotechnology Department, Iranian Research Organization for Science and Technology (IROST), ‎Tehran, Iran Jafar Hemmat Biotechnology Department. Iranian Research Organization for Science and Technology (IROST) Tehran, Iran Aliakbar Haddad-Mashadrizeh Industrial Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran 0000-0002-4597-4103 Nazanin Kazeminejad Biotechnology Department, Iranian Research Organization for Science and Technology (IROST), ‎Tehran, Iran Journal Article 2023 03 01 The stability of enzymes to heat and pH is one of the most important determining factors in their application. In this research, achieving thermostable and pH-stable cellulase was aimed by screening in one of the Hot springs of Garab in northeast Iran. The samples collected from one of the Hot springs of Garab, in Razavi Khorasan province in Iran, were used to isolate, screen, and identify bacteria capable of producing thermostable cellulase. The cultures were cultured at 45°C and under aerobic conditions. The bacteria were isolated on a CMC agar medium. Initial screening of the isolates was done based on specific culture medium, salt tolerance, and temperature changes. Then, their additional screening was done by evaluating endoglucanase activity at different temperatures and pH using qualitative and quantitative methods. Finally, the molecular identification of the selected isolate was done by 16S rRNA gene sequencing. The results of this study, led to the introduction of a selected strain identified primarily as Bacillus sp. G362, a moderate halophile growing in 6%(w/v) salt concentration. It showed high endoglucanase stability at 55°C and high stability in a wide range of acidic and alkaline pH. The studied Hot spring, like some other Iranian Hot springs, has cellulase-producing bacteria, which Bacillus sp. G362 is capable of producing endoglucanase showing the thermostablity and pH stability at a wide range of alkaline and Acidic pH. In addition, the specific endoglucanase gene of G362 Bacillus sp. can be identified through molecular methods and used for further research including structural studies. It may even be used as a protein template in protein engineering. The stability of enzymes to heat and pH is one of the most important determining factors in their application. In this research, achieving thermostable and pH-stable cellulase was aimed by screening in one of the Hot springs of Garab in northeast Iran. The samples collected from one of the Hot springs of Garab, in Razavi Khorasan province in Iran, were used to isolate, screen, and identify bacteria capable of producing thermostable cellulase. The cultures were cultured at 45°C and under aerobic conditions. The bacteria were isolated on a CMC agar medium. Initial screening of the isolates was done based on specific culture medium, salt tolerance, and temperature changes. Then, their additional screening was done by evaluating endoglucanase activity at different temperatures and pH using qualitative and quantitative methods. Finally, the molecular identification of the selected isolate was done by 16S rRNA gene sequencing. The results of this study, led to the introduction of a selected strain identified primarily as Bacillus sp. G362, a moderate halophile growing in 6%(w/v) salt concentration. It showed high endoglucanase stability at 55°C and high stability in a wide range of acidic and alkaline pH. The studied Hot spring, like some other Iranian Hot springs, has cellulase-producing bacteria, which Bacillus sp. G362 is capable of producing endoglucanase showing the thermostablity and pH stability at a wide range of alkaline and Acidic pH. In addition, the specific endoglucanase gene of G362 Bacillus sp. can be identified through molecular methods and used for further research including structural studies. It may even be used as a protein template in protein engineering.

https://bjm.ui.ac.ir/article_27632_46dccd3f81b91ddbe999ddc475e19d8b.pdf

University of Isfahan Journal of Microbial Biology 3060-7647 13 49 2024 03 20 Comparision of Rapid Detection of E. coli by Metallic Nanoparticles and Carbon Nanotubes Comparision of Rapid Detection of E. coli by Metallic Nanoparticles and Carbon Nanotubes 131 158 27740 10.22108/bjm.2023.136296.1517 FA Rafieh Merat Haghi Department of Microbiology, Faculty of Basic Sciences, Lahijan Branch, Islamic Azad University, Lahijan, Iran Khosro Issazadeh Islamic Azad university, Lahijan Branch, Faculty of Basic Sciences, Department of Microbiology , Lahijan, Iran Ali Abdolahzadeh Ziabari Nano Research Lab, Lahijan Branch, Islamic Azad University. Lahijan, Iran 0000-0001-6229-5789 Mohammad Faezi Ghasemi Department of Microbiology, Faculty of Basic Sciences, Lahijan Branch , Islamic Azad University, Lahijan, Iran Journal Article 2023 01 02 The development of rapid and highly sensitive diagnostic methods has received significant attention. Enzymatic nanobiosensors are a promising approach, offering fast results based on specific interactions between nanoparticles, enzyme, and target molecules. This study investigated the potential of ZnO nanoparticles, SnO, and multiwall carbon nanotubes (MWCNTs) as marker molecules in a system using beta-galactosidase enzyme for bacterial detection. Following nanoparticle synthesis, their structure was confirmed using XRD, FTIR, UV-DRS analyses. Enzyme activity was evaluated in the presence of the substrate ONPG. Different nanoparticle concentrations were tested with a constant amount of enzyme to determine the optimal concentration for inhibiting lactase activity. Subsequently, varying concentrations of Escherichia coli were introduced, and the ability of each nanoparticle type to detect bacteria was compared. Due to their small size, high surface area, and strong reactivity, these nanomaterials demonstrated the potential to detect low bacterial concentrations in the environment.Bacteria likely attached to the nanoparticles, hindering their intraction with the enzyme and consequently affecting enzyme activity. The results revealed MWCNTs to be most effective for bacterial identification, detecting as low as 10 CFU/mL bacteria within 15 min. This approach has promising applications in the food industry for rapid detection of low-level bacterial contamination. The development of rapid and highly sensitive diagnostic methods has received significant attention. Enzymatic nanobiosensors are a promising approach, offering fast results based on specific interactions between nanoparticles, enzyme, and target molecules. This study investigated the potential of ZnO nanoparticles, SnO, and multiwall carbon nanotubes (MWCNTs) as marker molecules in a system using beta-galactosidase enzyme for bacterial detection. Following nanoparticle synthesis, their structure was confirmed using XRD, FTIR, UV-DRS analyses. Enzyme activity was evaluated in the presence of the substrate ONPG. Different nanoparticle concentrations were tested with a constant amount of enzyme to determine the optimal concentration for inhibiting lactase activity. Subsequently, varying concentrations of Escherichia coli were introduced, and the ability of each nanoparticle type to detect bacteria was compared. Due to their small size, high surface area, and strong reactivity, these nanomaterials demonstrated the potential to detect low bacterial concentrations in the environment.Bacteria likely attached to the nanoparticles, hindering their intraction with the enzyme and consequently affecting enzyme activity. The results revealed MWCNTs to be most effective for bacterial identification, detecting as low as 10 CFU/mL bacteria within 15 min. This approach has promising applications in the food industry for rapid detection of low-level bacterial contamination.

https://bjm.ui.ac.ir/article_27740_1c9e7cf7ed189563a3a53e55b84c872a.pdf