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Environmental Microbiology (EM) | Microbial Ecology and Diversity
Microbiol. Biotechnol. Lett. 2022; 50(2): 255-269
Mohamed E. Osman1, Amany A. Abo El-nasr1, Hagar M Hussein1, and Moaz M Hamed2*
1Department of Botany and Microbiology, Faculty of Science, Helwan University, Cairo 11795, Egypt
2National Institute of Oceanography and Fisheries (NIOF), 84511, Egypt
Correspondence to :
Moaz M. Hamed, Moazfirstname.lastname@example.org
Actinomycetes isolated from marine habitats represent a promising source of bioactive substances. Here, we report on the isolation, identification, productivity enhancement and application of the bioactive compounds of Streptomyces qinglanensis H4. Eighteen marine actinomycetes were isolated and tested for resistance to seven bacterial diseases. Using 16S rRNA sequencing analysis (GenBank accession number MW563772), the most powerful isolate was identified as S. qinglanensis. Although the strain produced active compound(s) against a number of Gram-negative and Gram-positive bacteria, it failed to inhibit pathogenic fungi. The obtained inhibition zones were 22.0 ± 1.5, 20.0 ± 1, 16.0 ± 1, 12.0 ± 1, 22.0 ± 1 and 24.0 ± 1 mm against Bacillus subtilis ATCC 6633, Escherichia coli ATCC 19404, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231 and Staphylococcus aureus ATCC6538, respectively. To maximize bioactive compound synthesis, the Plackett-Burman design was used. The productivity increased up to 0.93-fold, when S. qinglanensis was grown in optimized medium composed of: (g/l) starch 30; KNO3 0.5; K2HPO4 0.25; MgSO4 0.25; FeSO4·7H2O, 0.01; sea water concentration (%) 100; pH 8.0, and an incubation period of 9 days. Moreover, the anticancer activity of S. qinglanensis was tested against two different cell lines: HepG2 and CACO. The inhibition activities were 42.96 and 57.14%, respectively. Our findings suggest that the marine S. qinglanensis strain, which grows well on tailored medium, might be a source of bioactive substances for healthcare companies.
Keywords: Red Sea, actinomycetes, ant-bacterial, anti-cancer
As one of Egypt's most magnificent coastal and marine habitats, the Red Sea has an abundance of wildlife. Numerous fish species and diverse microbial communities inhabit the sea's distinctive ecosystem, which includes seagrass beds and salt marshes as well as mangrove swamps and coral reefs . Actinomycetes are found in a wide range of aquatic and terrestrial environments, where they create a variety of bioactive constituents [2, 3]. A wide variety of marine animals, including corals, sponges, and jellyfish, belong to this phylum . Bioactive compounds found in marine ecosystems are produced mostly by actinomycetes .
There are numerous possible bioactivities of these substances isolated from marine actinomycetes, including antibacterial, antifungal, and ant parasitic, as well as antioxidant and immunomodulatory activities [6, 7].
The only option to solve this issue is to continue the hunt for new antibiotics, particularly in environments that have received less attention. Microorganisms originating from many marine sources provide potential antibacterial compounds that might be utilized to treat numerous infectious ailments and could eventually replace conventional medications . As the number of new bioactive compounds discovered by terrestrial actinomycetes decreases, a substantial amount of study has been focused on screening actinomycetes from various environments for their capacity to produce new secondary metabolites. Actinomycetes isolated from the marine are metabolically active and have adapted to life in the sea, according to researches .
Microorganisms' biosynthesis of secondary metabolites varies with the environment under which they thrive. Because of this, microbial isolates could produce more bioactive chemicals if nutritional and physical parameters were altered throughout the incubation phase. Surface approach and the Plackett-Burman experimental design are employed to construct the nutritional circumstances, and by optimizing all impacting parameters together, the feasibility of a single-factor optimization is removed .
These marine actinomycetes were isolated from the shallow Red Sea, Egypt and tested for their antibacterial efficacy against a variety of pathogenic microbes in this investigation. Plackett-Burman experimental design was used to establish the ideal culture conditions for maximum antibacterial agent production by the most effective isolate. In addition, these active compounds will be tested for their anti-cancer and antioxidant properties.
Sediment samples (10 samples) were collected from the shallow areas of the Red Sea during summer, 2019. Five locations were selected for the present study included Hurghada city, Safaga city, Mangrove area, Al- Quseir city and Marsa allam city. They are distributed along the Red Sea as shown in (Fig. 1). The Surfer programmed version 15.2.305 was used at the Marine Physics Laboratory (NIOF) in the Red Sea to get the coordinates and map work. Samples were collected at ~10 m depths below the water surface and kept at 4℃ for further working up .
Samples of 0.1 ml were spread on the starch nitrate agar plates (Starch, 20 g; KNO3, 1 g; K2HPO4, 0.5 g; MgSO4·7H2O, 0.5 g; FeSO4·7H2O, 0.01 g; Agar, 20 g/l) supplemented with tetracycline 50 μg/ml and 50 μg/ml nystatin, that help prevent other bacteria and fungi from growing. (After adjusting the pH of the medium to 7−7.2 and sterilized it in an autoclave at 121℃ for 15 min, it was cultured at 28℃ for 7−14 days until actinomycetes colonies grew). The plates were inspected after the incubation period for characteristic actinomycetes colonies, which were spherical, thin, opaque, compact, and sometimes colored with white, brown, gray-pink, or other colors. The isolated actinomycetes were re-cultured for further studies and stored at 4℃ .
The used bacterial indicators were
By using a well diffusion technique assay on (Mueller-Hinton agar), the acquired isolates were tested for antibacterial activity against pathogenic test pathogens. The isolates were cultivated on starch nitrate agar for 7 days at 30 ± 2℃ before being sliced into a disc of 8.0 mm diameter and aseptically placed into 250 ml Erlenmeyer flasks containing 50 ml of sterile starch nitrate broth medium. For seven days, the inoculated flasks were incubated on a rotary shaker at 30 ± 2℃ and 180 rpm. In order to sterilize the cultures, they were first filtered through Whatman filter paper No. 1 and then sterilized by filtration. A sterile cork borer made 8.0 mm holes in test organism-seeded plates. (In order to conduct the bioassay, reference microorganisms were first suspended in the appropriate liquid medium, and then the turbidity of the suspension was brought up to the 0.5 McFarland standard (OD625 = 0.08−0.13), 100 microliters of each filtrate was aseptically placed into each hole and incubated at 36 ± 2℃ for one day. The size of the inhibitory zone surrounding holes in millimeters was used to determine antagonism .
The fungal isolates were growing in Yeast extract peptone dextrose agar media (YEPD): (Peptone 20 g; Yeast extract; Dextrose 20 g and distilled water 1000 ml) at 25℃ for 7 days then, inoculums containing 105 spores/ cells ml-1 of fungi were prepared by harvesting spores from slants. For solidification media were add 1.5% (w/v) agar was added to the broth medium. Into plates that have been sterilized a soft agar medium chilled to 45℃ was poured over 100 ul of the indicator strain suspension and stirred. A culture supernatant (100 μl) was added into each well with a concentration of 100 mg ml-1. After incubation, the antifungal activities were determined by measuring the diameter of the inhibition zones (mm) .
Phenotypic characterization. The strain H4's morphological, biochemical, and physiological properties were explored in detail. Different NaCl concentrations (0− 13%), pH values (5−12), and temperatures (25−50℃) were used to cultivate the selected isolate, and the color of aerial mycelia, generation of diffusible pigment, as well as carbon source utilization, were then evaluated .
Genotypic characterization. The promising actinomycetes isolate was grown for seven days in a starch-nitrate liquid medium, and genomic DNAs were extracted using the Gene Jet genomic DNA purification Kit's genomic DNA extraction technique (Fermentas). PCR using Maxima Hot Start PCR Master Mix (Fermentas). Thermal cycler amplifications (Multigene Optimax, Labnet international, Inc.). This is how the PCR thermocycler was set up: 95℃ for 5 min to start denaturing, then 30 cycles of 95℃ for 1 min, 55℃ for 1 min, 72℃ for 2 min, and 72℃ for 10 min to finish extending. 50 L of 10X Standard Taq Reaction Buffer comprised 25 pmol of each primer, 10 ngchromosomal DNA, 200 mmol/LdNTPs, and 2.5 U of Taq Polymerase. Gene JETTM PCR Purification Kit (Fermentas) was utilized by Sigma Scientific Services Company in Egypt. GATC Company used ABI 3730xl DNA sequence with universal primers (16S 27F and 16S1492R), (5′AGAGTTTGATCCTGGCTCAG-3′ and 5′- GGTTACCTTGTTACGACTT-3′) to sequence the PCR product. 16S sequencing analysis was used to characterize the genotype. BioEdit was used to perform multiple alignments with sequences of the most closely related individuals and calculate levels of sequence similarity (software version 7). The National Center for Biotechnology Information (NCBI) database was used to collect rRNA gene sequences for comparison.
Electron microscopy study. The isolate of chosen actinomycetes was cultivated for 14 days at 30−32℃ on starch nitrate agar medium. An osmium tetroxide specimen was fixed in glutaraldehyde (2.5% v/v), washed, and then post-fixed in 1 percent w/v osmium tetroxide for one hour. The material was rinsed twice with water and dehydrated with ethanol (30−100%, v/v) in increasing concentrations. A scanning electron microscope (Jeol JSM 5400 LV, Japan) was used at Assiut University in Egypt to investigate the sample at 15−20 kV.
Plackett-burman design. The Plackett-Burman experimental design was used to assess the relative relevance of several parameters involved in the generation of antibiotic metabolites by the selected actinomycetes isolates. Table 1 shows the independent variables and their settings that were tested in this experiment. Table 2 shows the seven variables that were used. For each experiment, the rows in Table 3 show the number of participants and each column represents a specific variable. A high (+) or low (-) concentration was investigated for each nutritional variable. The main effect of each variable was determined with the following equation:
Mi+ and Mi- are the diameters of the clear zones around each well in the trials, where Exi is the variable main effect. The independent variable (xi) was present in both the high and low concentrations, and N is the total number of trials divided by two. Calculations of statistical t-values for equal samples of unpaired data were carried out in Microsoft Excel  determining the variable importance with Microsoft Excel An optimum medium was projected based on the primary effect data.
Verification experiment. The anticipated optimal values of the independent variables were checked and compared to the basal conditions setting in a verification experiment, and the average output of secondary metabolites was computed.
Effect of different temperature. Incubation temperatures of 25, 30, 35, and 40℃ were tested to find the perfect temperature. An orbital shaker at 150 rpm was used for 9 days to inoculate the isolated actinomycetes in the optimum medium, pH 8.0. After incubation, the antimicrobial agents tested against selected pathogen by well diffusion method .
Effect of salinity. By adding NaCl to selected medium (as indicated above), varying salinity concentrations (3, 6, 9, 12, and 15%) were achieved. The chosen isolate was injected and incubated for 9 days at 35℃. The extracts were produced and evaluated for antibacterial activity after incubation .
The most powerful isolates were chosen based on the main screening and cultured in customized starch nitrate broth as a production medium for crude compunds extraction. Furthermore, the chosen actinomycetes isolate were individually inoculated in starch nitrate broth and cultured for 9 days at 35℃ in a shaker incubator. After centrifuging the broth with actinomycetes for 20 min at 10,000 rpm, the supernatant was collected and mixed with ethyl acetate. After that, 1:1 (v/v) ethyl acetate was added to the filtrate and incubated in a shaker for 1 hour for full extraction. The bioactive substances were isolated from the aqueous phase after incubation. Finally, the isolated crude substances were dried using a heating mantle at 40℃ to produce dry powder .
Anticancer activity. First, the toxicity of culture filtrates was tested and evaluated against two cell lines: Hepatocellular carcinoma cells (HepG-2) and Intestinal carcinoma cells (CACO).
Cell line propagation. Cells were grown in DMEM with 10% heat-inactivated fetal bovine serum, 10% L-glutamine, HEPES buffer, and 50 g/ml gentamycin. All cells were kept at 37℃ in 5% CO2 and sub cultured twice a week. The effects of the test materials on cell morphology and viability were used to track the level of cell toxicity that was present.
Cytotoxicity evaluation using viability assay. For cytotoxicity assay, the cells were seeded in 96 well plate at a cell concentration of 1 × 104 cells per well in 100 μl of growth medium. After 24 h after seeding, fresh medium containing varied concentrations of the test sample was added. A multichannel pipette was used to administer serial two-fold dilutions of the tested chemical component to confluent cell monolayers distributed into 96 well, flat-bottomed microtiter plates (Falcon, USA). The microtiter plates were incubated for 48 h at 37℃ in a humidified atmosphere with 5% CO2. Each concentration of test sample was divided into three wells. Control cells were treated with or without DMSO in the absence of the test substance. The experiment was shown to be unaffected by the little amount of DMSO contained in the wells (maximum 0.1 percent). After incubation of cells at 37℃ for 24 h, various doses of sample were applied and the viable cell yield was evaluated using the acoloinetric technique. After the incubation period was completed, the medium was aspirated and the crystal violet solution (1%) was applied to each well for at least 30 min. The stain was removed, and the plates were cleaned with tap water to eliminate any remaining discoloration. The absorbance of the plates was measured after gently shaking on a microplate reader (TECAN, ink) using a test wavelength of 490 nm after glacial acetic acid (30%) was added to all wells and properly mixed. Background absorbance measured in wells without additional stain was used to adjust all findings. In the absence of the tested drug, treated samples were compared to cell controls. All of the experiments were done three times. Each compound's cytotoxic impact on cells was determined .
Determination of anti-MRSA activity. Anti-MRSA activity was tested by agar diffusion method against Methicillin-Resistant
Anti-inflammatory activity. The model of Dai and Liu was followed to evaluate membrane stabilization test in mice using the actinomycetes crude extract . The blood of rats (obtained with heparinized syringes through cardiac puncture) was washed three times with isotonic buffered solution (154 mM NaCl) in 10 mM sodium phosphate buffer (pH 7.4) and centrifuged for 10 min at 3000 g. Consisting of ninety six well plate, membrane stabilizing activity of the samples was assessed using hypotonic solution-induced erythrocyte hemolysis. The test sample include 0.5 ml of stock erythrocyte (RBCs) suspension mixed with 5 ml of hypotonic solution (50 mM NaCl) in 10 mM sodium phosphate buffered saline and actinomycetes crude extract in concentration range from 7.81 μg/ml to 1000 μg/ml in pH 7.4. Control sample consisted of 0.5 ml RBC mixed with hypotonic buffered saline solution. The table of actinomycetes concentration is described in Table 3.
After the sample inoculation, the mixtures were incubated for 10 min at room temperature and centrifuged for 10 min at 3000 g. the result were obtained by measuring the absorbance at 540 nm. The percentage of membrane stabilization was calculated according the following equation:
The IC50 value was defined as the concentration of the sample to inhibit 50% RBCs hemolysis under the assay conditions.
Ten sediment samples were obtained from the Red Sea at various places for this investigation. From the sediment samples, eighteen actinomycetes strains were recovered. The eighteen identified marine actinomycetes isolates showed antagonistic activity against the tested bacterial pathogens after being screened for antagonistic activity (Table 4). Seven isolates (39%) were active only against one bacterial pathogen, five isolates (27%) of actinomycetes isolates were capable of inhibiting two bacterial pathogens. Four isolates (22%) were active against three bacterial pathogens. Most promising candidate isolate H4 exhibited good activities against six bacterial pathogens. The inhibition zones ranged from 12 mm against
Extensive biochemical and physiological analysis of the isolate H4 was conducted to provide better knowledge and supplement the phylogenetic analysis. Phenotypic characters of isolate H4 were studied. It had yellow substrate mycelium with white aerial mycelium. It produced beige diffusible pigments. It grown at 25−32℃ and there is no grown at 40−50℃ also, its growth at pH 5−9 and tolerated up to 10% of NaCl. It utilized starch, lactose, dextrose, maltose and glycerol as carbon sources. The organism induced degradation of methyl red, gelatinase, protease, nitrate reductase and catalase. It can be hydrolyzed arginine dihydrolase, but negative for urease test, Voges-Proskaure test, indole test and sulphide production. Positive for citrate production and aesculin hydrolysis as shown in (Table 5), Fig. 2 illustrates the spore formation under scanning electron microscope.
Based on the obtained results, actinomycetes isolate H4 was selected for identification and molecular phylogenetic analysis. The amplicons generated by the chosen isolate were identified using agarose gel electrophoresis, as shown in Fig. 3. This methodology consisted 1500 base pair sequencing data (ABI 3730xl). As shown in (Fig. 4). This sequence was matched with other actinomycete sequences in the database to assess its phylogenetic connection to other actinomycete sequences. The 16S rRNA gene sequence analysis of isolate H4 was compared to those with the highest homology using the computer-based Blast search software. The resulting data indicated that, the isolate under study was similar to
Using a Plackett-Burman design, we evaluated the influence of starch nitrate medium on the synthesis of antimicrobial agents from
Based on these findings, the metabolites were supported by a positive (+) level of starch and sea water concentration, as well as a negative (-) level of KNO3 and K2HPO4. Furthermore, the t-value in Table 8 confirms this conclusion. This method validated the design that was used. A verification experiment was used to compare the basal and optimized mediums. Based on the Plackett-Burman results, the interaction effects of starch with K2HPO4 and sea water on the inhibition zone width of the generated antimicrobial agent(s) are depicted in three-dimensional graphs (Figs. 7A and B).
Verification experiment. According to the data obtained, a near optimum production medium was formulated as follows: (g/l) Starch 30; KNO3 0.5; K2HPO4 0.25; MgSO4 0.25; FeSO4·7H2O, 0.01; Sea water concentration (%) 100; pH 8 and the incubation period 9 days. A verification experiment was applied to compare the predicted optimum levels of the independent variables and the basal conditions. Cultivation of
Temperature effects on the growth of the isolate
The suitable concentration of NaCl for the production of antimicrobial compounds ranges 6−12 g/100 ml, the highest antimicrobial activity was observed at 9 g/100 ml.
Bioactive compounds production from
Mean inhibition zone in millimeter’s (mm) beyond the diameter of the well (6 mm) for a variety of pathogenic microorganisms. Results showed that, the active substances production from
Aspirin and actinomycetes extracts significantly inhibited the membrane stabilization for blood of rats. Aspirin used for comparison the activity of actinomycetes extract. The IC50 for hemolysis rate in actinomycetes was 47.6 which are higher than the aspirin (17.02). Actinomycetes are one of the more potent releasers of rat’s membrane stabilization and other mediators of inflammation compared to the aspirin extract. The result of the anti-inflammatory test demonstrated in (Table 11) and (Fig. 11). The figure and table showing that, actinomycetes extract was having gradually decreased in membrane stability percentage and reached to 61.63 percentages in concentration 125, unlike the aspirin extract which reached to 72.35% in the same concentration. The confirmation of anti-inflammatory test is calculated by IC50 range. Actinomycetes recorded the highest IC50 with 47.6.
As a tropical sea, the Red Sea has a distinct marine ecology with high salinity, high temperature, and great microbiological diversity . However, few studies reported the isolation of actinomycetes from Red Sea sediments [10, 24]. A good number of marine actinomycete isolates were obtained from sediment samples, and they were eighteen pure isolates. Marine sediments are a good and inspiring source for isolating actinomycetes. Marine sediments are a valuable source of novel actinomycetes with the potential to develop new bioactive compounds, as per literature [25, 26]. According to Sarika, primary antimicrobial activity screening was performed , by using well cut diffusion technique against previous bacterial and fungal pathogens. Examination of the antagonistic activity of the isolates obtained in this study revealed the presence of many marine isolates, which have the ability to inhibit the growth of pathogenic bacterial isolates. Through the previous table, it was noted that isolate No. H4 had a good ability to inhibit the growth of both
The current experiment's results validated the bioactive potential of marine actinomycetes strains. According to Patel, 79% of the actinomycetes isolates tested positive for at least one of the 18 pathogens tested . Forty one percent of the marine actinomycetes isolates were active against the tested pathogens . In vitro antibacterial activity of test isolates of marine actinomycetes isolates was shown to be particularly powerful against both phytopathogenic and other Gram +ve and Gram –ve bacteria in previous research [10, 29-31]. Five new compounds 15R-17,18-dehydroxantholipin (1), (3E,5E,7E)-3-methyldeca-3,5,7-triene-2,9-dione (2) and qinlactone A-C (3-5) were identified from mangrove
A combination of physiological parameters, phenotypic characterization, 16S rRNA gene sequences, and phylogenetic analysis (Fig. 6) revealed that the closest relatives of study isolate H4 are
In a multivariable scheme, statistical experimental designs are the most effective strategies for quickly identifying significant variables, and for reducing errors in assessing the impact of diverse factors [8, 33, 34]. We evaluated the influence of starch nitrate medium on the synthesis of antimicrobial agents from
Based on the data obtained from the statistical model and all calculations related to this experimental design, a condition close to optimum was suggested. This included a medium with the following composition: (g/l) Starch 30; KNO3 0.5; K2HPO4 0.25; MgSO4 0.25; FeSO4·7H2O, 0.01; concentration of sea water (%) 100; pH 8 and 9 days of incubation period. When compared to the basal medium, a verification experiment corroborated the acquired results and predicted near-optimum conditions, resulting in a 0.93-fold increase in antibacterial agents production of
The current results in this study are consistent with many recent studies that used statistical methods, especially Plackett-Burman design, to improve the production of active agents from marine actinomycetes. The statistical optimization using Plackett Burman design increased 1.3-fold bioactive compounds production by
In another step to improve the verified medium and obtain the best metabolic product of effective importance against pathogenic bacteria, the best temperature for incubation process was tested. The best temperature obtained was 35℃. Our findings are consistent with those of Aliero and his colleagues, who found that the minimum temperature for antagonistic activity generation from three actinomycete isolates (RF, M6 and SP) was 25℃, with the highest temperature being 35℃ . Increased productivity and increased generation of the antagonistic substance resulted from raising the incubation temperature . According to El-Sersy and Abou-elela (2006), a marine actinomycetes strain,
In another step to study the best concentrations of NaCl and after testing many of the concentrations used with verified medium, was 9% of NaCl concentration the best for the production of antimicrobial agents against
As a result, researchers working on nature products have done extensive work on the urgent need for new cancer medicines with new action modes and lower side effects, particularly in the area of marine re-sources. Natural compounds derived from marine microbial have been demonstrated to exhibit promise in vitro and in vivo anticancer efficacy with different tumor cell mechanisms of action . Actinomycetes secondary metabolites, especially
In the present study, the crude extract of
In the present investigation, the confirmation of antiinflammatory test is calculated by IC50 range.
A good source for desirable species to explore is the marine environment. The objective of this study was to explore our natural habitats and look for actinomycetes which produce bioactive compounds.
The authors have no financial conflicts of interest to declare.
Moaz M. Hamed*, Mohamed A.A. Abdrabo, and Asmaa M. YoussifMicrobiol. Biotechnol. Lett. 2021; 49(3): 356-366 https://doi.org/10.48022/mbl.2106.06007
Oh-Sung Kwon , Hae-Ryong Park , Bong-Sik Yun , Ji-Hwan Hwang , Jae-Chan Lee , Dong-Jin Park and Chang-Jin KimMicrobiol. Biotechnol. Lett. 2006; 34(4): 306-310 https://doi.org/10.4014/mbl.2006.34.4.306
박현주, 명지선, 박남실, 한규범, 김상년, 김응수Microbiol. Biotechnol. Lett. 2004; 32(3): 282-285 https://doi.org/10.4014/mbl.2004.32.3.282