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Screening and the Production of Myxopyronin A Antibiotic from different Soil Environments in Egypt

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01 May 2024

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07 May 2024

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Abstract
Background: Antibiotic resistance is an overwhelming serious difficulty globally. This necessitates the exploration of novel sources of antibiotics to overcome this challenge. Aim of the study: Bacterial Myxopyronin A production from various soil environments Egypt; as well as testing its antimicrobial activity in preclinical animal testing and randomized human clinical trials phases 1/2. Type of the study: Screening experimental study. Methodology: Different soil environments in Egypt were screened for the growth of bacterial isolates producing Myxopyronin A as an antimicrobial agent. Purification of Myxopyronin A was performed via reversed-phase HPLC. Paper disc diffusion assay, as well as broth dilution technique, were exploited to assess the invitro antimicrobial and minimum inhibitory concentration( MIC) of the test antibiotic. Furthermore, in vivo antimicrobial spectrum, adverse drug reactions, and pharmacokinetics were detected during animal model testing stages and human randomized clinical trials phases 1/2. Results: From the culture supernatant of the Myxobacterium Myxococcus fulvus 124B02 which was the predominant soil bacterial isolate grown on the Casein yeast peptone plate, Myxopyronin A was produced. The test antibiotic blocked the growth of many Gram +ve bacteria with MICs less than 100 mcg/ ml; whereas it inhibited the growth of a few Gram -ve bacteria such as Escherichia coli at MICs greater than 100 mcg/ ml. On the other hand, Eukaryotic cells such as fungal and human cells were not affected. Prokaryotic DNA-dependant-RNA polymerase( RNLP) was noticed to be inhibited via the test antibiotic suggesting its bactericidal action. Cmax was 7-8 mcg/ ml at Tmax 2 hours when 600 mg dose was orally administered in randomized human clinical trials phases 1/2; as well as T1/2 reached 2.5 hours following first-order kinetics of elimination. The duration of its action was nearly 12 hours after oral administration. Rare toxicity was detected during preclinical and randomized human clinical trials phases 1/2 in the form of mild diarrhea and cholestatic jaundice in less than 5 % of experimental candidates. Conclusion: The present study was promising due to the production of the bactericidal antibiotic Myxopyronin A from Myxococcus fulvus 124B02 isolated from different soil environments in Egypt.
Keywords: 
Subject: Medicine and Pharmacology  -   Medicine and Pharmacology

Introduction

An antibiotic needs to show selective toxicity in order to be therapeutically beneficial [1]. It must greatly suppress the activities of bacteria in comparison to those of human cells [2]. The hunt for new sources of antibiotics is necessary because antibiotic resistance is a significant, overwhelming problem that affects the entire planet [3]. The four primary targets of antibacterial medications are nucleic acids, cell walls, ribosomes, and cell membranes [4]. Since human cells lack a cell wall and contain distinct ribosomes, nucleic acid enzymes, and sterols in their membranes, these medications have no effect on human cells [5]. Bactericidal medications are well known to kill bacteria [6]. Bacteriostatic medications, on the other hand, stop germs from growing [7]. Bacteriostatic medications rely on the patient’s phagocytes to eradicate the pathogen [8].
Bacteriocidal medications should be administered to a patient if their neutrophil count is low [9]. Myxopyronins are a category of alpha-pyrone antibiotics [10]. A family of heterocyclic chemical compounds is known as pyrones or pyranones [11]. One oxygen atom and a ketone functional group are present in an unsaturated six-membered ring that they possess [12]. 2-pyrone and 4-pyrone are the names of the two isomers [13]. Since Myxopyronins do not cross-resistance with any other medicine, they might help address the increasing issue of drug resistance in TB [14]. Methicillin-resistant Staphylococcus aureus( MRSA) may also benefit from their therapy [15]. The current study’s objective was to evaluate the synthesis of Myxopyronin A, a new antibiotic, in several Egyptian soil environments. In phase 1/2 randomized human clinical studies, it was also tested for antibacterial activity.

Patients and Methods

Ethical statement:
All relevant institutional, national, and/or worldwide guidelines for the care and use of humans and animals were prioritized in the current survey. The Ethical Committee for Human and Animal Handling at Cairo University( ECAHCU), at the Faculty of Pharmacy, Cairo University, Egypt, approved all study procedures involving humans and animals by the recommendations of the Weatherall Report( approval number T-11-3-2023). The number of humans and animals included in the study, as well as their suffering, was minimized at all costs. The randomized human clinical trials phase 1/2 registration number was NCT00000714/ 2023.
Type of the study:
Screening experimental study.
Place and date of the study:
This study was finished at faculty of pharmacy, Cairo university, Egypt between March 2023 and January 2024.
Source of animal models:
They were obtained and legalized from the pharmacology and toxicology department of the faculty of pharmacy, Cairo university, Egypt.
Inclusion criteria for animal models:
Adult male obese rabbit animal models weighing about 2 kg; can be inoculated with different bacterial infections. Rabbits were acclimatized for one week before the experiment. At a humidity( 50 % ±5), light-dark cycle( 12/ 12 h), and a controlled temperature( 25±2 0C). The rabbits were fed with fresh grass.
Exclusion criteria for animal models:
Young and female rabbits; Non-obese rabbits weighing less than 2 kg.
Collection of 100 soil samples:
The samples were randomly selected grassland soils taken from various soil settings in Egypt at a depth of 30 cm. Before being processed, samples were kept at 4 ℃ in sterile containers. Each soil sample was weighed at one gram, and each 250 ml Erlenmeyer flask had 99 ml of sterile distilled water. The flasks were shaken at 400 rpm using a gyrator shaker for five minutes. Following dilutions from 10-1 to 10-6 in sterile distilled water, the soil suspensions were plated on selective Casein yeast peptone agar( CYP) medium( bought from Sigma-Aldrich, USA). 50 cc of nutrient broth liquid at PH 7 was added to 250 ml Erlenmeyer flasks to create the inoculum for the bacterial isolate under investigation.
The medium was autoclaved and then infected with a loopful of culture from a nutritional agar slant that had been left overnight. The inoculum was the inoculated flasks, which were shaken for a whole day at 150 rpm.
Instruments
Table 1. List of instruments.
Table 1. List of instruments.
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Material:
All chemical and biochemical substances were purchased from Algomhuria pharmaceutical company and Alnasr chemical company, Egypt. All chemical reagents used were of analytical grade.
Isolation of Myxococcus fulvus 124B02 producing Myxopyronin antibiotics:
The selective isolation of species of Myxococcus fulvus 124B02 from different soil samples was directly achieved using dilution plating. The technique comprised the suppression of competing bacteria exploiting antibiotics such as 10 mcg/ ml Vancomycin and/ or 10 mcg/ ml Chloramphenicol combined with wet heat treatment of soils and air drying. Fungi were eliminated via supplementing the plating medium with 2 mcg/ ml Terbinafine HCl. Swarming of Myxococcus fulvus 124B02 colonies was controlled with Casein Yeast Peptone( CYP) plates incubated at 30℃ and PH 7.2 for 5 days. The composition of CYP plate included 0.4 % Peptone from Casein, tryptically digested, 0.3 % CaCl2.2H2O, 0.1 % MgSO4.7H2O, PH 7.2. The potent bacterial isolate producing Myxopyronin was performed utilizing 16 S rRNA sequencing technique. The predominant bacterial isolate with high antibacterial activity was identified using 16S rRNA sequencing and other biochemical tests. Nucleic acid was extracted from a swab by bead-beating in a buffered solution containing Phenol, Chloroform and Isoamyl alcohol. Variable region of 16S rRNA gene was then amplified from the resulting nucleic acid using PCR. The genomic DNA was extracted from 120 hours cultured cells using a DNA purification kit[ PurreLinkTM Genomic DNA Mini Kit with Catalog number: K182002 was purchased from Invitrogen, USA] according to the protocol provided by the manufacturer of DNA purification kit. The 16S rRNA gene was amplified by PCR[ PCR SuperMix kit was purchased from Invitrogen,USA] using forward[ 5-AGAGTTTGATCCTGGCTCAG-3-] and reverse[ 5-GGTTACCTTGTTACGACTT-3-] primers. PCR amplicons from up to hundreds of samples were then combined and sequenced on a single run. The resulting sequences were matched to a reference database to determine relative bacterial abundances. Polymerase Chain Reaction ( PCR) was a powerful method for amplifying particular segments of DNA. PCR used the enzyme PlatinumTM Taq DNA polymerase with catalog number 10966018[ purchased from Invitrogen, USA] that directed the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase added nucleotides to the 3` end of a custom-designed oligonucleotide when it was annealed to a longer template DNA. Thus, if a synthetic oligonucleotide was annealed to a single-stranded template that contained a region complementary to the oligonucleotide, DNA polymerase could use the oligonucleotide as a primer and elongate its 3` end to generate an extended region of double stranded DNA. Denaturation was the initial PCR cycle stage The DNA template was heated to 94° C. This broke down the weak hydrogen bonds that held DNA strands together in a helix, allowing the strands to separate creating single stranded DNA. Annealing was the second PCR cycle. The mixture was cooled to anywhere from 50-70° C. This allowed the primers to bind (anneal) to their complementary sequence in the template DNA. Extension was the final step of PCR cycle. The reaction was ; then heated up to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extended the primers, adding nucleotides onto the primer in a sequential manner, using the target DNA as a template. With one cycle, a single segment of double-stranded DNA template was amplified into two separate pieces of double-stranded DNA. These two pieces were then available for amplification in the next cycle. As the cycles were repeated, more and more copies were generated and the number of copies of the template was increased exponentially. The amplified PCR product was sequenced using a genetic analyzer 3130XL[ purchased from Applied biosystems, USA]. DNA sequence homology search analysis of the predominant bacterial isolate was achieved using Blastn algorithm at NCBI website. Fruiting bodies were examined using a Stereomicroscope( dissecting microscope) MSC-ST45T( purchased from Infetik, China). Wet mounts from crushed fruiting bodies were prepared. The refaractility, shape and the size of Myxospores were determined victimizing phase contrast microscopy. On the other hand the plates were exposed to 360 nm wavelength ultraviolet light to assess the fruiting bodies fluoresced [16].
Identification Myxopyronin A producing bacterial isolates:
Gram stain:
It classified bacteria into two categories based on the makeup of their cell walls. The bacterial cells became purple after being treated with a solution of crystal violet and subsequently iodine on a microscope slide. When colored cells were treated with a solvent such as alcohol or acetone, gram-positive organisms kept the stain whereas gram-negative organisms lost the stain and turned colorless. With the addition of the counter-stain safranin, the clear, gram-negative bacteria became pink [17].
Spore shape:
This was discovered using the spore staining method. To get rid of any fingerprints, the slide was wiped with alcohol and a Kim-wipe. On the bottom of the slide, a Sharpie was used to create two circles. Each circle was filled with two tiny droplets of water using an inoculation loop. A very small amount of germs was taken out of the culture tube using an aseptic method. The water droplet on the slide had microorganisms on it. The slide was thoroughly dried by air. Bypassing the slide through the flame three to four times with the smear side up, the slide was heat-fixed. It took a while for the slide to completely cool. A piece of paper towel placed inside the slide’s border was used to hide the streaks. A beaker of heating water was situated over the slide. The slide was allowed to steam for three to five minutes; while the paper towel was covered with a malachite green liquid. Removed and thrown away was the discolored paper towel. To get rid of any stray paper towel bits, the slide was gently cleaned with water. The counter-stain was safranin for 1 minute. Before putting the slide on the microscope’s stage and seeing it via the oil immersion lens, the slide’s bottom was dried [18].
Spore site:
During the Gram stain test, the spore location was established [19].
Cell shape:
During the Gram stain test, the cell shape was assessed [20].
Blood haemolysis:
On blood agar media, the test antibiotic capacity to haemolyze the blood was tested [21].
Motility test:
It discriminated between motile bacteria and non-motile bacteria.
A sterile needle was used to penetrate the medium to within 1 cm of the tube’s bottom to select a well-isolated colony and test for motility. The needle was certainly retained in the same position as it was inserted and removed from the medium. It took 18 hours of incubation at 35°C, or until noticeable growth appeared [22].
Nitrate reduction test:
0.5 ml of nitrate broth was added in a clean test tube, was autoclaved for 15 minutes at 15 lbs pressure and 121°C, and was let to cool to room temperature. The tube was inoculated with a heavy inoculum of fresh bacterial culture and was incubated at 35°C for 2 hours. 2 drops of reagent A and 2 drops of reagent B were added and mixed well. The development of red color within 2 minutes was observed for. If no red color was developed, a small amount of zinc dust was added and observed for the development of the red color within 5 minutes [23].
Methyl red test:
In the Methyl Red test, an infected tube of MR broth was used before adding the methyl red PH indicator. The buffers in the medium were overcome by the acids when an organism used the mixed acid fermentation pathway and produced stable acidic end products, resulting in an acidic environment [24].
Catalase test:
A little inoculum of a specific bacterial strain was introduced to a 3% hydrogen peroxide solution to see if it might produce catalase. It was observed for the rapid emergence of oxygen bubbles [25].
Oxidase test:
The 1% Kovács oxidase reagent was applied to a tiny piece of filter paper, which was then allowed to air dry. A well-isolated colony was taken from a fresh (18 to 24-hour culture) bacterial plate using a sterile loop, and it was then rubbed onto prepared filter paper. Color alterations were noticed [26].
Citrate utilization:
Five milliliters of a Simmon Koser’s citrate medium were taken after it had been autoclaved at 15 pounds for 15 minutes. To create a clear slant and butt, the test tube containing melted citrate medium was slanted. Using sterilized wire and labeled tubes, the specified samples of microbe were injected on the media’s incline. For 24 hours, the tubes were incubated at 37°C. The medium’s color shift was watched for [27].
Starch hydrolysis:
For 48 hours at 37°C, the bacterium plates were injected. After incubation, a dropper was used to saturate the surface of the plates with an iodine solution for 30 seconds. Iodine that was in excess was afterward poured out. The area surrounding the bacterial growth line was looked at [28].
Tween 80 hydrolysis:
1% Tween 80 was used to create agar media. The supplied microorganism was added to the Tween 80 agar plates by utilizing an inoculating loop to create a single center streak in the plate. The plates were incubated for 24 hours at 37 °C. HgCl2 solution was poured over the plates. After a short while, the plates were examined. Positive test result; distinct halo-zone surrounding the injected region showed Tween 80 hydrolysis [29].
Growth at 10-45 0C:
On nutrient agar media, growth was observed to be possible at 45°C [30].
Indol test:
The test tube containing the microorganism for inoculation received 5 drops of the Kovács reagent directly. Within seconds after introducing the reagent to the media, the reagent layer formed a pink to red colour (cherry-red ring), which was a sign of a positive indol test [31].
Tolerance salinity test:
Its capacity to develop on nutrient agar while being responsive to 5% and 7 % NaCl was examined [32].
Voges-Proskauer(VP) test:
For the test, Voges-Proskauer broth, a glucose-phosphate broth loaded with microorganisms, was added to alpha-naphthol and potassium hydroxide. A successful outcome was indicated by a cherry red tint, whereas an unfortunate outcome was indicated by a yellow-brown color [33].
Casein hydrolysis test:
For testing the casein hydrolyzing activity of the test antibiotic, a single line streak of the given culture was made in the center of the skim milk agar plate under aseptic conditions and plate was incubated at 37°C in an incubator for 24-48 h [34].
Saccharide fermentation tests:
Glucose fermentation test:
The fermentation reactions of glucose were investigated using glucose purple broth. Peptone and the PH indicator bromcresol purple made up the purple broth. A 1% concentration of glucose was added. Isolated colonies from a 24-hour pure culture of microorganisms were added to the glucose purple broth as an inoculant. Parallel to the inoculation of the glucose-based medium, a control tube of purple broth base was used. The inoculated medium was incubated aerobically for 3 days at a temperature of 35–37 °C. The medium began to become yellow, which was a sign of a successful outcome. A poor carbohydrate fermentation response was indicated by the lack of yellow color development [35].
Fructose fermentation test:
A pure culture’s inoculum was aseptically transferred to a sterile tube of phenol red fructose broth. The infected tube was incubated for 18–24 hours at 35–37 °C. A color shift from red to yellow, signifying an acidic PH alteration, was a sign of a favorable response [36].
Maltose fermentation test:
A pure culture inoculum was aseptically transferred to a sterile tube containing phenol red maltose broth. The infected tube was incubated for 18–24 hours at 35–37 °C. A color shift from red to yellow, signifying an acidic PH alteration, was a sign of a favorable response [37].
Sucrose fermentation test:
A pure culture’s inoculum was aseptically transferred to a sterile tube containing phenol red sucrose broth. For 24 hours, the infected tube was incubated at 35–37 0C. A colour shift from red to yellow, signifying an acidic PH alteration, was a sign of a favourable response [38].
Purification of Myxopyronin A antibiotic:
This was achieved through reversed phase chromatography technique.
The aeration rate was 0.142 V/ V. min. The stirring rate was 500 rpm. PO2 was about 90 % of saturation; but decreased to about 20 % after 18 hours). The fermentation was stopped after 40 hours via centrifugation at 500 rpm in a gyrator shaker. The supernatants were collected; then tested for antimicrobial sensitivity using broth dilution technique to detect MICs and agar paper diffusion discs technique. The test antibiotic was extracted from the 2 liters of culture broth with 2/ 10 volume ethyl acetate. The ethyl acetate was then removed under the reduced pressure at 40℃. Afterwards, the residue was dissolved in 398 ml of methanol-water( 90: 10) and chromatographed on reversed phase HPLC. Methanol was the mobile phase. The eluent was 70 part methanol: 16 part water: 4 part acetic acid with flow rate 300 ml/ min. Detection of the antibiotic components was achieved exploiting refractive index. The main peak with retention time 5 minutes contained the biological antibiotic activity which was determined via agar diffusion assay using paper discs and Staphylococcus aureus as an indicator organism. On the other hand, the main peak was subjected to neutralization via NaHCO3. Myxopyronin A was extracted using 10 % V/ V Ethylene chloride. After the evaporation of the solvent, about 85 % of the remaining antibiotic substance was pure. It was noticed that the retention rime of Myxopyronin A was 11 minutes. Molecular formula of the purified Myxopyronin A was detected through mass spectrometer( Quadrupole mass spectrometer, Advion, USA) [39].
Procedure of Broth dilution assay for determination of MICs of Myxopyronin A:
During testing, multiple microtiter plates were filled with a certain broth, according to the needs of target bacteria. Varying concentrations of the antibiotics and the bacteria to be tested were then added to the plate. The plate was then placed into a non-CO2 incubator and incubated at thirty-seven degrees Celsius for sixteen to twenty hours. Following the allotted time, the plate was removed and checked for bacterial growth. When the broth became cloudy, bacterial growth occurred. The results of the broth microdilution method were reported in Minimum Inhibitory Concentration( MIC), or the lowest concentration of antibiotics that stopped bacterial expansion [40].
Agar diffusion assay with paper discs procedure for the determination of Myxopyronin A antimicrobial activity:
The disk diffusion method( DDM) was classified as an agar diffusion method( ADM) because the test antibiotic extract to be tested diffused from its reservoir through the agar medium seeded with the test microorganism. Generally, the reservoir was a filter paper disk, which was placed on top of an agar surface. When tested extracts compounds were microbiologically active, an inhibition zone developed around the filter paper disk after incubation. The diameter of the inhibition zone properly described the antimicrobial potency of test extract [41]. The test microbes were isolated using either selective or enrich growth media or broth( Table 2).
Estimation of Myxopyronin A effect on bacterial RNA synthesis:
The concentration of RNA isolated with RNeasy Kits( purchased from QIAGEN, USA) was determined by measuring the absorbance at 260 nm in a spectrophotometer. An absorbance of 1 unit at 260 nm corresponds to 40 µg of RNA per ml( A260 = 1 = 40 µg/ ml) [42].
Estimation of Myxopyronin A effect on bacterial protein synthesis:
Absorbance was measured at 205 nm to calculate the protein concentration by comparison with a standard curve. A( 205) method could be used to quantify total protein in crude lysates and purified or partially purified protein. The UV spectrophotometer was set to read at 205 nm allowing 15 min for the instrument to equilibrate. The absorbance reading was set to zero with a solution of the buffer and all components except the protein present. The protein solution was placed in the 1 mL cuvette and the absorbance was determined. The dilution and readings of samples were performed in duplicate.The matched cuvettes for samples and controls were utilized during the test procedure. The extinction coefficient of the protein was known, the following equation was employed. Absorbance = Extinction coefficient × concentration of protein × path length( 1 cm) to determine the concentration of the protein [43].
Estimation of pharmacodynamic and pharmacokinetic effects of Myxopyronin A during experimental animal testing in preclinical clinical trials:
In the present study, the pharmacokinetics and the pharmacodynamics of Myxopyronin A were evaluated after dosing in male rabbit animal models weighing about 2 kg. Furthermore, compound concentrations were determined in target compartments, such as lung, kidney and thigh tissue, using LC-MS/ MS. Based on the pharmacokinetic results, the pharmacodynamic profile of Myxopyronin A was assessed victimizing the standard neutropenic thigh and lung infection models [44].
Estimation of pharmacodynamic and pharmacokinetic effects of Myxopyronin A in randomized human clinical trials phases 1/2:
This study was conducted in 150 human volunteer subjects to show the bioavailability, pharmacokinetics and the pharmacodynamics of the test antibiotic. The study was designed as randomized, single-dose, 2-treatment, 2-period crossover trial with a washout period of 1 week. Blood samples were collected at 0( baseline), 10, 20, and 40 minutes and at 1, 1.5, 2, 3, 4, 6, 9, 12, and 24 hours postdose. Plasma concentrations of the 4 drugs were measured by using a rapid chromatography-tandem mass spectrometry method. Pharmacokinetic parameters were calculated by using noncompartmental methods. Bioequivalence was determined if the 90 % CIs of the log-transformed test/ reference ratios AUC( 0-24), AUC( 0-∞), and Cmax were within the predetermined range of 80% to 125%. Tolerability was assessed by using clinical parameters and subject reports Pharmacodynamic effects were evaluated through the determination of MICs via agar diffusion assay and broth dilution technique During randomized human clinical trials phases 1/2 all utilized infectious bacterial cell counts were estimated spectrophotometrically [45].
Estimation of of phototoxicity, mutagenicity and carcinogenicity of the test antibiotic:
The phototoxicity was determined via 3T3 neutral red uptake phototoxicity technique [46]. On the other hand, mutagenicity and carcinogenicity of the test antibiotic were assessed using Ames test [47].
The determination of toxokinetics and toxodynamic effects:
Up and down method for acute toxicity detection of Myxopyronin A was utilized for this purpose [48].
The determination of maximum bactericidal activity of Myxopyronin A:
A pure culture of a specified microorganism was grown overnight, then diluted in growth-supporting broth( typically Mueller Hinton Broth) to a concentration between 1 x 10^5 and 1 x 10^6 cfu/ ml. A stock dilution of the antimicrobial test substance was made at approximately 100 times the expected MIC. Further 1:1 dilutions were made in test tubes. All dilutions of the test antibiotic were inoculated with equal volumes of the specified microorganism. A positive and negative control tube was included for every test microorganism to demonstrate adequate microbial growth over the course of the incubation period and media sterility, respectively. An aliquot of the positive control was plated and used to establish a baseline concentration of the microorganism used.The tubes were then incubated at the appropriate temperature and duration. Turbidity indicated growth of the microorganism and the MIC was the lowest concentration where no growth was visually observed. To determine the MBC, the dilution representing the MIC and at least two of the more concentrated test product dilutions were plated and enumerated to determine viable CFU/ ml. The MBC was the lowest concentration that demonstrated a pre-determined reduction (such as 99.9%) in CFU/ ml when compared to the MIC dilution [49].
Determination of plasma protein binding capacity of Myxopyronin A:
Victimizing of an ultrafiltration technique, the protein binding( PB) extent and changeability of the test antibiotic medicates were settled when given simultaneously to 30 patients inoculated with infectious pneumococci inside hospitals in Egypt. Clinical samples used were routinely received by microbiological laboratory inside the faculty of Pharmacy, Cairo University, Egypt. Plasma proteins were likewise plumbed. A protein-free medium was used to determine the nonspecific binding. Plasma samples from 30 patients were enclosed, of which plasma proteins were deliberated for 24 patients.
Determination of liver, kidney and heart function tests:
These functional tests were performed to assess the vitality of liver, kidney and heart during the randomized human clinical trials phases 1/2. On the other hand, Urine, stool analyses were achieved in addition to estimation of blood complete counts to all experimental subjects received graded doses of Myxopyronin A.
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Figure 1. It demonstrates the structure of Myxopyronin A extracted from bacterial isolates Myxococcus fulvus strain Mx f50 collected from different soil environments in Egypt. Molecular formula of the purified test antibiotic was noticed to be C23H31NO6 determined through mass spectrometer.
Figure 1. It demonstrates the structure of Myxopyronin A extracted from bacterial isolates Myxococcus fulvus strain Mx f50 collected from different soil environments in Egypt. Molecular formula of the purified test antibiotic was noticed to be C23H31NO6 determined through mass spectrometer.
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Figure 2. It represents docking of Myxopyronin A ligand on Bacterial RNA polymerase. Myxopyronin showed high affinity and inhibitory effect towards the switch region.
Figure 2. It represents docking of Myxopyronin A ligand on Bacterial RNA polymerase. Myxopyronin showed high affinity and inhibitory effect towards the switch region.
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Figure 3. It demonstrates 3D structure of bacterial prokaryotic RNA polymerase comprising the switch binding site to which Myxopyronin A Ligand strongly bound inhibiting bacterial RNA polymerase activity selectively leading to the inhibition of mRNA transcription and subsequently the mortality of the microbe. The secondary structure of RNA polymerase enzyme consisted of spiral alpha and beta sheets. Its molecular mass was approximately 198 amino-acids.
Figure 3. It demonstrates 3D structure of bacterial prokaryotic RNA polymerase comprising the switch binding site to which Myxopyronin A Ligand strongly bound inhibiting bacterial RNA polymerase activity selectively leading to the inhibition of mRNA transcription and subsequently the mortality of the microbe. The secondary structure of RNA polymerase enzyme consisted of spiral alpha and beta sheets. Its molecular mass was approximately 198 amino-acids.
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Figure 4. It shows the impact of various concentrations of Soluble starch on the production of Myxopyronin A.
Figure 4. It shows the impact of various concentrations of Soluble starch on the production of Myxopyronin A.
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Figure 5. It shows the effects0 of different Peptone concentrations as nitrogen growth factor on the productivity of Myxopyronin A.
Figure 5. It shows the effects0 of different Peptone concentrations as nitrogen growth factor on the productivity of Myxopyronin A.
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Figure 6. It refers to the estimation of effect of Myxopyronin A on microbial mRNA productivity. mRNA synthesis was detected to be diminished proportionately up on employment of exploding doses of Myxopyronin A antibiotic.
Figure 6. It refers to the estimation of effect of Myxopyronin A on microbial mRNA productivity. mRNA synthesis was detected to be diminished proportionately up on employment of exploding doses of Myxopyronin A antibiotic.
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Figure 7. It demonstrates the influence of Myxopyronin A on protein synthesis using UV spectrophotometer absorption at 205 nm. Protein synthesis was noticed to be decreased dramatically up on utilization of increasing doses of Myxopyronin A antibiotic.
Figure 7. It demonstrates the influence of Myxopyronin A on protein synthesis using UV spectrophotometer absorption at 205 nm. Protein synthesis was noticed to be decreased dramatically up on utilization of increasing doses of Myxopyronin A antibiotic.
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Figure 8. It shows AUC of Myxopyronin A following IM administration in clinical trials stages 1/2. Efficacious dose ranged from 5-6 mg/ kg of body weight. Onset of action was observed following closely 15 minutes. It followed first order of elimination kinetics.
Figure 8. It shows AUC of Myxopyronin A following IM administration in clinical trials stages 1/2. Efficacious dose ranged from 5-6 mg/ kg of body weight. Onset of action was observed following closely 15 minutes. It followed first order of elimination kinetics.
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Figure 9. Area under the curve( AUC) following oral administration of Myxopyronin A during clinical trials phases 1/2. Efficacious dose ranged from 7-8 mg/ kg of body weight. Onset of action was observed following nearly 30 minutes. It followed first order of elimination kinetics.
Figure 9. Area under the curve( AUC) following oral administration of Myxopyronin A during clinical trials phases 1/2. Efficacious dose ranged from 7-8 mg/ kg of body weight. Onset of action was observed following nearly 30 minutes. It followed first order of elimination kinetics.
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Figure 10. Quantal dose response curve for the determination of toxokinetics of Myxopyronin A. LD50 % was found to be 200 mg / kg; while LD99 % was nearly 300 mg/ kg.
Figure 10. Quantal dose response curve for the determination of toxokinetics of Myxopyronin A. LD50 % was found to be 200 mg / kg; while LD99 % was nearly 300 mg/ kg.
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Statistical Analysis

All cultures were conducted in triplets. Their presentation was by means and standard deviation. One way analysis of variance( p value≤ 0.05) was used as means for performing statistical analysis and also, statistical analysis was based on excel-spreadsheet-software. The F statistical analysis test was utilized during the present study.
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Figure 11. It demonstrates the major Gram negative bacterial isolates producing Myxopyronin A antibiotic using Stereomicroscope.
Figure 11. It demonstrates the major Gram negative bacterial isolates producing Myxopyronin A antibiotic using Stereomicroscope.
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Figure 12. It shows Myxococcus fulvus colonies on CYP isolation plates secreting Myxopyronin A antibiotic.
Figure 12. It shows Myxococcus fulvus colonies on CYP isolation plates secreting Myxopyronin A antibiotic.
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Results

From the culture supernatant of the Myxobacterium Myxococcus fulvus 124B02 which was the predominant soil bacterial isolate( 19 isolates, Table 3) grown on Casein yeast peptone( CYP) plate, Myxopyronin A was was produced. The test antibiotic blocked the growth of many Gram +ve bacteria with MICs less than 100 mcg/ ml; whereas it inhibited the growth of few Gram -ve bacteria such as Escherichia coli at MICs greater than 100 mcg/ ml. On the other hand, Eukaryotic cells such as fungal and human cells were not affected. Prokaryotic DNA-dependant-RNA polymerase( RNLP) was noticed to be inhibited via the test antibiotic suggesting its bactericidal action. Cmax was 10 mcg/ ml at Tmax 2 hours when 600 mg dose was orally administered in randomized human clinical trials phases 1/2; as well as T1/2 reached 2.5 hours following first order kinetics of elimination. Duration of its action was nearly 12 hours after oral administration. Rare toxicity was detected during preclinical and randomized human clinical trials phases 1/2 in the form of mild diarrhea and cholestatic jaundice in less than 5 % of experimental candidates. Myxopyronin A was the predominating compound after reverse phase HPLC technique was utilized for the refinement and the purification of of the test antibiotics( Table 4). The phototoxicity was determined via 3T3 neutral red uptake phototoxicity technique which showed no phototoxicity. On the other hand, mutagenicity and carcinogenicity of the test antibiotic were assessed using Ames test which demonstrated no carcinogenicity and genotoxicity at all.
Table 3. It shows the distribution of Myxopyronin A producing bacterial isolates.
Table 3. It shows the distribution of Myxopyronin A producing bacterial isolates.
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Table 4. It demonstrates the degree of purity of test antibiotics following the purification via reversed phase HPLC technique.
Table 4. It demonstrates the degree of purity of test antibiotics following the purification via reversed phase HPLC technique.
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Table 5. It demonstrates 16 S rRNA detection of Myxopyronin A producing isolates using BLASTn software.
Table 5. It demonstrates 16 S rRNA detection of Myxopyronin A producing isolates using BLASTn software.
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Table 6. It shows the estimation of zones of inhibition and minimum inhibitory concentrations of Myxopyronin A via Agar diffusion assay using paper discs.
Table 6. It shows the estimation of zones of inhibition and minimum inhibitory concentrations of Myxopyronin A via Agar diffusion assay using paper discs.
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2 The initial density of each organism during Agar diffusion assay for the determination of minimum inhibitory concentrations and zones of inhibition of gtowth was nearly 10^5/ ml.
Table 7. It demonstrates MICs of Myxopyronin A on different microorganisms using broth microdilution technique.
Table 7. It demonstrates MICs of Myxopyronin A on different microorganisms using broth microdilution technique.
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Table 8. It demonstrates Minimum bactericidal concentrations( MBCs) of Myxopyronin A on different microorganisms using broth microdilution technique.
Table 8. It demonstrates Minimum bactericidal concentrations( MBCs) of Myxopyronin A on different microorganisms using broth microdilution technique.
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Table 9. It shows the estimation of mRNA quantity via UV spectrophotometer at 260 nm after addition of Myxopyronin A.
Table 9. It shows the estimation of mRNA quantity via UV spectrophotometer at 260 nm after addition of Myxopyronin A.
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Table 10. It shows the effect of Myxopyronin A on the microbial protein synthesis using UV spectrophotometer at 205 nm.
Table 10. It shows the effect of Myxopyronin A on the microbial protein synthesis using UV spectrophotometer at 205 nm.
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Protein synthesis and mRNA synthesis were decreased significantly with increasing the dose Myxopyronin A, as demonstrated in tables 10 and 9 respectively. Docking studies using MCULE and SWISS DOCK softwares demonstrated that the mechanism of action of the test antibiotic was probably due to the inhibition of RNA Polymerase through binding with its switch region. High ∆G of the test antibiotic was observed to be approximately 17 J/ mole as determined via SWISS model software. On the other hand, low Kd of the test antibiotic towards the switch region was found to be approximately 250 nM using SWISS MODEL software. The biochemical profile and the morphology of the potent bacterial isolates producing the test antibiotic in the present study was summarized in Table 11. The morphology and biochemical reactions indicated that the predominant bacterial isolates secreting the extracellular test antibiotic were Myxococcus fulvus. A total of 150 human subjects( mean SD age, 27.3[ 9.8] years were enrolled and completed the study. For the test antibiotic 90% CIs for the long transformed ratios of Cmax, AUC( 0-24) and AUC( 0-∞) were 85.1to 96.4, 87.2 to 97.3 and 91.7 to 98.4 respectively. The mean PB was observed for Myxopyronin A which approximated 70%. The major protein binding for Myxopyronin A and rifampicin was detected to be albumin. The unbound fraction was found to be responsible for the therapeutic activity.

Discuss

The in vitro and in vivo antimicrobial activity of Myxopyronin A, a novel antibiotic was evaluated in the present study. It demonstrated excellent bactericidal activity against a broad spectrum of G +ve bacteria with MICs did not exceed 20 mcg/ ml. On the other hand It showed few bactericidal activities against G -ve bacteria.with minimal inhibitory concentrations were greater than 100 mcg/ ml. Its mechanism of action was realized during the investigation of RNA synthesis to be via the inhibition of prokaryotic DNA-dependant-RNA polymerase; whereas no inhibitory impact was observed for Eukaryotic one. Docking studies through SWISS DOCK software confirmed this as well. The antibiotic activities Myxopyronin A and B were isolated from the culture supernatant of 29 bacterial isolates of Myxobacterium Myxococcus fulvus 124B02 detected molecularly using 16 S rRNA technique( Table 3).
The antibiotic activity did not inhibit the growth or kill eukaryotic cells such as human and fungal cells reflecting selectivity towards the inhibition of the growth of prokaryotic bacterial cells. This selectivity effect minimized the adverse effects noticed during the present study. Docking studies via SWISS DOCK software revealed that desmethylation of either Myxopyronin A or B enhanced its biological activity. Purification was performed through reversed phase HPLC. Myxopyronin A was the main refined antibiotic. Its purity degree reached approximately 80 %; while, the remaining purified antibiotic was detected to be Myxopyronin B. The antibacterial activity was assessed via the determination of MICs of the test antibiotics using the agar diffusion technique utilizing paper discs 5 mm in diameter and the broth dilution assay. The initial density of each test microorganism was about 105/ ml of the culture suspension. The MICs of test antibiotic against G +ve bacteria ranged from 5 to 20 mcg/ ml; Whereas MICs reached above 100 mcg/ ml against some selected G -ve bacteria. On the other hand no effect was detected against the growth of fungi and yeasts. ( Irschik H et al., 1983) stated that myxovalargin A was a novel peptide antibiotic isolated from the culture supernatant of the myxobacterium Myxococcus fulvus strain Mx f65. It was active against Gram-positive bacteria( MIC 0.3 approximately 5 micrograms/ ml), at higher concentrations also against Gram-negative ones( MIC 6 approximately 100 micrograms/ ml), and not at all against yeasts and molds. Its mechanism of action involved the inhibition of the bacterial protein synthesis [50]. According to( Glaus F et al., 2018) Ripostatin, a novel antibiotic, isolated from the culture supernatant of Myxobacterium, Sorangium cellulosum strain So ce377. On the other hand it interfered of the bacterial RNA synthesis [51]. On the other hand, Myxopyronin A was found to be structurally related to α-pyrone antibiotics from myxobacteria. Its ability to inhibit RNA polymerase was through interaction with the switch region of RNA polymerase; while Rifampicin inhibited the same enzyme through different region [52]. Myxopyronin showed no phototoxicity and mutagenicity in rabbit animal models during the preclinical trials stage, in the present study. Rare adverse effects including mild diarrhea and cholestatic jaundice were reported in less than 5 % of the experimental subjects received the test antibiotics during randomized human clinical trials phases 1/2. The biological half life of Myxopyronin A reached approximately 2.5 hours. 0.4 % peptone and 5 % soluble starch were detected to be the optimal nitrogen and carbon growth factors for bacterial isolates producing the test antibiotics, respectively( figures 4 and 5). High ∆G of the test antibiotic was observed to be approximately 17 J/ mole as determined via SWISS MODEL software reflecting high catalytic activity of the test antibiotic towards the switch region. On the other hand, low Kd of the test antibiotic towards the switch region was found to be approximately 250 nM using SWISS MODEL software indicating high affinity and binding capacity. Bioavailability studies were performed using HPLC during randomized human clinical trials phases 1/2 revealed that Myxopyronin A reached nearly 70% oral bioavailability, 82% IM bioavailability and 100% IV bioavailability. Metabolic studies using HPLC revealed that the test antibiotic showed no in vivo induction of hepatic metabolizing Cytochrome P450 enzymatic system; while rifampicin induced CYP3A4 hepatic metabolizing enzyme potently. Up and down procedure intended for the evaluation of acute toxicity profile of the test antibiotic showed that LD50% was about 200 mg/ kg body weight; while LD99% reached 300 mg/ kg. On the other hand, therapeutic margin of the test antibiotic ranged from 7 mcg/ ml to 100 mcg/ ml. Myxopyronin A producing bacterial isolates were gram negative, spore forming obligate aerobes and chemoorganotrophic. They were elongated rods with tapered ends. No flagella were present; but the cells moved via gliding. They fermented Tween 80, starch and casein. On the other hand they were positive for catalase while negative for oxidase tests. They reduced nitrates
And were able to grow at 10-37 ℃. A total of 150 human subjects( mean SD age, 27.3[ 9.8] years were enrolled and completed the study. For the test antibiotic 90% CIs for the long transformed ratios of Cmax, AUC( 0-24) and AUC( 0-∞) were 85.1to 96.4, 87.2 to 97.3 and 91.7 to 98.4 respectively. The point estimates for Cmax in the present study were outside the limit for bio-equivalence for rifampicin standard drug. The mean PB was observed for Myxopyronin A which approximated 70% while that of rifampicin reached 88% [53]. It was noticed that plasma protein binding was proportionally increased with increasing the doses of the test antibiotic. The plasma protein binding participated in extending the Myxopyronin A duration of action. The major protein binding for Myxopyronin A and Rifampicin was noticed to be albumin. The unbound fraction was detected to be responsible for the therapeutic activity.

Conclusion

Antibiotic resistance is a global challenge that the current study shows promise in solving. According to the findings of the current study, Myxopyronin A, which was isolated from the bacterial isolates Myxococcus fulvus 124B02 that were collected from various soil environments in Egypt, exhibited significant antibiotic activity both in vitro and in vivo against a moderate range of pathogenic bacteria, particularly G+ve varieties. Future research is recommended to investigate pharmacological interactions of the synergism type between Myxopyronin A and different antibiotic classes.

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Table 2. It demonstrates different isolation media for different pathogenic m.os. utilized in the Broth microdilution test and agar diffusion assay using paper discs.
Table 2. It demonstrates different isolation media for different pathogenic m.os. utilized in the Broth microdilution test and agar diffusion assay using paper discs.
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Table 11. The resolution of biochemical reactions.
Table 11. The resolution of biochemical reactions.
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