Spiramycin antibiotic produced by Streptomyces pulveraceus, Am 52: Fermentation, Purification and biological activities

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Spiramycin antibiotic produced by Streptomyces pulveraceus, Am 52: Fermentation, Purification and biological activities

https://doi.org/10.21203/rs.3.rs-2069083/v1

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We are presenting here the synthesis of specific bioactive substances that have demonstrated inhibitory effects against pathogenic microorganisms from actinomycetes strains. Ninety-two Streptomyces strains were isolated from soil sample collected from Sharkia province, Egypt. All these isolates were screened for their antimicrobial activity against Gram positive and negative bacteria. Among these 92 Streptomyces samples, the broad-spectrum strain Streptomyces sp. Am 52 was isolated from soil cultivated with wheat in Abo-Hamad and selected for compound characterization and purification. The nucleotide sequence of the 16s RNA gene (1.5 Kb) of the most potent strain evidenced 98% similarity with Streptomyces pulveraceus. From the taxonomic features, the actinomycetes isolate Am 52 matches with Streptomyces pulveraceus in the morphological, physiological and biochemical characters. Thus, it was given the suggested name Streptomyces pulveraceus, (Am 52). The active metabolite was extracted using ethyl acetate (1:1, v/v) at pH 7.0. The separation of the active ingredient and its purification was performed using both thin layer chromatography (TLC) and column chromatography (CC) techniques. The physicochemical characteristics of the purified antibacterial substance have been investigated. The study indicates a suggested empirical formula of C₄₃H₇₄N₂O₁₄. The minimum inhibition concentrations "MICs" of the purified antimicrobial agent were also determined. The purified antimicrobial agent was suggestive of being belonging to 16-membered Macrolide group (Spiramycin antibiotic) produced by Streptomyces pulveraceus, Am 52.

Antibacterial agent

Streptomyces pulveraceus

MIC

TLC

The genus Streptomyces are remarkable, and merit special consideration with regard to the morphological and metabolic differentiation phenomena they manifest during later stages of development (El-Naggar, 2007). Streptomyces species generally synthesize a sizeable number of diverse natural secondary metabolites, the best known of which antibiotics are currently used worldwide as veterinary and pharmaceutical industry (Saadoun and Gharaibeh, 2003). The macrolides are a group of antibiotics whose activity comes from the presence of a macrolide ring, a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. Macrolides belong to the polyketide class of natural products (Omura et al., 2002). The chemical structure of these antibiotics was characterized by a ring consisting of not fewer than 12 carbon atoms and closed condensation of a number of acetate and propionate provided an alternative source of n butyrate 2- methylmalonate and propionate building unites of the aglycones of the macrolide antibiotics monesin (Sathi et al., 2001). The mechanism of action of the macrolides is inhibition of bacterial protein biosynthesis by binding reversibly to the subunit 50S of the bacterial ribosome, thereby inhibiting translocation of peptidyl tRNA (Zhang et al., 2007). This action is mainly bacteriostatic but can also be bactericidal in high concentrations. The clinically useful macrolide antibiotics could be conveniently classified into three groups based on the number of atoms in the lactone nucleus. Erythromycins A, B, C, D, E and F, oleandomycin, roxithromycin, dirithromycin, clarithromycin and flurithromycin are 14-membered macrolides whereas azithromycin is a 15-membered compound. 16-Membered macrolides include josamycin, rosaramicin, rokitamycin, kitasamycin, mirosamicin, spiramycin, cirramycin and tylosin (Min and Chang-Qin, 2005). The 16-membered macrolide antibiotics may be divided into three classes depending on the number and position of carbonyl groups in the molecule. The first class represented by leucomycins, spiramycins, possesses an aldehyde group attached to the six positions of the ring structure through a methylene group. The second class includes the magnamycins, cirramycin, tylosin, and is similarly characterized by the aldehyde group attached to carbon 6 through a methylene group but also by a carbonyl group in position 9. The antibiotics of the third class, illustrated by chalcomycin and neutramycin, have only one carbonyl group, the keto function at position 9 (Nakagawa et al., 1972).

In the present study, the production of the bioactive substances that demonstrated inhibitory affects against microbial pathogenic, from Streptomyces pulveraceus, Am 52 were reported, along with some physico-chemical properties of secondary metabolites with high biological activities.

2.1 Microorganism: Ninty two actinomycete strains were isolated from soil sample collected from Sharkia province, Egypt; using the soil dilution technique (Williams and Davies, 1965).

2.2 Screening for Antimicrobial Activity: The anti-microbial activity was determined by cup method assay (Kavanagh, 1972). The antibiotic was determined against Gram positive as (Staphylococcus aureus, Listeria monocytogenes and Actinobacter baumaunnii) and Gram-negative bacteria as (Klebsiella pneumonia, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa and Proteus mirabilis).

2.3 Taxonomic Studies of Actinomycete Isolate

2.3.1 Morphological Characteristics: Morphological characteristics of the most potent produce strain Am 52 grown on starch nitrate agar medium at 30 °C for 6 days was examined under light and scanning electron microscopy (JEOL Technics Ltd.).

2.3.2 Molecular Identification of bacterial isolate (Am 52)

The Am 52 isolate employed herein was further identified via 16S rRNA cataloging analysis. The total DNA sample was extracted from expontially growing bacterial cells (Sambrook and Russel, 2001). The 16S rRNA gene was amplified for the isolate (Am 52) by PCR using the forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3' and also using the reverse primer 5'-GGTTACCTTGTTACGACTT-3' (Chénbey, et al., 2000 and Turner, et al., 1999). The PCR product was subjected to agarose gel electrophoresis (Sambrook and Russel, 2001) and band of amplified 16S rRNA gene was cleaned up using Gene Purification Kit (Fermentas). Amplified DNA fragments was partially sequenced at GATC Biotech AG (Konstanz, Germany) using ABI 373 OXI DNA sequencer. Sequence analysis and their comparison to deposited data in GenBank was made using Basic Local Alignment search Tool (BLAST) programm at (http://www.ncbi.nlm.nih.gov/blast) (Altschul, et al., 1997). Phylogenetic tree of isolated bacteria was constructed on the base of 16S rDNA data using Clustal W software (http://www.clustal).

2.4 Physiological and Biochemical Characteristics:

lecithinase was conducted on egg-yolk medium (Nitsh and Kutzner, 1969); lipase (Elwan et al., 1977); Protease (Chapman, 1952); pectinase (Hankin et al., 1971); á-amylase, hydrogen sulphide production, citrate utilization, coagulation of milk and oxidase test (Cowan, 1974); catalase test (Jones, 1949); melanin pigment Pridham et al., 1957); degradation of both esculin and xanthine (Gordan et al., 1974), Nitrate reduction (Gordan, 1966), The utilization of different carbon and nitrogen sources (Pridham and Gottlieb, 1948), Cell wall was performed by the method of (Becker et al., 1964; Lechevalier and Lechevalier, 1968). The cultural characteristics were studied in accordance with the guidelines established by the International Streptomyces Project (Shirilling and Gottlieb, 1966). Colors characteristics were assessed on the scale developed by (Kenneth and Deane, 1955)

2.4.1 Fermentation: The isolate, Am 52 was inoculated into 250 ml Erlenmeyer flasks containing 75 ml of liquid starch (seven flasks). The flasks were incubated at 30 °C for 6 days. A twenty-liter total volume was filtered through Whatman No.1 filter paper and followed by centrifugation at 5000 rpm for 20 minutes.

2.4.2 Extraction: The clear filtrate was adjusted at different pH values (4 to 9) and extraction process was carried out using different solvents separately at the level of 1:1 (v/v). The organic phase was concentrated to dryness under vacuum using a rotary evaporator. The residual material was dissolved in the least amount of DMSO and filtered. The filtrates were test for their antimicrobial activities. The antimicrobial agent was precipitated by petroleum ether (bp. 60-80°C) and centrifuged at 4000 rpm for 15 minutes where a white powdered precipitate could be obtained.

2.4.3 Purification by TLC: Separation of the antibacterial substance into individual components was carried out by thin-layer chromatography using a solvent system composed of chloroform and methanol (24:1, v/v). Among five bands developed, only one band at R_f 0.74 showed antimicrobial activity.

2.5 Physico-Chemical Properties

2.5.1 Elemental Analysis: The elemental analysis C, H, O, N and S was carried out at the Micro analytical Center, Cairo University, Egypt.

2.5.2 Spectroscopic Analysis: The IR, UV, Mass spectrum and NMR spectrum were determined at the Micro analytical Center of Cairo University, Egypt.

2.5.3 Reaction of the Antifungal Agent with Certain Chemical Test: For this purpose, the following reactions were carried out: Molish’s, Fehling, Sakaguchi, ninhydrin, Ehrlish, Nitroprusside, Ferric chloride and Mayer (Atta, 1999).

2.5.4 Biological Activity of the Antibacterial Agent: The minimum inhibitory concentration (MIC) has been determined by the dilution method assay.

2.5.5 Characterization of the Antibacterial Agent: The antibiotic produced by Streptomyces pulveraceus, Am 52 was identified according to the recommended international references (Umezawa, 1977).

3.1 Screening for the Antimicrobial Activities: The active metabolites produced by actinomycete culture, Am 52 exhibited various degrees of activities against as inhibition of growth of both Gram positive and negative bacteria in Table 1 of supporting information

3.2 Morphological, physiological, biochemical and Molecular Identification of the Actinomycete Isolate, Am 52:

The vegetative mycelia grew abundantly on both synthetic and complex media and show fragmentation into bacillary elements. The isolate 52 failed to grow on tryptone-yeast extract liquid medium (ISP-1) and yeast extract-malt extract agar medium (ISP-2). Good growth on oatmeal agar (ISP-3), aerial mycelium is yellowish white, substrate mycelium is yellowish brown and there is no diffusible pigment. The aerial mycelia grew abundantly on Starch-nitrate agar medium; Oatmeal agar medium (ISP-3) and Glycerol-Asparagine agar medium (ISP-5). The results presented in Fig.1 (a,b) revealed that the aerial hyphae bear spore chains of spiral (S) type and smooth spore surface. Color and Culture Characteristics: It was found that the isolate Am 52 shows the aerial mycelium is yellowish white, substrate mycelium is yellowish brown and there is no diffusible pigment (Table 1). The cell wall hydrolysate contains LL-diaminopimelic acid (LL-DAP) and sugar pattern were not detected. The physiological and biochemical characteristics of actinomycete isolate Am 52 were summarized in Table 2 of supporting information

Table (1)

Cultural characteristics of the isolate No. 52

Medium	Growth	Aerial Mycelium	Substrate mycelium	Diffusible pigment
Tryptone Yeast Extract Liquid Medium (ISP-1)	-	-	-	-
Yeast malt extract agar (ISP-2)	-	-	-	-
Oat meal agar (ISP-3)	++	Yellowish white	Yellowish brown	-
Inorganic salts starch agar (ISP-4)	++	Gray	Light brownish gray	-
Glycerol-Asparagine agar (ISP-5)	+++	Yellowish white	Yellowish	-
Tyrosine agar (ISP-7)	+	Light gray	Yellowish brown	-
Glucose- Asparagine agar	++	Whitish gray	Pale brown	-
Starch nitrate agar	+++	Gray	Yellowish brown	-
Glucose nitrate agar	+	Light gray	Pale brown	-

3.3 Identification of Actinomycete Isolate: This was performed basically according to the recommended international Key’s (Buchanan and Gibson, 1974; Williams, 1989; Hensyl, 1994). On the basis of the previously collected data and in view of the comparative study of the recorded properties of Am 52 in relation to the closest reference strain, viz. Streptomyces pulveraceus, it could be stated that actinomycetes isolate, AZ-SH514 is suggestive of being likely belonging to Streptomyces pulveraceus, Am 52. Further concentrated to confirm their identification by 16S rRNA cataloging analysis. DNA sample was extracted from the bacteria and the primers for given in material and methods to this DNA sample and then PCR was carried out. The PCR product of the amplified 16S rRNA gene was electrophoresed via agarose gel. The result was given in Fig. 1c showed that the 16S rRNA gene of the bacterial under identification studies was amplified successfully as clear band was cut from agarose gel by gene clean kits and was sequenced and 16S rRNA sequence is given in the supporting information figure 1.. The sequence was sent to GenBank under the accession number BankIt1786699 KP324825. Isolate is closely related to or belong to the species Streptomyces pulveraceus. The Basic Local Alignment Search Tool was used to obtain the similarity of the sequence in Fig. 2 with the stored ones in data base. As a given in Fig. 1a, the cluster analysis also showed a similarity to Streptomyces pulveraceus in figure 2 of supporting information.

3.4 Purification by Column Chromatography: The purification process through column chromatography packed with silica gel indicated that the most active fractions against the tested organisms ranged between 22 to 28 in Fig. 3.

3.5 Physicochemical characteristics of the antimicrobial agent: The purified antibacterial substance is a white powder; its melting point is 137°C. The compound produced by S. pulveraceus, Am 52 was soluble in methanol, ethanol, ethyl acetate, n-butanol, chloroform and acetone and insoluble in water, petroleum ether, n-hexane and benzene.

3.6 Elemental analysis: The elemental analytical data of the antibacterial substance revealed the following: C= 51.26%, H= 7.73%, N= 3.29% and S= 0.0%. This analysis indicates a suggested empirical formula of C₄₃H₇₄N₂O₁₄

3.7 Spectroscopic characteristics: The ultraviolet (UV) absorption spectrum of the antimicrobial agent recorded a maximum absorption peak at 242.50 nm (Fig. 4a). The infrared (IR) spectrum of the antibacterial substance shows broad absorption band at 3466 cm^-1 corresponding to hydroxyl group (OH) and absorption band at 2971 cm^-1 corresponding to aliphatic group (C-H, aliphatic). However, other absorption bands, 1724 cm^-1and 1643 cm^-1 corresponding to (2 C=O ester and aldehyde) and 1163 cm^-1corresponding to (C-O- ether) group (Fig. 4b). The Mass spectrum revealed that the molecular weight is 843 (Fig. 4c). NMR spectrum of antibacterial substance produced by S. pulveraceus has peaks in the region 0.68, 1.06, 1.12, 1.15 and 1.24 ppm (d, 15 H, 5 CH₃), 2.13 ppm (s, 3H, CH₃), 2.36 (s, 3H, CH₃O), 3.07 (s, 12 H, 4 CH₃(N(CH₃)₂), 1.7- 3.4 ppm (m, 32 H, aliphatic protons), 4.03, 4.4, 4.94 and 4.95 ppm (4 t, 4 OH, exchanged with D₂O), 5.62, 6.12 and 6.21 ppm (4 t, CH= CH, olefenic protons) and 9.7 (s, 1H, CHO, aldehydic proton).

3.8 Biochemical reaction of the antimicrobial agent: The reactions revealed the detection of certain groups in the investigated agents. The antibacterial agent exhibited positive results with Molish’s and Fehling and negative results with Ninhydrin, Sakaguchi, Nitroprusside, ferric chloride reaction, Ehrlish and Mayer reaction. This indicated the presence of sugar moiety and aldehyde or keto group in Table 3 of supporting information.

3.9 Biological activities of the antimicrobial agent: Data of the antimicrobial agent spectrum indicated that the agent is active against Gram-positive and Gram negative bacterial (Table 2).

Table (2)

MIC and MBC values of antibacterial substance by S. pulveraceus (S. 52) against different target bacteria.

Tested bacteria	MIC (µg/ml)	MBC (µg/ml)
Escherichia coli	0.8	6.4
Staphylococcus aureus	0.4	6.4
Listeria monocytogenes	6.4	12.8
Pseudomonas aureginosa	12.8	-

3.10 Identification of the antimicrobial agent: Based on the recommended keys for the identification of antibiotics and in view of the comparative study of the recorded properties of the antimicrobial agent, it could be stated that the antimicrobial agent is suggestive of being belonging to Macrolide group (Spiramycin antibiotic) (Table 3).

Table (3)

A comparative study of the characteristic properties of the purified antibacterial substance by S. pulveraceus (S. 52) in relation to Reference antibiotic (Spiramycin).

Characteristics

Purified antibiotic

Spiramycin

1- Melting point

2- Molecular weight

3- Chemical analysis

4- Ultra violet

5- Formula

6- Active aganist

137⁰C

843.20

51.55

5.21

2.92

0.06

242.5

C₄₃H₇₄N₂O₁₄

Gram positive and negative bacteria

134-138⁰ C

843

51.55

5.21

2.90

0.00

242.5

C₄₃H₇₄N₂O₁₄

Gram positive and negative bacteria

The active metabolites were extracted by ethyl acetate at pH 7. Similar results were obtained by (Criswell et al., 2006 and Sekiguchi et al., 2007). The organic phase was collected and evaporated under reduced pressure using rotary evaporator. The extract was concentrated and treated with petroleum ether (b.p. 60-80°C) for precipitation process, where only one active fraction was obtained in the form of white powder. The purification process through a column chromatography packed with silica gel and eluting solvents composed of Toluene: ethyl acetate: formic acid (9:1, v/v), indicated that fractions activities was recorded from fraction22 to 28. Many workers used a column chromatography packed with silica gel. Similar results were obtained by (Hitchens and Kell, 2003). The physico-chemical characteristics of the purified antibiotic revealed that, their melting point is 137 °C. The compound is freely soluble in methanol, ethanol, ethyl acetate, n-butanol, chloroform and acetone and insoluble in water, petroleum ether, n-hexane and benzene. A study of the elemental analysis of the antibacterial agent leads to an empirical formula of C₄₃H₇₄N₂O₁₄.The spectroscopic characteristics of the antimicrobial agent under study revealed the presence of a maximum absorption peak in UV at 242.50 nm, infra-red absorption spectrum represented by 11 peaks. The Mass spectrum revealed that the molecular weight is 843.20 and NMR-spectrum was determined, which is attributable to a conjugated carbonyl grouping. The biochemical tests of the antimicrobial agent gave positive reaction with Molish’s and Fehling reactions. The MIC of antibiotic was determined, and the results showed that the minimum inhibitory concentration (MIC) of the compound against Escherichia coli NCTC 10416, was 0.8 µg/ ml, Staphylococcus aureus, was 0.4 µg/ ml, Listeria monocytogenes was 6.4 µg/ml and for Pseudomonas aeruginosa ATCC 10415 was 12.8 µg/ ml.

Identification of the antimicrobial agent according to recommended international keys indicated that the antibiotic is suggestive of being likely belonging to Macrolide group (Spiramycin antibiotic).

It could be concluded that: The CirramycinB antibiotic produced by Streptomyces pulveraceus, Am 52 demonstrated obvious inhibitory affects against pathogenic microorganisms (Gram positive and Gram negative bacteria)

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Competing interests: The authors have declared that no competing interests exist and that no experiments were carried out on animals.

Authors' contributions

MEFA-H and EAE conceptualized the Work, AIA and EAE contributed in methodology and data curation, RA, AA and MEFA-H contributed in visualization and writing, review and editing. AA helped in funding acquisition.

Funding

The APC will be paid from the University of Salford, United Kingdom, grant number (SERA5002).

Availability of data and materials

All relevant data are within the paper and its Supporting Information files.

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Spiramycin antibiotic produced by Streptomyces pulveraceus, Am 52: Fermentation, Purification and biological activities

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1. Introduction

2. Material And Methods

3. Results

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