Abstract

The Philippine archipelago is geographically positioned in the tropics with rich areas of marine biodiversity. Its marine sediments harbor actinomycetes that exhibit antibacterial activity. Screening of actinomycetes isolated from marine sediments collected near the coast of Islas de Gigantes, Iloilo showed one isolate that exhibited high activity against the multidrug-resistant Staphylococcus aureus (MRSA) strain carrying the Staphylococcal Cassette Chromosome mec (SCCmec) type 1 gene, a biomarker for drug resistance. The isolate was identified as Streptomyces sp. strain DSD011 based on its 16s rRNA and protein-coding genes (atpD, recA, rpoB, and trpB) sequences, and was found to be a new species of salt-tolerant marine Streptomyces. Further, the strain harbors both non-ribosomal peptide synthetase (NRPS) and type II polyketide synthase (PKS) in its genome. The targeted chromatographic isolation and chemical investigations by Liquid Chromatography Mass Spectrometry-Time of Flight (LCMS-TOF), tandem mass spectrometry (MS/MS), and Global Natural Product Social molecular networking (GNPS) of the antibiotics produced by the strain afforded the two polycyclic aromatic polyketide angucycline glycosides, fridamycin A (1) and fridamycin D (2), which are products of type II PKS biosynthesis. Compounds 1 and 2 displayed antibacterial activity against MRSA with minimum inhibitory concentration (MIC) of 500 μg/mL and 62.5 μg/mL, respectively. These results suggest that the underexplored marine sediments near the coast of Islas de Gigantes, Iloilo offer access to undiscovered Streptomyces species that are invaluable sources of antibiotic leads.

Highlights

  • The seafloor covers about 70% of the Earth’s surface and is carpeted by marine sediments, which are mixtures of complex organic and inorganic particles that accumulated due to accretion and erosion of the continents, oceanic biological activities, volcanic eruptions, and chemical processes within the ocean [1,2]

  • This is an Iberian clone of multidrug-resistant S. aureus (MRSA) containing Staphylococcal Cassette Chromosome mec (SCCmec) type 1 gene and resistant to various types of antibiotics such as ampicillin, amoxicillin/clavulanic acid, ciprofloxacin, cephalothin, doxycycline, gentamicin, erythromycin, imipenem, methicillin, penicillin, tetracycline, oxacillin, azithromycin, clindamycin, ceftriaxone, rifampin, amikacin, and tobramycin (Staphylococcus aureus subsp. aureus (ATCC® BAA-44TM) [59]

  • Our results revealed that Streptomyces sp. strain DSD011 harbors both non-ribosomal peptide synthetase (NRPS) and type II polyketide synthase (PKS) in its genome

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Summary

Introduction

The seafloor covers about 70% of the Earth’s surface and is carpeted by marine sediments, which are mixtures of complex organic and inorganic particles that accumulated due to accretion and erosion of the continents, oceanic biological activities, volcanic eruptions, and chemical processes within the ocean [1,2]. Our group discovered the antibiotic anthracycline shunt metabolites bisanhydroaklavinone and 1-hydroxybisanhydroaklavinone from a Philippine marine sediment-derived Streptomyces griseorubens strain DSD069, which destroys the cell membrane integrity of multidrug-resistant Staphylococcus aureus [24]. These anthracyclines shunt metabolites are precursors of anticancer anthracyclines such as doxorubicin, daunorubicin, and cinerubins. They were accumulated in the growth medium during the fermentation of Streptomyces griseorubens strain DSD069, which is a rare event occurring in a non-genetically modified Streptomyces strain [24] This recent finding shows that Philippine marine sediment-derived actinomycetes are a potentially rich source of new or novel secondary metabolites. Thheemmaarirnineesseeddimimeennttssaammpplleesswweerreeccoolllleecctteeddbbyy sseellff--ccoonnttaaiinneedd uunnddeerrwwaatteerr bbrreeaatthhiinngg apappapraartautsus(S(CSCUUBBAA) )ddivivininggfrforommaaddeeppththooffaapppprrooxxiimmaatteellyy2200ttoo3300 mm aanndd aa ddiissttaannccee ooff 330000 ttoo 550000 mm awawayayfrformomthteheshsohroereofotfhtehenneaeraersetstisilsalanndd. .SSaammpplelessooffmmaarriinneesseeddiimmeennttss wweerree ccoolllleecctteedd bbyy ddiiggggiinngg aa shsahlalollwowtrternecnhchofo0f .03.3mmusuisninggaahahnanddtrtorwoweleltetcehchnniqiquuee. .TThheehhaabbiittaattss eennccoouunntteerreedd aatt eeaacchh ssaammpplliinngg lolcoactaiotinonwewreerceacteagteogroizreizdeadsassansadn, dsi,lts,ioltr, orererfe. eTfh. eThseedsiemdeimntesnwtserweecroellceocltleedctiendsitnersilteer5i0lem5L0 mtuLbetsuabneds keapntdokneipcteounnitcilepurnotciel spsreodceinsstehdeilnabthoeralatobroyr.atory

Culture-Dependent Isolation
PCR-Based Screening for Secondary Metabolite Biosynthetic Genes
Salt Tolerance
Carbon Utilization Test
Scanning Electron Microscopy
Cultivation and Extraction of Biomass
Antibacterial Assay
Disk Diffusion Assay of the Crude Extract
Antibacterial Activity by Microbroth Susceptibility Assay
Minimum Inhibitory Concentration
Gel Filtration Chromatography
Flash Column Chromatography
Isolation and Phenotypic Characterization of Actinomycete Strain DSD011
Isolation and Identification of Antibiotic Compounds 1 and 2
Conclusions
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