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Antibacterial Activity of Marine Culturable Bacteria Collected from a Global Sampling of Ocean Surface Waters and Surface Swabs of Marine Organisms

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Abstract

The purpose of the present study was to isolate marine culturable bacteria with antibacterial activity and hence a potential biotechnological use. Seawater samples (244) and 309 swab samples from biotic or abiotic surfaces were collected on a global Danish marine research expedition (Galathea 3). Total cell counts at the seawater surface were 5 × 105 to 106 cells/ml, of which 0.1–0.2% were culturable on dilute marine agar (20°C). Three percent of the colonies cultured from seawater inhibited Vibrio anguillarum, whereas a significantly higher proportion (13%) of colonies from inert or biotic surfaces was inhibitory. It was not possible to relate a specific kind of eukaryotic surface or a specific geographic location to a general high occurrence of antagonistic bacteria. Five hundred and nineteen strains representing all samples and geographic locations were identified on the basis of partial 16S rRNA gene sequence homology and belonged to three major groups: Vibrionaceae (309 strains), Pseudoalteromonas spp. (128 strains), and the Roseobacter clade (29 strains). Of the latter, 25 strains were identified as Ruegeria mobilis or pelagia. When re-testing against V. anguillarum, only 409 (79%) retained some level of inhibitory activity. Many strains, especially Pseudoalteromonas spp. and Ruegeria spp., also inhibited Staphylococcus aureus. The most pronounced antibacterial strains were pigmented Pseudoalteromonas strains and Ruegeria spp. The inhibitory, pigmented Pseudoalteromonas were predominantly isolated in warmer waters from swabs of live or inert surfaces. Ruegeria strains were isolated from all ocean areas except for Arctic and Antarctic waters and inhibitory activity caused by production of tropodithietic acid.

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Acknowledgments

The Carlsberg Foundation and the Danish Center for Food Research (LMC) are thanked for donations covering international travel. Funding from the Programme Committee for Food, Health and Welfare under the Danish Strategic Research Council is acknowledged. We express our thankfulness to Drs. Jørn Smedsgaard and Kristian Fog Nielsen and technician Ellen Kirstine Lyhne for participation in and sampling done during the cruise. We thank Kerstin Geitner and Andreas Espersen for help with Fig. 1 and Matthias Wietz for help with Fig. 3. The present work was carried out as part of the Galathea 3 expedition under the auspices of the Danish Expedition Foundation. This is Galathea 3 contribution no. P50.

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  1. Jesper Bartholin Bruhn

    Present address: Chr. Hansen A/S, Bøge Allé 10-12, 2970, Hørsholm, Denmark

Authors and Affiliations

  1. National Institute of Aquatic Resources, Technical University of Denmark, Søltofts Plads Building 221, 2800, Kgs. Lyngby, Denmark

    Lone Gram, Jette Melchiorsen & Jesper Bartholin Bruhn

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Correspondence to Lone Gram.

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Work was done at National Institute of Aquatic Sciences and on board the vessel “Vædderen” during a global marine research cruise.

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Supplementary Table 1

Distribution of 122 seawater samples. Samples were taken from CTD (conductivity temperature depth) casts and water analyzed from surface (SUR) and deep chlorophyll maximum (DCM) samples (114 samples). Three samples were from coastal waters and five from the stern tube inlet (5 m below surface). Samples were analyzed raw and divided into particle-associated (PA) bacteria on filters >5 µm and non-associated (NA) bacteria (filtrate from 5-µm filtration). The level of inhibitory bacteria was determined by replica plating onto an agar cast with Vibrio anguillarum strain 90-11-287 (serotype O1). Red strain number indicates that strain was sequenced (16S rRNA). Blue indicates that sequence was attempted but failed. nd = not done; "0" indicates no inhibitory colonies on replica (PDF 42 kb)

Supplementary Table 2

Distribution of 217 surface samples from 54 locations. One hundred and seventy four samples are from live marine surfaces (fish, snakes, mussels, sea weed, sponges, etc.) and 43 samples from inert surface (wood, plastic, stones, and sediment). Colony-forming units (CFUs) have been determined using marine agar (Difco 2216) diluted 50% with local seawater. Red strain number indicates that strain was sequenced (16S rRNA). Blue indicates that sequencing was attempted but failed. nd = not done; 0 = no inhibitory colonies (PDF 26 kb)

Supplementary Table 3

Distribution of 92 sea chest filter samples from 18 locations. Seventy two samples are from live marine surfaces (sea weed, crustaceans, fish, etc.) and 20 samples from inert surfaces (plastic, feathers). Colony-forming units (CFUs) have been determined using marine agar (Difco 2216) diluted 50% with local seawater. Red strain number indicates that strain was sequenced (16S rRNA). Blue indicates that sequencing was attempted but failed. (PDF 17 kb)

Supplementary Table 4

Grouping/identification by 16S rRNA gene sequence homology of 519 marine strains isolated from seawater or surfaces as potentially inhibitory towards Vibrio anguillarum strain 90-11-287 (serotype O1). Re-testing of the strains against V. anguillarum and testing against Staphylococcus aureus NCTC 8325 was done by streaking the isolates on agar cast with the pathogens. All marine strains were grown in marine broth at 25°C and the presence of antibacterial compounds in the culture supernatant tested in a well diffusion assay against the two target organisms. Species names of strains identified as Vibrio sp. are only tentative. Ruegeria pelagis and Ruegeria immobilis are not easily distinguished based on 16S rRNA gene sequence. Numbers on sub-samples refer to supplementary Tables 1, 2, and 3 in which the original water and surface samples are described. (PDF 39 kb)

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Gram, L., Melchiorsen, J. & Bruhn, J.B. Antibacterial Activity of Marine Culturable Bacteria Collected from a Global Sampling of Ocean Surface Waters and Surface Swabs of Marine Organisms. Mar Biotechnol 12, 439–451 (2010). https://doi.org/10.1007/s10126-009-9233-y

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