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Bioactive bacteria from Arctic marine environments

Authors:

Abstract

Bacterial antagonism plays a key role in microbial interactions, being a competitive advantage but also mean of communication. Bacterial metabolites with antagonistic activity could also have biotechnological applications. Here, we isolated marine bacteria from high Arctic environments (84 to 89º N) using Marine Agar 2216, chitin agar, two low-nutrient agars and an actinobacterial-selective mixture at different temperatures. This was applied to fifty-eight samples, including 17 seawater, 20 sea ice, 11 zooplankton, 3 sediment, and 7 freshwater-related samples (snow, meltwater and frost flowers). Culturable counts for ice samples were above 10e4 CFU/mL, compared to 102 CFU/mL for water and zooplankton and below 10e1 CFU/mL for all other samples. Counts on nutrient limited agar did not significantly differ from counts on nutrient rich agar. Also the incubation temperature did not influence the culturable count. 81 strains (representing 0.3% of all tested colonies) antagonized Vibrio anguillarum 90-11-287, of which 9 strains also inhibited Staphylococcus aureus 8325. The most pronounced bioactive strains are presently characterized with respect to identity and phenotypic characteristics. In addition, several strains with chitinolytic activity were isolated and are also being identified. In conclusion, this study contributes to the knowledge of bacterial antagonism and the diversity of culturable bioactive bacteria in polar environments. The antagonistic isolates are implemented in an ongoing research project on bioactive bacteria from the World’s Oceans.
Bioactive bacteria from Arctic marine environments
Background
Bioactive bacterial metabolites that antagonize other microbes play a key role in
microbial interactions and may have biotechnological potential. The present
study describes the isolation of marine bacteria from high Arctic environments,
their screening for inhibitory activity against pathogenic bacteria, and chemical
profiling of produced antimicrobials.
Results
Experimental approach
Bacteria were isolated from environmental samples at 20 and 5 °C using Marine,
Actinomycete (Oxoid), chitin, and two low-nutrient agars. Bioactivity was
assessed by growth inhibition of pathogenic bacteria. Active culture extracts
were analyzed by liquid chromatography-mass spectrometry (LC-MS).
Fig 1: Sampling stations
Acknowledgements Thanks to the crew and scientific party of the LOMROG-II expedition, particularly
those kindly sharing samples: Jeff Bowman, Jody Deming (University of Washington), Kajsa
Tönnesson, Rasmus Swalethorp (University of Gothenburg), Steffen Olsen, Leif Pedersen (Danish
Meterological Institute), Ludwig Löwemark, Markus Karasti, Åsa Wallin (University of Stockholm)
Matthias Wietz, Maria Månsson, Nete Bernbom, Yoke Yin Ng, Lone Gram
National Food Institute, Technical University of Denmark, Søltofts Plads, 2800 Lyngby, Denmark
Strain Source Gram Catalase
Oxidase Glucose
O/F
PP12 Ice positive +/- -/-
RR12 Amphipods negative +/+ +/-
TT4 Meltwater positive +/- -/-
LM7 Surface water positive +/- -/-
WX11 Deep water positive +/- -/-
MB33 Copepods negative +/+ -/-
MB182 Ice positive +/- -/-
Table 1: Strains with considerable inhibition of
Vibrio anguillarum and Staphylococcus aureus
Compound
extraction Activity test
LC-MS
Original plate Testing against
pathogen
Conclusions
The marine Arctic harbours bioactive bacteria that
antagonize bacterial pathogens
Sea ice highest in viable counts and bioactive strains
Bioactivity likely attributed to novel antimicrobials
Fig 4: LC-MS chromatogram of extract from WX-11. Peaks
relate to different compounds (candidates for bioactivity)
Contact: Matthias Wietz (mwi@aqua.dtu.dk)
Fig 3: Origin of 112 bioactive isolates and inhibition
of Vibrio anguillarum by bioactive culture extracts
Fig 2: Viable counts of 58 samples, ranging from 101
for sediment and 104 CFU/mL for ice samples. No
difference between agars or incubation temperatures
Time
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
%
10020.55
19.22
17.91
17.75
16.24
12.70
9.79 14.92
14.24
17.23 18.70
19.36
23.42
21.65
21.43 22.02
0
386 Da
738 Da
766 Da
1024 Da
1052 Da
1080 Da
1080 Da
1108 Da
1102 Da
Sediment
Snow/melt
Ice
Seawater
Zoo-
plankton
4x104
CFU/mL
2x102
CFU/mL
3x102 CFU/mL
4x101
CFU/mL
101 CFU/mL
20
17
11
73
... Chemical analyses indicated the presence of novel antimicrobials (Biondi et al. 2008), including alkaloids from Pseudomonas (Jayatilake et al. 1996), aromatic nitro compounds from Salegentibacter (Al-Zereini et al. 2007), a naphthalene compound from the cyanobacterium Nostoc (Asthana et al. 2009), and a novel angucyclinone antibiotic, designated frigocyclinone, from Streptomyces (Bruntner et al. 2005).36The present PhD study provides the first description of bioactive strains from high Arctic environments(Wietz et al. 2011b), describing seventeen bioactive strains affiliating with the Actinobacteria (8 strains), Pseudoalteromonas (4 strains), the Vibrionaceae (3 strains), and ...
Full-text available
Thesis
The purpose of the present study was to analyze the composition of marine bacterial communities around the world, and to investigate bacterial isolates regarding the production of antibiotics. This included molecular analyses of marine bacterioplankton, as well as culture-based studies of marine bacterial isolates with antagonistic activity. The work was based on samples collected during the Galathea 3 and LOMROG-II marine research expeditions that have explored many different oceanic regions worldwide. A molecular survey of marine bacterioplankton at 24 worldwide stations investigated the abundance of major bacterial groups, potential biogeographical patterns, and their relation to environmental parameters. The original aim was to determine whether the composition of the total microbiota correlates with the occurrence of culturable bioactive bacteria. No such correlation was found. Quantitative community analyses showed latitudinal patterns in bacterial distribution, revealing significantly different relative abundances of Bacteroidetes, unclassified Bacteria and Vibrio between warmer and colder oceans. Absolute cell numbers of most bacterial groups were positively correlated with nutrient concentrations in warmer oceans, and negatively with oxygen saturation in colder oceans. The finding of differing communities in warmer and colder oceans underlined the presence of biogeographical patterns among marine bacteria and the influence of environmental parameters on bacterial distribution. Studies of antagonistic isolates focused on six bioactive Vibrionaceae isolated during Galathea 3. The six strains were identified as Vibrio coralliilyticus (two strains), V. neptunius (two strains), V. nigripulchritudo (one strain), and Photobacterium halotolerans (one strain) by sequencing of housekeeping genes. Chemical metabolite profiling underlined genetic relationships by showing highly similar production of secondary metabolites for each species. Two known antibiotics were purified; andrimid from V. coralliilyticus and holomycin from P. halotolerans. In addition, two novel cyclic peptides from P. halotolerans and a novel siderophore-like compound from V. nigripulchritudo were isolated. All three compounds interfere with quorum sensing in S. aureus. During LOMROG-II further seventeen strains with antagonistic activity were isolated, affiliating with the Actinobacteria (8 strains), Pseudoalteromonas (4 strains), the Vibrionaceae (3 strains), and Psychrobacter (2 strains). Seven of the eight bioactive Actinobacteria, being isolated from different sources throughout the Arctic Ocean, were related to Arthrobacter davidanieli. Its broad antibiotic spectrum was likely based on production of the known arthrobacilin antibiotics. The eighth actinomycete, tentatively identified as Brevibacterium sp., produces a potentially novel antimicrobial compound. Most studies of antagonistic marine bacteria have been conducted with the aim of isolating novel antimicrobials with potential clinical applications. However, little is known about production and role of these compounds in the natural environment. This thesis took one step in this direction and demonstrated that V. coralliilyticus S2052 produced its antibiotic andrimid when grown with chitin as the sole carbon source. Whilst the strain produced an array of secondary metabolites in laboratory media, it focused on andrimid production with chitin. This indicates that the antibiotic is likely produced in the natural habitat and may serve an ecophysiological function. The finding that two related strains from public culture collections do not produce andrimid and have different biosynthetic temperature optima suggested that V. coralliilyticus may comprise different subspecies with different niches. In summary, the present study shows biogeographical patterns of marine bacterioplankton on a global scale. In addition, the thesis work has demonstrated that marine Vibrionaceae and polar Actinobacteria are a resource of antibacterial compounds and may have potential for future natural product discovery.
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