1. No category

Novel bioactive compounds from Antarctic bacteria targeting
opportunistic human pathogens
P. Tedesco1,2, F. Palma Esposito1,2, M. Fondi3, I. Maida3, E. Perrin3, J. Tabudravu4, M. Jaspars4, R. Fani3, D. de Pascale1
1- Institute of Protein Biochemistry, CNR, Naples, Italy
2- Department of Chemistry, University of Naples, Naples, Italy
3- Department of Biology, University of Florence, Florence, Italy
4- Marine Biodiscovery Center, University of Aberdeen, UK
Life thriving in extreme environments has raised some of the most intriguing microbiological questions and
has continued to challenge our understanding of biochemistry, biology and evolution. As more new
extremophiles are brought into laboratory culture, they provide a multitude of potential applications for
biotechnology. In particular, microorganisms from extreme environments have proved to be a very good
source of novel antimicrobials to counteract MDR infections. Here we report the set-up of a biodiscovery
pipeline for novel antimicrobial compounds targeting human opportunistic pathogens using Antarctic sub-sea
sediments.
1.Collection of
environmental
samples
PYG medium
plates for 15
days at 4°C
2.Isolation
of bacteria
Antarctic sub-sea sediments
Mario Zucchelli Station, Baia
Terranova Ross sea, Antarctica
24 isolates from Antarctic
sub-sea sediments
4.Generation
of a microbial
extracts
library
3.Cell-based
bioassays
Organic solvent
extractions
Pathogens panel
Organism
Staphylococcus
aureus
Escherichia coli
Pseudomonas
aeruginosa
Klebsiella
pneumoniae
Burkholderia
cenocepacia
Burkholderia
metallica
Strains
10 strains
6538P
K12
PA01
Cross-streaking assay targeting
opportunistic human pathogens
5 days of growth at 20°C
K6
LMG J2315
LMG 24068
5.Bioassaydriven
purification
5.1 Extracts library screening
targeting Bcc strains
In collaboration with Marine Biodiscovery Center,
Aberdeen, UK
5.2 SPE Fractionation and HPLC on active extracts
C9
[1 mg/mL] Pseudomonas Pseudoalteromonas Psychrobacter Arthrobacter %inhibiJon BTN 1 BTN 2 BTN 15 BTN 3 BTN 19 BTN 21 BTN 4 BTN 5 B. diffusa 90 75 77 43 45 70 63 77 B. metallica 90 70 71 32 30 53 64 77 B. cenocepacia 90 78 87 84 64 60 55 84 B. latens 90 53 75 55 43 40 68 56 B. anthina 90 38 60 30 25 46 50 38 C8
C7
C10
C6
strong inhibiJon weak inhibiJon Selection of the 3 best strains: BTN1-BTN5-BTN1
Isolation of 11 peaks from BTN1
5.3 Pure compounds bioassay
O
H
Burkholderia cenocepacia
200 ug/mL C5 0,118 C6 -­‐0,009 C7 -­‐0,002 C8 -­‐0,005 C9 -­‐0,009 C10 -­‐0,002 C11 0,109 DMSO 0,285 100 ug/mL 50 ug/mL 25 ug/mL 12,5 ug/mL Cells Only 0,224 0,281 0,368 0,361 0,336 -­‐0,007 -­‐0,007 -­‐0,004 -­‐0,002 0,238 -­‐0,003 -­‐0,003 -­‐0,002 -­‐0,003 0,282 -­‐0,002 -­‐0,001 -­‐0,001 0,000 0,280 -­‐0,006 -­‐0,005 -­‐0,004 -­‐0,001 0,036 0,057 0,140 0,229 0,158 0,217 0,235 0,242 0,291 0,301 0,304 0,303 6.Identification
of the bioactive
compounds
NMR and LC-MS on the
bioactive compounds
Staphylococcus aureus
C5 C6 C7 C8 C9 C10 C11 DMSO 100 ug/mL 50 ug/mL 25 ug/mL 12,5 ug/mL Cells Only 0,257 0,199 0,202 0,205 0,176 0,008 0,005 0,006 0,006 0,243 0,011 0,009 0,009 0,008 0,251 0,015 0,012 0,035 0,056 0,227 0,012 0,008 0,008 0,009 0,069 0,139 0,113 0,140 0,116 0,247 0,262 0,347 0,228 0,253 0,275 0,353 Future Task
N
HN
HO
H
Preliminary chemical
analysis suggest the
bioactivity relies on a
synergistic effect of
different 2,5
Diketopiperazines
O
-Determination of MIC against several
human pathogens
-Analysis of the mutation frequency
-In vivo proof of effecacy using the
nematode C. elegans