MAGE-TAB Version 1.1 Investigation Title Various evolutionary trajectories lead to loss of activity of tobramycin-potentiating compounds in Burkholderia cenocepacia biofilms Experimental Design Experimental Design Term Source REF Experimental Design Term Accession Number Experimental Factor Name compound sampling time point Experimental Factor Type compound sampling time point Experimental Factor Term Source REF EFO Experimental Factor Term Accession Number Person Last Name Sass Slachmuylders Person First Name Andrea Lisa Person Mid Initials Person Email andrea.sass@ugent.be elisabeth.slachmuylders@ugent.be Person Phone Person Fax Person Affiliation Ghent University Department of Pharmaceutical Sciences Laboratory pharmaceutical microbiology - Ghent university Person Address Ottergemsesteenweg 460 9000 Gent Belgium Ottergemsesteenweg 460, 9000 Ghent, Belgium Person Roles submitter submitter Person Roles Term Source REF Person Roles Term Accession Number Quality Control Type Quality Control Term Source REF Quality Control Term Accession Number Replicate Type Replicate Term Source REF Replicate Term Accession Number Normalization Type Normalization Term Source REF Normalization Term Accession Number Date of Experiment 2017-10-22 Public Release Date 2018-07-22 PubMed ID 30670425 Publication DOI 10.1128/aac.02092-18 Publication Author List Sass A, Slachmuylders L, Van Acker H, Vandenbussche I, Ostyn L, Bové M, Crabbé A, Chiarelli LR, Buroni S, Van Nieuwerburgh F, Abatih E, Coenye T. Publication Title Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms. Publication Status Publication Status Term Source REF Publication Status Term Accession Number Experiment Description Antibiotic adjuvants are commonly described as an alternative approach to overcome bacterial resistance towards conventional antibiotics. In this experiment, we investigated this statement for tobramycin (TOB) in combination with three adjuvants; baicalin hydrate (BH), a quorum sensing inhibitor, econazole (ECO) and miconazole (MICO), two antifungal agents that are repurposed as antibiotic adjuvants. We repeatedly exposed mature (24 hour old) Burkholderia cenocepacia J2315 biofilm cells to TOB alone (768ug/ml), or a combination of TOB with either BH (250uM), ECO (1uM) or MICO (1uM). We also included an untreated control. After a treatment, the remaining cells were quantified and the cells were allowed to regrow for another 48 hours. This process is one cycle. At the end of the experiment, biofilm cells were exposed to 15 cycles. DNA extraction was performed on the evolved cells and of cells from the start population. DNA sequencing was performed on these samples and single nucleotide polymorphisms compared to the start population were evaluated. Protocol Name P-MTAB-69526 P-MTAB-69531 P-MTAB-69530 P-MTAB-76887 P-MTAB-76886 P-MTAB-69527 P-MTAB-69528 P-MTAB-69529 Protocol Type sample collection protocol treatment protocol growth protocol nucleic acid sequencing protocol nucleic acid library construction protocol nucleic acid extraction protocol nucleic acid library construction protocol nucleic acid sequencing protocol Protocol Term Source REF EFO EFO EFO EFO EFO EFO EFO EFO Protocol Term Accession Number EFO_0005518 EFO_0003969 EFO_0003789 EFO_0004170 EFO_0004184 EFO_0002944 EFO_0004184 EFO_0004170 Protocol Description Start population: this sample was collected from an overnight culture. Evolved populations: after 15t cycles of biofilm formation - treatment with TOB alone, TOB+BH, TOB+ECO or TOB+MICO - planktonic regrowth (48h), cells were allowed to regrow for 48 hours. These cells were used to extract DNA from. After 24 hours, biofilms were treated with either tobramycin (TOB) (768ug/ml) alone or in combination with baicalin hydrate (BH) (250uM), econazole (ECO) (1uM) or miconazole (MICO) (1uM). An untreated control was also included. After 24 hours of treatment, the remaining cells were divided in two, where one part was used to quantify the remaining cells and the other part was allowed to regrow planktonically. This process was repeated 15 times. Three independent lineages of biofilms were set up, to avoid contamination. Biofilms were formed on beads, starting from a planktonic culture with approximately 10^6 cells. Biofilms were allowed to grow for 24 hours Illumina Nextseq 500, generating 150 bp paired-end reads Nextera kit DNA was extracted using a modified bead-beater protocol. Briefly, the pellets were resuspended in 200 µl TE-buffer (10 mM Tris-HCl (Roche Diagnostics, Mannheim, Germany), 1 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich), pH 8.0). 100 µl of this suspension was transferred to a 2 ml Eppendorf tube containing approximately 0.5 ml of 0.1-mm-diameter glass beads (Sigma-Aldrich), 0.5 mg/ml pronase (Roche) and 500 µl lysis buffer (50 mM Tris-HCl, 70 mM EDTA, 1% sodium dodecyl sulfate (SDS; Sigma-Aldrich), pH 8). After 10 sec of vigorous vortexing, the samples were incubated for 1 hour at 37°C. Afterwards, the tubes were briefly centrifuged and 200 µl saturated ammonium acetate was added to the lysate. The tubes were, again, vigorously vortexed for 10 sec and centrifuged (13000 rpm, 2 min). Subsequently, 600 µl chloroform (Sigma-Aldrich) was added and the tubes were mixed vigorously. Proteins and polysaccharides were removed by centrifugation (13000 rpm, 5 min), and nucleic acids were collected from the lysate by ethanol precipitation. The pellet was washed with 70% ethanol, and dissolved in 300 µl Low-TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8) containing 0.5 µg/ml RNase A (Sigma-Aldrich) to inhibit the remaining RNA. After 1 hour incubation at 37°C, DNA was quantified using the BioDrop μLITE (BioDrop, Cambridge, UK). NEBNext kit Illumina Nextseq 500, generating 150 bp paired-end reads Protocol Parameters Protocol Hardware NextSeq 500 NextSeq 500 Protocol Software Protocol Contact Term Source Name EFO ArrayExpress Term Source File http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/arrayexpress/ Term Source Version 2.38 SDRF File E-MTAB-6236.sdrf.txt Comment [Submitted Name] Various evolutionary trajectories lead to loss of activity of tobramycin-potentiating compounds in Burkholderia cenocepacia biofilms Comment [SecondaryAccession] ERP105327 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2697196-ERR2697197 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2202043-ERR2202052 Comment [AEExperimentType] DNA-seq Comment[ArrayExpressAccession] E-MTAB-6236