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MB Sample ID: SA244722

Local Sample ID:Ad CP36_2_29_1_985
Subject ID:SU002536
Subject Type:Bacteria
Subject Species:Pseudomonas aeruginosa

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Subject:

Subject ID:SU002536
Subject Type:Bacteria
Subject Species:Pseudomonas aeruginosa

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Ad CP36_2_29_1_985SA244722FL030679CP36Group

Collection:

Collection ID:CO002529
Collection Summary:The Department of Wildlife and National Parks (PERHILITAN), Malaysia, allowed use of crocodile material. Additionally, the use of animals was approved by Sunway Research Ethics Committee, Sunway University, Malaysia (SUNREC 2019/023). A convention on international trade in endangered species (CITES) of wild fauna and flora registered crocodile farm, provided the saltwater crocodile, Crocodylus porosus. Management of crocodile including anesthesia and dissection of the internal organs were carried out by qualified personnel at the farm who routinely perform these procedures. Additionally, we also confirmed that all the experiments were carried out in agreement with appropriate protocols and guidelines as formerly defined (Akbar et al., 2019a). The whole gut was removed aseptically and culturable bacteria were isolated using sterile cotton swabs (Akbar et al., 2019b). Next, the culture were streaked on blood agar plates. The plates were incubated for overnight at 37°C with 5% CO2 and 95% humidity. Several bacterial species were observed and further differentiated based on their colony shape, appearance, colour and texture blood agar plates. Unlike bacterial colonies were sub-cultured on nutrient agar plates and incubated for overnight at 37°C. Finally, bacterial identification was done using microbiological as well as 16S rRNA gene amplification and sequencing (Akbar et al., 2019a).
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR002548
Treatment Summary:NA

Sample Preparation:

Sampleprep ID:SP002542
Sampleprep Summary:A volume of 1 mL of the extraction solvent (methanol +0.1% formic acid) was added to the cell extract. The cell extract was then vortexed for 2 min to ensure the quantitative extraction of the metabolites and stored on ice for 1 h, during which the samples were vortexed every 15 min. After this, the insoluble cell matrices were collected and transferred to centrifuge tubes, intermittent ultrasonication using the COPLEY sonicator (QSONICA SONICATOR, USA) under 30% amplifier and for 30 seconds with an ice bath employed throughout the process. Following that, cells debris were then centrifuged (15000 rpm, 10 minutes, - 4 °C) and the sample supernatants were collected and transferred to LC vials for drying in the EZ-2 Plus (GeneVac-Ipswich, UK) at 37 ± 1 °C. Dried samples were resuspended with 200 µL (water + 0.1% formic acid), and vortexed for 2 min. Finally, the samples were filtered using hydrophilic Nylon Syringe Filter of 0.45µm pore size and analyzed by Q-TOF MS.

Combined analysis:

Analysis ID AN003988
Analysis type MS
Chromatography type Reversed phase
Chromatography system Bruker Elute
Column Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 µm, 90 Å)
MS Type ESI
MS instrument type QTOF
MS instrument name Bruker timsTOF
Ion Mode POSITIVE
Units AU

Chromatography:

Chromatography ID:CH002948
Instrument Name:Bruker Elute
Column Name:Hamilton Intensity Solo 2 C18 (100 x 2.1 mm, 1.8 µm, 90 Å)
Column Temperature:35
Flow Gradient:1%B to 99%B in 15 min
Flow Rate:250 uL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS003722
Analysis ID:AN003988
Instrument Name:Bruker timsTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Samples were chromatographically separated by inline reversed-phase chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8 µm beads) was maintained at 35℃ (metabolomics analyses) For metabolomics, 10 µL was injected twice for each sample and eluted using a 30-minute gradient as follows: 1% ACN was held for 2 minutes, ramping to 99% ACN over 15 minutes, held at 99% ACN for 3 minutes before re-equilibrating to 1% ACN for 10 minutes. Flow rates were 250 µL/min for elution and 350 µL/min for re-equilibration. The MS analysis was performed using a TimsTOF (Bruker, Darmstadt, Germany) with Apollo II electrospray ionization (ESI) source. The drying gas was set to flow at 10 L/min and the drying temperature to 220℃ and the nebulizer pressure to 2.2 bar. The capillary voltage was 4500 V and the end plate offset 500V. For proteomics, the scan range was 150 – 2200 m/z, and for metabolomics 20-1300 m/z. For metabolomics the collision energy was set to 20 eV, the cycle time to 0.5 seconds with a relative minimum intensity threshold of 400 counts per thousand and target intensity of 20,000. Sodium formate was injected as an external calibrant in the first 0.3 minutes of each LC-MS/MS run. MetaboScape® 4.0 software was used for metabolite processing and statistical analysis (Bruker Daltonics). The following parameters for molecular feature identification and ‘bucketing’ were set in the T-ReX 2D/3D workflow: For peak detection, a minimum intensity threshold of 1,000 counts is required, as well as a minimum peak duration of 7 spectra, with feature quantification determine using peak area. The file masses were recalibrated based on the external calibrant injected between 0-0.3 min. Only features found in at least three of the twelve samples (per treatment), with retention time between 0.3 and 25 minutes and m/z between 50 and 1,000 were considered and the MS/MS import method was set to be averaged.
Ion Mode:POSITIVE
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