Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA218333

Local Sample ID:	SE_22
Subject ID:	SU002363
Subject Type:	Bacteria
Subject Species:	Staphylococcus epidermidis
Taxonomy ID:	1282
Genotype Strain:	Staphylococcus epidermidis 19N

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Subject:

Subject ID:	SU002363
Subject Type:	Bacteria
Subject Species:	Staphylococcus epidermidis
Taxonomy ID:	1282
Genotype Strain:	Staphylococcus epidermidis 19N

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
SE_22	SA218333	FL026459	N55	class

Collection:

Collection ID:	CO002356
Collection Summary:	The Staphylococcus epidermidis 19N strain was collected from the anterior nares of a healthy person in Portugal in 2001. This strain was previously characterised by whole genome sequencing and belongs to clonal lineage B. A single colony from a S. epidermidis 19N strain culture grown O/N at 37ºC (TSA, BactoTM), was used to pre-inoculate Tryptic Soy Broth (TSB) medium with two different pH (5.5 and 7.4) that was incubated overnight at 37ºC under agitation. Pre-inoculums were adjusted either to pH 5.5 or pH 7.4, with hydrochloric acid. In this work, three pH transitions from pre-inoculum to inoculum were assayed. S. epidermidis pre-inoculums and the growth were performed at medium with pH 7.4, to mimic the blood pH; and pH 5.5, to mimic the skin pH. The pre-inoculum cellular density was adjusted to 0.06 (OD600) (aprox.1.5x108 CFU/mL) and used to inoculate fresh medium in the three conditions depicted in Figure 1, simulating S. epidermidis at skin and blood and a pH shock endured by S. epidermidis during the infection process from skin to blood transition. The cell cultures incubated at 37ºC with 225 rpm were followed by OD600 and recovered at mid-exponential phase for further analysis.
Sample Type:	Staphylococcus epidermidis intracellular
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002375
Treatment Summary:	In this work, three pH transitions from pre-inoculum to inoculum were assayed. S. epidermidis pre-inoculums and the growth were performed at medium with pH 7.4, to mimic the blood pH; and pH 5.5, to mimic the skin pH. The pre-inoculum cellular density was adjusted to 0.06 (OD600) (aprox.1.5x108 CFU/mL) and used to inoculate fresh medium in the three conditions depicted in Figure 1, simulating S. epidermidis at skin and blood and a pH shock endured by S. epidermidis during the infection process from skin to blood transition. The cell cultures incubated at 37ºC with 225 rpm were followed by OD600 and recovered at mid-exponential phase for further analysis. Pre-inocula were prepared in TSB medium at pH 5.5 or 7.4. Inocula at pH 5.5 was used for the cultures grown at 5.5 (N55) and 7.4 (N57), and the inoculum at pH 7.4 for the culture at the same pH (N77). The cells were harvested at mid-exponential phase.

Treatment ID:

TR002375

Treatment Summary:

In this work, three pH transitions from pre-inoculum to inoculum were assayed. S. epidermidis pre-inoculums and the growth were performed at medium with pH 7.4, to mimic the blood pH; and pH 5.5, to mimic the skin pH. The pre-inoculum cellular density was adjusted to 0.06 (OD600) (aprox.1.5x108 CFU/mL) and used to inoculate fresh medium in the three conditions depicted in Figure 1, simulating S. epidermidis at skin and blood and a pH shock endured by S. epidermidis during the infection process from skin to blood transition. The cell cultures incubated at 37ºC with 225 rpm were followed by OD600 and recovered at mid-exponential phase for further analysis. Pre-inocula were prepared in TSB medium at pH 5.5 or 7.4. Inocula at pH 5.5 was used for the cultures grown at 5.5 (N55) and 7.4 (N57), and the inoculum at pH 7.4 for the culture at the same pH (N77). The cells were harvested at mid-exponential phase.

Sample Preparation:

Sampleprep ID:	SP002369
Sampleprep Summary:	Cells were recovered at mid-exponential phase from 100 mL cultures following a protocol adapted from Somerville & Powers (Somerville and Powers 2014). Eight biological replicates of each independent growth condition were obtained. Cells were harvested by centrifugation at 5000 x g for 5 min at 4ºC. Cells were washed with 20 mM phosphate buffer pH 7.2-7.4 and centrifuged for 1 min at 13,000 rpm. Cell pellet was suspended in the same buffer with a final OD600 of 20 and stored at -80ºC for further metabolite extraction. Cells were thawed in a water bath at room temperature and 750 µL of 60% methanol were added and subjected to three freeze-thaw cycles using liquid nitrogen. Extracted samples were centrifuged at 21,000 g for 5 min at 4ºC. The extraction process on the pellets was repeated twice. The supernatants were kept and stored together at -20ºC overnight and dried in a SpeedVac. Dried samples were dissolved in: 750 µL phosphate buffer (33 mM, pH 7.0 in D2O with 2 mM of sodium azide) with 0.21 mM of 3-(trimethylsilyl)propionic-2,2,3,3-d4 (TSP). The suspensions were centrifuged at 21,000 g for 5 min at 4ºC and the resulting supernatants were then transferred to 5 mm NMR tubes.

Sampleprep ID:

SP002369

Sampleprep Summary:

Cells were recovered at mid-exponential phase from 100 mL cultures following a protocol adapted from Somerville & Powers (Somerville and Powers 2014). Eight biological replicates of each independent growth condition were obtained. Cells were harvested by centrifugation at 5000 x g for 5 min at 4ºC. Cells were washed with 20 mM phosphate buffer pH 7.2-7.4 and centrifuged for 1 min at 13,000 rpm. Cell pellet was suspended in the same buffer with a final OD600 of 20 and stored at -80ºC for further metabolite extraction. Cells were thawed in a water bath at room temperature and 750 µL of 60% methanol were added and subjected to three freeze-thaw cycles using liquid nitrogen. Extracted samples were centrifuged at 21,000 g for 5 min at 4ºC. The extraction process on the pellets was repeated twice. The supernatants were kept and stored together at -20ºC overnight and dried in a SpeedVac. Dried samples were dissolved in: 750 µL phosphate buffer (33 mM, pH 7.0 in D2O with 2 mM of sodium azide) with 0.21 mM of 3-(trimethylsilyl)propionic-2,2,3,3-d4 (TSP). The suspensions were centrifuged at 21,000 g for 5 min at 4ºC and the resulting supernatants were then transferred to 5 mm NMR tubes.

Analysis:

MB Sample ID:	SA218333
Analysis ID:	AN003721
Analysis Type:	NMR
Software Version:	Bruker TopSpin 3.2
Num Factors:	3
Num Metabolites:	43
Units:	nanomoles

NMR:

NMR ID:	NM000248
Analysis ID:	AN003721
Instrument Name:	Bruker Avance II+ 800 MHz
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
Spectrometer Frequency:	800
NMR Probe:	5 mm TXI-Z H/C/N/-D
NMR Solvent:	D2O
NMR Tube Size:	5 mm
Shimming Method:	Topshim
Temperature:	25
Number Of Scans:	256
Dummy Scans:	4
Chemical Shift Ref Std:	TSP
NMR Results File:	NMR_SE