#METABOLOMICS WORKBENCH wangchao_20250318_185134 DATATRACK_ID:5741 STUDY_ID:ST003866 ANALYSIS_ID:AN006354 PROJECT_ID:PR002422
VERSION             	1
CREATED_ON             	April 16, 2025, 5:42 pm
#PROJECT
PR:PROJECT_TITLE                 	Mechanisms of Exogenous Metabolites Alter the Bactericidal Efficiency of
PR:PROJECT_TITLE                 	Antibiotics by Affecting the Metabolic State of Bacteria
PR:PROJECT_SUMMARY               	Antibiotic resistance is a serious global crisis. Different from the traditional
PR:PROJECT_SUMMARY               	drug resistance mechanism, metabolic regulation can change bacteria from
PR:PROJECT_SUMMARY               	antibiotic resistant state to sensitive state, which provides a new way for
PR:PROJECT_SUMMARY               	antibiotics to treat the infection of resistant bacteria. In previous studies,
PR:PROJECT_SUMMARY               	we found that arginine can significantly improve the bactericidal efficiency of
PR:PROJECT_SUMMARY               	antibiotics, but the mechanism was not clear. We plan to study the effects of
PR:PROJECT_SUMMARY               	arginine on the global metabolic pathway from the perspective of metabolomics,
PR:PROJECT_SUMMARY               	and finally determine how arginine changes the bactericidal efficiency of
PR:PROJECT_SUMMARY               	antibiotics.
PR:INSTITUTE                     	Chinese Academy of Sciences
PR:LAST_NAME                     	Wang
PR:FIRST_NAME                    	Chao
PR:ADDRESS                       	NO.162 Jiefang Road, Jinan, China
PR:EMAIL                         	wangchao850303@163.com
PR:PHONE                         	13075330325
#STUDY
ST:STUDY_TITLE                   	Impact of Exogenous Arginine Addition on the Bacterial Metabolome
ST:STUDY_SUMMARY                 	The interaction between bacterial metabolic state modulation and antibiotic
ST:STUDY_SUMMARY                 	susceptibility has gained increasing attention as a strategy to combat
ST:STUDY_SUMMARY                 	antibiotic resistance. This study investigated the mechanism by which L-arginine
ST:STUDY_SUMMARY                 	enhanced the bactericidal efficiency of antibiotics. The metabolites in the
ST:STUDY_SUMMARY                 	exogenous Arg group and the control group were analyzed using LC-MS. KEGG
ST:STUDY_SUMMARY                 	enrichment analysis identified three significantly altered pathways:
ST:STUDY_SUMMARY                 	phenylalanine metabolism, degradation of aromatic compounds, and the TCA cycle.
ST:STUDY_SUMMARY                 	The results confirmed that arginine can change multiple metabolic pathways of
ST:STUDY_SUMMARY                 	bacteria, providing theoretical support for the study of arginine to change the
ST:STUDY_SUMMARY                 	bactericidal efficiency of antibiotics.
ST:INSTITUTE                     	Chinese Academy of Sciences
ST:LAST_NAME                     	Wang
ST:FIRST_NAME                    	Chao
ST:ADDRESS                       	NO.162 Jiefang Road, Jinan, China
ST:EMAIL                         	wangchao850303@163.com
ST:PHONE                         	13075330325
#SUBJECT
SU:SUBJECT_TYPE                  	Bacteria
SU:SUBJECT_SPECIES               	Escherichia coli
SU:TAXONOMY_ID                   	Escherichia coli BW25113
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	control 1	Sample source:Wild-type | Treatment:Control	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054189-1A
SUBJECT_SAMPLE_FACTORS           	-	control 2	Sample source:Wild-type | Treatment:Control	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054190-1A
SUBJECT_SAMPLE_FACTORS           	-	control 3	Sample source:Wild-type | Treatment:Control	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054191-1A
SUBJECT_SAMPLE_FACTORS           	-	control 4	Sample source:Wild-type | Treatment:Control	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054192-1A
SUBJECT_SAMPLE_FACTORS           	-	control 5	Sample source:Wild-type | Treatment:Control	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054193-1A
SUBJECT_SAMPLE_FACTORS           	-	sample 1	Sample source:Wild-type | Treatment:sample	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054195-1A
SUBJECT_SAMPLE_FACTORS           	-	sample 2	Sample source:Wild-type | Treatment:sample	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054196-1A
SUBJECT_SAMPLE_FACTORS           	-	sample 3	Sample source:Wild-type | Treatment:sample	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054197-1A
SUBJECT_SAMPLE_FACTORS           	-	sample 4	Sample source:Wild-type | Treatment:sample	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054198-1A
SUBJECT_SAMPLE_FACTORS           	-	sample 5	Sample source:Wild-type | Treatment:sample	RAW_FILE_NAME(Raw file name)=HFX11_CN1_FZTM240054199-1A
#COLLECTION
CO:COLLECTION_SUMMARY            	Random colonies of E. tarda strain ATCC 15947 were cultured overnight at 37°C
CO:COLLECTION_SUMMARY            	in TSB medium, and cells were diluted 1:100 into fresh medium under the same
CO:COLLECTION_SUMMARY            	incubation condition. The bacteria were harvested when OD600 was 1.0. The
CO:COLLECTION_SUMMARY            	equivalent cells were quenched with 60% (v/v) cold of methanol at −40°C for
CO:COLLECTION_SUMMARY            	5 s and collected by centrifugation at 1000g at 4°C for 10 min. After removing
CO:COLLECTION_SUMMARY            	the supernatant, the cells were stored at −80°C.
CO:SAMPLE_TYPE                   	Bacterial cells
#TREATMENT
TR:TREATMENT_SUMMARY             	E.tarda cells diluted in M9 media with or without exogenous Arg incubated at
TR:TREATMENT_SUMMARY             	37°C for 5 h. Then, the metabolic reaction is terminated by cold methanol
TR:TREATMENT_SUMMARY             	(-80°C), and bacterial cells were harvested rapidly by centrifugation at 5000g
TR:TREATMENT_SUMMARY             	for 5 min. After washed twice by cold sterile saline, the bacterial cells were
TR:TREATMENT_SUMMARY             	frozen by liquid nitrogen, and stored at −80°C.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	After equivalent cells resuspended by 0.1 mL of precooled methanol at -20°C and
SP:SAMPLEPREP_SUMMARY            	incubated for 60 min, the samples were collected by centrifugation at 14,000 g
SP:SAMPLEPREP_SUMMARY            	for 15 min, and the supernatants were dried in a centrifugal evaporator. The
SP:SAMPLEPREP_SUMMARY            	dried samples were redissolved by 80% methanol and analyzed.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Normal phase
CH:INSTRUMENT_NAME               	Vanquish UHPLC system
CH:COLUMN_NAME                   	Agilent ZORBAX Eclipse Plus C18 (100 x 2.1 mm, 3.5 µm)
CH:SOLVENT_A                     	95% Acetonitrile; 5% Water
CH:SOLVENT_B                     	50% Acetonitrile; 50% Water
CH:FLOW_GRADIENT                 	2%B for 1min, 2−50%B for 16.5min, and 50−2%B for 2.5 min
CH:FLOW_RATE                     	0.3 mL/min
CH:COLUMN_TEMPERATURE            	25°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6470A
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The mass spectrometer was operated with a spray voltage of 3.2 kV, capillary
MS:MS_COMMENTS                   	temperature of 320°C, sheath gas flow rate of 35 arb, and aux gas flow rate
MS:MS_COMMENTS                   	of 10 arb. Full MS scan acquired in the Orbitrap mass analyzer was in the
MS:MS_COMMENTS                   	70−1050 m/z range with resolution of 60 000, ACG target 2.00 E+05 ions, and
MS:MS_COMMENTS                   	fill time of 100 ms. The resolution of tandem mass spectra was 15,000 with the
MS:MS_COMMENTS                   	ACG target of 2.00 E+05 ions and fill time of 100 ms. The raw data of MS files
MS:MS_COMMENTS                   	were processed with the Compound Discoverer (version 3.0, Thermo Fisher
MS:MS_COMMENTS                   	Scientific) software. The peak alignment of different samples was conducted
MS:MS_COMMENTS                   	according to the retention time deviation of 0.2 min and the mass deviation of 5
MS:MS_COMMENTS                   	ppm. The peak with signal-to-noise ratio >3 and signal strength extraction >100
MS:MS_COMMENTS                   	000 was extracted to be quantified.
MS:MS_RESULTS_FILE               	ST003866_AN006354_Results.txt	UNITS:peak area	Has m/z:Yes	Has RT:No	RT units:No RT data
#END