#METABOLOMICS WORKBENCH chenqr_20250302_230752 DATATRACK_ID:5691 STUDY_ID:ST003777 ANALYSIS_ID:AN006204 PROJECT_ID:PR002355 VERSION 1 CREATED_ON March 6, 2025, 8:11 am #PROJECT PR:PROJECT_TITLE Metabolomics of Staphylococcus epidermidis HE23 isolated from the hospital PR:PROJECT_TITLE environment PR:PROJECT_SUMMARY Metabolomic analysis of Staphylococcus epidermidis HE23 isolated from the PR:PROJECT_SUMMARY hospital environment to investigate its metabolic profile and potential hazards PR:INSTITUTE Anhui Medical University PR:LAST_NAME Chen PR:FIRST_NAME Qingru PR:ADDRESS 81 Meishan Road, Hefei, Anhui 230032, China PR:EMAIL chenqr95@foxmail.com PR:PHONE 18156455228 #STUDY ST:STUDY_TITLE Separation and Characterization of a Clinically-Derived Staphylococcus ST:STUDY_TITLE epidermidis Strain HE23: Revealing Its Antibiotic Resistome and Metabolic ST:STUDY_TITLE Potential ST:STUDY_SUMMARY Neonatal incubators, while essential for infant care, are high-risk environments ST:STUDY_SUMMARY for nosocomial infections due to microbial colonization. Staphylococcus ST:STUDY_SUMMARY epidermidis, a biofilm-forming opportunistic pathogen, frequently contaminates ST:STUDY_SUMMARY medical devices, yet its metabolic adaptation mechanisms and virulence potential ST:STUDY_SUMMARY in incubator settings remain poorly understood. This study integrates genomics ST:STUDY_SUMMARY and metabolomics to comprehensively profile a multidrug-resistant S. epidermidis ST:STUDY_SUMMARY HE23 strain isolated from neonatal incubators, aiming to reveal its antibiotic ST:STUDY_SUMMARY resistance determinants and toxic metabolite production linked to clinical ST:STUDY_SUMMARY pathogenicity. A bacterial strain (HE23) was isolated from a neonatal incubator ST:STUDY_SUMMARY and identified as Staphylococcus epidermidis by 16S rRNA sequencing. Whole ST:STUDY_SUMMARY genome sequencing was performed using Illumina and Oxford Nanopore platforms, ST:STUDY_SUMMARY and a high-quality genome was obtained by hybrid assembly (Unicycler). And ST:STUDY_SUMMARY metabolomic analysis was performed by liquid mass spectrometry detection. ST:STUDY_SUMMARY Metabolomic profiling of Staphylococcus epidermidis HE23 revealed 47 ST:STUDY_SUMMARY differentially expressed metabolites (15 upregulated, 32 downregulated; VIP > ST:STUDY_SUMMARY 1.0, p < 0.05). Among upregulated metabolites, two clinically significant toxins ST:STUDY_SUMMARY were identified:Harmaline (m/z 446.2529 ), Cinobufagin (m/z 460.2686). Pathway ST:STUDY_SUMMARY enrichment highlighted glutathione metabolism (VIP = 2.1, p = 0.008) and ST:STUDY_SUMMARY branched-chain amino acid degradation (VIP = 1.8, p = 0.015), suggesting redox ST:STUDY_SUMMARY stress adaptation and nutrient scavenging strategies. ST:INSTITUTE Anhui Medical University ST:DEPARTMENT College of Life Sciences ST:LABORATORY College of Life Sciences, Anhui Medical University ST:LAST_NAME Chen ST:FIRST_NAME Qingru ST:ADDRESS 81 Meishan Road, Hefei, Anhui 230032, China ST:EMAIL chenqr95@foxmail.com ST:PHONE 18156455228 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Staphylococcus epidermidis SU:TAXONOMY_ID 1282 SU:GENOTYPE_STRAIN Staphylococcus epidermidis HE23 #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 - C1 Sample source:Blank | Sample_type:control RAW_FILE_NAME=NEG_Hc18LS_C1.mzML/POS_Hc18LS_C1.mzML SUBJECT_SAMPLE_FACTORS - C2 Sample source:Blank | Sample_type:control RAW_FILE_NAME=NEG_Hc18LS_C2.mzML/POS_Hc18LS_C2.mzML SUBJECT_SAMPLE_FACTORS - C3 Sample source:Blank | Sample_type:control RAW_FILE_NAME=NEG_Hc18LS_C3.mzML/POS_Hc18LS_C3.mzML SUBJECT_SAMPLE_FACTORS - H1 Sample source:Bacteria | Sample_type:Bacterial cells RAW_FILE_NAME=NEG_Hc18LS_H1.mzML/POS_Hc18LS_H1.mzML SUBJECT_SAMPLE_FACTORS - H2 Sample source:Bacteria | Sample_type:Bacterial cells RAW_FILE_NAME=NEG_Hc18LS_H2.mzML/POS_Hc18LS_H2.mzML SUBJECT_SAMPLE_FACTORS - H3 Sample source:Bacteria | Sample_type:Bacterial cells RAW_FILE_NAME=NEG_Hc18LS_H3.mzML/POS_Hc18LS_H3.mzML SUBJECT_SAMPLE_FACTORS - QC01 Sample source:QC | Sample_type:QC(Internal Standard) RAW_FILE_NAME=NEG_Hc18LS_QC01.mzML/POS_Hc18LS_QC01.mzML SUBJECT_SAMPLE_FACTORS - QC02 Sample source:QC | Sample_type:QC(Internal Standard) RAW_FILE_NAME=NEG_Hc18LS_QC02.mzML/POS_Hc18LS_QC02.mzML SUBJECT_SAMPLE_FACTORS - QC03 Sample source:QC | Sample_type:QC(Internal Standard) RAW_FILE_NAME=NEG_Hc18LS_QC03.mzML/POS_Hc18LS_QC03.mzML #COLLECTION CO:COLLECTION_SUMMARY The Staphylococcus epidermidis HE23 strain was isolated from the inner surface CO:COLLECTION_SUMMARY of a neonatal incubator in the Neonatal Intensive Care Unit (NICU) at the First CO:COLLECTION_SUMMARY Affiliated Hospital of Anhui Medical University (Hefei, China). Post-sampling, CO:COLLECTION_SUMMARY the isolate was purified via streak-plating on LB agar. Taxonomic classification CO:COLLECTION_SUMMARY was confirmed through 16S ribosomal RNA gene sequencing (primers 27F/1492R). For CO:COLLECTION_SUMMARY experimental analyses, HE23 was cultured in standard LB broth (10 g/L tryptone, CO:COLLECTION_SUMMARY 5 g/L yeast extract, 10 g/L NaCl; pH 7.2) under aerobic conditions. Pre-inoculum CO:COLLECTION_SUMMARY was prepared by incubating at 37°C with 200 rpm orbital shaking for 16–18 CO:COLLECTION_SUMMARY hours to reach mid-log phase (OD₆₀₀ ≈ 0.8). Cells were harvested by CO:COLLECTION_SUMMARY centrifugation (4,000 ×g, 10 min, 4°C) and washed twice with sterile CO:COLLECTION_SUMMARY phosphate-buffered saline (PBS) prior to downstream assays. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY NA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Accurately pipette 200 μL of sample into a 1.5 mL centrifuge tube; Add 400 μL SP:SAMPLEPREP_SUMMARY of extraction solution (methanol:acetonitrile=1:1 (v:v)) containing four SP:SAMPLEPREP_SUMMARY internal standards (e.g., L-2-chlorophenylalanine (0.02 mg/mL), etc.); vortex SP:SAMPLEPREP_SUMMARY and mix for 30 s, and then ultrasonic extraction was carried out for 30 min at SP:SAMPLEPREP_SUMMARY low temperature (5°C, 40 KHz); the sample was left at -20°C for 30 min; SP:SAMPLEPREP_SUMMARY centrifuged for 15 min (13,000g, 4°C), and the supernatant was removed and The SP:SAMPLEPREP_SUMMARY samples were allowed to stand at -20℃ for 30 minutes, centrifuged for 15 SP:SAMPLEPREP_SUMMARY minutes (13000g, 4℃), the supernatant was pipetted and blown dry with SP:SAMPLEPREP_SUMMARY nitrogen; 120 μL of reconstituted solution (acetonitrile:water=1:1) was added SP:SAMPLEPREP_SUMMARY to reconstitute the sample; the samples were vortexed again for 30 seconds, and SP:SAMPLEPREP_SUMMARY then sonicated at low temperature for 5 minutes (5℃, 40KHz); the samples were SP:SAMPLEPREP_SUMMARY then centrifuged for 10 minutes (13000g, 4℃), and the supernatant was pipetted SP:SAMPLEPREP_SUMMARY to the feeder vials with the cannulae for analysis. In addition, 20 μL of SP:SAMPLEPREP_SUMMARY supernatant was pipetted separately for each sample and mixed as a quality SP:SAMPLEPREP_SUMMARY control sample. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 95% water/5% acetonitrile; 0.1% formic acid CH:SOLVENT_B 47.5% acetonitrile/47.5% isopropanol/5% water; 0.1% formic acid CH:FLOW_GRADIENT 0–0.5 min: 40% B (isocratic) 0.5–3.5 min: 40% B → 55% B (linear) 3.5–6.0 CH:FLOW_GRADIENT min: 55% B → 70% B (linear) 6.0–7.0 min: 70% B → 100% B (linear) 7.0–8.0 CH:FLOW_GRADIENT min: 100% B (isocratic) Mobile Phase B gradient changes throughout the process. CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The samples were ionized by electrospray ionization, and the data were collected MS:MS_COMMENTS in negative ion scanning mode; the scanning range was 70-1050 m/z; the sheath MS:MS_COMMENTS gas flow rate was set at 50 arb, and the auxiliary gas flow rate was 13 arb; the MS:MS_COMMENTS heating temperature was set at 425 °C, and the capillary tube temperature was MS:MS_COMMENTS 325 °C; the spray voltage (in negative mode) was -3500 V; and the S-Lens RF MS:MS_COMMENTS Level was set at 50; The normalized collision energies were 20%, 40%, and 60%, MS:MS_COMMENTS respectively; and the full-scan resolution was set to 60,000. MS:MS_RESULTS_FILE ST003777_AN006204_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END