{
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"PROJECT":{"PROJECT_TITLE":"Identifying and mathematically modeling the time-course of extracellular metabolic markers associated with resistance to ceftolozane/tazobactam in Pseudomonas aeruginosa","PROJECT_TYPE":"Biomedical research","PROJECT_SUMMARY":"Extracellular bacterial metabolites have potential as markers of bacterial growth and resistance emergence, but have not been evaluated in dynamic in vitro studies. We investigated the dynamic metabolomic footprint of a multidrug-resistant hypermutable Pseudomonas aeruginosa isolate exposed to ceftolozane/tazobactam as continuous infusion (4.5g/day, 9g/day) in a hollow-fiber infection model over 7-9 days in biological replicates (n=5). Bacterial samples were collected at 0, 7, 23, 47, 71, 95, 143, 167, 191 and 215h, the supernatant quenched and extracellular metabolites extracted. Metabolites were analyzed via untargeted metabolomics, including hierarchical clustering and correlation with quantified total and resistant bacterial populations. The time-courses of five metabolites were mathematically modeled. These five (of 1921 detected) metabolites were from enriched pathways (arginine and central carbon metabolism). Absorbed L-arginine and secreted L-ornithine were highly correlated with the total bacterial population (r -0.79 and 0.82 respectively, p<0.0001). Ribose-5-phosphate, sedoheptulose-7-phosphate and trehalose-6-phosphate correlated with the resistant subpopulation (0.64, 0.64 and 0.67, respectively, p<0.0001), and were likely secreted due to resistant growth overcoming oxidative and osmotic stress induced by ceftolozane/tazobactam. Using PK/PD-based transduction models, these metabolites were successfully modeled based on the total or resistant bacterial populations. The models well described the abundance of each metabolite across the differing time-course profiles of biological replicates, based on bacterial killing and, importantly, resistant regrowth. These proof-of-concept studies suggest further exploration is warranted to determine the generalizability of these findings. The metabolites modeled in this work are not exclusive to bacterial cells. Future studies may use this approach to identify bacteria-specific metabolites correlating with resistance, which would ultimately be extremely useful for clinical translation.","INSTITUTE":"Monash Institute of Pharmaceutical Sciences","DEPARTMENT":"Monash Institute of Pharmaceutical Sciences","LABORATORY":"Cornelia Landersdorfer","LAST_NAME":"Landersdorfer","FIRST_NAME":"Cornelia","ADDRESS":"399 Royal Pd","EMAIL":"dovile.anderson@monash.edu","PHONE":"0448671141","PUBLICATIONS":"Identifying and mathematically modeling the time-course of extracellular metabolic markers associated with resistance to ceftolozane/tazobactam in Pseudomonas aeruginosa","CONTRIBUTORS":"Jessica R. Tait, Dovile Anderson, Roger L. Nation, Darren J. Creek, Cornelia B. Landersdorfer"},

"STUDY":{"STUDY_TITLE":"Identifying and mathematically modeling the time-course of extracellular metabolic markers associated with resistance to ceftolozane/tazobactam in Pseudomonas aeruginosa - Part 1","STUDY_SUMMARY":"Extracellular bacterial metabolites have potential as markers of bacterial growth and resistance emergence, but have not been evaluated in dynamic in vitro studies. We investigated the dynamic metabolomic footprint of a multidrug-resistant hypermutable Pseudomonas aeruginosa isolate exposed to ceftolozane/tazobactam as continuous infusion (4.5g/day, 9g/day) in a hollow-fiber infection model over 7-9 days in biological replicates (n=5). Bacterial samples were collected at 0, 7, 23, 47, 71, 95, 143, 167, 191 and 215h, the supernatant quenched and extracellular metabolites extracted. Metabolites were analyzed via untargeted metabolomics, including hierarchical clustering and correlation with quantified total and resistant bacterial populations. The time-courses of five metabolites were mathematically modeled. These five (of 1921 detected) metabolites were from enriched pathways (arginine and central carbon metabolism). Absorbed L-arginine and secreted L-ornithine were highly correlated with the total bacterial population (r -0.79 and 0.82 respectively, p<0.0001). Ribose-5-phosphate, sedoheptulose-7-phosphate and trehalose-6-phosphate correlated with the resistant subpopulation (0.64, 0.64 and 0.67, respectively, p<0.0001), and were likely secreted due to resistant growth overcoming oxidative and osmotic stress induced by ceftolozane/tazobactam. Using PK/PD-based transduction models, these metabolites were successfully modeled based on the total or resistant bacterial populations. The models well described the abundance of each metabolite across the differing time-course profiles of biological replicates, based on bacterial killing and, importantly, resistant regrowth. These proof-of-concept studies suggest further exploration is warranted to determine the generalizability of these findings. The metabolites modeled in this work are not exclusive to bacterial cells. Future studies may use this approach to identify bacteria-specific metabolites correlating with resistance, which would ultimately be extremely useful for clinical translation.","INSTITUTE":"Monash Institute of Pharmaceutical Sciences","DEPARTMENT":"Monash Institute of Pharmaceutical Sciences","LABORATORY":"Cornelia Landersdorfer","LAST_NAME":"Landersdorfer","FIRST_NAME":"Cornelia","ADDRESS":"399 Royal Pd","EMAIL":"dovile.anderson@monash.edu","NUM_GROUPS":"6 groups with time points","TOTAL_SUBJECTS":"NA","NUM_MALES":"NA","NUM_FEMALES":"NA","PUBLICATIONS":"Identifying and mathematically modeling the time-course of extracellular metabolic markers associated with resistance to ceftolozane/tazobactam in Pseudomonas aeruginosa","STUDY_TYPE":"Biomedical research","PHONE":"0448671141"},

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"Sample ID":"M_Control_000_2",
"Factors":{"treatment":"M_Control_0h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_000_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_000_3",
"Factors":{"treatment":"M_Control_0h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_000_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_007_1",
"Factors":{"treatment":"M_Control_7h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_007_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_007_2",
"Factors":{"treatment":"M_Control_7h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_007_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_007_3",
"Factors":{"treatment":"M_Control_7h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_007_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_023_1",
"Factors":{"treatment":"M_Control_23h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_023_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_023_2",
"Factors":{"treatment":"M_Control_23h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_023_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_023_3",
"Factors":{"treatment":"M_Control_23h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_023_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_047_1",
"Factors":{"treatment":"M_Control_47h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_047_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_047_2",
"Factors":{"treatment":"M_Control_47h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_047_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_047_3",
"Factors":{"treatment":"M_Control_47h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_047_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_071_1",
"Factors":{"treatment":"M_Control_71h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_071_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_071_2",
"Factors":{"treatment":"M_Control_71h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_071_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_071_3",
"Factors":{"treatment":"M_Control_71h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_071_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_095_1",
"Factors":{"treatment":"M_Control_95h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_095_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_095_2",
"Factors":{"treatment":"M_Control_95h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_095_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_095_3",
"Factors":{"treatment":"M_Control_95h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_095_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_143_1",
"Factors":{"treatment":"M_Control_143h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_143_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_143_2",
"Factors":{"treatment":"M_Control_143h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_143_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_143_3",
"Factors":{"treatment":"M_Control_143h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_143_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_167_1",
"Factors":{"treatment":"M_Control_167h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_167_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_167_2",
"Factors":{"treatment":"M_Control_167h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_167_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_167_3",
"Factors":{"treatment":"M_Control_167h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_167_3"}
},
{
"Subject ID":"-",
"Sample ID":"M_Control_191_3",
"Factors":{"treatment":"M_Control_191h"},
"Additional sample data":{"RAW_FILE_NAME":"M_Control_191_3"}
},
{
"Subject ID":"-",
"Sample ID":"BR_Control_215_1",
"Factors":{"treatment":"BR_Control_215h"},
"Additional sample data":{"RAW_FILE_NAME":"BR_Control_215_1"}
},
{
"Subject ID":"-",
"Sample ID":"BR_Control_215_2",
"Factors":{"treatment":"BR_Control_215h"},
"Additional sample data":{"RAW_FILE_NAME":"BR_Control_215_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_ControlWS_000_1",
"Factors":{"treatment":"M_ControlWS_0h"},
"Additional sample data":{"RAW_FILE_NAME":"M_ControlWS_000_1"}
},
{
"Subject ID":"-",
"Sample ID":"M_ControlWS_000_2",
"Factors":{"treatment":"M_ControlWS_0h"},
"Additional sample data":{"RAW_FILE_NAME":"M_ControlWS_000_2"}
},
{
"Subject ID":"-",
"Sample ID":"M_ControlWS_000_3",
"Factors":{"treatment":"M_ControlWS_0h"},
"Additional sample data":{"RAW_FILE_NAME":"M_ControlWS_000_3"}
},
{
"Subject ID":"-",
"Sample ID":"QC_1",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_1"}
},
{
"Subject ID":"-",
"Sample ID":"QC_10",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_10"}
},
{
"Subject ID":"-",
"Sample ID":"QC_11",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_11"}
},
{
"Subject ID":"-",
"Sample ID":"QC_12",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_12"}
},
{
"Subject ID":"-",
"Sample ID":"QC_2",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_2"}
},
{
"Subject ID":"-",
"Sample ID":"QC_3",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_3"}
},
{
"Subject ID":"-",
"Sample ID":"QC_4",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_4"}
},
{
"Subject ID":"-",
"Sample ID":"QC_5",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_5"}
},
{
"Subject ID":"-",
"Sample ID":"QC_6",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_6"}
},
{
"Subject ID":"-",
"Sample ID":"QC_7",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_7"}
},
{
"Subject ID":"-",
"Sample ID":"QC_8",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_8"}
},
{
"Subject ID":"-",
"Sample ID":"QC_9",
"Factors":{"treatment":"Pooled_QC"},
"Additional sample data":{"RAW_FILE_NAME":"QC_9"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For metabolomic footprint analysis, the supernatant from centrifuged bacterial samples was collected. These bacterial samples were obtained from the hollow-fiber cartridges for all untreated control and treated arms for each of the five biological replicates at 7, 23, 47, 71, 143, and 167 h. Additional bacterial samples were collected from the cartridges at 0 and 215 h for replicates 3 to 5. Matrix blank samples (of CAMHB not containing any bacteria) were collected from the diluent media bottles every 48 h for all untreated control and treated arms.","SAMPLE_TYPE":"Bacterial cells","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Ceftolozane-tazobactam was administered to simulate steady-state concentrations of ceftolozane predicted to occur in the epithelial lining fluid of the lung in patients with CF, following daily doses of 3 g/1.5 g and 6 g/3 g via continuous infusion (10.6 and 21.3 mg/L, respectively) (21-23). Total bacterial populations were quantified on antibiotic-free CAMHA, and resistant subpopulations on CAMHA containing ceftolozane-tazobactam (12 and 20 mg/L).","TREATMENT":"continuous infusion","TREATMENT_COMPOUND":"Ceftolozane-tazobactam","TREATMENT_DOSE":"3 g and 6 g","TREATMENT_DOSEVOLUME":"10.6 and 21.3 mg/L"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Each sample (25 µL) was added to 100 µL of pre-chilled methanol containing the internal standards (CHAPS, CAPS, TRIS and PIPES) at 1 µM. This mixture was vortexed, and subsequently centrifuged at 14800 x g and 4°C for 10 min. The final supernatant samples containing the extracted extracellular metabolites were stored at -80°C until LC-MS analysis was performed.","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"ZIC-pHILIC chromatography at ph9, biological replicates 3-5","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Merck SeQuant ZIC-pHILIC (150 x 4.6mm,5um)","SOLVENT_A":"20 mM ammonium carbonate","SOLVENT_B":"acetonitrile","FLOW_GRADIENT":"0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B","FLOW_RATE":"0.3 ml/min","COLUMN_TEMPERATURE":"25 C","METHODS_FILENAME":"Metabolomics_pHILIC_Parkville_v1.meth","COLUMN_PRESSURE":"60 bar at starting conditions. 180 bar at %A","INJECTION_TEMPERATURE":"4 C","SAMPLE_INJECTION":"10 uL","ANALYTICAL_TIME":"32 min","CAPILLARY_VOLTAGE":"4 kV","WASHING_BUFFER":"syringe wash 50% IPA","SAMPLE_LOOP_SIZE":"25 uL","SAMPLE_SYRINGE_SIZE":"25 uL","RANDOMIZATION_ORDER":"yes"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Monash Proteomics and Metabolomics Platform","OPERATOR_NAME":"Dovile Anderson","DETECTOR_TYPE":"Full scan","SOFTWARE_VERSION":"XCalibur 4.6","ACQUISITION_DATE":"29/10/2021","ANALYSIS_PROTOCOL_FILE":"PQMS3-MBPF-WIN-0501 LCMS data acquisition for untargeted metabolomics analysis.pdf","ACQUISITION_PARAMETERS_FILE":"Metabolomics_pHILIC_Parkville_v1.pdf","DATA_FORMAT":".RAW"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Full scan LCMS data acquired using polarity switching. raw data processed using IDEOM workflow ( 10.1007/978-1-0716-0239-3_21).","CAPILLARY_TEMPERATURE":"300 C","CAPILLARY_VOLTAGE":"3.5 kV","COLLISION_ENERGY":"NA","COLLISION_GAS":"NA","DRY_GAS_FLOW":"50","DRY_GAS_TEMP":"120","ION_SOURCE_TEMPERATURE":"120 C","IONIZATION":"ESI","MASS_ACCURACY":"3 ppm","PRECURSOR_TYPE":"[M-H]-","SOURCE_TEMPERATURE":"300"},

"MS_METABOLITE_DATA":{
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}

}