{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001966","ANALYSIS_ID":"AN003204","VERSION":"1","CREATED_ON":"November 9, 2021, 7:32 pm"},

"PROJECT":{"PROJECT_TITLE":"NMR Hydrophilic Metabolomic Analysis of Bacterial Resistance Pathways using Multivalent Antimicrobials with Challenged and Unchallenged Wild Type and Mutated Gram Positive Bacteria","PROJECT_SUMMARY":"Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both gram-positive and gram-negative bacteria. Nuclear Magnetic Resonance (NMR) metabolomics is an important method for studying resistance development in bacteria since it is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. The objectives are to determine the likely metabolic differences between antimicrobially challenged and unchallenged growth and wild type and antimicrobially mutated Bacillus cereus (B. cereus) samples by using NMR hydrophilic metabolomics. Proton (1H) NMR hydrophilic metabolite analysis was conducted using B. cereus wild type and B. cereus that was mutated with C16-DABCO and mannose functionalized poly(amidoamine) dendrimers (DABCOMD). Both the wild type and the mutated sample types were grown in low levels of DABCOMD (challenged samples) or without the addition of DABCOMD to the growth media (unchallenged samples) for sample collection at the mid log and stationary phases and for growth curve procurement. Hierarchical clustering of only the challenged sample type showed that both the stationary phase sample types (mutant and wild type) clustered together while the both the mid log phase sample types were distinct. Hierarchical clustering of the unchallenged samples showed complete separation of all sample types. There were statistically significant (p-value and fold change) changes in the concentrations of metabolites in both energy related pathways and peptidoglycan synthesis between all sample types, especially with mutants and especially the challenged sample types have more N-acetylglucosamine (as much as a 94.2-fold increase). The mid log phase sample types showed a larger difference between sample types than their stationary phase counter parts. The challenged and unchallenged mutant samples showed a larger difference between sample types in comparison to the differences between the challenged and unchallenged wild type sample types. There was a larger metabolite difference when comparing the challenged mutant samples to the challenged wild type samples than when comparing the unchallenged mutant samples to the unchallenged wild type samples. The metabolomic analysis of wild type and multivalent DABCOMD mutated B. cereus under both challenged and unchallenged conditions indicated that the mutants, especially the challenged mutants, are likely changing their peptidoglycan layer to protect themselves from the high positive charge on the membrane disrupting DABCOMD. This membrane fortification most likely led to the slow growth curve of the mutated and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for these changes to occur as well as to the decreased diffusion of nutrients across the membrane.","INSTITUTE":"Montana State University","DEPARTMENT":"Chemistry and Biochemistry","LABORATORY":"Dr. Mary Cloninger","LAST_NAME":"Aries","FIRST_NAME":"Michelle","ADDRESS":"103 Chemistry and Biochemistry Building, Bozeman, Montana, 59717, USA","EMAIL":"p49k881@msu.montana.edu","PHONE":"406-994-3051","FUNDING_SOURCE":"NIGMS, grant number 62444","CONTRIBUTORS":"Dr. Mary Cloninger"},

"STUDY":{"STUDY_TITLE":"NMR Hydrophilic Metabolomic Analysis of Bacterial Resistance Pathways using Multivalent Antimicrobials with Challenged and Unchallenged Wild Type and Mutated Gram Positive Bacteria","STUDY_TYPE":"NMR Hydrophilic Metabolomics","STUDY_SUMMARY":"Multivalent membrane disruptors are a relatively new antimicrobial scaffold that are difficult for bacteria to develop resistance to and can act on both gram-positive and gram-negative bacteria. Nuclear Magnetic Resonance (NMR) metabolomics is an important method for studying resistance development in bacteria since it is both a quantitative and qualitative method to study and identify phenotypes by changes in metabolic pathways. Determine the likely metabolic differences between antimicrobially challenged and unchallenged growth and wild type and antimicrobially mutated Bacillus cereus (B. cereus) samples by using NMR hydrophilic metabolomics. Proton (1H) NMR hydrophilic metabolite analysis was conducted using B. cereus wild type and B. cereus that was mutated with C16-DABCO and mannose functionalized poly(amidoamine) dendrimers (DABCOMD). Both the wild type and the mutated sample types were grown in low levels of DABCOMD (challenged samples) or without the addition of DABCOMD to the growth media (unchallenged samples) for sample collection at the mid log and stationary phases and for growth curve procurement. Hierarchical clustering of only the challenged sample type showed that both the stationary phase sample types (mutant and wild type) clustered together while the both the mid log phase sample types were distinct. Hierarchical clustering of the unchallenged samples showed complete separation of all sample types. There were statistically significant (p-value and fold change) changes in the concentrations of metabolites in both energy related pathways and peptidoglycan synthesis between all sample types, especially with mutants and especially the challenged sample types have more N-acetylglucosamine (as much as a 94.2-fold increase). The mid log phase sample types showed a larger difference between sample types than their stationary phase counter parts. The challenged and unchallenged mutant samples showed a larger difference between sample types in comparison to the differences between the challenged and unchallenged wild type sample types. There was a larger metabolite difference when comparing the challenged mutant samples to the challenged wild type samples than when comparing the unchallenged mutant samples to the unchallenged wild type samples. The metabolomic analysis of wild type and multivalent DABCOMD mutated B. cereus under both challenged and unchallenged conditions indicated that the mutants, especially the challenged mutants, are likely changing their peptidoglycan layer to protect themselves from the high positive charge on the membrane disrupting DABCOMD. This membrane fortification most likely led to the slow growth curve of the mutated and especially the challenged mutant samples. The association of these sample types with metabolites associated with energy expenditure is attributed to the increased energy required for these changes to occur as well as to the decreased diffusion of nutrients across the membrane.","INSTITUTE":"Montana State University","DEPARTMENT":"Chemistry and Biochemistry","LABORATORY":"Dr. Mary Cloninger","LAST_NAME":"Aries","FIRST_NAME":"Michelle","ADDRESS":"103 Chemistry and Biochemistry Building","EMAIL":"p49k881@msu.montana.edu","PHONE":"406-994-3051"},

"SUBJECT":{"SUBJECT_TYPE":"Bacteria","SUBJECT_SPECIES":"Bacillus cereus","TAXONOMY_ID":"11778","GENDER":"Not applicable"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"WT_1_1",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_1_1 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_1_2",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_1_2 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_1_4",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_1_4 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_1_5",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_1_5 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_1_6",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_1_6 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_2_22",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_2_22 - S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_2_23",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_2_23 - S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_2_25",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_2_25 - S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_2_27",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_2_27 - S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_2_173",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_2_173 - S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_3_9",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_3_9 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_3_42",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_3_42 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_3_43",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_3_43 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_3_44",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_3_44 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_3_45",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_3_45 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_3_46",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_3_46 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"WT_4_65",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_4_65 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_4_67",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_4_67 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_4_68",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_4_68 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_4_69",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_4_69 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"WT_4_70",
"Factors":{"Genotype":"Wild-type","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - WT_4_70 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_1_10",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_1_10 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_1_11",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_1_11 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_1_99",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_1_99 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_1_100",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_1_100 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_1_101",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_1_101 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_1_102",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_1_102 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_1_175",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_1_175 - ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_2_111",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_2_111 - S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_2_112",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_2_112 - S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_2_113",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_2_113 - S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_2_114",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_2_114 - S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_2_115",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"None"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_2_115 - S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_3_57",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_3_57 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_3_58",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_3_58 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_3_60",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_3_60 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_3_61",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_3_61 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_3_63",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_3_63 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_3_64",
"Factors":{"Genotype":"Mutant","Growth Phase":"Mid Log","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_3_64 - D ML"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_4_81",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_4_81 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_4_82",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_4_82 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_4_83",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_4_83 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_4_84",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_4_84 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_4_85",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_4_85 - D S"}
},
{
"Subject ID":"-",
"Sample ID":"Mut_4_86",
"Factors":{"Genotype":"Mutant","Growth Phase":"Stationary","Treatment":"DABCOMD"},
"Additional sample data":{"RAW_FILE_NAME":"fid - Mut_4_86 - D S"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Samples were collected in screw-cap conical centrifuge tubes for all sample types in both the mid log and stationary phases. They were centrifuged, the supernatant was discarded, they were rinsed with cold 1x PBS and transferred to glass centrifuge tubes. They were centrifuged, the supernantant was discarded and the cell pellets were frozen at - 80 C.","SAMPLE_TYPE":"Bacterial cells","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Unchallenged samples of both WT and Mut bacteria were grown in Brodo Mueller Hinton II Media (BMHII), the challenged samples (WT D and Mut D) were grown in BMHII with 33 % of the MIC value DABCOMD added to the media. All other procedures were the same between the two groups."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Hydrophilic metabolites were obtained from the aqueous layer of the methanol/water/chloroform extraction. An acetone precipitation was used to extract the proteins. Metabolite samples were prepared by taking the supernatant obtained after acetone precipitation, speed vacuuming them down and adding 700 µL of the NMR buffer. The mixture was vortexed and transferred to capped glass NMR tube."},

"ANALYSIS":{"ANALYSIS_TYPE":"NMR"},

"NM":{"INSTRUMENT_NAME":"Bruker Avance III 600 MHz NMR","INSTRUMENT_TYPE":"FT-NMR","NMR_EXPERIMENT_TYPE":"1D-1H","SPECTROMETER_FREQUENCY":"600 MHz","NMR_PROBE":"5 mm triple resonance (1H, 15N, 13C) liquid-helium cooled TCI NMR CryoProbe"},

"NMR_METABOLITE_DATA":{
"Units":"mM",

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}

}