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Supporting Information 1 Supporting Information 2 3 Automated High-Throughput Identification and Characterisation of Clinically 4 Important Bacteria and Fungi using Rapid Evaporative Ionisation Mass 5 Spectrometry (REIMS) 6 7 Frances Bolt (1)†; Simon JS Cameron (1)†; Tamas Karancsi (2); Daniel Simon (2); Richard Schaffer (2); 8 Tony Rickards (3); Kate Hardiman (1); Adam Burke (1); Zsolt Bodai (1); Alvaro Perdones-Montero (1); 9 Monica Rebec (3); Julia Balog (2); Zoltan Takats (1)*. 10 11 (1) Section of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial 12 College London, London, SW7 2AZ, United Kingdom; (2) Waters Research Centre, 7 Zahony Street, 13 Budapest, 1031, Hungary; (3) Department of Microbiology, Imperial College Healthcare NHS Trust, 14 Charing Cross Hospital, London, W6 8RF, United Kingdom 15 16 † Joint first authors 17 18 * Corresponding Author: [email protected] 19 20 21 22 This supporting material is provided to give more information on the experimental methodology 23 employed in this study and to include additional experimental data referred to in the primary 24 manuscript. S-1 25 Table S1. Taxonomic classification and culture conditions of 25 microbial species analysed using handheld bipolar probe and high-throughput REIMS. 26 Taxonomic classifications and culture requirements of microbial species analysed in this study. Abbreviations: ‘+’ Gram-stain positive; ‘-‘ Gram-stain 27 negative; ‘CBA’ Columbia Blood Agar; ‘Choc’ Chocolate Agar; Taxonomic Classifications Culture Conditions Gram Domain Phylum Class Order Family Media Temperature Atmosphere Duration Candida albicans N/A Fungi Ascomycota Saccharomycetes Saccharomycetales Debaryomycetaceae CBA 30 °C Aerobic 48 hrs Candida parapsilosis N/A Fungi Ascomycota Saccharomycetes Saccharomycetales Debaryomycetaceae CBA 30 °C Aerobic 48 hrs Corynebacterium amycolatum + Bacteria Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae CBA 37 °C Aerobic 48 hrs Micrococcus luteus + Bacteria Actinobacteria Actinobacteria Actinomycetales Micrococcaceae CBA 37 °C Aerobic 24 hrs Enterococcus faecalis + Bacteria Firmicutes Bacilli Lactobacillales Enterococcaceae CBA 37 °C Aerobic 24 hrs Enterococcus faecium + Bacteria Firmicutes Bacilli Lactobacillales Enterococcaceae CBA 37 °C Aerobic 24 hrs Lactobacillus jensenii + Bacteria Firmicutes Bacilli Lactobacillales Lactobacillaceae CBA 37 °C CO2 24 hrs Streptococcus agalactiae + Bacteria Firmicutes Bacilli Lactobacillales Streptococcaceae CBA 37 °C CO2 24 hrs Streptococcus pneumoniae + Bacteria Firmicutes Bacilli Lactobacillales Streptococcaceae CBA 37 °C CO2 24 hrs Streptococcus pyogenes + Bacteria Firmicutes Bacilli Lactobacillales Streptococcaceae CBA 37 °C CO2 24 hrs Staphylococcus aureus + Bacteria Firmicutes Bacilli Bacillales Staphylococcaceae CBA 37 °C Aerobic 24 hrs Staphylococcus epidermidis + Bacteria Firmicutes Bacilli Bacillales Staphylococcaceae CBA 37 °C Aerobic 24 hrs Staphylococcus haemolyticus + Bacteria Firmicutes Bacilli Bacillales Staphylococcaceae CBA 37 °C Aerobic 24 hrs Staphylococcus hominis + Bacteria Firmicutes Bacilli Bacillales Staphylococcaceae CBA 37 °C Aerobic 24 hrs Clostridium difficile + Bacteria Firmicutes Clostridia Clostridiales Clostridiaceae CBA 37 °C Anaerobic 48 hrs Enterobacter cloacae - Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae CBA 37 °C Aerobic 24 hrs Escherichia coli - Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae CBA 37 °C Aerobic 24 hrs Klebsiella oxytoca - Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae CBA 37 °C Aerobic 24 hrs Klebsiella pneumoniae - Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae CBA 37 °C Aerobic 24 hrs Morganella morganii - Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae CBA 37 °C Aerobic 24 hrs Proteus mirabilis - Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae CBA 37 °C Aerobic 24 hrs Serratia marcescens - Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae CBA 37 °C Aerobic 24 hrs Haemophilus influenza - Bacteria Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae Choc 37 °C CO2 24 hrs Pseudomonas aeruginosa - Bacteria Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae CBA 37 °C Aerobic 24 hrs Stenotrophomonas maltophilia - Bacteria Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae CBA 37 °C Aerobic 24 hrs 28 S-2 29 Table S2. Instrument Operational Conditions for Xevo G2-XS Q-ToF Instrument 30 The operational conditions for the Xevo G2-XS Q-ToF instrument employed in this study are given 31 here. Parameter Setting Scan Time 1000 ms Scan Mode Sensitive Mass Analyser Time of Flight Ionisation Mode Negative Ion Mode Mass Range 50 to 2500 Sampling Cone 80 V Source Offset 50 V Source Temperature 100 °C 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 S-3 53 Table S3. Individual Species Cross-Validation Scores for All Species Analysis 54 The individual species-level cross-validation scores for the preliminary all species analysis model are 55 given for handheld bipolar probe REIMS and high-throughput REIMS, both with and without 2- 56 propanol (IPA) infusion. For both REIMS approaches, infusion with 2-propanol improves species-level 57 accuracy. Handheld Bipolar REIMS High-Throughput REIMS No IPA With IPA No IPA With IPA Average 84.27% 90.40% 88.80% 89.87% Candida albicans (CALB) 86.7% 100.0% 100.0% 100.0% Candida parapsilosis (CALP) 100.0% 100.0% 100.0% 93.3% Corynebacterium amycolatum (CAMY) 93.3% 100.0% 86.7% 100.0% Micrococcus luteus (MLUT) 93.3% 100.0% 93.3% 86.7% Enterococcus faecalis (EFAC) 73.3% 60.0% 80.0% 86.7% Enterococcus faecium (EFAM) 66.7% 66.7% 73.3% 86.7% Lactobacillus jensenii (LACJ) 93.3% 86.7% 93.3% 100.0% Streptococcus agalactiae(SAGA) 80.0% 93.3% 73.3% 93.3% Streptococcus pneumoniae (SPNE) 86.7% 100.0% 73.3% 93.3% Streptococcus pyogenes (SPYO) 86.7% 100.0% 60.0% 93.3% Staphylococcus aureus (SAUR) 93.3% 100.0% 100.0% 93.3% Staphylococcus epidermidis (SEPI) 60.0% 80.0% 86.7% 73.3% Staphylococcus haemolyticus (SHAM) 60.0% 73.3% 86.7% 73.3% Staphylococcus hominis (SHOM) 80.0% 86.7% 86.7% 73.3% Clostridium difficile (CDIF) 100.0% 100.0% 93.3% 100.0% Enterobacter cloacae (ECLO) 53.3% 100.0% 80.0% 86.7% Escherichia coli (ECOL) 73.3% 46.7% 86.7% 60.0% Klebsiella oxytoca (KOXY) 93.3% 100.0% 93.3% 86.7% Klebsiella pneumoniae (KPNE) 93.3% 86.7% 100.0% 86.7% Morganella morganii (MMORG) 80.0% 86.7% 93.3% 93.3% Proteus mirabilis (PMIR) 93.3% 100.0% 100.0% 93.3% Serratia marcescens(SMAR) 80.0% 93.3% 93.3% 93.3% Haemophilus influenza (HINF) 93.3% 100.0% 86.7% 100.0% Pseudomonas aeruginosa (PAER) 93.3% 100.0% 100.0% 100.0% Stenotrophomonas maltophilia (SMAL) 100.0% 100.0% 100.0% 100.0% 58 59 S-4 60 Figure S1. REIMS Interface with T-Piece Set-Up 61 To allow infusion with leu-enkaphaline containing 2-propanol (IPA), the analyte vapour is mixed with 62 the matrix solvent prior to entry into the REIMS interface. This is accomplished through a T-piece set- 63 up whereby the analyte vapour inlet (a) is positioned between the IPA inlet (b) and the REIMS interface 64 (c) allowing for its mixture prior to entry. 65 c 66 67 68 S-5 69 Figure S2. Groupings used in Taxonomic Groupings Models Approach 70 For each level of the taxonomic hierarchy present with the 25 microbial species analysed in this study, 71 a PCA/LDA model was created to measure the cross-validation accuracy within each taxonomic 72 grouping. A total of 13 taxonomic grouping models were constructed in this approach, for each REIMS 73 approach, with each of the groupings indicated by a separate colour. 74 Domain Phylum Class Order Family Genus Species albicans Fungi Ascomycota Saccharomycetes Saccharomycetales Debaryomycetaceae Candida parapsilosis Corynebacteriaceae Corynebacterium amycolatum Actinobacteria Actinobacteria Actinomycetales Micrococcaceae Micrococcus luteus faecalis Enterococcaceae Enterococcus faecium Lactobacillaceae Lactobacillus jensenii Lactobacillales agalactiae Streptococcaceae Streptococcus pneumoniae Bacilli Firmicutes pyogenes aureus epidermidis Bacillales Staphylococcaceae Staphylococcus haemolyticus hominis Bacteria Clostridia Clostridiales Clostridiaceae Clostridium difficile Enterobacter cloacae Escherichia coli oxytoca Klebsiella Enterobacteriales Enterobacteriaceae pneumoniae Morganella morganii Proteobacteria Gammaproteobacteria Proteus mirabilis Serratia marcescens Pasteurellales Pasteurellaceae Haemophilus influenzae Pseudomonadales Pseudomonadaceae Pseudomonas aeruginosa Xanthomonadales Xanthomonadaceae Stenotrophomonas maltophilia 75 76 77 78 79 80 81 82 83 84 85 S-6 86 Figure S3. Gram-Stain, Genus, and Species-Level Accuracy for all REIMS Approaches 87 The Gram-stain, genus, and species-level cross-validation accuracy from an all species PCA/LDA model 88 is shown for handheld bipolar probe REIMS and high-throughput REIMS, both with and without 89 infusion with leu-enkaphaline containing 2-propanol (IPA). Gram-stain level accuracy is comparable 90 between all four REIMS approaches, but substantial differences are evident at the genus and species 91 level of classifications. 92 Gram Stain Genus Species 100 99 99 98 97 95 94 93 91 90 88 87 86 86 85 Cross Validation Score (% Accuracy) (% Score Validation Cross 80 77 75 No IPA With IPA No IPA With IPA HandheldBipolar Bipolar Forceps Probe REIMS REIMS HighHigh-Throughput-Throughput REIMS REIMS 93 94 95
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