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1 1 Supporting Information Automated High-Throughput Identification and Characterisation of Clinically Important Bacteria and Fungi using Rapid Evaporative Ionisation Mass Spectrometry (REIMS) Frances Bolt (1) ; Simon JS Cameron (1) ; Tamas Karancsi (2); Daniel Simon (2); Richard Schaffer (2); Tony Rickards (3); Kate Hardiman (1); Adam Burke (1); Zsolt Bodai (1); Alvaro Perdones-Montero (1); Monica Rebec (3); Julia Balog (2); Zoltan Takats (1)* (1) Section of Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College London, London, SW7 2AZ, United Kingdom; (2) Waters Research Centre, 7 Zahony Street, Budapest, 1031, Hungary; (3) Department of Microbiology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, W6 8RF, United Kingdom Joint first authors * Corresponding Author: z.takats@imperial.ac.uk This supporting material is provided to give more information on the experimental methodology employed in this study and to include additional experimental data referred to in the primary manuscript. S-1
2 Table S1. Taxonomic classification and culture conditions of 25 microbial species analysed using handheld bipolar probe and high-throughput REIMS. Taxonomic classifications and culture requirements of microbial species analysed in this study. Abbreviations: + Gram-stain positive; - Gram-stain 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 CO 2 24 hrs Streptococcus agalactiae + Bacteria Firmicutes Bacilli Lactobacillales Streptococcaceae CBA 37 C CO 2 24 hrs Streptococcus pneumoniae + Bacteria Firmicutes Bacilli Lactobacillales Streptococcaceae CBA 37 C CO 2 24 hrs Streptococcus pyogenes + Bacteria Firmicutes Bacilli Lactobacillales Streptococcaceae CBA 37 C CO 2 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 CO 2 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 S-2
3 Table S2. Instrument Operational Conditions for Xevo G2-XS Q-ToF Instrument The operational conditions for the Xevo G2-XS Q-ToF instrument employed in this study are given 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 S-3
4 Table S3. Individual Species Cross-Validation Scores for All Species Analysis The individual species-level cross-validation scores for the preliminary all species analysis model are given for handheld bipolar probe REIMS and high-throughput REIMS, both with and without 2- propanol (IPA) infusion. For both REIMS approaches, infusion with 2-propanol improves species-level 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).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%.7% 100.0% Micrococcus luteus (MLUT) 93.3% 100.0% 93.3%.7% Enterococcus faecalis (EFAC) 73.3% 60.0% 80.0%.7% Enterococcus faecium (EFAM) 66.7% 66.7% 73.3%.7% Lactobacillus jensenii (LACJ) 93.3%.7% 93.3% 100.0% Streptococcus agalactiae(saga) 80.0% 93.3% 73.3% 93.3% Streptococcus pneumoniae (SPNE).7% 100.0% 73.3% 93.3% Streptococcus pyogenes (SPYO).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%.7% 73.3% Staphylococcus haemolyticus (SHAM) 60.0% 73.3%.7% 73.3% Staphylococcus hominis (SHOM) 80.0%.7%.7% 73.3% Clostridium difficile (CDIF) 100.0% 100.0% 93.3% 100.0% Enterobacter cloacae (ECLO) 53.3% 100.0% 80.0%.7% Escherichia coli (ECOL) 73.3% 46.7%.7% 60.0% Klebsiella oxytoca (KOXY) 93.3% 100.0% 93.3%.7% Klebsiella pneumoniae (KPNE) 93.3%.7% 100.0%.7% Morganella morganii (MMORG) 80.0%.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%.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% 59 S-4
5 Figure S1. REIMS Interface with T-Piece Set-Up To allow infusion with leu-enkaphaline containing 2-propanol (IPA), the analyte vapour is mixed with the matrix solvent prior to entry into the REIMS interface. This is accomplished through a T-piece setup whereby the analyte vapour inlet (a) is positioned between the IPA inlet (b) and the REIMS interface (c) allowing for its mixture prior to entry. 65 c S-5
6 Figure S2. Groupings used in Taxonomic Groupings Models Approach For each level of the taxonomic hierarchy present with the 25 microbial species analysed in this study, a PCA/LDA model was created to measure the cross-validation accuracy within each taxonomic grouping. A total of 13 taxonomic grouping models were constructed in this approach, for each REIMS approach, with each of the groupings indicated by a separate colour. 74 Domain Phylum Class Order Family Genus Species Fungi Ascomycota Saccharomycetes Saccharomycetales Debaryomycetaceae Candida albicans parapsilosis Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae Corynebacterium amycolatum Micrococcaceae Micrococcus luteus Enterococcaceae Enterococcus faecalis faecium Lactobacillales Lactobacillaceae Lactobacillus jensenii agalactiae Streptococcaceae Streptococcus pneumoniae Bacilli Firmicutes pyogenes aureus Bacillales Staphylococcaceae Staphylococcus epidermidis 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 75 Xanthomonadales Xanthomonadaceae Stenotrophomonas maltophilia S-6
7 Cross Validation Score (% Accuracy) Figure S3. Gram-Stain, Genus, and Species-Level Accuracy for all REIMS Approaches The Gram-stain, genus, and species-level cross-validation accuracy from an all species PCA/LDA model is shown for handheld bipolar probe REIMS and high-throughput REIMS, both with and without infusion with leu-enkaphaline containing 2-propanol (IPA). Gram-stain level accuracy is comparable between all four REIMS approaches, but substantial differences are evident at the genus and species level of classifications. 92 Gram Stain Genus Species No IPA With IPA No IPA With IPA Handheld Bipolar Bipolar Forceps Probe REIMS REIMS High-Throughput REIMS S-7
8 Figure S4. Handheld Bipolar Probe REIMS Taxonomic Groupings Probability Matrix The taxonomic groupings probability matrix for handheld bipolar probe REIMS shows that high levels of accuracy is achieved at the domain, phylum, class, order, and family level of classifications, but that slightly reduced levels of accuracy are achieved at the genus and species level of classification. Each figure displays the percentage correct classification for each class within each constructed model. The overall score is the figure calculated from multiplication of each model accuracy for the specific taxonomic groupings of each microbial species. 110 Domain Phylum Class Order Family Genus Species Species Overall Score Candida albicans 100% 112 Candida parapsilosis 100% Corynebacterium amycolatum 100% Micrococcus luteus 100% 0.93 Enterococcus faecalis 91% 0.93 Enterococcus faecium 91% Lactobacillus jensenii 98% 0.93 Streptococcus agalactiae 91% 0.99 Streptococcus pneumoniae 98% Streptococcus pyogenes 98% Staphylococcus aureus 98% 0.93 Staphylococcus epidermidis 91% 0.80 Staphylococcus haemolyticus 78% 0.93 Staphylococcus hominis 91% Clostridium difficile 99% Enterobacter cloacae 99% 0.93 Escherichia coli 92% Klebsiella oxytoca 99% 0.99 Klebsiella pneumoniae 99% Morganella morganii 99% 0.93 Proteus mirabilis 92% Serratia marcescens 99% Haemophilus influenza 100% Pseudomonas aeruginosa 100% Stenotrophomonas maltophilia 100% S-8
9 Figure S5. High-Throughput REIMS Taxonomic Groupings Probability Matrix The taxonomic groupings probability matrix for high-throughput REIMS shows that high levels of accuracy are achieved at the domain, phylum, class, order, and family level of classifications, but that slightly reduced levels of accuracy are achieved at the genus and species level of classification. Each figure displays the percentage correct classification for each class within each constructed model. The overall score is the figure calculated from multiplication of each model accuracy for the specific taxonomic groupings of each microbial species Domain Phylum Class Order Family Genus Species Species Overall Score Candida albicans 100% Candida parapsilosis 100% Corynebacterium amycolatum 97% Micrococcus luteus 97% Enterococcus faecalis 78% 0.87 Enterococcus faecium 84% 0.93 Lactobacillus jensenii 93% 0.93 Streptococcus agalactiae % 0.93 Streptococcus pneumoniae 93% Streptococcus pyogenes 93% 0.93 Staphylococcus aureus 93% Staphylococcus epidermidis 100% 0.80 Staphylococcus haemolyticus 80% Staphylococcus hominis 100% Clostridium difficile 100% 0.93 Enterobacter cloacae 92% 0.80 Escherichia coli 79% Klebsiella oxytoca 99% Klebsiella pneumoniae 99% Morganella morganii 99% Proteus mirabilis 99% 0.93 Serratia marcescens 92% Haemophilus influenza 99% Pseudomonas aeruginosa 99% Stenotrophomonas maltophilia 99% 122 S-9
10 Figure S6. Comparison of Mass Spectra from Combined or Individual Analytical Repeats To measure the intra-sample variability of each REIMS approach, an all species PCA/LDA model was created using either mass spectra from combined or individual analytical repeats. Five analytical repeats were taken from each culture plate of the 375 isolates analysed Combined Vs. Individual S-10
11 Cross Validation Score (% Accuracy) Figure S7. Intra-Sample Variation Measured through All Species Models All species analysis PCA/LDA models were constructed comparing the species-level cross-validation accuracy of mass spectra from combination of the five analytical repeats and mass spectra from each individual analytical repeat. Models were built using both the 50 to 2500 m/z range and the 600 to 900 m/z range, with mass binning to 0.1. Minimal differences were observed between species-level accuracy in models built using both the 50 to 2500 m/z and 600 to 900 m/z ranges for both REIMS approaches; suggesting minimal intra-sample and technical variation exists to 2500 m/z Individual 50 to 2500 m/z Combined 600 to 900 m/z Individual 600 to 900 m/z Combined Handheld Bipolar Bipolar Forceps Probe REIMS REIMS High-Throughput REIMS 138 S-11
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