Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection

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1 SUPPLEMENTARY INFORMATION Articles In the format provided by the authors and unedited. Nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection Themoula Charalampous 1,8, Gemma L. Kay 1,2,8, Hollian Richardson 1,8, Alp Aydin 2, Rossella Baldan 1,3, Christopher Jeanes 4, Duncan Rae 4, Sara Grundy 4, Daniel J. Turner 5, John Wain 1,2, Richard M. Leggett 6, David M. Livermore 1,7 and Justin O Grady 1,2 * 1 Bob Champion Research and Educational Building, University of East Anglia, Norwich Research Park, Norwich, UK. 2 Quadram Institute Bioscience, Norwich Research Park, Norwich, UK. 3 CIDR, King s College London, St Thomas Hospital, London, UK. 4 Microbiology Department, Norwich and Norfolk University Hospital, Norwich, UK. 5 Oxford Nanopore Technologies, Gosling Building, Oxford Science Park, Oxford, UK. 6 Earlham Institute, Norwich Research Park, Norwich, UK. 7 AMRHAI, Public Health England, London, UK. 8 These authors contributed equally: Themoula Charalampous, Gemma L. Kay, Hollian Richardson. * justin.ogrady@quadram.ac.uk Nature Biotechnology

2 Supplementary Information (A) TTGTTGCTTCGTTCAGTCGGGTGGGTGTTTATGGGACCGTAAGCCTACCGTGACGTTACCAGTAGGAATGAATCCTTCGTT TTTCGTGCGCCGCTTCACTCTATAACTTGTTCCACAGCCATTTTTTGAAGATCTGGTGGACCATATTTCTTTAATAGGCGTCT AACTTGAACTTCTCATTTTAGCTTTAGCACTGTCTCGTTTGAACCAGTCAACACTCATGTTATCATAACTGTTTTTAGTTAACT CATGTGCAATGGCTCTTAACTCTTTATCGCCAAAGCTTCTTTAGCTGTTCATGTGAAGCTCGATACTACATACAATATACGC AACAATGCGCTTGTTTCGATTAGGTTTAGGCATTGAATCATAAAGTGCAATATATACTTAACACGTTCTTTAATATTTGAATTA AGACCTAGTAAATATTGTGCCATTATTTATGTGCTGAGATTTTAAAATAGAACATTTTAATATCCCGATTCTACGCAGTATAT GTTCATGGCGATTCAAAATTTTTTAAAACGTTTCTATCACATACCTTTTCACAGTCATTGTCCATCATTGTTTTTACTTATATAT AGTGCAGGTTAAACCAGAATATCATTTTAAAAACAACATATAAAGCTGGTTGAATATTAAGTCATGACTTTTCGCATGTGATA GTTCATGTAGAATAATATATTCAATTTCTTTGTCATTCATGGTTTCATTACGACAGTTGGTATTGGTACAATTGGGATTTCACT AAACCAAATACCATCGGATTATCAATGTTTTGAACTATAACTAATTGTTATATGCTTTTTTGTGCGTTACATCTTACTTTGACA TACTTTAAGTCGTTCATTAAGATATGACGATTCCAATGACAGACTTTAATAACATCCAATTTGTCGGAATGCCTTAATCATAT AAAAATAAGCACAACAAACTACCGAATACCATATCAAAAGAATCATATACGTTATATTTGAGGTCTCGGCAGTTAACATTAAT TTTCAAGTCTTTCGTAACAGATGATTGTTGACCATCTAACATATGACTAACCAGAAAGAAGTCGTGTCAGATACATTTCGATT CATCATATCTTTGAAAATGGAATTCGATATTTTGTAAAATGGGTATTAATGGAATTAACGTGGAGACGAGCACCTAATAACCA AATCTTATGTGACATAATATTTTGAGTATATTTTATATAGAACATTCTCACTAAAAAATTACATATCGTGAGCAATGAACTGAT TATACTTAACATTAAAAAGGTATTAACATATTCTACACCTCCATATCACAAAATTGTAACATTATTTTGACATAAATACTACATT TGTAATATACTACAAATGTGGTCTTATATAAGAGTATATTGATGAAAAGAAGATAAAAATTGTTCACACTTATTTTTAATAGT TGTAGTTGTCGGGTTTGGTATATATTTTATGCTTCAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAA TTTCAAACAAGTTTATAAGATAACAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATAATGACTGAACGTCCGAT AAAAATATATATAATAGTTAGGCGTTAAAGATATAAACATTCAGGATCCGTAAAATAAAAAAGTATCTAAAATAAAAACA GTAGATGCTCAATATAAAATTAAAACAAACTGGTAGCATTGATCCGCAGCGTTCCGTTTAATTTTGTTAAAAGATGGTAT GTGGAAGTTAGGTGGGATCATAGCGTCATTATTCAGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCA GAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGAGATAGGCATCGTTCCAA AGAATGTATCTAAAAGATTATAAAACAATCGCTAAAGAACTAAGTATTTCTGAAGACTATATCAAACCAACAAATGGATC AAAATTGGGTACAAAGATGATACCTTCGTTCTTTTAAAACCGTTAAAAATGGATGAATATTTAAGTGATTTCGCAAAATTT CATCTTACAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGAAAAACGACTTCACATCTATTAGGTTATGTTGGTCCC ATTAACTCTGAAGAATTAAAACAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAAAAGGGACTCGAAAA ACTTTACGATAAAAAACCAACATGAAGATGGCTATCGTGTCACAATCGTTGACGATAATAGCAATACAATCGCACATAC ATTAATAGAGAAAAAGAAAAAGATGGCAAAGATATTCAATAACTATTGATGCTGAAGTTCAAAAGTGACTATAACAACA TGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTATTAGCACTTGTAAGCACACCTTCA TATGACGTCTATCCATTTTATGTATAGCATGAGTAACGAAAAGAATATAATATAAATTAACCGAAGATGAAAGAAAAGAA CCTCTGCTCCAACAAGTTCCAGATTACAACTTCACCGAGTTCAACTCAAAAATATTAACAGCAATGATTGGGTTAAATAA CAAAACATTAGACGATAAAACAAGTTATAAAATCGATGGTAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACG CGTTACAAGATATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTTTGC TAGAGTAGCACTCAGATTAGGCAGTGAAATTTGAAAAGGCATGAAAAACTAGGTGTTGGTGAAGATATACCAAGTGATT ATCCATTTTATAATGCTCAGAATTTCAAACAAAATTTAGATAATGAAATATTGATAGCTGATTCAGGTTACGGACAAGGT GAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGCATTAGAAAGAAATAATAGCAATATTAACGCACCTCA TGATAAAAGAACTTACGAAAAACAAAGTTTGGAAGAAAAATATTTCAATGAAGCGGCGCACGAAAACAGAGGATTCATTC CACGGTAACACGTCACC (B)

3 (C) ATGGTTCCGTGGTGTTTAACAGTCATGTAAATGACCTTGTCCAAGTTTGTAACCTTCGTTTTTCGTGCGCCGCTTCCAATCG CGTCCTTTGTTTACACGAATGTCTTTTGCTCTTAATATTAATTCCTGCTTCCTCATCTATAATCGCATGTGCTTTCATTTTGCT TTGTATGCGGGAAAACCCTGCGATTTTAGCAGGAAGAGTTGTAATTAAAATATAATATTCCTCCAATATTGTTTATTTTAAAT CCCTATTTTCTAACCTCAGTTATCATTTTGGCAAACTATAGCAATGTAATGAGTCTAAAATACCTGATAGTAAAACCAATG ATTTACACCGATTTTATCGGCAAAAGAATCCAAGAAGAATTATTAATTGTATGAAGTGACATGATACTTTAAATCAAGAA AATACACGTCCCTAAATATTCTAGACTTAATTTTCTCCTGAAAAAGAGCTGTTTACACCATATAAATATACCAGCCCAATG TGCACAACAAACTACGAATATTATAATCCCAGCTGGAGGAAGTATTCCGAAACGGCTAAACTGGTCCCCATAAAAAATG AACTTGTTATTGGTAATACATGCTTTTCAGAAGCCCCTAATCTTCAACCTAATAAGCCTCCTGCTGGCATCCCAAATACTG AGGAAATTTCCATGTAGCAAAGTAATATACGTTCCATTAAGAGTGTTCCATGCTTATTAAAGAAAATAATTGCATTGATTG GCATATAAACAAAGTATATAGTGTTCTAAAAGAGTATAAGGCAAACAAATCCCTTTGTTTTGTCTCCAGAACCACAACTC CTTCTTTCATCTCCCTTGTGAAATTTAGTTCAAACTTTGCATGATTACCCAGCTTGGTATACGGTACAATTTACCGTATAA ATCACAGTCTGGCACCCATACGTCGATGGCAATAATAGCATTTAAATCCCAAACGGAGGTATAAGAGTGCTGCAACTGC CGGACTATTTCATATAACATAGACTGCAGACTGACTATAGCTCGCATTTCGTTAATAATTTCTTCTGGTACTAAAATTGGT GTAACCGCATTGAGAGTGCTGGGGTGGCTGTTCAATGCTACGGATAACAATACTATCGCAAATCATCAGACAGGTAGCT CCCGTCAGCAACCGAAACACTGCACTGCTTCTGCGATATTAAATCGGCACCAATCATTCCTCTTCTTCCTATCGTGATCC ACTAACTTTTCAATGGCGGTCAAATCGCATGGGGTAAAAACCTACTAATGAAGCCATAGACAGGACAAAAATGCAGATC CTGTTTTTCTGTAGGTAAAAATAATCATAATTTACGGGATGGCACTGGTCATGATACTGCTTTGCCCACTTATGTGCATAG GTACTTCGTTTCCAATTGTTTGTATTTCCATTTATATTTATCTCACCATATTCTTTATGAATTTCTAATGGCCTGAATAGCATT CCAGGCAATAAAAATGCGAATGACCAAGTAAGCTTTTGGTCTTACCTTACGCTGTTAGGTATTCATATTAAAGACCTTGGAT TGAAATTAAAGGGTATGGTTAAATAACCAATGTCCTCACCGTAACTATGAATGCTCAATATCGTATAAATAAGCACAACAAAA AGCCTATCATCGGGTATAGATTCCTTTGTTGCCAGCCAAACGCATTAGAGGCTGTCATAGTTTCGATATCTATCTTTGTTTAT ATCAATTTATGAGTAACTTTAACAAGATGATATGTTCAATAAAGGATTATGGAATGGACTCATACTTTAATTCGATTAGTTTAA AGGATATGACAGAGTTTTCTTCATGAACCGTAACGCTCTAGTTATCTTCCTTAGTGAGCAAGCGATTAGCTTTCTAAAATCTT CTGTTTGTAGTTTAGAATTCATCAGGATTTTCTAACTCAACCTCAAATATTCATTTTACATTCCTATTTCATTTATTGATAAATT AGTTTGCAAAAAATTGGACAATTTTTGTCCTCTTAACCTTTAAATAAGTTTTTTTAATCGATTTAGGTTGCCTAAATTATTGCT TATTCGGTAAATGAAGTATTGCTTTCAACAGATTTCCTTCAACTACACTTCTTGATTCAAACAAGTTGTTTATTCTTCCACAAA CTCCTTGTCAATGGAATAAAACACATGCCACAGGTAAATGAATAGAACTGATGATTTCTAAACTATCGCTACCTCATTCCAG AATTTTCTCACTCTCTAGATGCCCACCCTGTAAAGGCAAAACTGCAATCAAACTCCATTATCCTTTCGTTCAAGGACCACAA GCTTTACAAACCGGGCGACCCACACTCAGCGGTTCGCTATCTCCGTTCTGTCACGCGTGCTAACTTGTCGTCACGGACGG CTATCGCATGGAGCGGAGTAAATGTAATCTTCGTCGTTTTGCTCCTTGACCGCCAAACTACCCGCCTCAGCATGCTAAATG CATCTCCTATATTAAAACGAAAGTAACTGAGTTGTTGTTTAAATTAAAATAGAATGAAACTATTTTATAGTTACCATTTTTCAA CACCAGCTGATATTCATCCGCCAAAAATTTACCCTTTAACTTACGGCAAACTTGTTGAGTCACATAGCACTTTTAACGTCCA ACTTCACCATTCTTCTTTAATCAACTCTTTTATTATTCAGCTTTTCTCAATCAAATGCCAACTAGGAATGGCCGTTGCTTTAAT ATCCATCGTACCATTCTCCATTCCCTGCGCTTGATTGCATTGCTGATATTTCTTCAGCTCCGTAGGAACGGGCCATGGAGC ACCAACTATGTTGTTGAAAGAATATAGTGAAGCGGCCGCACGAGAAAAACGAAGGTTACGCAAACCCTGGACAGGTGGG (D)

4 Supplementary Figure 1: The metagenomic pipeline with a turnaround time of approx. six hours (optimised) and approx. eight hours (pilot) from sample collection to sample result inferring antibiotic resistance gene/organism association using resistance gene flanking sequence. A: An example MinION read mapping to the meca gene (gene sequence in bold)* B: Identification of Staphylococcus aureus chromosome sequence using the meca gene 5 flanking sequence of the read*. C: An example of a MinION read mapping to the gene (gene sequence in bold)*. D: Identification of Streptococcus gordonii chromosome sequence using the gene 3 flanking sequence of the read*. *Three independent clinical samples were analysed (an example of a pathogen and a lung commensal are represented).

5 Supplementary Table 1a: Comparison of bacterial DNA extraction methods using qpcr. Sample Sample type Microbiology result O1 Sputum S. aureus O2 Sputum P. aeruginosa Condition 16S rrna gene V3-V4 fragment qpcr assay (Cq) Bacterial gain to standard depletion (DCq) Human qpcr assay (Cq) New DNA depletion compared to standard depletion (DCq) Standard depletion New depletion (enzyme cocktail) (3 fold) New depletion (bead beating) (21 fold) (2 fold) Standard depletion New depletion (enzyme cocktail) New depletion (bead beating)

6 Supplementary Table 1b: Comparison of the microbial community with altered PCR extension times for MinION sequencing. Sample Sample type Microbiology result PCR extension time (min) Average read length (Kb) Bacterial species from WIMP analysis ( 0.5% of classified reads) O1 Sputum S. aureus S. aureus Streptococcus agalactiae Streptococcus anginosus group Veillonella parvula S. aureus Streptococcus agalactiae Streptococcus anginosus group O2 Sputum P. aeruginosa P. aeruginosa group Pseudomonas P. stutzeri group P. aeruginosa group Pseudomonas Percentage of total classified reads (%)

7 Supplementary Table 2a: Limit of detection for Gram-positive and Gram-negative organisms using the optimised method in a high bacterial background. Sample Replicate Pathogen Approx. number of pathogen cells (CFU) DNA yield (ng/µl) Total raw read count Reads mapping to hg38 Nonhuman reads Classified from nonhuman reads Unclassified from non-human reads Total number of pathogen reads from total classified reads , (0.9%) 65,947 (99.1%) 50,922 15,015 12,658 (24.9%) SA , , (0.8%) 54,158 (99.2%) 42,612 11,524 4,286 (10.1%) , (0.6%) 36,064 (99.4%) 29,303 6,749 3,882 (13.2%) 1 S. aureus , (0.7%) 39,739 (99.3%) 29,311 10, ** (0.6%) SA , , (0.9%) 36,104 (99.1%) 27,233 8, (1.4%) , (0.9%) 44,428 (99.1%) 32,656 11, (2.1%) , (0.7%) 36,963 (99.3%) 28,559 8,398 1,346 (4.7%) EC , , (0.5%) 50,581 (99.5%) 38,924 11,637 2,030 (5.2%) , (0.5%) 44,959 (99.5%) 34,140 10,798 1,092 (3.2%) 1 E. coli , (0.4%) 27,916 (99.6%) 22,324 5,586 90** (0.4%) EC , , (0.7%) 44,469 (99.3%) 33,525 10, ** (0.5%) , (0.7%) 46,589 (99.3%) 34,319 12, ** (0.4%) *22Cq bacterial 16S rrna gene V3-V4 fragment qpcr assay **number of reads detected was below the ³1% of classified microbial reads and WIMP assignment q-score ³20 cut-off required for a sample to be considered positive.

8 Supplementary Table 2b: Limit of detection for Gram-positive and Gram-negative organisms using the optimised method in a low bacterial background*. Sample Replicate Pathogen Approx. number of pathogen cells (CFU) DNA yield (ng/µl) Total raw read count Reads mapping to hg38 Nonhuman reads Classified from nonhuman reads Unclassified from non-human reads Total number of pathogen reads from total classified reads** , (1.5%) 37,624 (98.5%) 36,271 1,343 23,514 (64.8%) SA , , (1.1%) 8,384 (98.9%) 8, ,928 (72.7%) , (4.1%) 13,719 (95.9%) 12,642 1,074 3,129 (24.8%) 1 S. aureus ,889 19,276 (31.1%) 42,613 (68.9%) 38,796 3,805 7,222 (18.6%) SA ,360 9,337 (29.8%) 22,023 (70.2%) 19,905 2,115 3,037 (15.3%) ,346 2,531 (10.0%) 22,815 (90.0%) 20,6802 2,120 2,499 (12.1%) , (3.0%) 28,443 (97.0%) 26,452 1,983 10,609 (40.1%) EC , , (2.5%) 7,139 (97.5%) 6, ,756 (41.5%) , (2.3%) 3,965 (97.7%) 3, ,072 (29.5%) 1 E. coli 0.8 2, (3.3%) 2,557 (96.7%) 2, (4.8%) EC , , (2.7%) 10,099 (97.3%) 8,736 1, (4.4%) , (3.6%) 14,473 (96.4%) 12,663 1, (4.4%) *27Cq bacterial 16S rrna gene V3-V4 fragment qpcr assay **The number of reads detected for all samples was above the 1% of classified reads and WIMP assignment q-score 20 required for a sample to be considered positive.

9 Supplementary Table 3: Mock community qpcr results in triplicate for spiked NRF samples with and without the optimised method. qpcr assay Sample Triplicate 1 (Cq) Triplicate 2 (Cq) Triplicate 3 (Cq) Average (Cq) Bacterial loss (DCq) Human Undepleted Depleted (10 3 fold) E. coli Undepleted Depleted H. influenzae Undepleted Depleted K. pneumoniae Undepleted Depleted P. aeruginosa Undepleted Depleted S. aureus Undepleted Depleted S. maltophilia Undepleted Depleted S. pneumoniae Undepleted Depleted (5.7 fold)

10 Supplementary Table 4: Sequencing metadata for all respiratory samples processed with the optimised method. Sample number Number of raw reads from 2hrs Number of reads minus hg38 Human reads Classified reads Unclassified reads Metagenomics output S E. coli S K. pneumoniae 1692 S P. aeruginosa S S. marcescens 3900 S K. oxytoca 3254 K. pneumoniae 906 S S. aureus Process extraction negative E. coli S. aureus S. pneumoniae S P. aeruginosa H. influenzae 232 S M. catarrhalis 7078 S. pneumoniae 504 S E. coli S H. influenzae S. pneumoniae 571 S S. pneumoniae 693 S M. catarrhalis H. influenzae Process extraction negative S. aureus H. influenzae E. coli S S. marcescens S S. aureus 9465 Number of pathogen reads ( 1% of classified reads and WIMP assignment q- score 20)

11 M. catarrhalis 8089 S S. aureus 6597 S. pneumoniae 390 S S. aureus S S16-undepleted S. aureus 6203 Process extraction negative 3 S H. influenzae S S H. influenzae 2827 S H. influenzae S. pneumoniae S S H. influenzae Process extraction negative S H. influenzae S H. influenzae S M. catarrhalis 3831 S H. influenzae S. pyogenes S. aureus S S. pneumoniae 464 S P. aeruginosa S. aureus Process extraction negative H. influenzae S. aureus E. coli S. pyogenes S1-undepleted E. coli

12 S P. aeruginosa S H. influenzae 1644 S E. coli S S Process extraction E. coli 10 negative 6 S E. coli S H. influenzae S P. aeruginosa S S. aureus P. aeruginosa S M. catarrhalis H. influenzae S S. aureus 6138 Process extraction negative E. coli H. influenzae M. catarrhalis P. aeruginosa S. aureus S S. aureus H. influenzae E. coli

13 Supplementary Table 5: Confirmatory qpcr analysis of additional pathogen detection. Discordant results compared to culture Sample qpcr gene target qpcr result (Cq) S Escherichia coli 1 S41 cyaa - S Haemophilus influenzae 1 S12 omp P6 - S S Klebsiella pneumoniae 1 S5 mdh - S Moraxella catarrhalis 1 S14 copb 22.0 S Pseudomonas aeruginosa* S9 oprl - Pseudomonas aeruginosa 1 S7 oprl 32.9 Staphylococcus aureus 1 S29 eap 32.7 S8 - S Streptococcus pneumoniae 1 S11 ply 32.2 S15 - S21 - S28 - Streptococcus pyogenes 1 S27 sdab Detected by metagenomics and not culture *Detected by culture but not metagenomics

14 Supplementary Table 6: Species-specific gene analysis for H. influenzae and S. pneumoniae Pathobionts detected by metagenomics pipeline Haemophilus influenzae Streptococcus pneumoniae Sample Pathobiont specific gene Number of reads aligned to pathobiont specific gene S7 5 S10 85 S12 0 S18 85 S20 67 S21 siat 49 S23 21 S24 61 S25 90 S27 63 S31 5 S36 60 S39 43 S41 2 S8 0 S10 5 S11 19 S15 ply 0 S21 0 S28 0

15 Supplementary Table 7: Microbiology antibiogram and ARMA output for all optimised method samples. Sample Microbiology Antibiogram culture result S1 E. coli Amoxicillin R, Gentamicin S, Co-amoxiclav R, Co-trimoxazole R, Tazocin I, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tigecycline S, Tobramycin S, Cefepime S S2 K. pneumoniae Amoxicillin R, Gentamicin S, Co-amoxiclav R, Co-trimoxazole S, Tazocin I, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tigecycline S, Tobramycin S, Cefepime S S3 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Ceftazidime S, Meropenem S S4 S. marcescens Gentamicin S, Co-trimoxazole S, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Amikacin S, Tigecycline S, Tobramycin S, Levofloxacin S, Colistin R, Cefepime S, Minocycline S, Ticarcillin S S5 K. oxytoca Gentamicin S, Co-trimoxazole S, Tazocin I, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Amikacin S, Tigecycline S, Tobramycin S, Levofloxacin S, Colistin S, Cefepime S, Imipenem S, Minocycline S, Ticarcillin R S6 S. aureus Flucloxacillin S, Erythromycin/clarithromycin S, Clindamicin S, Fuscidic acid S, Tetracycline/doxycycline S, Mupirocin S S7 H. influenzae Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S8 M. catarrhalis Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S9 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S E. coli Co-amoxiclav R, Co-trimoxazole R, Tazocin S, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tigecycline S, Tobramycin R, Cefepime S ARMA Output sul1 mpha dfra17 aada5 ACT-5 oqxb oqxa InuA OXA-50 catb7 APH(3 )- llb AAC(6 )- lc oqxb OXY-4-1 oqxb APH(3')- Ia vgac tet38 teto IsaC ermc sul1 aada2 AAC(3 )- lla mpha dfra12 AAC(3 )- llc S10 NSG ND

16 teto S11 NRF ND S12 NRF ND ErmB S13 S. marcescens Gentamicin S, Co-amoxiclav R, Co-trimoxazole S, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime S, Ceftriaxone S, Cefuroxime R, Amikacin S, Ertapenem S, Tigecycline I, Tobramycin S, Cefepime S S14 S. aureus Flucloxacillin S, Erythromycin/clarithromycin S, Clindamicin S, Fuscidic acid S, Tetracycline/doxycycline S, Mupirocin S S15 S. aureus Flucloxacillin S, Erythromycin/clarithromycin S, Clindamicin S, Fuscidic acid S, Tetracycline/doxycycline S, Mupirocin S S16 S. aureus/mrsa Penicillin R, Flucloxacillin R, Oxacillin R, Erythromycin S, Clindamycin S, Trimethoprim R, Gentamicin R, Ciprofloxacin R, Fusidic acid R, Mupirocin S, Rifampicin S, Vancomycin S, Teicoplanin S, Tigecycline S, Linezolid S AAC(6 )- lc oqxb tet38 tetq tet38 tetw tet38 meca ErmB S17 NRF ND tetq ErmF CfxA3 S18 H. influenzae Amoxicillin R, Tetracycline/doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Co-trimoxazole S TEM-70 TEM-33 TEM-105 TEM-104 TEM-208 S19 NRF ND tetw ErmX

17 S20 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S teto S21 NRF ND tet32 S22 NRF ND tetq S23 H. influenzae Amoxicillin S, Tetracycline/doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Co-trimoxazole S S24 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S25 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S26 M. catarrhalis Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S S27 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole S teto tetd tet32 tetw teta teto ermt tet38 S. aureus Flucloxacillin S, Erythromycin R, Clindamycin R, Fuscidic acid S, Tetracycline S, Mupirocin S S28 NRF ND lsac S29 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S S30 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S OXA-50 catb7 tet38 OXA-50 catb7 S31 NRF ND S32 NSG ND

18 AAC(3)- lla AAC(3)- llc vgac TEM-1 S33 NRF ND S34 NSG ND lsaa ErmB S35 E. coli Amoxicillin R, Gentamicin S, Co-amoxiclav R, Co-trimoxazole S, Tazocin R, Ciprofloxacin S, Meropenem S, Aztreonam S, Ceftazidime I, Ceftriaxone S, Cefuroxime S, Amikacin S, Ertapenem S, Tobramycin S S36 H. influenzae Amoxicillin R, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole R S37 P. aeruginosa Gentamicin S, Tazocin R, Ciprofloxacin S, Meropenem S, Ceftazidime R S38 P. aeruginosa Gentamicin S, Tazocin S, Ciprofloxacin S, Meropenem S, Ceftazidime S TEM-11 TEM-2 TEM-187 ACT-5 TEM-67 ErmB Mel catb7 OXA-50 tet38 S. aureus Flucloxacillin S, Erythromycin S, Clindamycin S, Fuscidic acid S, Tetracycline S, Mupirocin S S39 H. influenzae Amoxicillin S, Doxycycline S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole R S40 S. aureus (MRSA) Penicillin R, Flucloxacillin R, Oxacillin R, Erythromycin S, Doxycycline S, Clindamycin S, Trimethoprim S, Gentamicin S, Ciprofloxacin R, Fuscidic acid S, Rifampicin S, Vancomycin S, Teicoplanin S, Tigecycline S, Linezolid S, Daptomycin S, Chloramphenicol S S41 S. aureus Flucloxacillin S, Erythromycin S, Clindamycin S, Fuscidic acid S, Tetracycline S, Mupirocin S meca tet38

19 H. influenzae Tetracycline S, Amoxicillin S, Ceftriaxone S, Co-amoxiclav S, Ciprofloxacin S, Cotrimoxazole R R = resistant S = sensitive

20 Supplementary Table 8: All microorganisms identified in all samples tested using the optimised method (above our thresholds). Sample Organism cultured by microbiology Organisms identified from metagenomic pipeline ( 1% of classified reads and WIMP assignment q-score 20) S1 Escherichia coli Escherichia coli S2 Klebsiella pneumoniae Klebsiella pneumoniae S3 Pseudomonas aeruginosa Pseudomonas aeruginosa S4 Serratia marcescens Serratia marcescens Pantoea stewartii Citrobacter freundii Salmonella enterica Serratia plymuthica S5 Klebsiella oxytoca Klebsiella oxytoca Citrobacter freundii Klebsiella sp. M5al Klebsiella pneumoniae Klebsiella michiganensis S6 Staphylococcus aureus Staphylococcus aureus S7 Haemophilus influenzae Pseudomonas aeruginosa Veillonella parvula Rothia mucilaginosa Streptococcus sanguinis Haemophilus influenzae Haemophilus parainfluenzae Neisseria sicca S8 Moraxella catarrhalis Moraxella catarrhalis Streptococcus mitis Veillonella parvula Streptococcus salivarius Rothia mucilaginosa Streptococcus pseudopneumoniae Streptococcus pneumoniae Neisseria sicca Streptococcus gordonii S9 Pseudomonas aeruginosa & Escherichia coli Escherichia coli Lactobacillus paracasei Lactobacillus casei Candida albicans S10 NSG Haemophilus influenzae Streptococcus pseudopneumoniae Streptococcus sp. oral taxon 431

21 Streptococcus pneumoniae Streptococcus mitis S11 NRF Streptococcus sp. A12 Streptococcus mitis Rothia mucilaginosa Bifidobacterium longum Streptococcus pseudopneumoniae Streptococcus pneumoniae Streptococcus sp. I-P16 Streptococcus sp. I-G2 Haemophilus parainfluenzae S12 NRF Haemophilus parainfluenzae Moraxella catarrhalis Streptococcus gordonii Neisseria sicca Haemophilus influenzae S13 Serratia marcescens Serratia marcescens S14 Staphylococcus aureus Staphylococcus aureus Moraxella catarrhalis Rothia mucilaginosa Streptococcus constellatus Fusobacterium nucleatum Fusobacterium periodonticum Streptococcus anginosus Streptococcus intermedius S15 Staphylococcus aureus Staphylococcus aureus Streptococcus mitis Citrobacter koseri Rothia mucilaginosa Streptococcus salivarius Haemophilus parainfluenzae Streptococcus pseudopneumoniae Streptococcus pneumoniae Prevotella aninogenica S16 MRSA Staphylococcus aureus Lactobacillus rhamnosus S17 NRF Streptococcus salivarius Prevotella aninogenica Streptococcus sp. oral taxon 431 Streptococcus sp. A12 Streptococcus sp. NPS 308 Streptococcus sp.fdaargos_192 S18 Haemophilus influenzae Haemophilus influenzae

22 S19 NRF Streptococcus salivarius Streptococcus mitis Streptococcus sp.fdaargos_192 Streptococcus sanguinis Rothia mucilaginosa Veillonella parvula Streptococcus sp. oral taxon 431 Streptococcus gordonii Streptococcus constellatus S20 Haemophilus influenzae Haemophilus influenzae Rothia mucilaginosa Streptococcus mitis S21 NRF Haemophilus influenzae Streptococcus mitis Haemophilus parainfluenzae Streptococcus sp. oral taxon 431 Streptococcus salivarius Streptococcus pneumoniae S22 NRF Prevotella aninogenica Streptococcus mitis Streptococcus sanguinis Rothia mucilaginosa Veillonella parvula Haemophilus parainfluenzae Streptococcus salivarius Streptococcus sp. A12 Streptococcus cristatus S23 Haemophilus influenzae Haemophilus influenzae Streptococcus salivarius Veillonella parvula Streptococcus pseudopneumoniae Streptococcus mitis Streptococcus sp.fdaargos_192 S24 Haemophilus influenzae Haemophilus influenzae Haemophilus parainfluenzae Veillonella parvula Streptococcus salivarius Streptococcus sp. oral taxon 431 S25 Haemophilus influenzae Haemophilus influenzae S26 Moraxella catarrhalis Streptococcus mutans Moraxella catarrhalis Veillonella parvula

23 S27 Haemophilus influenzae & Staphylococcus aureus Streptococcus salivarius Streptococcus gordonii Rothia dentocariosa Lactobacillus salivarius Haemophilus influenzae Streptococcus pyogenes Staphylococcus aureus Streptococcus salivarius S28 NRF Streptococcus mitis Streptococcus salivarius Veillonella parvula Streptococcus sp. A12 Streptococcus sanguinis Rothia mucilaginosa Streptococcus sp. oral taxon 431 Streptococcus pneumoniae Streptococcus pseudopneumoniae Prevotella aninogenica Streptococcus sp. I-G2 Streptococcus sp. I-P16 Haemophilus parainfluenzae S29 Pseudomonas aeruginosa Pseudomonas aeruginosa Staphylococcus aureus S30 Pseudomonas aeruginosa Pseudomonas aeruginosa S31 NRF Haemophilus influenzae Streptococcus anginosus Prevotella intermedia Tannerella forsythia Bifidobacterium longum Rothia mucilaginosa Streptococcus gordonii Veillonella parvula Campylobacter concisus S32 NSG Escherichia coli S33 NRF Streptococcus salivarius Clavispora lusitaniae Streptococcus sp.fdaargos_192 Rothia mucilaginosa Saccharomyces cerevisiae S34 NSG Candida albicans Enterococcus faecalis S35 Escherichia coli Escherichia coli S36 Haemophilus influenzae Haemophilus influenzae Veillonella parvula

24 Streptococcus salivarius Streptococcus mitis S37 Pseudomonas aeruginosa Pseudomonas aeruginosa S38 Staphylococcus aureus & Pseudomonas aeruginosa Staphylococcus aureus Pseudomonas aeruginosa S39 Haemophilus influenzae Moraxella catarrhalis Haemophilus influenzae S40 MRSA Staphylococcus aureus Streptococcus constellatus Streptococcus anginosus Streptococcus intermedius S41 Haemophilus influenzae & Staphylococcus aureus Staphylococcus aureus Haemophilus influenzae Neisseria sicca Escherichia coli

25 Supplementary Table 9: qpcr gene targets and primer sequences. Organism Gene target Forward primer (5-3 ) Reverse primer (5-3 ) Probe (5-3 ) Reference Human Bacteria RNA polymerase A 16S rrna gene V3-V4 fragment TGAAGCCGTGCGGAA GG TCGTCGGCAGCGTCA GATGTGTATAAGAGAC AGCCTACGGGNGGCW GCAG E. coli cyaa CGATAATCGCCAGATG GC H. influenzae omp P6 AGCGGCTTGTAGTTCC TCTAACA K. pneumoniae mdh CGGGCGTAGCGCGTA A M. catarrhalis copb GGTGAGTGCCGCTTTT ACAAC P. aeruginosa oprl AGCCTTCCTGGTCCCC TTAC S. aureus eap ACTGTAACTTTGGCAC TGG S. maltophilia 23S rrna GCCGAAAGCCCAAGG TTT S. pneumoniae ply GCTTATGGGCGCCAA GTCTA ACAAGAGAGCCAAGTGTCG GTCTCGTGGGCTCGGAGATGTGTA TAAGAGACAGGACTACHVGGGTAT CTAATCC CCTAAGTTGCAGGAGATGG CAACAGAGTATCCGCCAAAAGTT GATACCCGCATTCACATTAAACAG TGTATCGCCTGCCAAGACAA CCTAATGAACCCCAGTGTATAAGT TTG GCAGATACCTCATTACCTGC CGACTTTCGTCCTCGCCTTA CAAAGCTTCAAAAGCAGCCTCTA [6FAM]TACCACGTCAT CTCCTTTGATGGCTCC TAT[BHQ1] Sybr Green Master Mix [6FAM]TAGAGCGCCTT CGGTGTCGGT[BHQ1] [6FAM]CGATGCTGCAG GCAATGGTGCT[BHQ1] [6FAM]CCCGGCATGGA TCGTTCCGA [BHQ1] [6FAM]TGCTTTTGCAG CTGTTAGCCAGCCTAA G[BHQ1] [6FAM]TGAACTGACGG TCGCCAACGGTT[BHQ 1] [6FAM]ATCGCAACGAC TGGCGCTA[BHQ1] Sybr Green Master Mix [6FAM]CTCAAGTTGGA AACCACGAGTAAGAGT GATGAA[BHQ1] Designed in house 56 Designed in house Designed in house 15 15

26 S. pyogenes sdab GGRACACGTACCCAA AATGTAGGA TCTTGAGCTCTTTGTTCGGTRTAG [6FAM]CGTGACCAAAA AGGCGGCATGC[BHQ1 ] 15