Andleeb, Saadia

Burnham, Carey-Ann D.

D\u27Souza, Alaric W.

Dantas, Gautam

Gul, Danish

Kwon, Jennie H.

Patel, Sanket

Potter, Robert F.

Shupe, Angela

Sun, Xiaoqing

Wallace, Meghan

English

Digital Commons@Becker

Supplementary Information 1 Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces 2 Alaric W. D’Souza1*, Robert F. Potter1*, Meghan Wallace2, Angela Shupe2, Sanket Patel1,2, Xiaoqing Sun1,2, 3 Danish Gul3, Jennie H. Kwon4, Saadia Andleeb3**, Carey-Ann D. Burnham2,4,5,6**, Gautam Dantas1,2,5,7** 4  5 *These authors contributed equally to this work. 6  7 **Corresponding Authors 8 (Saadia Andleeb: saadiamarwat@yahoo.com, Carey-Ann D. Burnham: cburnham@wustl.edu, Gautam Dantas: 9 dantas@wustl.edu) 10  11 1The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of 12 Medicine, St. Louis, MO, USA 13 2Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA 14 3Atta ur Rahman School of Applied Biosciences, National University of Sciences and Technology Islamabad, 15 Pakistan 16 4Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA 17 5Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA 18 6Departments of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA 19 7Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA  20 Providencia rettgeri (1)Shewanella putrefaciens (1)Brevundimonas diminuta (0)Achromobacter denitrificans (1)Alcaligenes faecalis (1)Pantoea dispersa (1)Ochrobactrum anthropi (2)Pseudomonas luteola (2)Acinetobacter spp. (0)Acinetobacter iwoffi (1)Acinetobacter johnsonii (2)Pandoraea apista (2)Empedobacter brevis (0)Pseudomonas saudiphocaensis (1)Pseudomonas oleovorans (1)Pseudomonas sp. o96/297 (2)Pseudomonas viridiflava (0)Pseudomonas putida (0)Acinetobacter junii (4)Stenotrophomonas maltophila (7)Citrobacter farmeri (1)Citrobacter freundii (1)No ID (0)Atlantibacter hermanii (0)Enterobacter cloacae (1)Klebsiella oxytoca (1)Escherichia coli (6)Serratia marcescens (13)Pseudomonas stutzeri (24)Pseudomonas aeruginosa (27)Klebsiella pneumoniae (33)Enterococcus faecium (47)Acinetobacter baumannii (74)Klebsiella quasipneumoniae (1)Acinetobacter soli (1)Pseudomonas xanthomarina (3)Pseudomonas sp. s199 (1)Enterobacter hormaechei (5)Atlantibacter subterranea (1)Atlantibacter subterranea (2)Superficieibacter electus (2)Stenotrophomonas sp. 278 (1)Pseudomonas fulva (1)Pseudomonas sp. p99/361 (2)Pseudomonas sp. p106 (1)Pseudomonas sp. v388 (1)Pseudomonas fluorescens (1)Pseudomonas moraviensis (1)Empedobacter falsenii (2)Acinetobacter variabilis (1)Erwinia sp. 198 (1)Halomonas stevensii (1)Alcaligenes aquatilis (1)Achromobacter aegrifaciens (1)Brevundimonas sp. 357 (1)Hadamard matrix valuesMALDI-TOFMASHANISpeciesSupplementary Figure 1: Taxonomic identification by WGS gives higher resolution than MALDI-TOF MS 21 Pairwise average nucleotide identity (ANI) between all PAK-H isolates. Axes of this graph are hierarchically 22 clustered and the MALDI-TOF MS identifications are shown as colored annotations next to the heatmap. MASH 23 ANI species identifications to type strains are shown as colored annotations where they differed from MALDI-24 TOF MS annotations. Source data is provided in the source data file.  25 US_6US_2US_10US_14013973963943913903893873823813743693683643593312722672612582302212031931861721701681611591561521491471451401261151071041021000980970940910880850830770740620560530430390360350290260250230200140023493471650951941913633532350000002000005100000010000050positionaRoomWeekICU.area386384375370367358354334313292275205181157143139132129127120111108086078054052051034033028006380264248155103048042238231166176171169325101092000005100000010000050positionRoomWeekICU.areab377376366257234211197151138134133131130128125110087075072066050027001167000005100000010000050positionRoomWeekICU.areac3463433283173103073011581231210632412151161090930440403202801841600892081440840170081951361350460300e+00 1e+06 2e+06 3e+06 4e+06positionRoomWeekICU.areae2982892862831821181171141130640610600590583042942541870320111851801791781120410380e+00 1e+06 2e+06 3e+06 4e+06 5e+06positionRoomWeekICU.areadAcinetobacter baumannii core genome recombinationPseudomonas aeruginosa core genome recombinationKlebsiella pneumoniae core genome recombinationEnterococcus faecium core genome recombinationPseudomonas stutzeri core genome recombinationLineageSpeciesA. baumanniiE. faeciumK. pneumoniaeP. aeruginosa1 54 7632LineageRoomPak-2Pak-1Pak-3US-1Pak-4ICU.areaalcohol foam dispensernursing call buttonroom sinkbedside light switchbedside railWeek1210268052642Supplementary Figure 2: Recombinant core genome positions for major species 26 Core-genome alignments with recombinant positions for (a) A. baumannii, (b) E. faecium, (c) P. stutzeri, (d) P. 27 aeruginosa, (e) K. pneumoniae. The x-axis for each plot is the position in the core genome and each row is an 28 isolate. Vertical bars in the plot show where recombination is predicted, and the colors correspond to the lineage 29 of origin. Isolate numbers are colored by hierBAPS lineage and each isolate is annotated on the left with room, 30 week, and surface information. Source data for all panels is provided in the source data file.  31 0100200300400density01002003000.000 0.001 0.002 0.003distcounta050010001500density01002003000.0000 0.0005 0.0010 0.0015 0.0020distcountb0100020003000density02460e+00 2e−04 4e−04 6e−04 8e−04distcountc01000200030004000density01020300e+00 5e−04 1e−03distcountd0100200300400500density02040600.000 0.005 0.010 0.015 0.020distcounteA. baumanniiE. faeciumK. pneumoniaeP. stutzeriP. aeruginosaSupplementary Figure 3: Histograms of pairwise core genome SNP distances for major species 32 Distribution of pairwise SNP distances between isolates for (a) A. baumannii, (b) E. faecium, (c) K. pneumoniae, 33 (d) P. aeruginosa, (e) P. stutzeri. The x-axis is the ratio of number of pairwise SNPs to core-genome length and 34 the y-axis is counts for the histograms and density for the density plots. Source data for all panels is provided in 35 the source data file.  36 1 2 3 4 5 6 7 8 9 10 11clonelineage7notclonal−0.10.00.10.20.0 0.2 0.434.08% variation15.18% variationA. baumannii accessory genesap-val = 0.009299−0.10.00.10.20.3−0.2 −0.1 0.0 0.146.82% variation19.15% variationA. baumannii accessory genesbp-val < 0.0001clone 1122334 5 64lineagenotclonal−0.4−0.20.00.20.4−0.2 0.0 0.2 0.443.42% variation15.57% variationE. faecium accessory genescp-val < 0.0001−0.20.00.20.40.0 0.2 0.4 0.649.61% variation19.82% variationE. faecium accessory genesdp-val < 0.0001significance from Permanova10000 permutationssignificance from Permanova10000 permutationsSupplementary Figure 4: Accessory genome ordination of A. baumannii and E. faecium 37 Principal coordinates analysis of accessory genome for (a) A. baumannii with non-clonal isolates included, (b) A. 38 baumannii without non-clonal isolates, (c) E. faecium with non-clonal isolates included, (d) E. faecium without 39 non-clonal isolates. Samples for each specific isolate are filled circles and centers of gravity for each clonal group 40 are open squares. Lines connect samples to their centers of gravity. PERMANOVA was used to determine was 41 used compare clonal groups’ centroids and dispersion. Source data for all panels is provided in the source data 42 file.  43 050010001500100 150 200 250temporal distancecountz−score = −8.18AB clone_1a050010000 5 10 15temporal distancecountz−score = −2.36AB clone_2b050010000 20 40 60temporal distancecountz−score = −5.26AB clone_3c050010000 5 10 15temporal distancecountz−score = −2.37AB clone_4d0500100020 40 60temporal distancecountz−score = −4.38AB clone_6e050010000 5 10 15temporal distancecountz−score = −0.97AB clone_7f02505007500 10 20temporal distancecountz−score = −3.33AB clone_8g050010000 5 10 15temporal distancecountz−score = −2.34EF clone_1h0500100015000 10 20 30temporal distancecountz−score = −3.31EF clone_2i025050075010000 10 20temporal distancecountz−score = −3.35EF clone_3j025050075010000 10 20 30temporal distancecountz−score = −1.39EF clone_4k0300600900500 550 600 650temporal distancecountz−score = −1.42EF clone_5l050010000 5 10 15temporal distancecountz−score = −2.33EF clone_6m0100020003000130 140 150 160 170spatial distancecountz−score = −4.84AB clone_1n010002000300040000 2 4 6spatial distancecountz−score = −4.05AB clone_2o010002000300015 20 25 30spatial distancecountz−score = −4.17AB clone_3p010002000300040000 2 4 6spatial distancecountz−score = −3.06AB clone_4q010002000300015 20 25 30spatial distancecountz−score = −2.87AB clone_6r010002000300040000 2 4 6spatial distancecountz−score = −3.11AB clone_7s0100020000 5 10spatial distancecountz−score = −3.64AB clone_8t010002000300040002 4 6spatial distancecountz−score = −4.14EF clone_1u010002000300040005.0 7.5 10.0 12.5spatial distancecountz−score = −3.69EF clone_2v0100020004 6 8 10 12spatial distancecountz−score = −3.68EF clone_3w0100020002.5 5.0 7.5 10.0 12.5spatial distancecountz−score = −4.36EF clone_4x05001000400 420 440 460spatial distancecountz−score = −2.91EF clone_5y010002000300040000 2 4 6spatial distancecountz−score = −3.05EF clone_6zobserved distanceA. baumannii expected distanceE. faecium expected distanceSupplementary Figure 5: Spatial and temporal linkage of core genome SNP identified clonal groups 44 Temporal and spatial linkage for A. baumannii and E. faecium clones during the first 3 months of collection. 45 Temporal distances are calculated as +1 for every 2 week span separating isolate collections. Spatial distances 46 are given as +0 if isolates were collected from the same surface and room, +1 if they were collected from the 47 same room, but different surfaces, and +2 if they were collected from different rooms. Expected distance 48 distributions for A. baumannii are shown in blue and expected distance distributions for E. faecium are shown in 49 red. Observed distances are shown as vertical black lines. Z-scores for the observed values compared to the 50 expected distribution are given for each graph. (a-g) Temporal distance distributions for A. baumannii clones. (h-51 m) Temporal distance distributions for E. faecium clones. (n-t) Spatial distance distributions for A. baumannii 52 clones. (u-z) Spatial distance distributions for E. faecium clones. Source data for all panels is provided in the 53 source data file.  54 Isolate 1 4 from referenceIsolate 2 3 from reference+0 +0+1 +1 +1variablepositions 3 pairwise distanceisolate1 isolate2Iso1 Iso2SNP count3Iso1 Iso3 1IsoN IsoN+1 3Supplementary Figure 6: Identification of pairwise variant distances from reference read mapping 55 Dark black horizontal lines represent reference genomes and colored short horizontal lines represent mapped 56 short reads. Vertical dashed red lines represent variant positions from the reference. Every position is checked 57 for differences from the reference and from all other isolates.   58 variant distancecore genome SNP distance02000040000600000 10000 20000 30000R2 = 0.9471p < 0.0001avariant distancecore genome SNP distance0500010000150000 500 1000 1500 2000R2 = 0.8696p < 0.0001bSupplementary Figure 7: Correlation between core genome SNP and variant distances 59 Comparison of pairwise core genome SNP distances to pairwise variant distances for (a) A. baumannii in blue 60 and (b) E. faecium in red. Each point shows the distance a pairwise comparison of two isolates with the x-axis 61 showing the distance by core genome SNPs and the y-axis showing the distance by variants called by mapping 62 quality filtered reads to the type strain. The density of the points for the core genome SNP and variant axis are 63 shown above and to the right of the scatterplots. The black line is the prediction given by the linear model (variant 64 distance ~ SNP distance) and the shaded grey area around the line is the 95% confidence interval for the 65 correlation. The adjusted R2 coefficient of determination and the p-value are given on the respective plots. Source 66 data for all panels is provided in the source data file.  67 For each cutoff:isolate1 isolate2Iso1 Iso2variant dist3Iso1 Iso3 1IsoN IsoN+1 31. filter pairwise distance table by cutoff2. find all maximally connected cliques4. remake graph without strongest clique3. sort cliques by highest minimum edgeweight0.50.50.90.80.70.75. recurse on steps 3 and 4Supplementary Figure 8: Identification of cliques at given cutoff value 68 In step 1, the pairwise distance table is filtered by the cutoff value. In step 2, this filtered table is then used to 69 generate a network. In step 3, the network is searched for fully connected cliques. The weakest connection in each 70 fully connected clique is identified and cliques are sorted in descending order based on their weakest connection. 71 In step 4, the clique with the highest value weakest connection is stored and its associated nodes are removed 72 from the filtered table. The network is then regenerated without the nodes. For step 5, steps 3 and 4are repeated 73 until all maximally connected cliques are identified.  74 A. johnsoniiA. lwoffiiA. juniiS. putrefaciens A. juniiAtlantibacter subterraneaAtlantibacter subterraneaAtlantibacter subterraneaP. rettgeriE. cloacaeC. freundii E. coli Alcaligenes faecalisA. variabilisKlebsiellapneumoniaeotherbacteriaab(−) blaOXA-23(+) blaOXA-2312340 2 4 6 8 10 12 26 52alcohol foam dispenserwashroom sinkbedside light switchbedside railnursing call buttonalcohol foam dispenserwashroom sinkbedside light switchbedside railnursing call buttonalcohol foam dispenserwashroom sinkbedside light switchbedside railnursing call buttonalcohol foam dispenserwashroom sinkbedside light switchbedside railnursing call buttonWeekSupplementary Figure 9: Spatial and temporal organization of high-risk resistance genes 75 (a) Overview of blaOXA-23 on PAK-H surfaces. Each horizontal grey panel represents a PAK-H room. Within each 76 room, the horizontal gridded white lines are the five sampled surfaces. Each vertical white line is one of the 77 collection weeks. Places where the horizontal and vertical white lines intersect represent a sampling. Filled black 78 squares are A. baumannii isolates positive for blaOXA-23 and unfilled black squares are A. baumannii isolates 79 negative for blaOA-23. 80 (b) Overview of blaNDM-1, blaNDM-5, and blaNDM-7 on PAK-H surfaces. Each horizontal grey panel represents a 81 PAK-H room. Within each room, the horizontal gridded white lines are the five sampled surfaces. Each vertical 82 white line is one of the collection weeks. Places where the horizontal and vertical white lines intersect represent 83 a sampling. Small circles are K. pneumoniae isolates with a blaNDM gene. Large boxes are other bacterial isolates 84 with a blaNDM gene. These boxes are annotated with the specific species identification. Circles and boxes are 85 colored by gene found in the isolate. blaNDM-1 is red, blaNDM-5 is green, and blaNDM-7 is blue. 86 Source data for all panels is provided in the source data file.  87 K. pneumoniae Susceptibility TestingKP_346KP_343KP_317KP_310KP_301KP_063KP_123KP_121KP_158KP_160KP_307KP_089KP_044KP_215KP_040KP_093KP_109KP_241KP_328KP_116KP_084KP_144KP_017KP_008KP_208KP_184KP_280KP_195KP_320KP_046KP_030KP_136KP_135AmpicillinCefazolinCefotetanCeftriaxoneCeftazidimeCefepimeMeropenemImipenemPipercillin.TazobactamCeftazidime.AvibactamAmpicillin.SulbactamCiprofloxacinLevofloxacinGentamicinAmikacinTrimethoprim.sulfaFosfomycinAztreonamDoxycyclineMinocyclineTigecyclineNitrofurantoinRoomICU.areaWeekLineageClassE. faecium Susceptibility TestingAmpicillinVancomycinNitrofurantoinRoomICU.areaWeekLineageSynercidGentamicin (high)ChloramphenicolDoxycyclineEF_238EF_231EF_166EF_034EF_078EF_380EF_358EF_176EF_264EF_048EF_042EF_248EF_354EF_375EF_171EF_169EF_155EF_139EF_334EF_028EF_086EF_033EF_111EF_143EF_157EF_181EF_205EF_275EF_292EF_052EF_051EF_054EF_132EF_129EF_103EF_370EF_367EF_127EF_120EF_313EF_386EF_384EF_325EF_092EF_108EF_101Daptomycin.E.TestClassP. aeruginosa Susceptibility TestingPA_064PA_060PA_059PA_058PA_061PA_118PA_114PA_113PA_117PA_185PA_179PA_178PA_182PA_298PA_289PA_286PA_283PA_180PA_112PA_041PA_038PA_187PA_011PA_254PA_032PA_294PA_304CeftazidimeCefepimeMeropenemImipenemPipercillin.TazobactamCeftolozane.TazobactamCeftazidime.AvibactamCiprofloxacinLevofloxacinGentamicinAmikacinFosfomycinColistinAztreonamRoomICU.areaWeekLineageClassab dcRoomPak-2Pak-1Pak-3US-1Pak-4Surfacealcohol foam dispensernursing call buttonroom sinkbedside light switchbedside railSusceptibilityResistantIntermediateSusceptibleAB_353AB_349AB_363AB_194AB_191AB_347AB_039AB_359AB_083AB_088AB_161AB_387AB_382AB_381AB_235AB_230AB_221AB_401AB_397AB_396AB_043AB_074AB_036AB_140AB_156AB_272AB_193AB_331AB_267AB_261AB_095AB_203AB_165AB_035AB_029AB_026AB_094AB_107AB_100AB_104AB_368AB_152AB_145AB_149AB_159AB_172AB_020AB_014AB_391AB_389AB_258AB_053AB_369AB_147AB_025AB_364AB_374AB_062AB_056AB_085AB_077AB_097AB_091AB_102AB_098AB_115AB_126AB_170AB_168AB_186AB_002AB_023AB_390AB_US_1AB_US_10AB_394AB_US_6AB_US_2CeftriaxoneCeftazidimeCefepimeMeropenemImipenemPipercillin.TazobactamAmpicillin.SulbactamCiprofloxacinLevofloxacinGentamicinAmikacinTrimethoprim.sulfaDoxycyclineMinocyclineRoomICU.areaWeekLineageClassA. baumannii Susceptibility TestingWeek1210268052642Classβ-lactamFluoroquinoloneAminoglycosideFolate pathway inhibitorTetracyclineAmphenicolVancomycin (Glycopeptid)Streptograminmisc.lineageA. baumanniiE. faeciumK. pneumoniaeP. aeruginosa1547632LineageSupplementary Figure 10: Phenotypic susceptibility results for major species 88 Phenotypic AST results using CLSI guidelines for (a) A. baumannii, (b) K. pneumoniae, (c) E. faecium, (d) P. 89 aeruginosa. Antibiotics are grouped by class on the y-axis and individual isolates are hierarchically clustered by 90 their resistance phenotypes on the x-axis. Red squares indicate phenotypic resistance in an isolate, pink 91 represents intermediate resistance, and white represents susceptibility. Colored annotations are added next to 92 antibiotics for drug class and above the charts for hierBAPS lineage, week, surface, and room. Source data for 93 all panels is provided in the source data file. 94 

Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces

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Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces

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