Microbiome Disturbance and Resilience Dynamics of 3 the Upper Respiratory Tract During Influenza a Virus 4 Infection 5

1 Supplemental information for

2 Microbiome disturbance and resilience dynamics of 3 the upper respiratory tract during influenza A virus 4 infection 5

6 Drishti Kaul1,8, Raveen Rathnasinghe2,8, Marcela Ferres2, Gene S. Tan1,3, Aldo Barrera2,4, Brett

7 E. Pickett5, Barbara A. Methe5, Suman Das5, Isolda Budnik2, Rebecca Halpin5, David

8 Wentworth5b, Mirco Schmolke6c, Ignacio Mena6, Randy A. Albrecht6, Indresh Singh5, Karen E.

9 Nelson5, Adolfo Garcia-Sastre6,7, Chris L. Dupont1*, Rafael A. Medina2,4,6*.

11 1J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA 92037, USA.

12 2Departmento de Enfermedades Infecciosas e Inmunología Pediátrica, Facultad de Medicina,

13 Pontificia Universidad Católica de Chile, Santiago, Chile.

14 3 Department of Infectious Diseases, University of California San Diego, La Jolla, CA 92037,

15 USA

16 4Millennium Institute on Immunology and Immunotherapy, Santiago, Chile

17 5J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, 14 USA.

18 6Department of Microbiology, Global Health and Emerging Pathogens Institute, Icahn School of

19 Medicine at Mount Sinai, New York, NY 10029, USA.

20 7Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029,

21 USA.

22 8These authors contributed equally to this work

23 b Present address: National Center for Immunization and Respiratory Diseases, Centers for

24 Disease Control and Prevention, Atlanta, GA, USA.

25 c Present address: Department of Microbiology and Molecular Medicine, University of Geneva,

26 Switzerland

27 *Corresponding author: E-mail: [email protected], [email protected]

29 Supplementary Figure 1. Diversity distance analyses of the microbiome of infected and

30 uninfected humans. Box and whisker plots for beta diversity distances within and between

31 influenza types for the human samples (P: Influenza positive, U: Influenza unknown, Flu

32 negative). The boxplots represent the diversity between the different infection types. All the

33 distances were calculated using the Bray-Curtis metric. The box represents the interquartile

34 range, the red line in the box indicates median for each of the sample groupings and the error

35 bars represent standard deviation. Dotted whiskers outside the box extend from the highest to the

36 lowest observation represented in the plot. Source data are provided as a Source Data file.

Fig. S#. Microbiome stratifcation for human patients according to two clinical factors. Clustering of data points for all human patients shown according to Age (A) and Vaccination status (B).

A PC 2 (7.21 %) B PC 2 (7.21 %) < 2yrs (2 patients) N 2 - 5yrs (2 patients)

6 - 18yrs (3 patients) NA

19 - 64 yrs (35 patients) Y > 65 yrs (10 patients)

PC 3 (6.13 %) PC 1 (25.34 %) PC 3 (6.13 %) PC 1 (25.34 %) 37

38 Supplementary Figure 2: Microbiome stratification for human patients according to two 39 clinical factors. Clustering of data points for all human patients shown according to A) different

40 age groupings and B) Vaccination status (NA: Not available/ applicable; Y: Yes; N: No). Source

41 data are provided as a Source Data file.

43 Supplementary Figure 3. Relative abundance for the top ten bacterial families in the URT

44 among infected and uninfected human subjects. The relative abundance values for the most

45 prevalent bacterial families among the three infected ecostates (a, b, and c, ) and the one

46 uninfected (d) ecostate from human samples based on the Bayesian posterior predictive

47 probabilities from the Infinite Dirichlet Multinomial mixture Models run over 2000 iterations

48 (top to bottom, (a)-(d)). Source data are provided as a Source Data file.

50 Supplementary Figure 4. Comprehensive taxonomic breakdown for influenza-infected

51 human subjects. The plot summarizes the relative abundances at the order level for taxonomic

52 groups that are present in greater than 1% of the samples. Each vertical column is an individual

53 subject. Source data are provided as a Source Data file.

NR_148643_1_269-518_Acinetobacter_equi_strain_114_16S_ribosomal_RNA__partial_sequence 69 NR_119114_1_249-498_Acinetobacter_johnsonii_strain_DSM_6963_16S_ribosomal_RNA__partial_sequence NR_117624_1_273-522_Acinetobacter_johnsonii_strain_ATCC_17909_16S_ribosomal_RNA__partial_sequence 88 NR_114074_1_253-502_Acinetobacter_radioresistens_strain_NBRC_102413_16S_ribosomal_RNA__partial_sequence 48 NR_026210_1_249-498_Acinetobacter_radioresistens_strain_FO-1_16S_ribosomal_RNA__partial_sequence 18 135 NR_153741_1_207-456_Acinetobacter_celticus_strain_ANC_3831_16S_ribosomal_RNA__partial_sequence 47 NR_025425_1_243-492_Acinetobacter_parvus_strain_LUH4616_16S_ribosomal_RNA__partial_sequence 0 NR_148846_1_253-502_Acinetobacter_proteolyticus_strain_NIPH_809_16S_ribosomal_RNA__partial_sequence 1000 NR_148845_1_253-502_Acinetobacter_modestus_strain_NIPH_236_16S_ribosomal_RNA__partial_sequence NR_042026_1_243-492_Acinetobacter_gyllenbergii_strain_RUH_422_16S_ribosomal_RNA__partial_sequence 57 NR_044975_1_263-512_Acinetobacter_johnsonii_strain_Mannheim_3865/60_16S_ribosomal_RNA__partial_sequence NR_117193_1_270-518_Acinetobacter_tjernbergiae_strain_DSM_14971_16S_ribosomal_RNA__partial_sequence 4 50 NR_148847_1_253-502_Acinetobacter_vivianii_strain_NIPH_2168_16S_ribosomal_RNA__partial_sequence 266 NR_157606_1_273-522_Acinetobacter_colistiniresistens_strain_NIPH_1859_16S_ribosomal_RNA__partial_sequence NR_117629_1_273-522_Acinetobacter_tjernbergiae_strain_DSM_14971_16S_ribosomal_RNA__partial_sequence 100 Otu000004_ferret|Pseudomonadales_Moraxellaceae_Acinetobacter NR_148843_1_253-502_Acinetobacter_courvalinii_strain_ANC_3623_16S_ribosomal_RNA__partial_sequence 74 NR_117931_1_223-472_Acinetobacter_nosocomialis_strain_RUH_2376_16S_ribosomal_RNA__partial_sequence 84 NR_152004_1_273-522_Acinetobacter_lactucae_strain_NRRL_B-41902_16S_ribosomal_RNA__partial_sequence 21 NR_042387_1_264-513_Acinetobacter_calcoaceticus_strain_NCCB_22016_16S_ribosomal_RNA__partial_sequence 44 NR_116774_1_274-523_Acinetobacter_pittii_DSM_21653_strain_CIP_70_29_16S_ribosomal_RNA__partial_sequence 15 NR_117621_1_273-522_Acinetobacter_pittii_DSM_21653_strain_ATCC_19004_16S_ribosomal_RNA__partial_sequence NR_117930_1_225-474_Acinetobacter_pittii_strain_LMG_1035_16S_ribosomal_RNA__partial_sequence 2167 NR_026207_1_249-498_Acinetobacter_haemolyticus_strain_DSM_6962_16S_ribosomal_RNA__partial_sequence 86 NR_117622_1_273-522_Acinetobacter_haemolyticus_strain_ATCC_17906_16S_ribosomal_RNA__partial_sequence NR_119359_1_263-512_Acinetobacter_haemolyticus_strain_ATCC_17906_16S_ribosomal_RNA__partial_sequence 16 NR_114216_1_237-486_Pseudomonas_mandelii_strain_NBRC_103147_16S_ribosomal_RNA__partial_sequence 10 NR_029042_2_255-504_Pseudomonas_lini_strain_DLE411J_16S_ribosomal_RNA__partial_sequence 7 74 NR_074597_1_250-499_Pseudomonas_syringae_strain_DC3000_16S_ribosomal_RNA__partial_sequence 10 NR_156815_1_264-513_Pseudomonas_silesiensis_strain_A3_16S_ribosomal_RNA__complete_sequence NR_132724_1_192-441_Pseudomonas_prosekii_strain_AN/28/1_16S_ribosomal_RNA__partial_sequence 89 NR_112075_1_235-484_Pseudomonas_veronii_strain_CIP_104663_16S_ribosomal_RNA__partial_sequence 75 NR_025587_1_243-492_Pseudomonas_meridiana_strain_CMS_38_16S_ribosomal_RNA__partial_sequence NR_025586_1_243-492_Pseudomonas_antarctica_strain_CMS_35_16S_ribosomal_RNA__partial_sequence NR_043716_1_205-454_Pseudomonas_syringae_strain_NCPPB_281_16S_ribosomal_RNA__partial_sequence 4 NR_024707_1_237-486_Pseudomonas_savastanoi_strain_ATCC_13522_16S_ribosomal_RNA__partial_sequence 0 05 NR_025174_1_243-492_Pseudomonas_extremorientalis_strain_KMM_3447_16S_ribosomal_RNA__partial_sequence 0 NR_114911_1_258-507_Pseudomonas_extremaustralis_14-3_16S_ribosomal_RNA__partial_sequence 2 NR_040798_1_243-492_Pseudomonas_fcuserectae_strain_JCM_2400_16S_ribosomal_RNA__partial_sequence 4 8 NR_028986_1_235-484_Pseudomonas_poae_strain_P_527/13_16S_ribosomal_RNA__partial_sequence NR_117820_1_209-458_Pseudomonas_syringae_strain_ICMP_3023_16S_ribosomal_RNA__partial_sequence 0 NR_028706_1_256-505_Pseudomonas_veronii_strain_CIP_104663_16S_ribosomal_RNA__partial_sequence

0 NR_025549_1_237-486_Pseudomonas_tremae_strain_TO1_16S_ribosomal_RNA__partial_sequence NR_146827_1_208-457_Pseudomonas_cerasi_strain_58_16S_ribosomal_RNA__partial_sequence 0 8 0 NR_028987_1_235-484_Pseudomonas_trivialis_strain_P_513/19_16S_ribosomal_RNA__partial_sequence 0 NR_042392_1_185-434_Pseudomonas_simiae_strain_OLi_16S_ribosomal_RNA__partial_sequence 0 NR_117821_1_207-456_Pseudomonas_marginalis_strain_ICMP_3553_16S_ribosomal_RNA__partial_sequence 0 NR_114749_1_255-504_Pseudomonas_protegens_strain_CHA0_16S_ribosomal_RNA__partial_sequence 2 NR_117822_1_209-458_Pseudomonas_savastanoi_strain_CFBP_1670_16S_ribosomal_RNA__partial_sequence NR_028985_1_237-486_Pseudomonas_congelans_strain_P_538/23_16S_ribosomal_RNA__partial_sequence Otu000003_human|Pseudomonadales_Pseudomonadaceae_Pseudomonas Otu000002_human|Pseudomonadales_unclassifed NR_113646_1_235-484_Pseudomonas_alcaligenes_strain_NBRC_14159_16S_ribosomal_RNA__partial_sequence 01 NR_121733_1_255-504_Pseudomonas_knackmussii_B13_16S_ribosomal_RNA__partial_sequence 1 NR_114194_1_235-484_Pseudomonas_citronellolis_strain_NBRC_103043_16S_ribosomal_RNA__partial_sequence 0 NR_114472_1_244-493_Pseudomonas_alcaligenes_strain_ATCC_14909_16S_ribosomal_RNA__partial_sequence 48 0 NR_112069_1_231-480_Pseudomonas_citronellolis_strain_ATCC_13674_16S_ribosomal_RNA__partial_sequence 824 NR_117827_1_207-456_Pseudomonas_alcaligenes_16S_ribosomal_RNA__partial_sequence 61 0 NR_026533_1_235-484_Pseudomonas_citronellolis_strain_DSM_50332_16S_ribosomal_RNA__partial_sequence NR_043731_1_257-506_Pseudomonas_delhiensis_strain_RLD-1_16S_ribosomal_RNA__partial_sequence NR_041702_1_271-519_Pseudomonas_knackmussii_B13_16S_ribosomal_RNA__partial_sequence 14 NR_117678_1_255-502_Pseudomonas_aeruginosa_strain_DSM_50071_16S_ribosomal_RNA__partial_sequence 4 NR_042435_1_251-498_Pseudomonas_nitroreducens_strain_IAM_1439_16S_ribosomal_RNA__partial_sequence 5 NR_119225_1_237-484_Pseudomonas_multiresinivorans_strain_ATCC_700690_16S_ribosomal_RNA__partial_sequence 1535 67 NR_114471_1_256-503_Pseudomonas_aeruginosa_strain_ATCC_10145_16S_ribosomal_RNA__partial_sequence NR_114975_1_251-498_Pseudomonas_nitroreducens_strain_DSM_14399_16S_ribosomal_RNA__partial_sequence 163 NR_113601_1_237-484_Pseudomonas_nitroreducens_strain_NBRC_12694_16S_ribosomal_RNA__partial_sequence 4 NR_113599_1_237-484_Pseudomonas_aeruginosa_strain_NBRC_12689_16S_ribosomal_RNA__partial_sequence 8 NR_114957_1_199-447_Pseudomonas_guezennei_strain_RA26_16S_ribosomal_RNA__partial_sequence NR_043289_1_259-507_Pseudomonas_otitidis_strain_MCC10330_16S_ribosomal_RNA__partial_sequence 6 2 NR_041715_1_243-492_Pseudomonas_stutzeri_ATCC_17588_=_LMG_11199_16S_ribosomal_RNA__partial_sequence 0 NR_116489_1_249-498_Pseudomonas_stutzeri_strain_VKM_B-975_16S_ribosomal_RNA__partial_sequence 02 NR_159318_1_242-491_Pseudomonas_fuvialis_strain_ASS-1_16S_ribosomal_RNA__partial_sequence 9 NR_103934_2_262-511_Pseudomonas_stutzeri_ATCC_17588_=_LMG_11199_16S_ribosomal_RNA__complete_sequence 13 NR_113652_1_235-484_Pseudomonas_stutzeri_strain_NBRC_14165_16S_ribosomal_RNA__partial_sequence NR_114751_1_249-498_Pseudomonas_stutzeri_strain_DSM_5190_16S_ribosomal_RNA__partial_sequence NR_118798_1_232-481_Pseudomonas_stutzeri_strain_CCUG_11256_16S_ribosomal_RNA__partial_sequence NR_024909_1_248-497_Pseudomonas_orientalis_strain_CFML_96-170_16S_ribosomal_RNA__partial_sequence EF055454_S000752001__Root_Archaea_Euryarchaeota_Halobacteria_Halobacteriales_Halobacteriaceae_Halosarcina 90 100 EF028067_S000842633__Root_Archaea_Euryarchaeota_Halobacteria_Halobacteriales_Halobacteriaceae_Halorubrum 50 EF077639_S000769054__Root_Archaea_Euryarchaeota_Halobacteria_Halobacteriales_Halobacteriaceae_Halorubrum 31 40 EF077631_S000769046__Root_Archaea_Euryarchaeota_Halobacteria_Halobacteriales_Halobacteriaceae_Natronococcus EF077633_S000769048__Root_Archaea_Euryarchaeota_Halobacteria_Halobacteriales_Halobacteriaceae_Haloterrigena EF016285_S000824488__Root_Archaea_Euryarchaeota_Methanobacteria_Methanobacteriales_Methanobacteriaceae_Methanobacterium

54 0.8

55 Supplementary Figure 5: Phylogenetic inference of the diagnostic Pseudomonadales OTUs

56 relative to reference sequences from cultivated strains. Source data are provided as a Source

57 Data file.

Figure S7. Comprehensive time-course taxonomic breakdown for human subjects. The plot summarizes the relative taxonomic abundances at the order level across all timepoints for taxonomic groups that are present in greater than 1% of the four infuenza infected subjects (2 for each virus subtype, A-D clockwise) and 2 healthy subjects (E-F). Pseudomonadales (pink) is prevalent among the infected subjects (red panel on top), whereas Actinobacteria/Flavobacteria is among the most abundant in uninfected humans (blue panel on the bottom). A Unhealthy Patient #2 | Subtype: H1N1 | Year of Collection: 2011 B Unhealthy Patient #18 | Subtype: H1N1 | Year of Collection: 2011

1.00 1.00

0.75 Bacteria;Actinobacteria;Actinobacteria;Actinomycetales 0.75

Bacteria;Bacteroidetes;Bacteroidia;Bacteroidales

Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales

Bacteria;Firmicutes;Bacilli;Bacillales

Bacteria;Firmicutes;Bacilli;Lactobacillales

0.50 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales 0.50 undance undance b b Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales axa A axa A T

Bacteria;Proteobacteria;Gammaproteobacteria;Enterobacteriales T

Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales

Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales 0.25 Bacteria; Other 0.25

Other

Unclassifed;Other

0.00 0.00

Day 1 Day 3 Day 5 Day 9 Day 1 Day 2 Day 3 Day 5 Day 22 Time after infection Time after infection

D Unhealthy Patient #4 | Subtype: H3N2 | Year of Collection: 2012 C Unhealthy Patient #15 | Subtype: H3N2 | Year of Collection: 2012

1.00 1.00

0.75 0.75 Bacteria;Actinobacteria;Actinobacteria;Actinomycetales Bacteria;Firmicutes;Bacilli;Bacillales Bacteria;Firmicutes;Bacilli;Lactobacillales Bacteria;Firmicutes;Clostridia;Clostridiales Bacteria;Other;Other;Other undance 0.50 Bacteria;Proteobacteria;Alphaproteobacteria;Rhizobiales undance 0.50 b b Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales axa A axa A

T Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales T Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales Other Unclassifed;Other;Other;Other 0.25 0.25

0.00 0.00

Day 0 Day 1 Day 2 Day 3 Day 7 Day 21 Day 0 Day 1 Day 2 Day 3 Day 7 Day 21

Time after infection Time after infection

E Healthy Patient #2 | Year of Collection: 2014 F Healthy Patient #21 | Year of Collection: 2014

1.00 1.00

0.75 0.75

Bacteria;Actinobacteria;Actinobacteria;Actinomycetales; Corynebacteriaceae Bacteria;Actinobacteria;Actinobacteria;Actinomycetales; Propionibacteriaceae Bacteria;Bacteroidetes;Flavobacteriia;Flavobacteriales Bacteria;Firmicutes;Bacilli;Bacillales 0.50 undance Bacteria;Firmicutes;Bacilli;Lactobacillales undance 0.50 b b Bacteria;Firmicutes;Clostridia;Clostridiales axa A

Bacteria;Proteobacteria;Betaproteobacteria;Burkholderiales axa A T T Bacteria;Proteobacteria;Betaproteobacteria;Rhodocyclales Bacteria;Proteobacteria;Gammaproteobacteria;Pseudomonadales Bacteria;Proteobacteria;Gammaproteobacteria;Xanthomonadales 0.25 Other 0.25

0.00 0.00

Day 1 Day 2 Day 3 Day 5 Day 8 Day 28 Day 1 Day 2 Day 3 Day 5 Day 8 Day 28

Time after infection Time after infection 58

59 Supplementary Figure 6. Comprehensive temporal taxonomic breakdown for human

60 subjects. The plot summarizes the relative taxonomic abundances at the order level across all

61 timepoints for taxonomic groups that are present in greater than 1% of the four influenza infected

62 subjects (2 for each virus subtype, A-D clockwise) and 2 healthy subjects (E-F).

63 Pseudomonadales (pink) is prevalent among the infected individuals (to 4), whereas inconsistent

64 taxa are seen among the healthy control individuals (bottom 2). Source data are provided as a

65 Source Data file.

67 Supplementary Figure 7. Diversity distance analyses of the microbiome of infected and

68 uninfected ferrets. Box and whisker plots for beta diversity distances within and between

69 influenza types for samples obtained for groups of 7 Infected (grouped by time points as Inf_T0,

70 Inf_others and Inf_T14) and 7 Uninfected (U) ferrets. The boxplots, sorted by median, represent

71 the diversity between and within the different infection types; boxplots identifying individual

72 time point-specific within diversity have been colored. Diversity within infected ferrets from

73 T=14 (red) being the least, and diversity between all uninfected (U) and infected ferrets from the

74 acute viral timepoints (T=1,3,5,7 dpi) being the highest, followed closely by diversity between

75 all infection states (all Infected vs all Uninfected) being the second highest. All the distances

76 were calculated using the Bray-Curtis metric. The box represents the interquartile range, the

77 horizontal line in the box indicates median for each of the sample groupings and the error bars

78 represent standard deviation. Dotted whiskers outside the box extend from the highest to the

79 lowest observation represented in the plot. Source data are provided as a Source Data file.

80 Enterobacteriaceae

Firmicutes, Bacillales 81 Clostridiaceae_1

Pseudomonadales,Acinetobacter 82 Enterobacteriaceae

Leuconostocaceae 83 Clostridiaceae_1 Top 10 OTU taxa10 OTU Top Peptostreptococcaceae 84 Pseudomonadales,Pseudomonas

Pseudomonadales,Acinetobacter 85 0 0.02 0.04 0.06 0.08 0.1 0.12

86 Porphyromonadaceae

Pseudomonadaceae

87 Leuconostocaceae

Clostridiales

88 Clostridiales

Clostridiaceae_1

89 Lachnospiraceae

Clostridiaceae_1 Top 10 OTU taxa10 OTU Top 90 Clostridiales

Peptostreptococcaceae

91 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 Relative abundance 92 Supplementary Figure 8. Relative abundance for the top ten most prevalent bacterial

93 families in the URT among infected and uninfected ferrets. The relative abundance was

94 determined based on the Bayesian posterior predictive probabilities from the Infinite Dirichlet

95 multinomial mixture models run over 1000 iterations. Analysis were performed on

96 pyrosequencing data obtained for the V1-V3 region of the 16S rRNA of nasal wash samples

97 obtained from 7 ferrets infected (top) with the A/Netherlands/602/2009 H1N1 virus and from

98 uninfected ferrets (bottom) at the time points indicated on Fig. 4. Source data are provided as a

99 Source Data file.

100

101 Supplementary Figure 9. Comprehensive taxonomic breakdown for all 14 ferrets. The plot

102 summarizes the relative taxonomic abundances at the order level across all timepoints for

103 taxonomic groups that are present in greater than 5% of the samples (see legend below).

104 Pseudomonadales (pink) is prevalent among the infected ferrets (bottom 7), whereas

105 Clostridiales (dark blue) is the most abundant among uninfected ferrets (top 7). Source data are

106 provided as a Source Data file.

Supplementary fgure. Changes in alpha diversity within the infected cohort (red) and uninfected cohort (blue) among ferrets during IAV infection. A decrease in alpha diversity was observed among the infected ferrets over the peak infection time course (3 to 7 dpi), with eventual recovery, in tune with the Pseudomonas bloom observed and the peak IAV titer collected from the same time points (Fig 4B). No such decrease at peak time points was observed for the healthy uninfected cohort.

Healthy Infected

Kruskal-Wallis (all groups), p-value =0.0322 Kruskal-Wallis (all groups), p-value = 0.00097

140 ic r

120 TUs met O ed v r 100 Obse

T0 T1 T3 T5 T7 T14 T0 T1 T3 T5 T7 T14 Time after infection

Healthy Infected

Kruskal-Wallis (all groups), p-value = 0.0023 Kruskal-Wallis (all groups), p-value = 0.00018

5 ic r ersity met v 4 Shannon di

T0 T1 T3 T5 T7 T14 T0 T1 T3 T5 T7 T14 107 Time after infection

108 Supplementary Figure 10. Temporal diversity distance analyses of the microbiome of

109 infected and uninfected ferrets. Changes in alpha diversity within the uninfected ferrets (n=7,

110 blue) and infected ferrets (n=7, red) during IAV infection. A decrease in alpha diversity was

111 observed among the infected animals during the acute phase of viral infection (3 to 7 dpi), with

112 an eventual recovery. This was in agreement with the Pseudomonas bloom observed and the

113 peak IAV titers collected from the same time points. The boxplots represent the diversity

114 between the different time points. Alpha diversity was calculated using the observed OTUs

115 metric (A) and shannon diversity index (B). Statistical analysis and significance testing was

116 done using the one-way Kruskal-Wallis method (significance level of 0.05). The box represents

117 the interquartile range, the horizontal line within the box indicates the median for each sample

118 grouping, observations are indicated by dots, and the whiskers outside the box extend from the

119 highest to the lowest observation represented in the plot. Source data are provided as a Source

120 Data file.

121 Supplementary Table 1. Clinical-epidemiological characteristics of the hospitalized human

122 patients diagnosed with Influenza A-like illness, and healthy controls.

Hospitalized patients Healthy Characteristic Total H1N1 positive H3N2 positive controls (n=28) (n=13) (n=15) (n=22)* Age < 2 years 2 1 1 0 2 - 65 years 17 9 8 22 > 65 years 9 3 6 0 Gender Male 13 7 6 10 Female 15 6 9 12 Clinical severity factors Hospitalized by Influenza 23 8 13 N/A CCU by Influenza 11 5 6 N/A O2 supply 20 8 10 N/A MV supply 7 5 1 N/A VAD supply 5 4 1 N/A

Vaccination and Treatments

Influenza Vaccine 6 2 4 5 Antibiotics 27 12 13 N/A Antiviral 29 12 15 N/A Comorbidities Asthma 2 0 2 N/A COPD/Respiratory pediatric disease 3 2 1 N/A Diabetes 8 3 4 N/A Obesity 7 3 4 N/A Cancer 4 3 1 N/A Cronical cardiovascular disease 12 5 6 N/A Cronical renal disease 2 2 0 N/A Neurological disorder 5 2 3 N/A Severe inmunological compromise 9 5 4 N/A Symptoms Fever 24 12 10 N/A Runny nose 20 9 10 N/A Throat pain 4 1 3 N/A Expectoration 22 11 10 N/A Myalgia 16 8 8 N/A Conjunctivitis 5 5 0 N/A Nasopharyngeal samples sequenced a 2 days 3 1 0 0 3 days 4 3 0 0 4 days 6 5 0 1 5 days 12 4 8 1 6 days 5 0 5 20 7 days 3 0 2 0 22 days 18 1 15 22 CCU: Clinical Care Unit, MV: Mechanical ventilation, VAD: Vasoactive drugs, COPD: Chronic obstructive pulmonary disease. N/A: Not applicable. a Days since onset of symptoms. * Healthy controls were obtained at the outpatient clinic. Source data are provided as a Source Data file.

123 Supplementary Table 2. Two-sided Student’s two sample t test results for human samples. 124 Comparison of every pair of boxplots (Supplementary Figure 1) to determine if they are 125 significantly different from each other. The significance indicates that samples within the same 126 infection state are significantly more similar to each other than samples across or between 127 infection states. Source data are provided as a Source Data file.

Parametric p-value Parametric p- Group 1 Group 2 t statistic (Bonferroni- value corrected)

Flu negative vs. Flu negative All within InfectionResult -48.874895 0 0 Flu negative vs. Flu negative P vs. P -62.752559 0 0 Flu negative vs. Flu negative P vs. U -125.7211 0 0 Flu negative vs. Flu negative U vs. U -144.01251 0 0 Flu negative vs. Flu negative Flu negative vs. P -184.67231 0 0 Flu negative vs. Flu negative All between InfectionResult -239.67117 0 0 Flu negative vs. Flu negative Flu negative vs. U -255.04846 0 0 All within InfectionResult P vs. P -20.460236 1.27E-91 3.55E-90 All within InfectionResult P vs. U -51.51848 0 0 All within InfectionResult U vs. U -63.077323 0 0 All within InfectionResult Flu negative vs. P -82.583982 0 0 All within InfectionResult All between InfectionResult -144.77785 0 0 All within InfectionResult Flu negative vs. U -135.79499 0 0 P vs. P P vs. U -5.3872418 7.56E-08 2.12E-06 P vs. P U vs. U -10.356331 7.40E-25 2.07E-23 P vs. P Flu negative vs. P -19.193782 1.54E-79 4.31E-78 P vs. P All between InfectionResult -21.916458 3.11E-105 8.71E-104 P vs. P Flu negative vs. U -29.715216 3.14E-187 8.80E-186 P vs. U U vs. U -8.413988 4.74E-17 1.33E-15 P vs. U Flu negative vs. P -21.393038 7.71E-99 2.16E-97 P vs. U All between InfectionResult -30.462617 8.55E-200 2.39E-198 P vs. U Flu negative vs. U -42.0745 0 0 U vs. U Flu negative vs. P -12.041826 3.94E-33 1.10E-31 U vs. U All between InfectionResult -19.628053 4.38E-85 1.23E-83 U vs. U Flu negative vs. U -31.556094 1.14E-211 3.20E-210 Flu negative vs. P All between InfectionResult -5.4887437 4.09E-08 1.14E-06 Flu negative vs. P Flu negative vs. U -19.243122 1.34E-81 3.77E-80 All between InfectionResult Flu negative vs. U -18.618746 6.05E-77 1.69E-75

128 Supplementary Table 3. Non-parametric multivariate analysis using Anosim and Adonis

129 tests. Examining the effect of clinical parameters (gender, age and antibiotic usage) on the

130 infected human URT microbiomes. Source data are provided as a Source Data file.

df df Adonis test Variable Anosim test (permutations=999) (n-1) (permutations=999) (n-1)

Gender R statistic= 0.0101 R2 statistic= 0.009 1 1 (n=2; M/F) p-value < 0.118 p-value < 0.017

Antibiotic Usage R statistic= 0.242 R2 statistic= 0.04231 1 1 (n=2; Y/N) p-value < 0.001 p-value < 0.001

Age R statistic=0.402 R2 statistic= 0.427 37 37 (n=38) p-value < 0.001 p-value < 0.001

Vaccination status R statistic=0.631 R2 statistic= 0.2456 2 2 (n= 3; Y/N/NA) p-value < 0.001 p-value < 0.001

131

132 Supplementary Table 4: Random forest analysis results for the human microbiomes. Ranks

133 range from the first few attributes predictive of the infection state, followed by the attributes that

134 are most predictive of the data (maximum accuracy displayed in bold). Source data are provided

135 as a Source Data file.

Rank Ranked attributes (OTUs) OTU taxonomya Accuracy (%) (1-667) 1st Otu000002 Bacteria;Proteobacteria; Gammaproteobacteria; 64.00 Pseudomonadales

2nd Otu000002; Otu000001 Bacteria;Proteobacteria; Alphaproteobacteria; 64.00 Rhizobiales; Brucellaceae; Ochrobactrum

3rd Otu000002; Otu000001; Otu000003 Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; 62.00 Pseudomonadaceae; Pseudomonas

137th Otu000002; Otu000001; Otu000003; Otu000006; Otu000055; Otu000035; Otu000005, etc (130 71.00 other OTUs)

a 136 Taxonomy presented for most predictive OTU identified in bold

137 Supplementary Table 5. Two-sided Student’s two sample t test results for ferrets.

138 Comparison of every pair of boxplots (Supplementary Figure 4). The significance indicates that

139 samples within the same infection state are significantly more similar to each other than samples

140 across or between infection states. Source data are provided as a Source Data file.

Group 1 Group 2 t statistic Parametric p- Parametric p-value value (Bonferroni-corrected)

Inf_T14 vs. Inf_T14 All within Infection -9.8555 3.79E-22 2.50E-20

Inf_T14 vs. Inf_T14 All between Infection -11.5323 6.32E-30 4.17E-28

Inf_others vs. All within Infection -4.39922 1.16E-05 0.000763 Inf_others

Inf_others vs. All between Infection -20.6832 4.63E-88 3.05E-86 Inf_others

All within Infection Inf_T14 vs. Inf_T0 -1.65626 0.097905 1

All within Infection U vs. U -3.93485 8.59E-05 0.00567

All within Infection Inf_T0 vs. Inf_T0 0.572274 0.567235 1

All within Infection Inf_T14 vs. U -8.18941 5.37E-16 3.54E-14

All within Infection Inf_T0 vs. Inf_others -7.7768 1.39E-14 9.16E-13

All within Infection U vs. Inf_T0 -11.5317 1.36E-29 8.99E-28

All within Infection Inf_T14 vs. Inf_others -10.7143 7.57E-26 5.00E-24

All within Infection All between Infection -28.681 1.34E-162 8.85E-161

All within Infection U vs. Inf_others -37.1721 2.51E-240 1.66E-238

U vs. U All between Infection -21.4854 1.69E-95 1.12E-93

Inf_T0 vs. Inf_T0 All between Infection -4.62237 4.01E-06 0.000265

All between Infection U vs. Inf_others -10.5276 1.59E-25 1.05E-23 141

142 Supplementary Table 6. Random forest analysis results for the ferret microbiomes. Ranks

143 range from the first few attributes predictive of the infection state, followed by the attributes that

144 are most predictive of the data (maximum accuracy displayed in bold). Source data are provided

145 as a Source Data file.

Rank Accuracy Ranked attributes (OTUs) OTU taxonomya (1-259) (%) 1st Otu000004 Bacteria;Proteobacteria; 79.79 Gammaproteobacteria; Pseudomonadales; Moraxellaceae;Acinetobacter

2nd Otu000004; Otu000028 Bacteria;Proteobacteria; 91.69 Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae;Enterobacter

3rd Otu000004; Otu000028; Otu000017 Bacteria;Firmicutes; 89.26 Bacilli;Bacillales; Family_XII;Exiguobacterium

7th Otu000004; Otu000028; Otu000017; Otu000001; 96.47 Otu000027; Otu000170; Otu000008

a 146 Taxonomy presented for most predictive OTU identified in bold