Supplementary Materials for

Staphylococcus aureus toxin potentiates opportunistic bacterial lung

infections

Taylor S. Cohen, Jamese J. Hilliard, Omari Jones-Nelson, Ashley E. Keller,

Terrence O’Day, Christine Tkaczyk, Antonio DiGiandomenico, Melissa Hamilton,

Mark Pelletier, Qun Wang, Binh An Diep, Vien T. M. Le, Lily Cheng, JoAnn Suzich,

C. Kendall Stover, Bret R. Sellman*

*Corresponding author. E-mail: sellmanb@medimmune.com

Published 9 March 2016, Sci. Transl. Med. 8, 329ra31 (2016)

DOI: 10.1126/scitranslmed.aad9922

This PDF file includes:

Materials and Methods

Fig. S1. MEDI4893* protects against mixed infection with diverse S. aureus

strains.

Fig. S2. MEDI4893* prevents mortality associated with mixed infection of S.

aureus and either K. pneumoniae or A. baumannii.

Fig. S3. AT potentiates infection with either K. pneumoniae or A. baumannii.

Fig. S4. Mixed infection does not result in excessive tissue damage or

inflammation.

Fig. S5. Immune cell populations in the lung.

Fig. S6. AT neutralization increases phagocytosis of S. aureus by AMs.

Fig. S7. AT reduces phagocytosis of S. aureus by AMs.

Fig. S8. Anti-NK1.1 depletion of NK cells.

Fig. S9. AT prevents colocalization of S. aureus with acidic lysosomes.

Fig. S10. AT reduces lysosomal acidification in human cells.

Fig. S11. Contribution of immune cells to the clearance of P. aeruginosa or K.

pneumoniae from the lung.

Fig. S12. Confocal imaging analysis of P. aeruginosa internalization by hPBMCs.

Fig. S13. Flow cytometry gating strategy and isotype controls.

Table S1. Exact P values for CFU data in Fig. 1.

Table S2. Bacterial strains.

Other Supplementary Material for this manuscript includes the following:

www.sciencetranslationalmedicine.org/cgi/content/full/8/329/329ra31/DC1

(available at www.sciencetranslationalmedicine.org/cgi/content/full/8/329/329ra31/DC1)

Table S3. Source data for all figures (Excel).

Materials and Methods:

Study design

Experiments were designed to test the hypothesis that S. aureus increases the pathogenesis

associated with sub-lethal Gram negative pneumonia. Experts in statistics and experimental design were

consulted to validate the design of all in vivo experiments before execution. All animal studies were

approved by the MedImmune Institutional Animal Care and Use Committee and were conducted in an

Association for Accreditation and Assessment Laboratory Animal Care (AAALAC)-accredited facility in

compliance with U.S. regulations governing the housing and use of animals. Guidelines for humane

endpoints were strictly followed for all in vivo experiments; moribund animals were immediately

euthanized by CO2 asphyxiation and recorded as a nonsurvivor. Sample sizes in all animal studies for

each model were estimated using log-rank test with 5% type I error rate and 80% power. The

hypothesized effect size for each comparison was derived from historical data or pilot study data. Sample

sizes were calculated using nQuery Advisor software. All animals were randomly assigned to treatment

groups using a randomization tool implemented in MS Excel. Animal allocations were not blinded to

study scientists for containment and personnel considerations. Pathologists that evaluated tissue sections

were blinded to study groups. All experiments were repeated 3 times unless noted in figure legend.

Reagents

Bacterial strains described (table S1). Monoclonal antibodies (mAbs) were prepared fresh daily

from concentrated, refrigerated stocks. The anti-alpha toxin neutralizing monoclonal antibody (mAb)

MEDI4893* and anti-P. aeruginosa mAb MEDI3902 were previously described 1,2. Purification and

characterization of native AT (AT) and the toxoid H35L (ATH35L) were previously described 3. Isotype

control human IgG1 (c-IgG) was included as a control for these studies.

Pneumonia Model

SF8300 frozen stock cultures were thawed and diluted to the appropriate inoculum in sterile PBS,

pH 7.2 (Invitrogen) 2. Pseudomonas aeruginosa, Acinetobacter baumanni or Klebsiella pneumoniae were

inoculated onto trypticase soy agar (TSA) plates (BBL, Becton, Dickinson Laboratories) and incubated

overnight at 37°C. Pseudomonas aeruginosa and Klebsiella pneumoniae were removed from plates and

suspended in PBS, pH 7.2 to an OD650nm of 0.5. Acinetobacter baumanni was inoculated into 2xYT broth

and grown to an OD650nm of 0.5. Colony forming units (CFU) in the challenge inoculum were confirmed

by serial dilution. All bacterial suspensions and toxins were administered in 50 µL of PBS. Each

individual pathogen was titrated to determine lethal dose 100 (LD100) and non-lethal challenge inocula,

followed by titration of the non-lethal dose of each Gram-negative pathogen with the non-lethal S. aureus

concentration (table S2). In select experiments, MEDI4893*, MEDI3902 or c-IgG were administered in

0.5 mL intraperitoneally (IP) at the time described in the text. Anakinra (Kinaret®, SOBI) was

administered (10 mg/kg) IP for 4 days and the animals were infected on day 4, as previously described

RW.ERROR - Unable to find reference:139.

Specific-pathogen-free 7- to 8-week-old female C57BL/6J mice (The Jackson Laboratory) were

briefly anesthetized and maintained in 3% isoflurane (Butler Schein™ Animal Health) with oxygen at 3

L/min and infected intranasally with individual bacterial strains, recombinant AT, toxoid control, or a

combination of bacteria or bacteria and AT. In select experiments NOD-scid IL2Rgnull-3/GM/SF (NSG-

SGM3) mice were engrafted with human CD34+ cells at Jackson Laboratories prior to delivery, and

subsequently infected. Animals were euthanized with CO2 at the indicated time points and bronchial

alveolar lavage fluid (BALF), lung tissue or spleens were collected for analysis. Histopathology was

performed as previously described 2. The bacterial load in BALF, lung, and spleen was determined by

plating serial dilutions on TSA (mono-infection), Pseudomonas isolation agar, mannitol salt agar or

CHROMagar, (Difco/BBL) for differential isolation of A. baumannii and K. pneumoniae in co-infection

models.

Cellular Depletion Models

Alveolar macrophages were depleted 24h prior to infection via intra-nasal inoculation with 50 µL

clodronate liposomes as previously described; control mice received PBS filled liposomes

(clodronateliposomes.com) 4,5. Neutrophils were depleted with anti-Ly6G antibody delivered IP (500

µg/mouse) 24h prior to infection 5. Natural killer cells (NK) were depleted by IP injection of anti-NK1.1

(250 µg/mouse) 24 and 48h prior to infection 6. Control mice were given rat IgG2a (neutrophil) or mouse

IgG2a (NK) at the same concentrations. Depletion antibodies and control IgG were from Bio X Cell (Bio

X Cell).

Cell Culture

RAW 264.7 cells and RAW 264.7 cells expressing m-cherry-LC3 were cultured in RPMI 1640

with 10% fetal bovine serum and penicillin and streptomycin (100U/mL). NK cells, neutrophils, and

monocytes were isolated from peripheral blood of healthy volunteers. Macrophage like cells were

isolated by collecting adherent monocytes following 7 days in culture 7. AT toxicity was determined by

incubating human cells (200k cells per well of a 96 well plate) with increasing concentrations of AT for

4h. Dead cells were stained with Zombie live/dead stain (Biolegend), and the percent of cells positive for

the dead stain was determined by flow cytometry.

Confocal Imaging

For imaging, 200k cells were allowed to adhere to each well of a chamber slide (Corning)

overnight, and loaded with Lysotracker (1 µM, Life Technologies) for 1h prior to bacterial inoculation. In

experiments involving AT neutralization, MEDI4893* (10 µg/mL), or c-IgG (10 µg/mL) was added along

with bacteria, similarly for experiments involving co-incubation with recombinant AT or ATH35L. In

select experiments cells were pretreated (1h) with Calpain Inhibitor VI (100 µM, EMD Millipore),

equivalent volume DMSO control. Cells were incubated (MOI 10) with S. aureus, P. aeruginosa, or S.

aureus+P. aeruginosa (1:10 ratio) for 1h, washed 3x with PBS, fixed in 10% formalin (10 min, VWR

International), the cell membrane permeabilized with 0.1% Triton X-100, and the actin cytoskeleton

stained with phalloidin (AF488 or AF568 conjugated, Life Technologies). Slides were sealed with

Vectashield (with or without DAPI, Vector Laboratories) and imaged with a Leica TCS SP5 X confocal

microscope (Leica Microsystems CMS Gmb). In each of three independent experiments at least 3 images

per group were acquired and representative images are presented.

Cells recovered from BALF were adhered to poly-L-lysine (Sigma-Aldrich) coated slides and

fixed with 10% formalin. Fixed cells were washed 3X with PBS and macrophages were labeled with

anti-CD11c (FITC conjugated, clone N418). Cell membranes were permeablized with 0.1% TritonX-100

and intracellular S. aureus were labeled with human anti-LTA mAb SAC55 (Humabs) followed by

AF647 goat anti-human IgG (Life Technologies). Slides were sealed and imaged as described above.

Flow Cytometry

Lungs were homogenized through a 40 µm filter (Corning, Inc.), pelleted (500 x g, 5 min) and

washed twice in FACs buffer (PBS with 5% fetal bovine serum, and 0.1% sodium azide). Red blood cells

were removed with ACK Lysing Buffer (Life Technologies), Fc receptors were blocked with anti-mouse

CD16/CD32 (eBioscience) and cells were stained with antibodies against mouse CD11c (APC-Cy5.5 or

FITC conjugated, clone N418), CD11b (BV605 conjugated, clone M1/70), Ly6-G (BV421 or PE-Cy7

conjugated, clone 1A8), NK1.1 (PerCP-Cy5.5 or eflour450 conjugated, clone PK136), CD3 (APC-Cy7

conjugated, clone C17A2), CD200R (APC conjugated, clone 0X110), CD274 (PD-L1, PerCP-eflour710

conjugated, clone MIHS), or IFNγ (APC conjugated, clone XMG1.2) from eBioscience or BioLegend.

Cells were imaged using the LSR II Flow Cytometer (BD Biosciences) and analyzed with FlowJo

(FlowJo). Gating strategy (fig. S13). A known concentration of counting beads (Bangs Laboratories)

was added to each sample to calculate the number of cells.

To measure in vivo phagocytosis of bacteria, S. aureus SF8300 was incubated with Calcein-AM

(5μM, Life Technologies) for 1h prior to infecting mice. GFP expressing P. aeruginosa was used to

monitor uptake of P. aeruginosa. Animals were sacrificed 4h post infection and the lungs processed as

described above. Mean fluorescence intensity (MFI) of macrophage and neutrophil populations was

determined as a measure of in vivo bacterial uptake.

Cytokine Expression and BALF Protein

Mice were infected and dosed as described above. At 8h or 24h post-infection, groups of 5

animals were euthanized, and the BALF was collected and processed as described above under CFU

determination. BALF was spun in a micro centrifuge at 500 x g for 5 min to remove cellular debris. The

supernatants were aliquoted and stored at -80oC until assayed. Cytokines were quantitated using the V-

PLEX Proinflammatory mouse cytokine multiplex kit (Mesoscale Diagnostics). Protein concentrations

were quantified using Pierce 660nm Protein Assay (ThermoFisher Scientific).

Bacterial Killing Assay

RAW 264.7 cells or human peripheral blood monocytes or neutrophils were plated (200k cells/96

well). RAW 264.7 and human monocytes were allowed to adhere overnight in culture media while

neutrophils were used on the day of isolation. Cells were washed with antibiotic free media and

incubated with S. aureus or P. aeruginosa (MOI-0.1). In select experiments cells were pretreated (1h)

with Calpain Inhibitor VI (100 µM, EMD Millipore) or equivalent volume DMSO control. In

experiments involving AT neutralization, MEDI4893* (10 µg/mL), or c-IgG (10 µg/mL) was added along

with bacteria, similarly for experiments involving co-incubation with recombinant AT or ATH35L.

Bacteria were prepared in the same manner as those for in vivo study, and one hour following bacterial

inoculation, the entire contents of the well (cells and media) were removed and bacterial CFU enumerated

by serial dilution. Percent killed was calculated by subtracting the number of recovered bacteria from the

innocula and dividing that number by the innocula.

Bacterial pH Measurement

SF8300 S. aureus or Δhla S. aureus were labeled by incubation with FITC ‘Isomer I’ and Alexa

Fluor 647 NHS Ester (Succinimidyl Ester) (2.5 mg/ml, 1h, Life Technologies). Bacteria were washed

three times in fresh PBS to remove unbound dye, and were frozed until use. RAW 264.7 cells were

prepared as described above and incubated with bacteria at an MOI-0.1. Following a 2h incubation, cells

were placed on ice, washed with cold PBS to remove free bacteria, and scraped from the well. FITC

intensity (MFI) was immediately measured by FACS, gating on bacterial positive macrophages

(determined by FSC and SSC, and AF647+).

Calpain Assay

Calpain activation was monitored as previously described 8. Briefly, RAW 264.7 cells were

stimulated with increasing concentrations of AT or AT-H35L for 1h, lysed with 0.1% TritonX-100, and the

lysate incubated with the calpain substrate N-Suc-LY-AMC (10 µM, Sigma-Aldrich). Fluorescence was

measured at 340/460 nm with an EnVision Multilabel plate reader (PerkinElmer).

NK Activation Assay

Human NK cells, isolated from peripheral blood by RoboSep Human NK Cell Enrichment Kit

(Stemcell Technologies), were seeded (200k cells/96 well) and incubated for 48h with recombinant hIL-

12 (100 U/mL, Peprotech) and increasing concentrations of AT. IFNγ production was measured in cell

supernatant using the human IFN gamma ELISA Ready-SET-Go kit (eBioscience).

Statistical analyses.

Data were analyzed using two-tailed t tests, Mann-Whitney tests, analysis of variance (ANOVA)

followed by Dunnett’s test, Kruskal-Wallis followed by Dunn’s test, or log-rank test where appropriate.

All statistical analyses were performed using GraphPad Prism version 6.0. A P value of ≤ 0.05 was

considered statistically significant.

Supplemental Figures:

Fig. S1. MEDI4893* protects against mixed infection with diverse S. aureus strains. (A) Survival of

mice treated with MEDI4893* (15 mg/kg) or c-IgG 24h prior to infection with S. aureus-NRS387+P.

aeruginosa. (B) Survival of mice treated with MEDI4893* (15 mg/kg) or c-IgG 24h prior to infection

with S. aureus-NRS261+P. aeruginosa. S. aureus (Sa), P. aeruginosa (Pa). Data representative of at

least 3 independent experiments.

Fig. S2. MEDI4893* prevents mortality associated with mixed infection of S. aureus and either K.

pneumoniae or A. baumannii (A) Survival of mice treated with MEDI4893* (15 mg/kg) or c-IgG 24h

prior to infection with S. aureus+K. pneumoniae. (B) K. pneumoniae CFU recovered from the lungs of

mice treated with MEDI4893* (15 mg/kg) or c-IgG 24h prior to infection with S. aureus+K. pneumoniae.

(C) Survival of mice treated with MEDI4893* (45 mg/kg) or c-IgG 24h prior to infection with S.

aureus+A. baumannii. (D) Numbers of A. baumannii recovered from the lungs of mice treated with

MEDI4893* (45 mg/kg) or c-IgG 24h prior to infection with S. aureus+A. baumannii. S. aureus (Sa), K.

pneumoniae (Kp), A. baumanii (Ab). Significance determined by (A, C) log-rank test, (B, D) Mann-

Whitney test. Data representative of at least 3 independent experiments.

Fig. S3. AT potentiates infection with either K. pneumoniae or A. baumannii (A) Survival of mice

infected with K. pneumoniae, S. aureus, S. aureus+K. pneumoniae, or Δhla S. aureus+K. pneumoniae. (B)

S. aureus and K. pneumoniae CFU recovered from the lungs of mice 24h following infection with S.

aureus or Δhla S. aureus+K. pneumoniae. (C) Survival of mice infected with A. baumannii, S. aureus, S.

aureus+A. baumannii, or Δhla S. aureus+A. baumannii. (D) S. aureus and A. baumannii CFU recovered

from the lungs of mice 24h following infection with S. aureus or Δhla S. aureus+A. baumannii. (E)

Survival of mice infected with AT or ATH35L + K. pneumoniae. (F) K. pneumoniae CFU recovered from

the lungs of mice 24h following infection with AT or ATH35L + K. pneumoniae. (G) Survival of mice

infected with AT or ATH35L + A. baumannii. (H) A. baumannii CFU recovered from the lungs of mice 24h

following infection with AT or ATH35L + A. baumannii. S. aureus (Sa), K. pneumoniae (Kp), A.

baumanii (Ab). Significance determined by (B, D, F, H) Mann-Whitney test. Data representative of at

least 3 independent experiments.

Fig. S4. Mixed infection does not result in excessive tissue damage or inflammation (A) Multiplex

ELISA measurement of cytokine expression 8h following infection with S. aureus (5e7 CFU), P.

aeruginosa (1e5 CFU), P. aeruginosa (1e5 CFU) + AT (0.055 μg), or S. aureus+P. aeruginosa in mice

treated with MEDI4893* (15 mg/kg) or c-IgG. (B) Survival of mice treated with Anakinra prior to

infection with S. aureus+P. aeruginosa. (C) Hematoxylin and eosin stained lung sections from mice 24h

following infection with S. aureus, P. aeruginosa or S. aureus + P. aeruginosa. (D) Protein

concentrations in the BALF of naïve mice or mice 24h following infection with S. aureus, P. aeruginosa

or S. aureus + P. aeruginosa. S. aureus (Sa), P. aeruginosa (Pa). Significance determined by (D)

Kruskal-Wallis followed by Dunn’s test. Data representative of at least 3 independent experiments.

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Fig. S5. Immune cell populations in the lung. (A) Numbers of alveolar macrophages in the lungs of

mice 24h following treatment with control (PBS) or depleting (clodronate) liposomes. (B) Numbers of

neutrophils in the lungs of mice 24hours following treatment with c-IgG or anti-Ly6G antibody. (C) PD-

L1 expression on alveolar macrophages in the lungs of mice treated with MEDI4893* or c-IgG 24h prior

to infection (24h) with S. aureus. (D) CD200R expression on alveolar macrophages in the lungs of mice

treated with MEDI4893* or c-IgG 24h prior to infection (24h) with S. aureus. (E) PD-L1 expression on

neutrophils in the lungs of mice treated with MEDI4893* or c-IgG 24h prior to infection (24h) with S.

aureus. (F) CD200R expression on neutrophils in the lungs of mice treated with MEDI4893* or c-IgG

24h prior to infection (24h) with S. aureus. Data representative of at least 3 independent experiments.

Supplemental Fig. 6

Alveolar Macrophage

Neutrophil

c-IgGMEDI4893*

Fig. S6. AT neutralization increases phagocytosis of S. aureus by AMs. Representative histograms

from FACs analysis of S. aureus association with macrophages and neutrophils in the lungs of mice

treated (-24h) with c-IgG or MEDI4893* and infected with S. aureus (5e7 CFU, 4h).

Fig. S7. AT reduces phagocytosis of S. aureus by AMs. FACs analysis of S. aureus or Δhla S. aureus

association with macrophages and neutrophils in the lungs of mice infected with S. aureus (5e7 CFU, 4h).

Significance determined by Mann-Whitney test. Data representative of 3 independent experiments.

NK1.1

CD

3c-IgG anti-NK1.1

Supplemental Fig. 7

Fig. S8. Anti-NK1.1 depletion of NK cells. Representative FACs plot demonstrating depletion of

NK1.1+CD3- cells.

WT S. aureus Δhla S. aureusMEDI4893* S. aureus

Fig. S9. AT prevents colocalization of S. aureus with acidic lysosomes. Representative images of

RAW 267.4 cells following incubation with calcein-blue labeled S. aureus. Acidic lysosomes are labeled

with lysotracker (red). Pink indicates lysosomes containing bacteria.

c-IgG MEDI4893*

Fig. S10. AT reduces lysosomal acidification in human cells. Confocal images of lysosomal

acidification (red) in human PBMCs infected with S. aureus (MOI 10, 1h) in the presence of MEDI4893*

or c-IgG, S. aureus are labeled blue.

Fig. S11. Contribution of immune cells to the clearance of P. aeruginosa or K. pneumoniae from the

lung (A) K. pneumoniae CFU recovered from the lungs of control (PBS) or alveolar macrophage

(Clodronate) depleted mice 24h following infection with K. pneumoniae (1e4 CFU). (B) K. pneumoniae

CFU recovered from the lungs of control (c-IgG) or neutrophil (anti-Ly6G) depleted mice 24h following

infection with K. pneumoniae (1e4 CFU). (C) K. pneumoniae CFU recovered from the lungs of control

(IgG) or NK cell (anti-NK1.1) depleted mice 24h following infection with K. pneumoniae (1e4 CFU). (D)

FACs analysis of P. aeruginosa association with macrophages in the lungs of MEDI4893* (15mg/kg) or

c-IgG treated (24h prior) mice infected with S. aureus (5e7 CFU, 4h). (E) FACs analysis of P. aeruginosa

association with neutrophils in the lungs of MEDI4893* (15mg/kg) or c-IgG treated (24h prior) mice

infected with S. aureus (5e7 CFU, 4h). (F) Percent of P. aeruginosa killed following 1h incubation with

human PMNs (MOI 1) in the presence of AT (0.1µg/mL) or ATH35L (0.1µg/mL). (G) Percent of K.

pneumoniae killed following 1h incubation with human PMNs (MOI 1) in the presence of AT (0.1µg/mL)

or ATH35L (0.1µg/mL). (H) Survival of macrophage depleted (clodronate) mice treated with MEDI4893*

(15 mg/kg) or c-IgG 24h prior to infection with S. aureus (5e7 CFU) + K. pneumoniae (1e2 CFU). S.

aureus (Sa), P. aeruginosa (Pa), K. pneumoniae (Kp). Significance determined by (A-E) Mann-Whitney

test, (F,G) t test, (H) log-rank test. Data representative of at least 3 independent experiments.

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Fig. S12. Confocal imaging analysis of P. aeruginosa internalization by hPBMCs. Confocal images of

human macrophages infected with P. aeruginosa or S. aureus+P. aeruginosa (1:10 ratio) for 1h. DNA

(blue), Actin (magenta), P. aeruginosa (green), Lysotracker (yellow).

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NK1.1+CD3-

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A

Alveolar Macrophage (AM), Neutrophil (PMN), and Inflammatory Monocyte (IM) Gate

Fig. S13. Flow cytometry

gating strategy and

isotype controls. (A) Flow

cytometry gating strategies.

(B) Isotype control

antibody FACs plots.

BCD11c CD11b Ly6G NK1.1

CD3 CD200R PDL1 IFNγ

Isotype Antibody

Lung

Sa Pa Sa + Pa (Sa) Sa + Pa (Pa)

Time (h) Mean SD Mean SD Mean SD Mean SD

P-value Pa vs. Sa + Pa (Pa)

4 7.816 0.250 3.856 0.223 7.616 0.258 4.110 0.271 0.1446

8 7.504 0.220 3.594 1.260 7.372 0.157 4.268 0.568 0.3073

24 6.716 0.907 2.212 0.747 7.068 1.028 5.870 0.669 <0.0001

48 4.330 2.093 1.690 0.000 6.210 0.641 6.527 0.300 <0.0001

Sa Kp Sa + Kp (Sa) Sa + Kp (Kp)

Time (h) Mean SD Mean SD Mean SD Mean SD

P-value Kp vs. Sa + Kp (Kp)

4 7.192 0.227 2.690 0.000 7.196 0.205 3.277 1.048 0.2461

8 6.868 0.209 2.690 0.000 7.106 0.338 3.727 0.747 0.0146

24 5.556 1.050 2.690 0.000 5.639 0.418 5.105 1.016 <0.0001

48 3.500 0.664 3.598 1.769 3.506 0.714 6.158 1.733 0.0049

Sa Ab Sa + Ab (Sa) Sa + Ab (Ab)

Time (h) Mean SD Mean SD Mean SD Mean SD

P-value Ab vs. Sa + Ab (Ab)

4 7.358 0.079 6.076 0.126 7.374 0.063 6.108 0.138 0.7083

8 7.230 0.137 6.074 0.048 7.154 0.102 6.304 0.123 0.0079

24 6.888 0.218 5.166 0.183 6.778 0.099 5.526 0.423 0.1186

48 4.388 1.567 3.366 0.569 6.258 0.096 6.848 0.411 <0.0001

Spleen

Pa Sa + Pa (Pa)

Time (h) Mean SD Mean SD P-value Pa vs. Sa + Pa (Pa) 4 1.300 0.000 1.300 0.000 NA

8 1.300 0.000 1.300 0.000 NA

24 1.300 0.000 1.420 0.268 0.347

48 1.300 0.000 3.077 0.895 0.003

Kp Sa + Kp (Kp)

Time (h) Mean SD Mean SD P-value Kp vs. Sa + Kp (Kp) 4 1.300 0.000 1.443 0.464 0.3414

8 1.300 0.000 1.297 0.000 NA

24 1.300 0.000 1.299 0.002 0.0132

48 1.646 0.659 3.108 0.428 <0.0001

Ab Sa + Ab (Ab)

Time (h) Mean SD Mean SD P-value Ab vs. Sa + Ab (Ab)

4 1.576 0.383 1.744 0.610 0.6169

8 2.286 0.581 2.664 0.354 0.2495

24 2.698 0.423 2.297 0.951 0.4146

48 1.300 0.000 5.166 0.888 <0.0001 Table S1. Exact P values for CFU data in Fig. 1.

Strain Phenotype/Genotype LD100 (CFU/mouse) Fraction of LD100

used in mixed

infections

Source

S. aureus SF8300 WT- USA300-0117 2e8 1/4 B. A. Diep

S. aureus SF8300 ΔagrA NA B. A. Diep

S. aureus SF8300 ΔclfA NA B. A. Diep

S. aureus SF8300 Δhla NA B. A. Diep

S. aureus SF8300 Δhla comp:hla 2e8 1/4 B. A. Diep

S. aureus SF8300 ΔisdH NA B. A. Diep

S. aureus SF8300 Δpsm alpha type

1,2,3,4

NA B. A. Diep

S. aureus SF8300 Δpvl NA B. A. Diep

S. aureus SF8300 ΔspA NA B. A. Diep

S. aureus NRS 261 WT - CC30 2e8 1/3 NARSA*

S. aureus NRS 386 WT - CC5 2e8 1/4 NARSA*

P. aeruginosa 6077 ExoU+ 5e5 1/5 Bascom-Palmer

Eye Institute

P. aeruginosa GFP-

PAK

ExoS+ A. Prince

K. pneumoniae subsp.

pneumoniae

(ATCC® 8045™)

Type 1 1e3 1/20 ATCC

A. baumannii 18500 1e7 1/10 J. Fernandez

Table S1. Bacterial strains Description of each strain of bacteria, its source, and its LD100 in

a murine, mono-infection pneumonia model.

* - Network on Antimicrobial Resistant S. aureus

Table S2. Bacterial strains Description of each strain of bacteria, its source, and its LD100 in a murine,

mono-infection pneumonia model.