SUPPLEMENTARY MATERIAL for manuscript

Antibiotic-driven dysbiosis mediates intraluminal agglutination and alternative segregation

of Enterococcus faecium from the intestinal epithelium

Antoni P. A. Hendrickx1,3, Janetta Top1, Jumamurat R. Bayjanov1, Hans Kemperman2, Malbert

R. C. Rogers1, Fernanda L. Paganelli1, Marc J.M. Bonten1 and Rob J.L. Willems1

1Department of Medical Microbiology and 2Clinical Chemistry and Haematology, University

Medical Center Utrecht, Utrecht, The Netherlands. 3To whom correspondence should be

sent: Dr. Antoni P.A. Hendrickx, Heidelberglaan 100, 3584CX Utrecht, Room G04.614, The

Netherlands, Phone: +31887557627, E-mail: A.P.A.Hendrickx-4@umcutrecht.nl

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Supplementary Methods

Bacterial strains. A commensal (E980) and a clinical hospital associated (E1162) E. faecium

isolate were used in this study. Bacterial strains were grown aerobically at 37°C on

Trypticase soy agar II (TSA) plates supplemented with 5% sheep blood. For selective

enumeration of E. faecium E980 and E1162 from mouse faeces and/or intestines Slanetz-

Bartley (Tritium Microbiologie B.V., Netherlands) agar plates were used. The MICs of

ceftriaxone and cefoxitin for E980 and E1162 are 8 and >512 µg/ml and 32 and 256 µg/ml,

respectively.

Mice. Thirty specific pathogen-free 10 week old female wild-type Balb/c mice were

purchased from Charles River Laboratories (USA). All mice were housed in the animal facility

of the Academic Medical Center (Amsterdam, The Netherlands) in rooms with a controlled

temperature and a 12-hour light-dark cycle. Animals were acclimated for 1 week prior the

experiment and received standard rodent chow and water ad libitum.

Mouse intestinal colonization model. Intestinal colonization by E980 and E1162 was

performed as previously described(1, 2). Eight mice were two days prior inoculation of

bacteria (on day -2, -1 and 0) injected subcutaneously with ceftriaxone (Roche, The

Netherlands; 100 µl per injection, 12 mg/ml; 5 injections total) two times daily and one time

at the day of inoculation (on day 0) and left on ad libitum cefoxitin (0.125 g/l) supplemented

sterile drinking water. In addition, 8 mice received 0.9% NaCl injections two days prior

inoculation of bacteria (on day -2, -1 and 0; 5 injections total) and were left on normal sterile

drinking water without antibiotics. Of the 8 antibiotics treated or 8 0.9% NaCl treated mice,

4 mice were inoculated on day 0 with E980 and 4 mice with E1162. The inoculum of 2 ×

2

109 CFU of E980 or E1162 was prepared as described previously(2, 3). As a control, 3 mice

were treated with ceftriaxone/cefoxitin antibiotics alone as described above, but did not

receive E. faecium. As another control group, 3 mice were left untreated. Collection of faecal

samples (2-3 faecal pellets/ml 0.9% NaCl) was done on days -2, 0, 1, 3, 6 and 10.

Determination of E. faecium outgrowth was performed by enumeration of CFU´s from

Slanetz-Bartley agar. After 10 days of colonization mice were anesthetized with ketamine (75

mg/kg) and medetomidine (1 mg/kg) followed by cervical dyslocation. Small intestines,

cecum and colon were removed by necropsy, weighed, photographed, sectioned in parts

and fixed in 2% glutaraldehyde for SEM and 4% formalin for histopathology. In addition, this

mouse intestinal colonization experiment was repeated for E. faecium E1162 to confirm the

reduction of the microbiota layer, mucus layer and colon wall. The experimental set-up was

identical as above with 2 animals/group for untreated mice, untreated mice inoculated with

E. faecium E1162, mice treated with antibiotics only, mice treated with antibiotics and

inoculated with E. faecium E1162 (thus, 8 additional mice). These animals were sacrificed on

day 1 and intestines were fixed in Carnoy's fixative. In total, in the two experiments, 30 mice

were included, 4 mice were treated with antibiotics and inoculated with E. faecium E980 and

6 with E. faecium E1162. In addition, 4 mice were untreated (injected with 0.9% NaCl) and

inoculated with E. faecium E980 and 6 with E. faecium E1162. Five mice were treated with

antibiotics only and 5 mice were left untreated (injected with 0.9% NaCl).

Ethics statement. The Animal Care and Use Committee (DEC) of the University of

Amsterdam, The Netherlands reviewed and approved the mouse intestinal colonization

experiment (number 102902). The experiment was conducted following the Dutch law on

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'Experiments on Animals' (Wet op de Dierproeven, WOD), in which the European Union

guideline 2010/63/EU is implemented per 18/12/2014.

Antibodies and recombinant proteins. Polyclonal Goat anti-rabbit IgG antibody directed

against mouse E-cadherin, Polyclonal Goat-anti human E-cadherin IgG antibody and Rabbit

anti-mouse pIgR IgG were purchased from R&D Systems (France). Goat anti-mouse IgA and

Donkey anti-Goat IgG-HRP were purchased from Southern Biotech (USA). Rabbit polyclonal

anti-Muc-2 IgG antibody was purchased from Abcam/ITK Diagnostics (The Netherlands).

Alexafluor 488 Goat-anti Rabbit IgG (H+L), Alexafluor 488 donkey anti-Goat IgG (H+L) and

TO-PRO-3 iodide 642/661 were all purchased from Molecular Probes/Life technologies (The

Netherlands). For immuno fluoresence experiments primary antibodies were used at a 1:100

dilution and the secondary antibodies at 1:500 dilution, while for Western blots dilutions

were 1:2500 and 1:5000, respectively. Recombinant mouse E-cadherin (CDH1-Met 1- Val

709) and recombinant mouse pIgR (Met 1- Lys 645) were from Sino Biological/Life

technologies (China) and human natural IgA protein was from Abcam. Bovine serum albumin

(BSA) was purchased from Serva. All antibodies, conjugates and proteins were reconstituted

by the manufacturer's instructions if necessary.

16S rRNA gene sequencing. Multiplexed 16S rRNA gene sequencing of DNA of bacteria

isolated from faeces of mice obtained from day -2, 0, 1 and 10 during the course of the

experiment was performed using the Illumina MiSeq platform method as described by

Fadrosh et al. using an improved dual indexing approach(4). First, DNA was extracted from

fecal pellets (0.01-0.1 gram) as described by Godon et al. with minor modifications(5).

Briefly, the homogenized faecal pellets used for E. faecium enumeration were thawed on ice

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and pelleted by centrifugation for 15 min. at 14,000 x g. The pellet was resuspended in 250

µl 4M guanidine thiocyanate–0.1 M Tris (pH 7.5) and 40 µl of 10% N-lauroyl sarcosine as

described previously(5). After DNA isolation, 5 µl of the sample was loaded on an agarose gel

to check the DNA and 2 µl were used in a PCR using E. faecium specific primers based on the

housekeeping gene ddl (D-Ala-D-Ala ligase) as described previously(6). DNA concentrations

were determined using the Qubit (Life Technologies) according to manufacturer’s

instructions and diluted to 5 ng/µl for 16S rRNA gene PCR. PCR products were only obtained

after a 1:10 dilution of the 5 ng/µl diluted sample. PCR and normalization was performed as

described and samples were pooled(4). After pooling the sample was purified using

Agencourt AMPure XP beads (Beckman Coulter Genomics), i.e. beads were added to the

sample in a 1:0.9 ratio, mixed and incubated for 5 min at RT. Beads were pelleted using a

magnetic stand and the DNA was eluted in a total volume of 52.5 µl. The concentration was

determined using the Qubit. The sample and PhiX Control (5%) were prepared according to

the Illumina protocol (15039740-d) for v3 chemistry and low diversity libraries and were

sequenced on 300PE MiSeq run.

16S rRNA sequence data analysis. Untrimmed paired-end reads were assembled using the

FLASH assembler, which performs error correction during the assembly process(7). Pooled

sequence data were de-multiplexed using split_libraries_fastq.py script of Qiime microbial

community analysis pipeline (version 1.8.0)(8). There were 8 samples for which no or only a

few sequences could be assigned, which were not used in further analysis. For remaining

samples, a median number of sequences per sample was 61103. After removal of the

barcodes, heterogeneity spacers, and primer sequences there were nearly 4.9 million

sequences with a median length of 424 bases. Sequences with a minimum of 97% similarity

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were assigned into operational taxonomic units (OTUs) using Qiime's open-reference OTU

calling workflow. This workflow was used with the “-m usearch61” option, which uses the

USEARCH algorithm for OTU picking and UCHIME for chimeric sequence detection(9, 10).

Taxonomic ranks for OTUs were assigned using the Greengenes database (version 13.8) with

the default parameters of the script pick_open_reference_otus.py(11). Representative OTU

sequences were aligned to the Greengenes core reference database using the PyNAST

aligner (version 1.2.2)(12, 13). Highly variable parts of alignments were removed using the

filter_alignment.py script, which is part of the pick_open_reference_otus.py workflow.

Subsequently, filtered alignment results were used to create an approximate maximum-

likelihood phylogenetic tree using FastTree (version 2.1.3)(14). For more accurate taxa

diversity distribution, OTUs with a number of sequences less than 0.005% of total number of

sequences were discarded using the filter_otus_from_otu_table.py script with the

parameter “--min_count_fraction 0.00005”(15). The filtered OTU table and generated

phylogenetic tree were used to assess within-sample (alpha) and between sample (beta)

diversities using Qiime's core_diversity_analyses.py workflow. For rarefaction, the

subsampling depth threshold of 4605 was used. The UniFrac distance was used to calculate

beta-diversity of samples(16). In addition to alpha- and beta-diversity analysis and

visualizations, this workflow also incorporates principal coordinates analysis and

visualization of sample compositions using Emperor(17).

Histopathology. Overnight formalin fixed or Carnoy's fixed cecum and colon tissue was

embedded in paraffin and sectioned and sections were prepared for immune fluorescence as

described elsewhere(18). Cecum and colon sections were either stained with Hematoxylin

and Eosin (H&E), Gram or Periodic acid-Schiff (PAS) stain according to standard procedures

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of the Department of Pathology of the University Medical Center Utrecht. Sections were

analyzed by light microscopy after training and instruction by a pathologist. From each of the

6 groups of the mouse experiment, H&E-, Gram and PAS-stained thin sections of at least 2

animals were analyzed. Microscopy data was confirmed by repeating the animal experiment

(except for E. faecium E980) and analysis of an additional 2 animals/group (thus 4

mice/group) of which the intestines were fixed in Carnoy's fixative. Integrity of the

epithelium of the cecum and colon was determined from at least 50 randomly chosen sites.

Microbiota diameters were measured in Gram-stained thin sections as indicated in

supplemental figure 2. Mucus-layer diameters were measured in PAS-stained thin sections

and colon barrier (comprising the crypts of Lieberkühn and underlying muscularis externae

and serosa) diameters from H&E stained thin sections. Diameters were randomly measured

along the microbiota layer/mucus layer/colon wall using ImageJ and plotted using Graphpad

Prism 6.0 software and relevant groups were compared.

Immunohistochemistry. Cecum and colon sections were prepared for immune fluorescence

as described elsewhere(18). In brief, 4 µm cecum and/or colon thin sections on glass slides

were deparaffinized using 100% xylene and rehydrated by consecutive incubations in 100%

ethanol, 90% ethanol, 70% ethanol and water. Antigen was retrieved by boiling slides at 95°C

for 20 minutes in 0.01 M Na-citrate buffer, pH = 6) and a cooling down in the same buffer for

20 minutes at room temperature, followed by rinsing with water. Immuno staining was

performed by incubating slides with 150 µl of 1:100 primary Goat anti-rabbit polyclonal IgG

antibodies directed against mouse E-cadherin/pIgR/IgA for 1 hr in Hank's Balanced salt

solution containing 1% bovine serum albumin (HBSS-BSA). After incubation, slides were

washed with 50 ml of HBSS-BSA and subsequently incubated for 1 hr in 200µl 1:100 diluted

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Donkey anti-Goat IgG-Alexa Fluor 488 and 1:1000 diluted TOPRO3 nuclei stain in the dark,

followed by another 50 ml wash with HBSS-BSA. Finally, bacteria were stained with 1 µg/ml

FM5-95 in H2O for 30 seconds and glass coverslips mounted using Prolong Gold anti-fade

reagent (Invitrogen). Tissue sections of untreated mice (n = 4), untreated mice inoculated

with E. faecium E1162 (n = 4) or E. faecium E980 (n = 2), mice treated with antibiotics only (n

= 4), mice treated with antibiotics and inoculated with E. faecium E1162 (n = 4), mice treated

with antibiotics and inoculated with E. faecium E980 (n = 2) were analyzed. Negative control

sections for all antibodies were processed in an identical manner after omitting the primary

antibody and showed no staining. Slides were analyzed by confocal microscopy as described

below. Labeled samples were stored at 4°C to allow reanalysis.

HT-29 cell culture and adherence assay. Human colorectal adenocarcinoma HT-29 cells

(obtained from the collection of Dr. S. van Mil, Wilhelmina Children's Hospital Utrecht) were

maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal

calf serum (FCS) and 1% penicillin/streptomycin at 37°C in a 5% CO2/air atmosphere.

Monolayers grown to 100% confluence were maintained for 2 weeks and culture medium

was refreshed daily. For E. faecium recovering assays, HT-29 monolayers were washed twice

with DMEM + FCS and incubated for 1 hr with DMEM + FCS. Subsequently, monolayers were

washed twice with DMEM and incubated for another 1 hr in DMEM. E. faecium E1162 was

added to the monolayers (MOI of 50 bacteria per cell) with or without varying

concentrations of (0, 5, 10, 20 mM) of CaCl2, MgCl2, or KCl in a final volume of 1 mL DMEM.

Monolayers with bacteria were spun down for 5 minutes at 1500 rpm to allow E. faecium to

bind and were incubated for 1 hr at 37°C in a 5% CO2/air atmosphere. Media and unbound

bacteria were removed and monolayers washed 3x with phosphate buffered saline (PBS),

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prior trypsinizing and homogenizing the HT-29 cells. Bound E. faecium was enumerated by

plating CFU's from serial dilutions onto TSA agar. Recovering of E. faecium from HT-29 cells

was plotted as % recovered (CFU recovered/CFU inoculum x 100%). Experiments were

repeated independently for at least 3 times.

HT-29 protein extraction. To analyze E-cadherin cleavage in the presence of cations, HT-29

cells were grown to 100% confluence and monolayers were maintained for 2 weeks.

Monolayers were washed as described above and incubated with varying concentrations of

(0, 5, 10, 20 mM) of CaCl2, MgCl2, or KCl in a final volume of 1 mL DMEM for 1 hr at 37°C in a

5% CO2/air atmosphere. After 1 hr incubation, monolayers were washed and HT-29 cells

lysed in lysis buffer (1% Triton X-100 in PBS supplemented with Complete mini protease

inhibitor cocktail [Roche]) for 10 minutes at 4°C. Cell debris was removed by centrifugation

for 1 min at 14,000 rpm and cell lysate was boiled for 5 minutes in sample buffer and

analyzed by Western blotting as described below.

Cation determination assays. Faecal extracts of the antibiotics treated and E980 or E1162

inoculated groups and untreated control mice obtained during the course of the experiment

were spun down for 10 minutes at 14,000 rpm, supernatant was transferred to a new tube

and spun down again to remove debris and bacteria. Calcium, Magnesium and Potassium

were measured on an AU 5811 routine chemistry analyzer (Beckman Coulter, Brea, USA).

Calcium and Magnesium concentrations were determined with colorimetric assays and

Potassium with an ion-selective electrode. Data obtained in mmol/l were corrected for

faecal weight and plotted in mM using Graphpad Prism 6.0 software and relevant groups

were compared.

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Immuno fluorescence and confocal microscopy. From each of the 6 groups of the mouse

experiment, cecum contents of 2 animals were analyzed. Mouse cecum contents was

harvested by puncturing the cecum and contents was post fixed for 15 min onto poly-L-

lysine covered glass slides and air dried. HT-29 monolayers grown on poly-l-lysine covered

glass slides were incubated with varying concentrations of cations and E1162 as described

above and fixed in formalin. Samples were washed with 15 mL PBS and incubated with

primary Goat anti-Rabbit IgG antibody (1:100 dilution) in HBSS for 30 min followed by

another 15 mL PBS wash. Subsequently, samples were incubated for 30 min with 1:500

diluted Goat anti-rabbit Alexa fluor 488 conjugate in the dark. Bacteria were stained with

1 µg/ml FM5-95 in H2O for 30 seconds. After incubation, the stain was removed and

coverslips were transferred to glass microscope slides with Prolong Gold anti fade reagent

(Invitrogen) and analyzed by a confocal laser scanning microscope (CLSM, Leica SP5),

equipped with an oil plan-Neofluar ×63/1.4 objective. AF488 was excited at 488 nm, and

FM5-95 at 633 nm. Images were analyzed using the LAS AF software (Leica).

Scanning electron microscopy. E. faecium cells were fixed for 15 min with 1% (v/v)

glutaraldehyde (Sigma, The Netherlands) in PBS at room temperature onto poly-L-lysine

covered glass slides. HT-29 monolayers on poly-l-lysine glass slides were fixed in 4% formalin.

Mouse cecum and colon tissue was fixed in 2% glutaraldehyde directly after dissection and

stored at 4°C. Bacterial, cell line or tissue samples were washed twice with PBS to remove

excess fixative and were subsequently serially dehydrated by consecutive incubations in 1 to

5 ml of 25% (v/v) and 50% (v/v) ethanol-PBS, 75% (v/v) and 90% (v/v) ethanol-H2O, and 100%

ethanol (2x), followed by 50% ethanol-hexamethyldisilazane (HMDS) and 100% HMDS

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(Sigma, The Netherlands). The glass slides or tissue parts were removed from the 100%

HMDS and air-dried overnight at room temperature. After overnight evaporation of HMDS,

samples were mounted onto 12 mm specimen stubs (Agar Scientific, The Netherlands) and

bacteria coated with gold to 1 nm and tissue to 4 nm using a Quorum Q150R sputter coater

at 20 mA prior to examination with a Phenom PRO Table-top scanning electron microscope

(PhenomWorld, The Netherlands).

In vitro agglutination. Agglutination of E. faecium E1162 was achieved by two distinct ways.

Firstly, 20 µl of cleared faecal extracts of untreated mice or mice treated with antibiotics

were inoculated with 1x105 CFU/ml E. faecium E1162 and incubated for 30 min at 37°C.

Secondly, 10 µg/ml of recombinant E-cadherin, pIgR, IgA or BSA was added to PBS inoculated

with E. faecium and incubated for 30 minutes at 37°C. Then, bacteria were transferred to

poly-l-lysine covered glass slides and stained with either FM5-95 or subjected to immuno

fluorescence labeling using specific antibodies and analyzed by confocal microscopy.

SDS-PAGE and Western blot analysis. Cleared faecal protein samples, HT-29 cell extracts or

recombinant proteins in sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium

dodecylsulfate, 0.01% bromophenol blue) were separated through a 4-20% gradiënt SDS-

polyacrylamide gel (Biorad). Prior to electroblotting, the membranes were incubated for 10

min in blot buffer (20 mM Tris, 150 mM glycine, and 20% methanol, pH 8.3). Samples were

electroblotted using a Trans-Blot cell tank transfer unit at 100V onto PVDF membranes (Bio-

Rad Laboratories Inc., The Netherlands). The membranes were blocked with 4% skim milk

(ELK, Campina Holland, The Netherlands) in PBS-0.1% Tween 20 for 1 hr at 20°C. Incubation

with primary antibodies (all at a 1:2,500 dilution) was carried out for 1 hour in 1% ELK in PBS-

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1% Tween 20 at 37°C, followed by a wash of 30 min in PBS-0.1% Tween 20 at 20°C.

Subsequently, membranes were incubated for 1 hr with anti-Goat IgG (heavy plus light

chains)-horseradish peroxidase (1: 5000 dilution, Southern Biotech) in 1% BSA in PBS (PBSb)-

1% Tween 20 at 20°C. Membranes were washed twice with PBS-0.1%, Tween 20, and

proteins were visualized using the ECL plus Western blotting detection system (GE

Healthcare) and exposed using a LAS AF imager.

Statistical analysis. All data are expressed as mean ± SEM. Differences were analyzed with a

Student's t test and performed using Graphpad Prism version 6.0 software. Values of p <0.05

were considered statistically significant.

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Supplemental References

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Acknowledgements. The authors thank members of our laboratory for discussions and Dr.

Nina van Sorge and Dr. Willem van Wamel for critical reading of the manuscript. We also

thank Prof. Dr. Saskia van Mil for providing the HT-29 cell line. Prof. Dr. Tom van der Poll for

facilitating the animal experiments at the Academical Medical Center Amsterdam, Jan

Beekhuis for assistance with preparation of mice intestines for microscopy, and Joost

Daalhuizen and Marieke S. ten Brink for their expert technical assistance during the animal

experiment.

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