ex vivo assay by supporting growth of commensal microbiota at … · 2019-10-03 · click to edit...
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10/2/19 1
Novel Glycans Reduce Carbapenem-resistant Enterobacteriaceae (CRE) and Vancomycin-resistant Enterococcus (VRE) Colonization in an Ex Vivo Assay by Supporting Growth of Commensal Microbiota at the Expense of Multidrug-Resistant (MDR) Organisms
Gabby LeBlanc, BS1, Brandon Brooks, PhD1, Madeline Hartman, BS1, Max Hecht, BS1, Hoa Luong, BA1, Zheng Pan, PhD1, Stephen Sofen, PhD1, Jonathan Lawrence, PhD1, Kelsey Miller, MS1, Brian Meehan, PhD1, Michael Mahowald, MD, PhD1, Tanya Yatsunenko, PhD1
1Kaleido Biosciences, Inc, Lexington, MA
TABLE 1. INFORMATION ON PATIENTS IN ICU
INTRODUCTION: SUPPORTING GROWTH OF COMMENSAL GUT MICROBIOTA REDUCES THE RISK OF INFECTIONS WITH MDR ORGANISMS
Acknowledgements: Elizabeth L Hohmann/Mariam Torres Soto (Infectious Diseases Division, Massachusetts General Hospital), Jasmohan S Bajaj (VCU Medical Center and Hunter Holmes McGuire VA Medical Center-Richmond), and Raymond T Chung (Gastrointestinal Division, Massachusetts General Hospital) for providing clinical samples. ETHOS Health Communications provided editorial support. Reference: Data on file. Kaleido Biosciences Inc.Commercial: Authors have nothing to disclose.Company details: All authors are Kaleido employees and own equity.
ACKNOWLEDGEMENTS AND REFERENCES
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• KB109 was selected via a high throughput screening cascade for its ability to stimulate the growth of the gut commensal microbiota at the expense of MDR pathogens
• KB109 reduces the relative abundance of CRE and VRE in ex vivo studies of microbiota from healthy subjects, critically ill subjects receiving broad-spectrum antibiotics, and patients with end-stage cirrhosis at risk of infection
• These results suggest that KB109 is a promising candidate to reduce the risk of infection due to MDR pathogens in high-risk patients and is currently being evaluated in a randomized, open-label study (NCT03944369)
CONCLUSIONS
• We hypothesized that defined microbial community and complete microbiota in stool samples from healthy donors or patients represent relevant model systems that can be used to screen the effect of MMTs on pathogen relative abundance (further referred to as ex vivo assays).
• An ex vivo screening cascade identified a candidate MMT, KB109, that selectively favors the growth of commensal bacteria at the expense of MDR pathogens (Figure 3)
• A suite of single strain assays assessed the ability of individual pathogens to utilize the MMTs for growth in order to generate orthogonal data and further de-risk the approach
RESULTS: GLYCAN MMTS MODULATE LEVEL OF SPIKED MDR PATHOGENS IN GUT MICROBIOTA FROM HEALTHY SUBJECTS
FIGURE 4. PROPORTION OF ENTEROBACTERIACEAE DEPENDENT ON AN MMT
FIGURE 5. MMTS MAINTAIN EFFICACY REGARDLESS OF PATHOGEN
Patient IDa Principal diagnoses Concurrent antibiotics (within 7 days)
Pt. 1 Sepsis, cirrhosis Vancomycin, meropenem, metronidazole
Pt. 6 Oropharyngeal cancer, hyponatremia Piperacillin-tazobactam
Pt. 12 Hemothorax, chronic obstructive pulmonary disease, chronic bronchitis Vancomycin, cefepime, metronidazole
Pt. 2 Gastrointestinal bleeding, idiopathic pulmonary fibrosis, cirrhosis, congestive heart failure Vancomycin, cefepime, piperacillin-tazobactam
Pt. 7 Pericardial effusion, metastatic adenocarcinoma Vancomycin
Pt. 13 Acute respiratory distress syndrome, trauma Vancomycin, cefepime
Pt. 11 MRSA endocarditis Vancomycin
Pt. 4 Septic shock, congestive heart failure Vancomycin, cefepime, clindamycin
Pt. 10 Hypoxemic respiratory failure, morbid obesity Vancomycin, cefepime
Pt. 3 Altered mental status Vancomycin
Pt. 8 Subarachnoid hemorrhage, pneumonia Vancomycin, cefepime
Pt. 5 Acute pancreatitis, sarcoidosis, cirrhosis Ceftriaxone
Pt. 9 Acute respiratory failure, interstitial lung disease, type 2 diabetes Vancomycin, cefepime, trimethoprim-sulfamethoxazole
RESULTS: KB109 REDUCES LEVELS OF MDR PATHOGENS IN STOOL SAMPLES OBTAINED FROM ICU PATIENTS RECEIVING BROAD-SPECTRUM ANTIBIOTICS
RESULTS: KB109 DOES NOT SUPPORT THE GROWTH OF BACTERIAL AND FUNGAL PATHOGENS
RESULTS: KB109 REDUCED LEVELS OF MDR PATHOGENS IN STOOL SAMPLES OBTAINED FROM PATIENTS WITH END-STAGE LIVER DISEASE WHO ARE AT RISK OF INFECTION
• KB109 reduced spiked CRE (Figure 8a) and VRE (Figure 8b) in samples obtained from 44 patients suffering from end-stage liver disease (ESLD) who are at risk of infection (in contrast to the commercially available glycan FOS)
VITORA: CLINICAL STUDYb
• VITORA is an ongoing, randomized, open-label clinical study evaluating KB109 in subjects whose gastrointestinal tracts are colonized with MDR organisms, with the objective of evaluating the safety and tolerability of KB109, its effects on the structure and function of the gut microbiota, and rate of pathogen decolonization (NCT03944369)
bStudy is being conducted under regulations supporting research with food.
• We hypothesized that testing MMTs in samples from patients in the ICU who were receiving broad-spectrum antibiotics would be the most rigorous test for MMT activity, because these were expected to represent the most severely damaged patient microbiota available (with fewer commensals)
• Thirteen fecal samples from patients in the ICU receiving broad-spectrum antibiotic therapy (Table 1) were spiked with CRE E. coli and incubated with 55 MMTs in the ex vivo assay
FIGURE 7b. PATHOGEN REDUCTION RELATIVE TO CONTROL
Each row represents the fecal microbiota of a patient or healthy subject; columns represent genus-level bacterial taxa with proportions of each taxa shown in each cell.
• The microbiota of ICU patients is extremely depleted in taxa found in healthy subjects (Figure 7a)
• KB109 successfully reduced the relative abundance of pathogens in all microbial communities with commensal bacteria (Figure 7b)
P.1 P.6 P.2 P.7P.12 P.4P.11P.13 P.10 P.9P.5P.8P.3 Healthy
These patients had no commensal bacteria in their microbiota (see Figure 6a)
FIGURE 9. PROPORTION OF SPIKED CRE OR VRE IS DECREASED DUE TO GROWTH OF COMMENSAL TAXA (ORDER LEVEL, GROUPED BY PHYLUM)
Potential stereo- and regiochemical possibilities of carbohydrates make them the most structurally
diverse biopolymer known
MMTs are structurally diverse ensembles of complex carbohydrates (glycans), enabled by Kaleido’s
synthetic chemistry platform
8 possible head-tail dimers:a-1,2; a-1,3; a-1,4; a-1,6b-1,2; b-1,3; b-1,4; b-1,6
Glycosyl acceptors
Glycosyl donor
Mono-Oligo-Poly-
saccharide
Catalyst
Bond distributions
• Colonization is one of the strongest factors predicting infection with MDR organisms• The human gut microbiota normally prevents colonization by pathogenic bacteria and helps maintain the intestinal
barrier• Chemotherapy and antibiotic use reduces the diversity of the commensal microbiota, resulting in a decreased ability to
perform these protective functions
A
C
• A fecal community from a healthy donor was spiked with CRE K. pneumoniae to 70% initial concentration relative to the commensal microbiota, and incubated with 55 MMTs
• Community composition was determined at the end of the ex vivo incubation using 16S rDNA sequencing
• Data are shown for a subset of MMTs for clarity (Figure 4): rows are MMTs, columns are family level bacterial taxa, proportion in each sample is indicated in each cell
• The proportion of Enterobacteriaceae (includes spiked CRE K. pneumoniae) depends on an MMT• Without an MMT, pathogens represented 66% of the
community• Some MMTs supported the growth of pathogens
(top row), reaching 86% of the community• KB109 reduced pathogens to 27% (from an initial
70% concentration) and supported growth of several families of commensal microbiota
• MMTs perform similarly with CRE E. coli, CRE K. pneumoniae, and VRE E. faecium, which is consistent with the proposed mechanism of action(Figure 2 and 5)
B D
E
MRSA, methicillin-resistant Staphylococcus aureus.aPatient information presented in the same order as Figure 7.
46.9 1.1 027.4 0.4 5.6 180.6
5.1 1.7 0.133.4 0 11.6 47.20.9
6.2 0.3 0.651 0.1 9.4 30.61.810.4 0.3 0.440.2 2.2 24.1 20.32
25.2 0.8 0.133.3 4.1 13.8 20.91.9
7.1 0 6.966.2 0.2 14.1 0.25.3
5.3 25.3 0.457.9 3.5 7 0.40.327.7 0.6 065.5 1 4.8 00.3
8.7 0.4 086.6 0.1 3.9 0.10.214.4 5.6 0.672.3 0.1 5.8 0.11
KB109−1
No glycan
MMTs
65.8 0.9 0.323.2 1.3 6.4 1.60.528.5 0.6 1.318.4 0 17.8 32.40.9
24.7 1.4 0.323.4 0.1 11 38.20.8
80.4 0.5 1.59.5 0 4.7 2.80.6
Bact
eroi
dace
ae
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tridi
acea
e
Clos
tridi
ales
XI
Enterobacteriaceae
Erys
ipel
otric
hace
ae
Lach
nosp
irace
ae
Oth
er
Porp
hyro
mon
adac
eae
Family level taxa
0% community15% community80% community
• KB109 reduced spiked CRE (Figure 9a) and VRE (Figure 9b) abundance in an ex vivo assay by supporting growth of commensal microbiota at the expense of MDR pathogens. Shown is the proportion of each order-level taxa grouped by phylum
• Complex carbohydrates are the major energy source for gut commensal bacteria, which possess a rich repertoire of carbohydrate-degrading enzymes (glycosidases), unlike most MDR organisms (Figure 1)
• Novel synthetic glycans (Microbiome Metabolic Therapies [MMT]™) may take advantage of this difference by efficiently and exclusively supporting the growth of commensal bacteria, thus reducing colonization and subsequent infections by MDR pathogens (Figure 2)
(a) CRE
KB109 FOS
Log2
[com
poun
d/w
ater
]CRE
E. c
oli
(b) VRE
KB109 FOS
Log2
[com
poun
d/w
ater
]VRE
E. f
aeci
um
Pathogens have smaller number and encode distinct repertoire of glycosidases
Bacteroides ovatusBacteroides thetaiotaomicron
Bacteroides xylanisolvensBacteroides dorei
Bacteroides fragilisClostridium cellulovorans
Parabacteroides distasonisKlebsiella oxytoca
Enterobacter aerogenesEnterococcus faecium
Klebsiella pneumoniaeBifidobacterium infantis
Akkermansia muciniphilaEnterobacter cloacae
Salmonella entericaCitrobacter freundii
Escherichia coliRuminococcus sp.
Bifidobacterium breveClostridioides difficile
Bifidobacterium bifidumEnterococcus faecium
Citrobacter koseriEnterococcus faecalisRuminococcus obeum
Pseudomonas aeruginosaRuminococcus bromii
Proteus mirabilisStaphylococcus aureus
100 200 3000
PC2 (12% variation explained)
PC1
(42%
var
iatio
n ex
plai
ned)
CommensalsPathogens
Number of glycosidases
FIGURE 1. COMPLEX CARBOHYDRATES AND COMMENSAL GUT BACTERIA
FIGURE 2. MMTS MAY REDUCE THE LIKELIHOOD OF INFECTION WITH MDR ORGANISMS
FIGURE 3. KB109 WAS SELECTED VIA AN ASSAY CASCADE OF INCREASING COMPLEXITY AND DISEASE RELEVANCE
Defined community +Healthy communities
ICU patientcommunities
Single-strainassay
Process scale-up and
characterizationKB109
• Response in ≥10 of 13 fecal communities
• Highest microbiotadiversity
• No growth of CRE, VRE, C. difficile and Candidastrains on MMT
Selection Criteria:
• Best reduction ofMDR abundance
• Highest microbiotadiversity
423 MMTs
A
B
C
4 MMTs
• MMT scalability
55 MMTs
Ex vivo testing in additional patient
populations
D
Human study insubjects colonized
with MDR organisms
E
FIGURE 6. BACTERIAL PATHOGENS DO NOT GROW ON KB109
FIGURE 8. REDUCTION IN CRE (a) AND VRE (b) WHEN INCUBATED WITH KB109 vs FOS
The y-axis shows reduction of a pathogen relative to control (no MMT)
Bacteroidetes
Verrucomicrobia
Proteobacteria(not Enterobacteriaceae)
VRE E. faecium
Enterobacteriales
Actinobacteria
Firmicutes
a) CRE E. coli
100%
80%
60%
40%
20%
0%100%
80%
60%
40%
20%
0%
b) VREE. faecium
% o
f Tot
al R
eIat
ive
Abun
danc
e M
ean
% o
f Tot
al R
eIat
ive
Abun
danc
e M
ean
ESLD HealthyPathogen spiked
Initi
al E
SLD
com
mun
ity
KB10
9
FOS
Wat
er (n
o gl
ycan
)
KB10
9
FOS
Wat
er (n
o gl
ycan
)
FIGURE 7a. COMPOSITION OF MICROBIOTA OF ICU PATIENTS IN COMPARISON TO HEALTHY SUBJECTS
Pathogen colonization
↑ Likelihood of infection with MDR organisms
Glycan-based MMTs selectively favor growth of commensals
↓ Likelihood of infection with MDR organisms
Pathogen
Commensal
Genus-level bacterial taxa
3 out of 13 did not contain any detectable commensal taxa (patients 1, 6, 12) IC
U P
atie
nts
Taxa present in healthy subjects
Microbiota of ICU patients are highly enriched in pathogens
Common pathogens
present in ICU patients
0% community
99% community10% community
0.3 4.60.7 830.1 1.4 0.3 6.10.1 01 3.5 00 06.2 0.1 1.1 10.1 14.82.6 8.8
5.8 6.90 7.530.7 0.8 0.3 6.90 00 2.1 0.70 0.44.7 0 16.6 0 70.5 9.2
0 3.43.6 0.522.1 0.4 2.6 5.30 00.1 10.8 3.70 1.512.8 0.3 18.8 0.8 2.90.4 10.1
0 0.31.2 8.322.1 6.8 14.8 2.80 00.2 3.6 00 17.28.3 0.1 6.3 0 0.60.6 6.8
0.3 6.51.4 027.7 0.7 0.1 7.70 00.2 12.1 3.80.1 014.5 0.1 9.5 1.6 3.70.8 9.2
0 4.59.2 5.318 1 1.3 2.70 00.1 11.4 3.30 012.1 0 16.2 3.7 0.80.4 10
0 30.8 0.320.8 0.3 0.2 2.80 00 10.3 2.40 019.9 0.1 1.5 6.1 1.22.2 28.1
0 1.44 0.423.5 1 3.8 2.50 00 11.2 0.30 022.7 0.1 5 0.7 6.11 16.2
0 8.524 1.88.9 7.9 3.7 1.20 00 15.9 00 012.8 0 8 2.2 0.20 4.9
0 0.72.1 025.9 4.3 7 40 00.4 10.9 12.70 015.8 0.5 7.5 0 30.1 5.2
Healthy.1Healthy.2Healthy.3Healthy.4Healthy.5Healthy.6Healthy.7Healthy.8Healthy.9
Healthy.100 00 90.3 0 0.5 0 0.1 8.1 0 0 0 00 0 0 0.20.6
34 00 13.6 31.9 0 0 0 5.9 0 0 0 03.3 0 0 011.2
15 1.60 4.3 0 10 6.5 12.6 1.9 8.5 0.6 5.3 1.70 0 0 0.132
26.4 0.21.4 22.6 19.3 6 1 0.3 5.1 0 0 0.1 03.5 0 0 014.2
18.3 0.91.2 42.7 0.5 0 0 0 4.7 0 0 0 017 0 1.3 013.4
0 036.9 52.4 0 0 0 0 0.6 0 0 0 00 0 10.2 00
65.6 0.70 3.5 0 1.4 2.2 0.9 0.3 0 0 2.6 2.71.2 0 4.4 014.6
51 00.8 2.3 2.3 6.4 6.3 0.1 1.2 2.1 0 5.6 0.70 0 0 021.1
0.1 00.4 18.4 26.5 0 0 0 0 0 0 51.9 00 0 0 02.6
40.3 90 10.6 0 0 12.5 0 0.1 0 8.2 11.8 2.10 0 0 2.82.5
31.6 033.7 12.7 0 0 0 0 0 0 0 10.1 00 11.1 0 00.6
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0 20 40 60Ranking of glycans in terms of reduction of CRE E.coli or CRE K.pneumoniae
Rank
ing o
f glyc
ans i
n te
rms o
f red
uctio
n of
VRE
E.faecium
Ranking of glycans in terms of reduction of CRE E. coli or CRE K. pneumoniae
Rank
ing
of g
lyca
ns in
term
s of
redu
ctio
n of
VRE
E. f
aeci
um
• Single-strain assays of 16 CRE strains of K. pneumoniae, E. coli or E. cloacae (Figure 6a), 18 strains of VRE E. faecium (Figure 6b), 3 strains of C. difficile (including Nap1 strains) (Figure 6c), as well as 6 Candida strains (Figure 6d), were performed in order to screen for the absence of the ability of lead glycans to support growth
• KB109 was identified as the superior lead compound, as it did support no or minimal growth compared to no carbon-added controls for all strains tested
• In addition, KB109 did not support the growth of 3 Candidastrains. Representative results are shown in Figure 6d
C.difficile Nap 1 strain
0.00.20.40.60.81.01.2
Hour
OD
600
100 20 30 40
C. difficile Nap1(c)
Time (minutes)
CRE K. pneumoniae CRE E. coliCRE E. cloacae VRE E. faecium
Minimal growth media Rich growth media
(b)(a)
FIGURE 6d. FUNGAL PATHOGENS DO NOT GROW ON KB109
Water (no glycan)1.0
0.5
0 20 40 60
OD6
00
Glucose
0 20 40 60
KB109
0 20 40 60Hours
C. albicansC. glabrataC. tropicalis
KB109
Water (negative control – no added carbon)
Glucose (positive control)
ID Week. October 2-6 2019. Washington, DC
Y-axis shows reduction of all pathogens relative to control (no added MMT) in each patient’s microbiota (x-axis)