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KB174 Reduces Relative Abundance of Multidrug Resistant (MDR) Enterobacteriaceae in Fecal Samples From Patients With Cirrhosis in an Ex Vivo AssayJasmohan S. Bajaj,1 Kelsey Miller,2 Jonathan Lawrence,2 Jie Tan,2 Norma A. Palma,2 Gabrielle LeBlanc,3 Tanya Yatsunenko31Virginia Commonwealth University School of Medicine and Hunter Holmes McGuire VA Medical Center, Richmond, Virginia;2Kaleido Biosciences, Inc, Lexington, Massachusetts; 3Former employees of Kaleido Biosciences, Inc
INTRODUCTION• Patients with liver disease are at risk for spontaneous bacterial peritonitis and other infections1
• Infection occurs in 25% to 35% of patients at admission or during hospitalization (4-5x higher than in the general population), often with multidrug-resistant bacteria1,2
• Bacterial infection increases the probability of death 4-fold in patients with decompensated cirrhosis, with sepsis the number one cause of death in hospitalized patients with cirrhosis3,4
• Many multidrug-resistant infections, including spontaneous bacterial peritonitis, originate as gut-colonizing pathogens, which translocate to the ascitic fluid or other body sites1
• Microbiome Metabolic Therapies (MMTs™) are designed to target the glycan utilization potential of commensal microbiota and not pathogens (e.g. carbapenem-resistant Enterobacteriaceae [CRE]) (Figure 1)5
OBJECTIVE
• KB174 is not directly used by CRE E. coli but causes an enrichment of Parabacteroides, which contributes to the reduction of the relative abundance of E. coli in an ex vivo assay with samples from healthy subjects and patients with cirrhosis
• KB174 led to significant decreases in the relative abundance of multidrug-resistantEnterobacteriaceae as compared with water
PRECISIONscientia provided editorial support
FIGURE 4. RELATIVE ABUNDANCE OF ENTEROBACTERIACEAE IN CIRRHOTIC
SAMPLES AFTER INCUBATION WITH KB174 OR LACTULOSE
RESULTS (CONT)
CONCLUSIONS
ACKNOWLEDGEMENTS
• To determine whether KB174 can reduce the relative abundance of CRE E coli in fecal samples from patients with liver disease in comparison to lactulose
REFERENCES1. Piano S, et al. Infections complicating cirrhosis. Liver Int. 2018;38(suppl 1):126-133.2. Jalan R, et al. Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference 2013. J Hepatol. 2014;60(6):1310-1324.3. Aravanti V, et al. Infections in patients with cirrhosis increase mortality four-fold and should be used in determining prognosis. Gastroenterology. 2010;139(4):
1246-1256.4. Schmidt ML, et al. Decreasing mortality among patients hospitalized with cirrhosis in the United States from 2002 through 2010. Gastroenterology. 2015;148(5):
967-977.e2.5. LeBlanc G, et al. 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. Poster presented at: IDWeek 2019; October 2-4, 2019; Washington, DC.
6. Data on file. Kaleido Biosciences Inc.7. Bajaj JS, et al. Microbiome Metabolic Therapies reduce microbiota-associated ammonia in ex vivo fecal samples from healthy subjects and patients with minimal
hepatic encephalopathy and demonstrate improved tolerability over lactulose in a clinical study. Accepted for presentation at: EASL 2020 The International Liver Congress; April 15-19, 2020; London, United Kingdom (postponed to August 2020); and Digestive Disease Week; May 2-5, 2020; Chicago, IL (cancelled). Poster 4178.
8. Bhandari BR, et al. A randomized, double-blind study to evaluate the safety and tolerability of KB174, a novel synthetic glycan, in patients with well-compensated cirrhosis. Accepted for presentation at: EASL 2020 The International Liver Congress; April 15-19, 2020; London, United Kingdom (postponed to August 2020); and Digestive Disease Week; May 2-5, 2020; Chicago, IL (cancelled). Poster 4204.
Ex vivo assay: fecal samples from 25 patients with cirrhosis (collected in Kaleido study S005) who were not on lactulose or rifaximin and from 19 healthy controls were processed into suspensions. CRE E. coli*was added to each of the fecal slurries. Samples were incubated anaerobically for 45 hours at 37°C in mega medium without the addition of TWEEN 80 (Sigma-Aldrich) or meat extract. KB174 or lactulose was added as a carbon source, and samples incubated with water in the absence of added carbon source served as a control. Genomic DNA was extracted from all samples in the study and used for shallow shotgun sequencing*ID: Antibiotic Resistance Isolate Bank 0001. Resistance mechanisms: aac(6')Ib-cr, aadA5, dfrA17, KPC-3, mph(A), OXA-1, sul1, tet(A). Source: Centers for Disease Control and Prevention Enterobacterales Carbapenem Breakpoint panel
RESULTS• KB174 does not support the growth of CRE E. coli above what is measured in the background media
with no added carbon source, suggesting E. coli cannot use this MMT • Despite significant differences in community composition between healthy and cirrhotic fecal
communities, incubation with KB174 led to significant decreases in the relative abundance of multidrug-resistant Enterobacteriaceae as compared with water (mean percent decrease, 43% [P<.001] and 50% [P<.001], respectively) (Figure 3)
FIGURE 3. RELATIVE ABUNDANCE OF E COLI IN HEALTHY OR CIRRHOTIC SAMPLES AFTER INCUBATION WITH KB174, LACTULOSE, OR WATER
KB174 Lactulose
Structure
Glycan type Oligosaccharide mixture Dimer
Molecular formula H-[C6H10O5]n-OHWhere n=9 on average C12H24O12
Sugar types Glucose, galactose Galactose, fructose
Bond types Multiple alpha and beta 1,1-; 1,2-; 1,3-; 1,4-; and 1,6- Only 1 beta-1,4-
Branching Yes No
Amount of carbohydrate-activated enzymes required for fermentation Large Small
TABLE 1. STRUCTURAL COMPLEXITY OF KB174 AS COMPARED WITH LACTULOSE
FIGURE 5. PERCENT CHANGE IN PARABACTEROIDES RELATIVE ABUNDANCE IN HEALTHY OR CIRRHOTIC SAMPLES AFTER
INCUBATION WITH KB174 OR LACTULOSE
• A greater reduction in relative abundance of Enterobacteriaceae was seen in 68% (17 of 25) of samples tested from patients with cirrhosis when incubated with KB174 compared with lactulose (Figure 4)
• Reduction in abundance of Enterobacteriaceae was compensated by significant increases of Parabacteroides in samples from both healthy controls and patients with cirrhosis for KB174, which shows KB174 and lactulose reduce pathogen levels through different commensal pathways (Figure 5)
Healthy Cirrhosis
KB174 Lactulose KB174 Lactulose
Mean reduction (vs water)
-43% -41% -50% -33%
P value (vs water) <.0001 <.0001 <.0001 <.0001
P value (vs compound) 0.601 0.005
a19 healthy microbiomes and 25 cirrhosis microbiomes were tested (1 sample from each patient) with CRE E coli spiked in at start of experiment. Average reduction is relative to water (with no added carbon source).
FIGURE 1. MMTS ARE DESIGNED TO TARGET THE GLYCAN UTILIZATION POTENTIAL OF COMMENSAL BACTERIA
EASL | The Digital International Liver Congress; August 27-29, 2020. Poster 4178.
Pathogens have smaller number andencode distinct repertoire of
glycosidases
Potential stereochemical 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’ssynthetic chemistry platform
• KB174, an MMT, has previously been shown to:• Reduce the relative abundance of CRE E coli ex vivo in fecal samples from healthy subjects6
• Reduce the amount of ammonia produced ex vivo in fecal samples from patients with hepatic encephalopathy7
• Be safe and generally well-tolerated in patients with well-compensated cirrhosis, and reduce the urinary excretion of a biomarker of ammonia produced by the gut microbiome8
• Lactulose, a nonabsorbed disaccharide available to the gut microbiota, is the current first-line treatment for hepatic encephalopathy
• Certain prebiotic fibers (e.g. fructooligosaccharides) promote pathogen growth in ex vivo culture (internal data) so evaluation of lactulose and comparison to KB174 is a relevant study
• We hypothesize that commensal bacteria will outcompete pathogens in the gut when fed an MMT, leading to a reduced risk of infection for the host. Moreover, we hypothesize that KB174 will show increased performance over lactulose in part due to the larger structural complexity of KB174 vs. lactulose (Table 1)
Number of Glycosidases0
Bacteroides ovatusBacteroides thetaiotaomicron
Bacteroides xylanisolvensBacteroides dorei
Bacteroides fragilisClostridium cellulovorans
Parabacteroides distasonisKlebsiella oxytoca
Enterobacter aerogenes
Klebsiella pneumoniaeBifidobacterium infantis
Enterococcus faecium
Akkermansia muciniphilaEnterobacter cloacae
Salmonella entericaCitrobacter freundii
Escherichia coliRuminococcus sp
Bifidobacterium breveClostridioides difficile
Bifidobacterium bifidumEnterococcus faecium
Citrobacter koseriEnterococcus faecalisRuminococcus obeum
Pseudomonas aeruginosaRuminococcus bromii
Staphylococcus aureusProteus mirabilis
PC2 (12% VariationExplained)
PC1
(42%
Var
iatio
n E
xpla
ined
)
CommensalsPathogens
100 200 300
METHODSSingle strain assay: to confirm that KB174 is not used by CRE E. coli,* the CRE strain was incubated anaerobically with KB174 in monoculture, and OD600 was measured over time to generate growth curves
RORORO
RORO
RORO
RO
RORO
RO RO
RORORO
RORO
RO
RO
RO
RO
RO
RO
OR
OR
OR
OR
OR
OR
OR
OROROR
OROROROR
+
or
;
;
;
;
;
;OR
O
OO
O O
O
O
O
O
O
OROR OR
RO
O
OHO
HOHO
HOHO
OH
OH
OHO
a-1,2; a-1,3; a-1,4; a-1,6b-1,2; b-1,3; b-1,4; b-1,6
HOHO
Glycosylacceptors
8 Possible head-tail dimers:
Glycosyldonor
HO OHHO
O
METHODS (CONT)
Each R group is as defined above
• A reduction in the abundance of pathogenic bacteria in the gut (including decolonization) has the potential to reduce the risk of infection in susceptible populations. This ex vivo data supports, in conjunction with ammonia reduction7, a multi-pronged mechanism of action to treat HE
• The totality of data on KB174 activity supports its inclusion in a clinical study in people with cirrhosis (NCT03855956)
SUMMARY
Compound
Rel
ativ
e Ab
unda
nce
0.4
0.6
0.2
Healthy Cirrhosis
KB174Lactulose KB174LactuloseWater Water
Escherichia
Bond distributions
CatalystMono-/Oligo-/Poly-/
saccharide
25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 110
5
0
-5
-10
-15
-20
Patient
Cha
nge
in R
elat
ive
Abun
danc
e of
Ent
erob
acte
riace
ae, %
-25
-30
-35
-40
-45
-50
-55
-60
-65
-70
-80
-75 KB174Lactulose
-200
0
200
400
600
800
1000
1200
Healthy Cirrhosis
Perc
ent C
hang
ea
KB174
Lactulose