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USDA - ARS - National Center for Agricultural Utilization Research
Novel antimicrobial
compounds from
microbial sources
KM Bischoff, TD Leathers, NPJ Price,
CD Skory, S Liu, DM Donovan, JO Rich
National Center for Agricultural
Utilization Research
Flagship research lab of the USDA
270,000 sq. ft. (25,000 m2)
270 FTE research staff
100 Ph.D. scientists
7 research units
35 national priority projects
$32M budget
Internationally renowned
Bioenergy
Bio-oils
Crop Bioprotection
Functional Foods
Mycotoxin Prevention and
Applied Microbiology
Plant Polymer
Renewable Products
Technology
More than 100,000
strains of microbes
maintained at NCAUR
Largest of its kind
accessible to the public
Widely considered to be
the most useful in the
world
nrrl.ncaur.usda.gov
Microbial Culture Collection
• Revenue and
economic growth
• Broad spectrum of
new jobs
• Rural development
• Advanced
technologies and
manufacturing
• Reduced emissions
and Environmental
Sustainability
• Export potential of
technology and
products
• Positive societal
changes
• Investments and
new infrastructure
Biorefinery Concept
courtesy of Valerie Reed, USDA Office of Chief Scientist
Marketable Value-Added Bioproducts
Bioproducts oils carboxylic
acids
biopolymers proteins
Source plants, fungi fungi, bacteria fungi, bacteria yeast, bacteria,
phage
Uses antimicrobials,
biofuels,
flavorings and
consumer
products
commodity and
specialty
chemicals
materials, food
ingredients,
healthcare,
industrial and
consumer
products
antimicrobials,
food and feed
Alternatives to Antimicrobials
Concerns over the shortage of
antimicrobials
– commercially available or under
development
– efficacy against pathogens with
antibiotic-resistant genes
Restrictions on antibiotic uses in
production agriculture
Reservoirs of antibiotic resistance
genes
– antimicrobials with limited scope
– multiple products that work
synergistically
Seal et al (2013) Animal Health Res Rev doi:10.1017/S1466252313000030
Novel Antimicrobials
Antimicrobial Compound Source Target Specificity
Agricultural Production Problem
Liamocins Aureobasidiumpullulans
Streptococcus species
narrow Mastitis, septicemia, neonatal mortality
Laparaxin Lactobacillus paracasei
Gram positive bacteria
broad Food borne pathogens, drug resistant pathogens
Endolysins Various bacteriophage
Gram positive bacteria
narrow Antibiotic-contaminated animal feed
Unknown Bacillus sp. Lactobacillusspecies
narrow Infections of industrial fermentations
Renewable Product Technology, NCAUR, Peoria, IL
Bacterial contamination of fuel ethanol
production
Rich et al., Bioresour. Technol. (2015) 196:347-354
Corn
grinding slurry liquefaction
saccharification
heat
exchanger
combined
liquefaction
fermentation
distillation
yeast
propagation
backset
1 2
3
6 7
8
4
5
EtOH
DDGs
Survey of bacterial contaminants in
fuel ethanol plantsGenus % of totala
Wet mill Dry Grind #1 Dry-Grind #2
Bifidobacterium 0 – 20 1 – 2 0
Clostridium 0 – 9 0 0
Lactobacillus 44 – 60 37 – 39 69 – 87
Lactococcus 0 – 4 0 – 6 0
Leuconostoc 0 – 6 1 – 8 0 – 8
Pediococcus 0 – 6 19 – 24 0 – 4
Weisella 0 - 2 18 – 24 0 – 6
aValues represent a range over multiple samplings.
Skinner and Leathers (2004) J. Ind. Microbio. Biotechnol. 31:401-408.
LAB are not always friendlyBacterial contamination of fuel ethanol fermentations
Yeast fermentation is not a pure culture process
Survey contaminants
Develop new interventions
Reduce antibiotic use in ethanol industry.
Antibiotics used in the ethanol industry
Narendranath, (2003) In: The Alcohol Textbook, 4th Edition, pp 287-298.
Antibiotic Resistance
– Complicates management of contamination
– Creates a reservoir of resistance?
Potential for residues in co-products
Survey of Antibiotics in Distillers Grains
StudyPositive
samplesAntibiotics
Conc. range
(ppm)
FDA Survey1 4/46 (9%) VIR, ERY, PEN 0.16 – 0.58
Compart et al.2 20/159 (13%) VIR, ERY, PEN, TET 0.11 – 0.6
Bischoff et al.3 N/A VIR 0.69
VIR = Virginiamycin; ERY=Erythromycin; PEN=Penicillin; TET=Tetracycline
1 Luther, M. Report of FY 2010 Nationwide Survey of Distillers Products for Antibiotic Residues.
www.fda.gov/AnimalVeterinary/Products/AnimalFoodFeeds/Contaminants/ucm300126.htm.
2 Compart, D. M. P.; Carlson, A. M.; Crawford, G. I.; Fink, R. C.; Diez-Gonzalez, F.; DiCostanzo, A.;
Shurson, G. C. (2013) J. Animal Sci. 91:2395-2404.
3 Bischoff, K.M., Zhang, Y., Rich, J.O. (2016) World J Microbiol Biotechnol. 32:76.
Bacteriophage treatment of
contaminated fermentations
EtOH(g/L)
Glucose(g/L)
Lactic Acid(g/L)
Acetic acid(g/L)
Fermentation
control137 ± 2.4 0.44 ± 0.19 1.9 ± 0.05 0.85 ± 0.04
Contamination
control117 ± 1.6 28.7 ±5.7 5.3 ± 0.13 2.8 ± 0.07
Sau Φ treatment137 ± 1.0 0.47 ± 0.13 2.4 ± 0.01 0.76 ± 0.03
Inf Φ treatment 134 ± 0.9 0.42 ± 0.13 2.4 ± 0.07 0.84 ± 0.03
Sau + Inf
treatment136 ± 0.9 0.60 ± 0.09 2.4 ± 0.04 0.76 ± 0.03
Liu et al., Biotechnol Biofuels (2015) 8:132
Non-antibiotic interventions methodsPhage Endolysins
Explore bacteriophage genomes for
putative endolysins against lactic
acid bacteria.
Roach et al., Biotechnol. Biofuels 6:20 (2013).
See Donovan et al poster (p76)
Non-antibiotic interventions methodsPhage Endolysins
Khatibi et al., Biotechnol Biofuels 7:104 (2014)
Endolysin specificity
Activity
Strain LysA LysA2 LysgaY λSa2
Lactobacillus amylovorus B-4540 - ++ ++ ++
Lactobacillus brevis 0605-48 ++ ++ ++ ++
Lactobacillus delbrueckii B-763 - - + +
Lactobacillus fermentum 0605-B44 +++ + + +++
Staphylococcus warneri + - + +++
Staphylococcus xylocus ++ - ++ +++
Streptococcus agalactiae KU-MU-3B ++ - ++ +++
Streptococcus dysgalactiae ++ - ++ +++
Streptococcus pyogenes + - + +++
Streptococcus suis 531-668 +++ - +++ +++
Weisella viridescens B-1951 - - + +++
Roach et al., Biotechnol Biofuels (2013) 6:20
Biofilm Formation and Ethanol Inhibitionmajor contaminants
Rich et al., Bioresour. Technol. (2015) 196:347-354
Bacillus natural products
2° metabolites, including
lipopeptides, polypeptides,
enzymes, and non-peptide
products
Food preservation, animal
medicine, consumer products
Treatment for plant disease
(eg, fire blight)
Biofilms in ethanol production
Rich et al., Bioresour. Technol. (2011) 102:1124-30
Bacillus supernatant inhibits lactic acid bacteria
biofilm formationBacillusstrain
Media 1 Media 2
LAB X LAB Y LAB Z LAB X LAB Y LAB Z
1 0.64 0.87 0.65 1.09 0.95 0.04
2 0.84 0.66 0.70 0.54 0.50 -0.02
3 0.80 0.67 0.76 0.76 0.53 0.08
4 0.61 0.94 0.73 0.71 0.29 0.04
5 0.68 1.02 0.83 0.35 0.28 0.12
6 0.69 0.87 0.49 0.64 0.47 0.03
7 0.64 0.88 0.62 0.71 0.70 0.01
8 0.89 0.67 0.86 0.61 0.56 0.20
9 0.72 0.71 0.93 0.80 0.66 1.62
10 0.85 0.67 0.98 0.57 0.56 0.04
11 0.00 0.02 -0.01 2.20 1.63 1.88
12 0.10 0.12 0.34 0.64 0.64 0.09
13 0.10 0.17 0.47 0.55 0.48 0.38
14 0.84 0.83 0.71 0.73 0.60 0.31
15 0.71 0.86 0.76 0.59 0.68 0.22
16 0.85 0.74 0.86 0.39 0.46 0.17
17 1.11 0.70 0.45 0.55 0.40 0.22
18 0.96 0.97 0.86 0.49 0.54 0.41
19 1.17 0.69 0.36 0.63 0.28 0.13
20 0.77 0.94 0.79 0.74 0.46 0.04
21 0.61 0.93 0.85 0.56 0.38 0.21
22 1.16 0.71 0.88 0.09 0.12 0.09
23 0.80 0.74 0.86 0.47 0.41 0.07
24 1.43 0.84 0.54 0.25 0.19 0.06
25 1.46 0.73 0.73 0.13 0.13 0.16
26 0.78 0.81 0.95 0.16 0.20 0.40
27 1.32 0.71 0.84 0.25 0.18 0.19
Bacillusstrain
Media 1 Media 2
LAB X LAB Y LAB Z LAB X LAB Y LAB Z
28 1.09 0.83 0.76 0.31 0.12 0.09
29 0.62 1.06 0.79 0.92 0.58 0.07
30 0.79 0.88 1.01 0.67 0.53 2.08
31 0.66 0.90 0.61 0.30 0.28 0.06
32 1.10 0.66 0.50 0.47 0.12 0.07
33 0.84 1.26 0.56 0.50 0.25 0.15
34 0.65 0.88 0.69 0.78 0.59 0.21
35 0.63 0.91 0.64 1.07 0.89 0.04
36 0.73 0.78 0.98 0.96 0.80 0.31
37 0.74 0.95 1.12 0.81 0.61 0.17
38 0.65 0.93 0.69 0.14 0.13 0.12
39 0.65 0.82 0.67 0.33 0.32 0.06
40 1.08 0.66 0.41 0.21 0.08 0.06
41 0.66 0.89 0.64 0.36 0.35 0.21
42 0.55 0.50 0.60 0.91 0.71 0.05
43 0.73 0.66 0.35 1.19 0.97 0.62
44 1.28 0.58 0.34 1.21 0.70 0.46
45 0.90 0.64 0.40 0.92 0.27 0.09
46 1.17 0.77 0.71 0.56 0.54 0.66
47 0.23 0.10 0.46 0.34 0.23 0.04
48 0.58 0.54 0.44 0.08 0.39 0.13
49 0.91 0.75 0.87 0.63 0.56 0.31
50 0.76 0.70 0.93 0.31 0.28 0.13
51 0.76 0.90 0.74 0.73 0.85 0.33
52 0.66 0.92 0.73 0.38 0.31 0.04
53 0.66 0.94 0.66 0.50 0.42 0.05
54 0.78 0.93 1.04 0.38 0.36 0.23
Aureobasidium pullulans
Polymorphic fungus, considered to be a filamentous
ascomycete in class Dothideomycetes, subclass
Dothideomycetidae
– budding yeast cells or blastospores, swollen cells,
pseudohypha, hypha, and chlamydospores
Black yeasts, fungal melanin
Colonial characteristics
– Young: smooth, moist, yeast-like, creamy, pale pink or
yellow colonies
– Old: brown to black and velvety
Habitats
– Widespread and cosmopolitan
– decaying leaves, wood and aerial portion of plants
– bathroom wall, shower curtain, tile grout, and window sills
– soil and water
Heavy oils produced by
Aureobasidium pullulans
21/50 strains produced heavy oil.
Yields:
0.5 - 6.0 g oil / L culture media
0.01 – 0.12 g oil / g sucrose
Extracellular polyol lipids from
Aureobasidium was previously
reported by Kurosawa et al.,
(1994).
Surface active (biosurfactant).
Manitchotpisit et al., Biotechnol. Lett. 33:1151-1157 (2011)
Structural similarity to Exophilin A
Exophilin A reported by Doshida et al., (1996).
Product of Exophiala pisciphila (now Aureobasidium pullulans).
Antibacterial activity against Gram positives.
Possible source of dihydroxydecanoic acid
CH2OH
HHO
HHO
OHH
OHH
CH2O
O OH O
O OH O
O OH OH
CH2OH
HHO
HHO
OHH
OHH
CH2O
O OH O
O OH O
O OH O
O OH OH
Liamocins A1 and A2 Liamocins B1 and B2
(OAc)
(OAc)
Exophilin A
Structural similarity to Exophilin A
Exophilin A reported by Doshida et al., (1996).
Product of Exophiala pisciphila (now Aureobasidium pullulans).
Antibacterial activity against Gram positives.
Possible source of dihydroxydecanoic acid
Do liamocins inhibit bacterial contaminants of bioethanol fermentation?
CH2OH
HHO
HHO
OHH
OHH
CH2O
O OH O
O OH O
O OH OH
CH2OH
HHO
HHO
OHH
OHH
CH2O
O OH O
O OH O
O OH O
O OH OH
Liamocins A1 and A2 Liamocins B1 and B2
(OAc)
(OAc)
Exophilin A
Qualitative antibacterial assays
Lactobacillus fermentum
BR0315-1
Lactobacillus brevis
5-37
Lactobacillus plantarum
5-38
Agar diffusion assay. Bacteria (20 ml) at a density of 0.5 McFarland units were evenly spread on
agar media, and paper discs (6 mm diameter) were placed on the surface. Oil from A. pullulans
NRRL 50380 was dissolved to a concentration of 50 mg/ml in solvent (1:1 dimethylsulfoxide:2-
butanone), and 10 ml applied to each disc. Plates were incubated at 37°C overnight.
Qualitative antibacterial assays
Lactobacillus fermentum
BR0315-1
Lactobacillus brevis
5-37
Lactobacillus plantarum
5-38
Escherichia coli
ATCC 25922
Staphylococcus aureus
ATCC 29213
Psuedomonas aeruginosa
ATCC 27853
Enterococcus faecalis
ATCC 29212
Streptococcus agalactiae
KU-MU-3B
Agar diffusion assay. Bacteria (20 ml) at a density of 0.5 McFarland units were evenly spread on
agar media, and paper discs (6 mm diameter) were placed on the surface. Oil from A. pullulans
NRRL 50380 was dissolved to a concentration of 50 mg/ml in solvent (1:1 dimethylsulfoxide:2-
butanone), and 10 ml applied to each disc. Plates were incubated at 37°C overnight.
Qualitative antibacterial assays
S. uberis S. mitis NRRL B-14574
S. agalactiae NRRL B-1815
S. infantarius NRRL B-41208
S. salivarius NRRL B-3714
S. suis ATCC 43765
S. pneumoniae ATCC 55143
S. pyogenes ATCC 12344
S. mutans ATTC 25175
Broth dilution susceptibility
Strain Minimum Inhibitory Concentration
(mg liamocins / ml)
Streptococcus species
S. agalactiae KU-MU-3B 40
S. agalactiae NRRL B-1815 20
S. uberis 80
S. suis ATCC 43765 ≤ 10
S. pneumoniae ATCC 55143 ≤ 10
S. pyogenes ATCC 12344 16
S. mutans ATCC 25175 80
S. mitis NRRL B-14574 20
S. infantarius NRRL B-41208 80
S. salivarius NRRL B 3714 ≤ 10
MICs of oil from A. pullulans NRRL 50380 were determined by broth dilution method, with serial two-
fold dilutions of oil ranging from 1250 mg/ml to 10 mg/ml.
Carbon Source Minimum Inhibitory Concentration
(mg/ml)
Arabinose ≤ 20
Glucose 39
Sucrose 39
Xylose 39
Wheat straw 156
AHP-treated corn fiber 625
Oat spelt xylan 312
A. pullulans was grown in media containing the indicated carbon source, and oil extracted from the
culture was tested for antibacterial activity. MICs for Streptococcus agalactiae were determined by
broth dilution susceptibility testing.
Feedstocks for production of liamocins
Polyols determine the headgroupCarbon % Polyol Headgroups from Acid-Hydrolyzed Liamocins2
Source1 Gro Thr Ery Rib Ara Xyl Man Glc Gal
Sucrose - - - - - - 100 - -
Lactose - - - - - - 100 - -
D-fructose - - - - - - 100 - -
D-glucose - - - - - - 100 - -
D-mannose - - - - - - 100 - -
D-galactose - - - - - - 100 - -
D-arabinose - - - - - - 100 - -
L-arabinose - - - - - - 100 - -
D-xylose - - - - - - 100 - -___
D-mannitol - - - - - - 100 - -
D-glucitol - - - - - - 65 35 -
D-galactitol - - - - - - 73 8 19
D-arabitol - - - - 98 - 2 - -
L-arabitol - - - - 62 - 38 - -
D-xylitol - - - - 27 45 28 - -
D-ribitol - - - 18 63 - 19 - -
D/L-threitol - 78 - - - - 22 - -
erythritol - - 5 - - - 95 - -
D-glycerol 75 - - 8 - 17 - -
1Sole carbon source at 5% in PM medium (above the line the carbon sources are sugars, and below are polyols).2Analyzed by GC/MS. The dry weight yields of the total liamocins were 1.2 – 1.5 g/L.
L-threitol
MIC = 156 mg/ml
D-threitol
MIC = 156 mg/ml
Mannitol
MIC = 16 mg/ml
Solvent control
Mannitol
MIC = 16 mg/mlXylitol
MIC = 128 mg/ml
Arabitol
MIC = 64 mg/ml
Solvent control
Susceptibility of S. agalactiae to
structural analogs of liamocins
Biologically active components
MIC
(mg/ml)
>128
64
64
16
128
32
MALDI-MS Analysis of fractions
Oil from A.pullulans NRRL 50380 was fractionated on an RP18 HPLC column using a linear
gradient of 50 – 100 % acetonitrile in water. The collected fractions were assayed by
MALDI-MS, and tested for antibacterial activity by broth dilution.
Cell leakage assaysS. suis ATCC 43765
DNA concentration determined using Quant-iT PicoGreen dsDNA kit (Invitrogen).
0 10 20 39 78 156 312 No cells
1000-
500-
200-
Conclusions
Endolysins
Lactic acid bacteria reduce ethanol yields.
Antibiotics are effective, but drug resistance and regulatory constraints
may limit their use and effectiveness.
Antibiotic residues are found in the animal feed coproduct.
Biotechnology (phage endolysins) can supplement or replace
antibiotics for effective contamination control in fuel ethanol production.
Liamocins
Liamocins have antibacterial activity with specificity for Streptococcus.
Growth on polyols can alter the head group.
Mannitol liamocin B1 (the non-acetylated tetramer) appears to be the
most active type.
Liamocins treatment results in loss of membrane integrity.
Liamocins may be developed as a narrow spectrum antimicrobial agent
that targets streptococcal pathogens but avoids disruption of the
beneficial normal flora.
Acknowledgments Pen Manitchotpisit
Piyum Khatibi
Dwayne Roach
Lauren Saunders
Amber Anderson
Kristina Glenzinski
Trina Hartman
Eric Hoecker
Stephen Hughes
Melinda Nunnally
Ecolyse
– Liz Summer
– Mei Liu
– M.D. Mire-Criscione
NCERC
– John Caupert
– Yang Zhang
Funding from USDA NIFA AFRI 2010-65504-20377
NIFA AFRI 2010-65504-20420
ARS 3620-41000-164-00D
ARS 3620-41000-172-00D
ARS 3620-41000-173-00D
Nat’l Corn Growers
Association
BRDC
ADM
Cargill
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