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

Peoria is home to some familiar names

Estimated metro population is 400,000

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

Endolysin treatment resolves

contamination…

…and improves EtOH production

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