Antimicrobial activity of fatty acids against Bacillus larvae, the causative agent of American foulbrood disease

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Antimicrobial activity of fatty acids against Bacillus

larvae, the causative agent of American foulbrood

disease

Mf Feldlaufer, Da Knox, Wr Lusby, H Shimanuki

To cite this version:

Original

article

Antimicrobial

_activity

of

_fatty

acids

against

Bacillus

_larvae,

the causative

_agent

of American foulbrood disease

MF

Feldlaufer

DA

Knox

2

WR

_Lusby

H

Shimanuki

2

1 _Insect

Neurobiology

and Hormone

_{Laboratory, Bldg}

467 BARC-East, Beltsville, MD; 2

See Research Laboratory, Bldg 476 BARC-East, Beltsville, MD _20705, USA

(Received

15 October 1992;

accepted

4 December 1992)

Summary —

Various saturated and unsaturated free

_fatty

acids were tested for their antibiotic

activ-ity against

Bacillus larvae, the causative _agent of American foulbrood disease of

_honey

bee larvae. Saturated

compounds containing >

14 carbons were inactive. Antimicrobial

_activity

could be restored

to the

longer

chain

length

compounds

by

the addition of one or more double bonds. The antibiotic

activity

of

fatty

acids may make them suitable for use in the

_prevention

and control of foulbrood dis-eases of

_honey

bees.

foulbrood / antimicrobial

_activity

/

fatty

acids / Bacillus larvae

INTRODUCTION

The

_report

_by

Shimanuki et al

(1992)

that

an ethanolic extract of chalkbrood

mum-mies inhibited the

_growth

of

Melissococ-cus

_pluton

and Bacillus

_larvae,

the

causa-tive

_agents

of

_European

and American

foulbrood diseases of

_honey

bee

larvae,

respectively,

led us to seek the

_identity

of

the active

_compound(s).

In the

_previous

paper

(Feldlaufer

et al,

1993)

we

_reported

the isolation of an active antimicrobial

agent

identified as _{linoleic acid from}

my-celia and _spores of the chalkbrood fun-gus,

Ascosphaera apis.

In an extension of

this

_study,

we have tested various

saturat-ed and unsaturatsaturat-ed free

_fatty

acids for

an-tibiotic

_{activity against}

B larvae in an effort to correlate antimicrobial

_activity

with

_fatty

acid structure.

MATERIALS AND METHODS

Fatty

acids

Fatty

acids were obtained from Nu-Chek

_Prep,

Inc

(Elysian,

MN,

USA).

Stock solutions

(10 mg/ml)

and dilutions

(1:10,

1:100, and

Bioassay

Twenty-five μl

of the test solutions were

_applied

to _{paper discs with} an electronic

pipet,

supply-ing

final amounts of 250, 25, 2.5 and 0.25 μg of

fatty

acid per disc. After

drying,

the discs were

placed

in the center of agar

plates containing

B larvae spores. Discs treated with benzene served as controls. Active

_compounds

exhibited measurable zones of inhibition after 3 d. Full de-tails of the

bioassay

and incubation conditions have been

previously

described

(Feldlaufer

et

al, 1993).

Nomenclature

Common names for

fatty

acids are used where available

(Weete,

1974,

1980).

Abbreviated

symbols

are also used; the number

preceding

the colon indicates the number of carbon atoms in the

_fatty

acid, while that

_following

the colon

represents the number of double bonds. For

ex-ample,

18:0 would refer to stearic acid, while 18:2 would refer to linoleic acid.

Configuration

of double bonds is cis, unless otherwise stated.

RESULTS

The antimicrobial

_activity

of various

satu-rated and unsatusatu-rated

_fatty

acids

_against

B larvae is shown in table I.

_Among

the saturated

_fatty

acids,

activity

increased with chain

_length

from 6 carbons to 12

car-bons,

and then decreased.

_Activity

was

greatest

with lauric acid

(12:0),

and any saturated

_fatty

acid > 14 carbons in

_length

was inactive

(15:0

through

18:0).

Activity

could be enhanced or restored

to the

_longer

chain

_lengths

_by

the addition of a double bond. For

_example,

while myr-istic acid

_(14:0)

was

_{only slightly}

active

(10 mm

inhibition

_zone),

_myristoleic

acid

(14:1)

exhibited antimicrobial

_activity

_equal

to that of lauric acid

(80 mm).

Additionally,

while

_palmitic

acid

_(16:0)

was

inactive,

the

corresponding

unsaturated

_fatty

_acid,

pal-mitoleic

_(16:1)

was

_quite

active

_{(72 mm).}

However,

once

_fatty

acid chain

_length

con-sisted of or exceeded 18

_carbons,

more

than one double bond was necessary to

in-hibit bacterial

_{growth. Polyunsaturated}

fat-ty acids,

consisting

of

3,

4 or 6 double

bonds,

also showed

_activity

even when the

chain

_length

consisted of 22

carbons,

though

this

_activity

was somewhat reduced

(see

table

_I).

_Among

the active unsaturat-ed

_fatty

acids,

the cis

_{configuration}

was al-ways more active than the trans

configura-tion.

Since unsaturated

_fatty

acids are

sus-ceptible

to oxidation and are therefore

con-siderably

less stable than their saturated

counterparts,

we tested

_only

several of the active

_fatty

acids for antimicrobial

_activity

at lower concentrations

_{(table II).}

While all of those tested inhibited B larvae at a dose of 25 μg per

disc,

only

lauric

(12:0),

tridec-anoic

_(13:0),

_palmitoleic

_(16:1)

and linoleic

(18:2)

acid exhibited

_activity

at _{2.5 μg per}

disc. No

_fatty

acid tested was active at the lowest dose

_{(0.25 μg).}

DISCUSSION

Our isolation of linoleic acid as an

antimi-crobial

_agent

from the

_{fungus Ascosphaera}

apis (Feldlaufer

et

_{al, 1993),}

led us to

ex-amine a

_variety

of saturated and unsaturat-ed

_fatty

acids for antibiotic

_activity.

Our

re-sults are in

_general

_agreement

with others that have

_{investigated fatty}

acids as

antimi-crobial

_{agents (see Kabara, 1978;}

Kabara et

al,

1972).

We found that both lauric and

tridecanoic acid were the most active

satu-rated

_fatty

acids,

while

_palmitoleic

and

linoleic acid were most active of the unsat-urates at the lower doses tested.

Introduc-tion of a double bond or

_multiple

double bonds is _necessary to maintain antibiotic

activity

once the chain

_length

of the

_fatty

the cis

_{configuration}

_bring

the

_carboxyl

end of the molecule closer to the

_methyl

end

_{by bending}

the

molecule,

so the

over-all

_length

of the molecule is

_probably

im-portant

in

_maintaining

antibiotic action. The demonstration that certain

saturat-ed and unsaturatsaturat-ed

_fatty

acids inhibit the bacterium

_responsible

for American

foul-brood disease of

_honey

bees should

stim-ulate research directed at a

_practical

appli-cation. As control

_{agents, fatty}

acids would be safe and

_{environmentally-sound.}

In

ad-dition,

their

_relatively

low

cost would make them

_prime

candidates for use _in

preven-tion programs.

ACKNOWLEDGMENT

We thank KR Wilzer Jr for his technical exper-tise.

Résumé — Activité antimicrobienne d’acides _gras contre Bacillus

larvae,

agent

de la

_loque

américaine. L’activité

antibiotique

de l’acide

_linoléique

contre la bactérie

_responsable

de la

_loque

américai-ne chez les abeilles nous a conduit à exa-miner l’activité antimicrobienne d’autres

acides _{gras libres saturés et} insaturés. Le test

_biologique

a _{consisté à exposer} des spores de Bacillus larvae à des

disques

de

papier

traités avec _{250 μg des}

composés

étudiés et à mesurer la

zone

d’inhibition de

croissance au bout de

_{3 j}

_{(tableau I).}

Il en

ressort que l’activité antimicrobienne des acides gras saturés

augmente

avec la

lon-gueur de la chaîne

_jusqu’à

12 carbones

(acide laurique).

Les acides gras saturés dont la chaîne contient

_plus

de 14

carbo-nes sont inactifs. L’activité

_peut

être

ac-crue, ou restaurée chez les

_{composés qui}

ont les

_{plus grandes longueurs}

de

chaîne,

en

_ajoutant

une ou

_plusieurs

doubles

liai-sons. Certains des

_composés

les

_plus

ac-tifs ont été testés à des doses inférieures

(tableau II).

Tous les acides gras testés ont été actifs à 25 μg par

disque,

tandis

que seuls les acides

laurique,

tridécanoï-que,

palmitoléique

et

linoléique

sont restés actifs à la dose de

2,5

μg par

disque.

La preuve que les acides gras saturés et insa-turés inhibent la croissance de la bactérie

de la

_loque

américaine devrait encourager les recherches en vue d’une

_application

pratique.

Les acides gras sont sans dan-ger,

respectueux

de l’environnement et bon marché.

loque

américaine / Bacillus larvae / acide _gras / antimicrobien

Zusammenfassung —

Die antimikrobiel-le Aktivität von Fettsäuren _gegen Bacil-lus

larvae,

dem

_Erreger

der

Amerikani-schen Faulbrut. Die antimikrobielle

Aktivi-tät der Linolsäure _gegen die für Faulbrut-krankheiten der

_Honigbiene

verantwortli-chen Bakterien führte uns zur

ungesättig-ter Fettsäuren hinsichtlich ihrer mikrobiel-len Aktivität. Es wurden Biotests

durchge-führt,

bei denen

_Sporen

von Bacillus larvae mit

_{Papierscheiben}

in

_Berührung

_gebracht

wurden,

die mit 250 μg der Prüfsubstanz

getränkt

waren; nach 3

_Tagen

wurde die

Hemmzone gemessen

(Tabelle I).

Dabei

ergab

sich,

daß die antimikrobielle Aktivität der

_gesättigten

Fettsäuren mit der

Ketten-länge

bis zu 12 Kohlenstoff-Atomen

(Lau-rinsäure)

zunimmt.

_Gesättigte

Fettsäuren

von einer

_Kettenlänge

von mehr als 14

C-Atomen waren inaktiv. Die Aktivität konnte durch

_Einfügung

einer oder mehrerer

Dop-pelbindungen

gesteigert,

bzw bei den län-geren Ketten

wiederhergestellt

werden.

Einige

der stärker aktiven

_Verbindungen

wurden einem Biotest in

_niedrigeren

Dosen unterworfen

(Tabelle II).

Alle

gete-steten Substanzen waren _{bei 25 μg pro}

Scheibe

aktiv,

während

Laurin-Iridekan-,

Palmitolein- und Linol-Säure schon bei

2,5

μg pro Scheibe aktiv waren. Der Nachweis der

_{Hemmwirkung gesättigter}

_und

unge-sättigter

Fettsäuren auf das Wachstum des Bakteriums der Amerikanischen Faulbrut sollte

_Forschungen

in

_Richtung

der

prakti-schen

_{Anwendung ermutigen.}

Fettsäuren

sind

_{sicher, umweltverträglich}

und sie sind

billig.

Bacillus larvae / Faulbrut /

Antimikro-bielle Aktivität / Fettsäuren

REFERENCES

Feldlaufer M,

Lusby

WR, Knox DA, Shimanuki H

(1993)

Isolation and identification of linoleic acid as an antimicrobial _agent from chalk-brood

fungus. Apidologie24,

89-94

Kabara JJ (1978)

Fatty

acids and derivatives as antimicrobial

agents -

a review. In: The

Phar-macological

Effect of

_Lipids

_{(Kabara JJ,}

_ed)

Am Oil Chem _Soc,

_Champaign,

IL

Kabara JJ, Swieczkowski DM,

Conley

AJ,

Tru-ant JP (1972)

Fatty

acids and derivatives as antimicrobial agents. Antimicrob

_Agents

Chemother 2, 23-28

Shimanuki H, Knox DA, Feldlaufer MF ₍₁₉₉₂₎

Honey

bee disease interactions: the

_impact

of chalkbrood on other bee brood diseases.

Am Bee J 132, 735-736

Weete JD ₍₁₉₇₄₎

_{Fungal Lipid Biochemistry}

-Distribution and Metabolism. Plenum Press,

New York