Journal

A MEDIUM

FOR THE

EDWARDSIELLA Emmett

SELECTIVE

for

the

bacteria, Yersinia based enterococci.

allows ictaluri.

Key

Diseases, © Wildlife

26(2),

Disease

1990,

pp. 214-218

Association

1990

OF

ICTALURI

B. Shotts

and

W.

Douglas

Waltman,

Department of Medical Microbiology, College University of Georgia, Athens, Georgia 30602, 2 Present Address: Georgia Poultry Laboratory,

ABSTRACT:

ISOLATION

of Wildlife

111.2

of Veterinary Medicine, USA Oakwood, Georgia 30566,

USA

medium, called Edwardsiella ictaluri medium (ElM), has been formulated Edwardsiella ictaluri. The medium inhibits the growth of most gram-negative except Proteus sp., Serratia marcescens and some isolates of Aeromonas hydrophila and ruckeri. The bacteria that grow on the ElM are easily differentiated from E. ictaluri on colony morphology. The ElM inhibits gram-positive bacteria with the exception of The addition of fungizone to ElM suppressed the growth of most fungi. The ElM the evaluation of environmental reservoirs, levels of contamination and carrier states of E. A selective

isolation

of

words:

Edwardsiella

isolation,

experimental

ictaluri,

selective

medium,

INTRODUCTION

ictaluri

Edwardsiella of mortality nel catfish

by bacteria (Ictalurus

is the

major

in cultured punctatus).

studies have focused on characteristics and potential tors of E. ictaluri (Waltman

the

(Waltman positive evaluations collections chemical

cause chanRecent

biochemical virulence facet al., 1986b),

Media

g

in the catfish hindrance to

or other animals. these studies has

been the absence of a selective medium for isolating E. ictaluri. This organism is relatively slow growing, and may be overgrown by normal flora found in the same This

to formulate could be used

enteric

septicemia

of catfish,

study

a selective for studying

was

undertaken

medium which the presence of

E. ictaluri in the environment. The potential usefulness of this selective medium for the isolation of E. tarda also was evaluated. MATERIALS

and Shotts, 1986a, b). Other gramgram-negative bacteria for media were availbale from laboratory stock and were identified by standard bioprocedures (Krieg and Holt, 1984).

and

formulation

(Difco

Bacto-tryptone

Laboratories,

Detroit,

Michigan 48232, USA), 10 g yeast extract (Difco), 1.25 g phenylalanine (Fisher Scientific, Norcross, Georgia 30091, USA), 1.20 g ferric ammonium citrate (Fisher), 5 g sodium chloride (Fisher), 0.03 g brom thymol blue (Fisher), 17 g agar (Difco), and 990 ml distilled water. The components were dissolved, the pH was adjusted to 7.0 to 7.2, and the medium was autoclaved at 121 C for 15 mm. A 10 ml solution containing 3.5 g mannitol (Difco) and 10 mg colistin (Sigma Chemical Co., St. Louis, Missouri 63178, USA) was filter-sterilized and added to the medium prior to pouring the plates. Other additives, evaluated for their effectiveness, included bile salts (Difco) (0.1% and 1.0%), crystal violet (Fisher) (10 g/ml and 100 g/ml), bacitracin (Sigma) (10 IU/ml), and fungizone (Sigma) (0.5 tg/ml).

1986; Waltman and Shotts, 1986b). The environmental reservoir of E. ictaluri is unknown, as is the level of E. ictaluri in the environment and the presence and site

environments.

disease,

A medium (ElM) was formulated based on the biochemical and antimicrobial susceptibilities of E. ictaluri. The medium consisted of 10

the histopathobogy (Blazer et al., 1985) and the pathogenesis of the disease (Shotts et al., 1986), as well as possible vaccination and chemotherapy methods (Plumb et ab.,

of carriage The main

fish

study.

Other

media

used

included

MacConkey

(MAC) agar, trypticase soy agar (TSA), brain heart infusion (BHI) broth and thioglycollate (THIO) broth. These media were obtained commercially (Difco) and prepared as recommended by the manufacturer.

AND METHODS

Bacteria

Media

Over 100 isolates of E. ictaluri and E. tarda were collected and characterized biochemically (Waltman et al., 1986a, b) and antimicrobially

evaluations

The ElM was tested for ing E. ictaluri. Gram-poitive 214

its sensitivity in growand gram-negative

SHOTTS

bacteria

were

then

grown

inoculated

cubated 48

color

The ative

If bacteria

of the

colony

to

of

MAC

E. tarda

were

and

diluted

in

this in

solution triplicate.

the

bacteria numbers

each

plating

at

size,

saline

to

give

at

counted

30

C for

24

on each plate and were determined

of bacteria The

onto

each

medium.

differences

ElM.

in

After

bacteria

were

the

ratia

exception

determined

and

ElM

effectively

inhibited

gram-negative

ictaluri

the

Staphylococcus

tarda

be 10% medium and and

bile

sp.

(Table

percentage to cobistin (Waltman did not

bile salts (10 lU/mb)

Crystab effective

gram-positive salts

was

to be

1).

(10

All

(%)

with 0.1% bile salts

ElM

5

100

0

3

100

0

monocytogenes sp. sp.

Streptococcus Pseudomonas

sp. sp.

100

0

3

100

0

5

100

6

100

0 50

100

20

Edwardsiella

tarda

50

Edwardsiella

ictaluri

50

0

90

90

100

100

Proteus

(H2S+)

2

100

100

Proteus

(H2S-)

3

100

100

Serratia

marcescens

3

100

100

Yersinia

ruckeri

15

33

33

20

75

Aeromonas

hydrophila

Escherichia

coli

Enterobacter

sp.

Klebsiella sp. Citrobacter sp. Salmonella sp. Arizona sp. Shigella sp. Plesiomonas shigelloides Pseudomonas aeruginosa Pseudomonas putida Pseudomonas putrefaciens Acinetobacter sp. Alcaligenes sp. Bordetella bronchiseptica

of

1).

E.

ited

The bacj.g/ml

bacitracin inhibiting

However, effective

Ic-

No. strains tested

sp.

Micrococcus

Not

(0.1% and 1.0%) were tested as

violet and in selectively bacteria.

found

violet

and

applicable

since

0

NA’

3

0

NA

0

0

NA

6

0

NA

3

0

NA

0

NA

2

0

NA

0

0

NA

3

0

NA

0

NA

0

NA

0

NA

0

NA

0

NA

2

no growth

was

75

5

obtained

on

ElM

bile

salts

alone.

isolates,

growth

Shotts, 1986a). gram-positive

crystal

100 tg/ml) bacitracin

positive

Edwardsiella

iso-

of E. tarda isolates had been found to

and inhibit

Therefore,

additives. were not the

ruckeri

hydrophila the

bacteria

Ser-

grew on ElM, but only 90% of E. were able to grow. This was expected

since the susceptible

teria.

Yersinia

some

Aeromonas some Pseudomonas

some

lates,

gram

on

sp.

Listeria

on

sp.,

Bacillus

the for

incubation,

of Proteus

rnarcescens,

isolates,

species

Bacterial

Corynebacterium

RESULTS

With

selected

to grow

ElM

hr,

plates were observed for differences in colmorphology. The usefulness of the plating media for environmental sampling was determined by culturing samples of catfish pond water and mud and also intestinal contents of catfish. These samples were 10-fold serially diluted in sterile saline solution and plated onto MAC, TSA, and ElM. After incubation at 30 C for 24 hr, the of

(ElM).

ap-

sl of medium

the ony

numbers

taluri medium

of

100

species

total

bacteria

Survival

solution

incubation

plating

ability

negative

215

ICTALURI

shape

number of bacteria growing on these three media were analyzed using an analysis of variance. Significant differences in the stastistics were located using a Tukey’s test. The ability of the media to differentiate E. ictaluri and E. tarda from other bacteria was determined by mixing two or more bacterial and

The

1.

gram

determined relIsolates of E. ictaluri in BHI broth overnight

2,000 colonies/ml; added to each plating

medium.

TABLE

in-

OF EDWAROSIELLA

recorded.

to

were

mean

the

ISOLATiON

was

TSA.

After

and

were

determined

grew,

grown

sterile

200 was

proximately

broth

plates

was

was

ElM

and

and

The

growth

hours.

sensitivity

in BHI

ElM.

at 30 C and

24 and and

overnight

onto

AND WALTMAN-SELEC11VE

0.1% (Table

At

this all

concentration,

the

gram-poitive

for enterococci, Pseudomonas

exept many

having siella

a detrimental spp. isolates.

Since vironmental

ElM

might samples

bacteria and sp.

also isolates

effect

inhibtested

inhibited without

on

be important such as pond

Edwardfor enwater

and mud, we investigated the possibility of adding fungizone to ElM in order to inhibit the growth of fungi. The addition of 0.5 jLg/mb of fungizone to the medium did not

affect

isolates. ies with

the

growth

Furthermore, MAC and

of Edwardsiella

spp.

in comparison studTSA, it greatly inhibited

216

JOURNAL

2.

TABLE

OF WILDLIFE

Comparison

DISEASES,

of

the

VOL. 26, NO. 2, APRIL

sensitivity

of

Mac-

EdEd-

Conkey (MAC), Trypticase Soy Agar (TSA) and wardsiella ictaluri medium (ElM) for growing wardsiella ictaluri and E. tarda. Number

TABLE

teria Agar from

Total

of colonies’

Sample Pond

water

A

6

240.7

Pond

water

B

2 x 10

2 x 10

96.7

150.0

150.3

Pond

mud

7 x 108

3 x 10

219.3

204.0

187.3

Fish

intestine

A

8 x

10

884

191:3

230.7

171.3

Fish

intestine

B

6

10

3 3

915

30.7

38.3

32.7

:38.0

37.0

84-225

916

NDb

Edwardsiella

tarda

siella

spp.

28.0

43.0

40.3

entiated

23

17.3

18.0

20.0

75

94.3

112.7

115.3

87

123.7

105.0

118.3

morphology. lates overgrew

88

41.0

39.7

33.3

Mean

of triplicate

plates.

ly the growth water and

of fungi associated mud, allowing the

Edwardsiella

based taluri

with pond recovery of

spp.

ElM

of bacteria entiated

was

formulated

so that

species

growing on it could be differfrom Edwardsiella spp. isolates

on colony morphology. and E. tarda produced

mm green translucent Proteus sp. produced green colonies that rnarcescens produced

colonies. on ElM

Both 0.5

produced 2 to 5 opaque colonies; and 1 to 2 mm yellowish-

Enterococci produced

capable of tiny (approx.

mm) yellowish colonies. The relative ability of ElM growth assessed

of Edwardsiella relative to MAC

to support spp. isolates and TSA (Ta-

ble 2). There were no significant ences (at a 95% confidence level) growth

capabilities

of

Mixed isolates

cultures of Proteus

of Edward sp., A.

Y. ruckeri

were

onto

MAC,

differin the

ElM.

prepared. TSA,

E. icto 1.0

colonies after 48 hr. 2 to 3 mm brownishmight swarm. Serratia 2 to 3 mm reddish

colonies; A. hydrophila mm yellowish-green Y. ruckeri produced green growing

x

x

ElM

10

6

be

but

this