The Activity of Ceftiofur Sodium for Aeromonas spp. Isolated from Ornamental Fish Author(s): Beverly A. Dixon and Gerard S. Issvoran Source: Journal of Wildlife Diseases, 28(3):453-456. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/0090-3558-28.3.453 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-28.3.453

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Journal

The

Activity

Isolated

of Ceftiofur

from

Sodium

Ornamental

Our

ABSTRACT:

of

objective

ceftiofur

Department

was to determine

sodium was aeromonads the

42

were

determinations.

highly isolated

isolates

isolates

to ceftiofur

were

were

resistant

resistant

Key

ceftiofur

ornamental

sodium

the

pet

fish

hobbyist

emerged pathogen; and

tank.

Aeromonas

the most common it causes infections

opportunistic

of temperature

or poor monas

water spp.

quality. to

is an emerging industry;

monas

used

problem

in the

from

Singapore

were

cline, lesser

sulfa drugs, degree to (Dixon

resistance

et a!.,

also

bacteria isolated nad resistance

cm#{174}) and

has

been

thors Ganzhorn,

(Bullock,

1984;

of ceftiofur,

is

B-lactamase-resistant

Naxcel#{174} currently is marUpjohn Company (KalamaUSA) for treatment of bodiseases associated with and P. multocida also has excellent in Gram-negative bac-

as Escherichia coli, Salmonella and Actinobacillus pleuro(Yancey et a!., 1987).

and

imported

MATERIALS To determine 50

ornamental

fish.

tetracy-

and antibacterial

to a

a local

kidneys

or

streaked

of ceftiofur

fish

wholesaler

(Pan

California, fish included

any

sodium,

were

obtained

Ocean Aquarium, USA) over the course

representatives

Anabantidae, Cyprinidae.

evident

from

CallichthySamples

external

on Rimler-Shotts

lesions

medium

of

were

(Shotts

and

of Aeromonas spp., and incubated C. Oxidase positive isolates were identified to genus according to Fish Health Section Bluebook guidelines (Amos, 1985) using the following tests; motility, sensitivity to novobiocin (5 mcg), growth Rimler,

in

Aeromo(Terramy-

two antibiotics apfish in the United by numerous auMacMillan,

efficacy

the following families: idae, Cichlidae, and

Antimicrobial

fish.

AND METHODS

ornamental

Inc., Hayward, of 4 mo. The

imported to

the

freshwater

from

ormethoprim/sulfadimethoxine

(Romet-30#{174}), the only proved for use on food States, has been reported

efficacy

domestic

documented

from food to tetracycline

screening

for

the

antibiotics

fish

1990).

spectrum

of Aero-

resistant

erythromycin, quinolone

to begin

pounds

Our objective was to determine the in vitro activity of ceftiofur sodium (Naxcel#{174}) against Aeromonas spp. isolated from

ornamental isolates of Aero-

tropical

from

drugs

Resistance

example,

time

com

handling,

commonly for

spp.

following

change,

Hayward,

has

bacterial in wounds,

infections

stresses

broad

teria such typhimurium pneumoniae

in-

spp.

as

State University,

Pasteurella hemolytica infections. Ceftiofur vitro activity against

that bacterial infections are responfor heavy losses, from the farm level

to the

fish

71%

fish.

in

at this

icrobial

cephalospormn. keted by the zoo, Michigan, vine respiratory

Aero-

activity,

California

Aeromonas spp. Naxcel#{174}, the sodium salt

all

and

Sciences,

against a

however,

ampicillin,

It is well established dustry sible

Disease

the treatment of this Gram-negative pathogen (Dixon et a!., 1990). It may

antim

Ceftiofur

sodium; to

pp. 453-456 Association 1992

1992,

28(3),

spp.

be necessary

to tetracycline.

words:

monas,

for fish

effective in vitro against from ornamental fish. Of Aeromonas spp. tested, none

of

resistant

the

against

and A. sobria of domestic and imported antimicrobial drugs were against these aeromonads Disk Diffusion technique concentration

of Biological

Aeromonas isolated from a variety tropical fish. Twelve tested for effectiveness using the Kirby-Bauer and minimum inhib-

sodium

hydrophila

itory

for Aeromonas

Diseases, © Wildlife

Fish

Beverly A. Dixon and Gerard S. Issvoran, California 94542, USA

activity

of Wildlife

1973)

in salt-free of glucose spp.

1985;

1987;

Hastings and McKay, 1987; Taylor, 1987; Stamm, 1989). Many of the commonly used antimicrobial drugs may no longer prove efficacious

were

strip

(NFT)

view,

New

Once

were mel,

for

isolation at 30

nutrient gelatin, and fermentation on OF medium. Suspect Aeromonas inoculated

into

system York,

identified,

maintained Sacramento, grown for 24 453

primary

the

Nonfermenter

(Analytab USA) stock

for

species

cultures

Test

Products,

Plain-

identification. of

the

on tryptic soy agar slants California). Stock isolates hr at 30 C, matched to

isolates

(Rewere

a

0.5

454

JOURNAL

OF WILDLIFE

1. Activity spp. isolated ornamental

TABLE

rnonas imported

DISEASES,

VOL. 28, NO. 3, JULY

of Ceftiofur sodium for from freshwater domestic fish.

1992

Aeroand

Percent of Number resistant isolates

Antibiotic

of

resistant isolates (n 42)

Ampicillin

42

Tetracycline

30

71

11

26

sulfa

11

26

Erythromycin

8

19

8

19

Nalidixic

acid

Triple

Nitrofuradontoin

Romet-30#{174}

100

\

1

“

___!#{176}.!___

Aeromonas

1.

FIGURE

sobria

unexposed

24-hr

culture

grown

on

Mueller-Hinton

12

Note

uniform

rod

shaped

cells.

5

12

5

12

sodium samples

(ormetoprim-

sulfadimethoxine)

(

5

controls; agar

at 30 C.

Sulfamethoxazonetrimethoprim Oxolinic

acid

Neomycin

4

9.5

Trimethoprim

4

9.5

0

0

Ceftiofur

sodium

McFarland

Standard

and

streaked

onto

Mueller-

Hinton agar (Remel, Sacramento, California). In vitro antibiotic sensitivity to 12 antimicrobial drugs was determined using the Kirby-Bauer disk

diffusion

technique

(Bauer

et

al.,

sections were stained with uranyl acetate lead citrate, and examined using a Hitachi

1966).

Twelve

susceptibility disks were used. Erythromycin (0.015 mg), nalidixic acid (0.03 mg), neomycin (0.03 mg), and sulfamethoxazole (25 mcg)/trimethoprim (25 mcg) were obtained from General Diagnostics (Morris Plains, New Jersey, USA). Nitrofurantoin (0.3 mg), triple sulfa (0.3 mg), tetracycline (0.03 mg), trimethoprim (5 mcg), and ampicillin (10 mcg) were obtained from Difco Laboratories (Detroit, Michigan). Oxolinic acid (2 mcg) and ormetoprim (1.2 mcg)/sulfadimethoxine (23.8 mcg) (Romet-30#{174}) were sampled by Baltimore Biological Laboratories (Cockeysville, Maryland, USA). by

Ceftiofur the

gan).

at 30 measured. (MIC)

incubation

by the Veterinary University using

Sensititre

of

mcg)

was

supplied

(Kalamazoo,

zone sites were concentrations

System, fornia,

(30

Company

Following

inibition inhibitory

mined

sodium

Upjohn

Diagnostic California, susceptibility

Michi-

C for 24 hr, Minimum were deter-

Laboratory Davis,

Cali-

plates

cacodylate buffer for 10 mm each. The were stained for 30 mm with 2% uranyl acetate made up in 30% ethanol. Dehydration was completed with 50% ethanol for 10 mm, 70% ethanol for 10 mm, 95% ethanol twice for 10 mm each, and 100% ethanol three times for 10 mm each. The samples were embedded in Spurr’s resin (Ted Pella, Inc., Redding, California); sections were taken with a glass knife. The

(Ra-

transmission electron entific Instruments, nia).

Twenty-four of the 42 isolates were identified as A. sobria, 12 as A. hydrophila and six as Aeromonas spp. None isolates were resistant to ceftiofur as determined measurements.

to 35 mm. Minimum inhibitory concentrations ranged from 0.25 to 1.0 ig/ml, well within the range of susceptibility. However, all 42 isolates were resistant to ampicillin, tetracycline Control

twice

in

2%

also were 1).

cells appeared

bition of Aeromonas

rinsed

cells

and 30 (Table

1%

and

of the sodium

by zone of inhibition size Zone sizes ranged from 19

shaped

tetroxide,

microscope Mountain

RESULTS

diometer /Copenhagen Company, Westlake, Ohio, USA). We sampled bacteria from the zone of inhibition around the ceftiofur sodium disk. As a control, we sampled bacteria from the same plate in an area without antibiotic exposure. The samples were fixed in 2% glutaldehyde for 30 mm, rinsed twice in 0.05 M sodium cacodylate buffer for 10 mm each at pH 7.2, fixed for 60 mm in osmium

and

HS-8 (Hitachi SciView, Califor-

(Fig.

resistant

to

as uniform

rod-

1). In contrast,

aero-

monad cells exposed to ceftiofur sodium appeared elongate with several indentations along

the cell wall

(Fig. 2).

DISCUSSION

Ceftiofur

sodium

caused spp.

in vitro inhi-

isolated from

do-

SHORT

veterinary

N,4.4

COMMUNICATiONS

medicine,

to antibiotics domestic and industry.

bacterial

455

resistance

is a potential problem imported ornamental

Further

in vivo

in the fish

pet

study

to determine the potential for dium as an efficacious treatment monad infections of ornamental

is needed ceftiofur sofor aerofish.

ACKNOWLEDGMENTS

2. Aerornonas

2.

FIGURE

of ceftiofur Hinton

sodium;

sobria

24-hr

at 30 C. Note

agar

exposed

culture

to 30

grown

indentations

on

mcg

Mueller-

along

the

Funding for this study was Upjohn Company, Kalamazoo, The

authors

thank

for

providing

Steve

the

cell

K. H.

ASI0S,

and

fish. Naxfor ceftiofur sodium,

imported

cel#{174}, the trade

name

cey

et al., 1987).

Ceftiofur

sodium,

like

other cephalosporins, is bacteriocidal inhibiting cell wall synthesis. Aeromonad cells exposed to ceftiofur appeared gate with the presence of numerous dentations of the cell wall (Fig. 2) pared

with

In

1988,

Food and treatment

uniform

control

Naxcel#{174}

was

cells approved

Drug Administration of respiratory disease

(U.S. Food and Drug 1988). Its broad spectrum is attributed

(Fig. by

1). the

for the of cattle

to bacterial

1989). Aeromonas antimicrobial

been observed from including Chesapeake

of

pet

imported

fish

sistant

to

tested. Because tibiotics

natural Bay

(McNicol reported

isolates

12

isolated

Singapore antimicrobial

of the bacterial already

spp. rehave

environments, (Maryland)

spp. from

of

drugs

and

et al., 1980). Dixon that over half of 70

Aeromonas seven

Maryland,

observed

from were redrugs

resistance in

human

A.

W.,

Journal

J. H.

BROWN, in

to anand

disk

1984.

SHERRIS,

45:

Fish

Leaflet

American use

and

abuse

20: 34-43.

redmouth

disease

Wildlife

National

M.

AND

493-496.

Their

and

67,

Laboratory,

Be-

testing

Aquaculture

Enteric

U.S.

Society,

method.

Antibiotics:

World

C.

Fish

susceptibility

Pathology

1989.

salmonids. search

J. C.

single

aquaculture.

BULLOCK,

Fisheries

KIRBY,

Clinical

of

and

pathogens.

pp.

Antibiotic

by a standardized

Smith

for the detection fish

114

M.

USA.

Nancy

CITED

American

M.

by the

photomicrographs.

certain

1966.

TURCK.

Disease

elonincom-

B-lactamase (Jaglan et a!., 1989). Antibiotic resistant bacteria have been described from both food fish and shrimp

areas in Asia et a!., (1990)

Section,

by

Administration, activity partly

to its resistance

culture (Brown, sistant to several

Health BAUER,

and

Procedures of

thesda,

ornamental

previously was shown to be very effective in the control of Gram-positive and Gramnegative bacterial pathogens of veterinary importance both in vivo and in vitro (Yan-

electron

1985.

identification

mestic

Scott

LITERATURE

wall.

provided Michigan,

Fish

Health

Re-

Fish

Kearneysville,

of

Service West

Virginia,

2 pp.

B. A., J.

DIXON,

Antibiotic

YAMASHITA,

from

of

Aquatic

EVELYN.

Aeromonas

of

fish

tropical

Journal

F.

AND

resistance

imported Animal

1990.

spp.

from Health

isolated

Singapore. 2: 295-297.

J. 1987. Aeromonas salmonicida, apparent drug resistance to ormetoprim-potentiated sulfadimethoxine. Fish Health Section Newsletter 15: 4, American Fisheries Society, Bethesda,

GANZHORN,

Maryland,

pp.

T., Aerornonas culture

1-12.

A. MCKAY. salmonicida

HASTINGS,

1987.

AND

61:

to

Resistance

oxolinic

acid.

of Aqua-

165-171.

P. 5., M. F. KUBICEK, T. S. ARNOLD, B. L. Cox, R. H. ROBBINS, D. B. JOHNSON, AND T. J.

JAGLAN,

GILBERTSON.

Nature

of urinary

and

cattle.

istry

37:

nel

Journal

catfish

of

Metabolism of plasma metabolites Agricultural

Food

ceftiofur. in rats Chem-

1112-1118.

J.

MACMILLAN,

1989. and

1985. culture,

Amsterdam,

Infectious C.

Netherlands,

diseases.

S. Tucker pp.

(ed).

In ChanElsevier,

405-496.

L. A., K. M. S. AzIz, I. HUG, J. B. KAPAR, E. F. REMMERS, W. M. SPIRA, VOLL, AND R. R. COLwELL. 1980. Isolation of drug resistant Aeromonas hydrophila from aquatic environments. Antimicrobial Agents and Chemotherapy 17: 477-483. SHOTTS, E. B., AND R. RIMLER. 1973. Medium for MCNICOL,

H. A. M. J.

LOCKMAN,

JOURNAL

456

OF WILDLIFE

DISEASES,

VOL. 28, NO. 3, JULY

isolation of Aeromonas hydrophila. Applied Microbiology 26: 550-553. STAMM, J.M. 1989. In vitro resistance by fish pathogens to aquacultural antibacterials, including the qumnolones difloxacin (A-56619) and sarafloxacin (A-566120). Journal of Aquatic Animal Health the

1:

P.

1987.

Antibiotic

resistance

bacterial infections of catfish. Section Newsletter 15: 4, American ciety, Bethesda, Maryland, 12 pp.

30#{174}in

U.S.

AND

FOOD

imal

drugs,

sterile

R.

to

Romet-

Fish Health Fisheries So-

DRUG

ADMINISTRATION.

feeds,

and related

powder.

R. J.,

YANCEY,

JR.,

Federal

M. L.

1987.

Ceftiofur

American

sodium,

Evaluation Journal

1988.

products;

Registry

53:

B. J.

KINNEY,

J.C.

GOODENOUCH,

alosporin:

135-141.

TAYLOR,

1992

HAMEL,

AND

5369-5370.

C. W.

and in vivo

of Veterinary

for

publication

21 October

1991.

FORD.

cephin mice.

Research

1050-1053.

Received

K.

ROBERTS,

a broad-spectrum

in vitro

An-

ceftiofur

48: