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In this study, we evaluated the susceptibility of C. pecorum in vitro to macrolides, tetracyclines, quinolones and plactam antibiotics in cell cultures. The antibiotics.
Microbiol. Immunol., 42( 1), 61-63, 1998

In Vitro Susceptibility of Chlamydia pecorum Macrolides, Tetracyclines, Quinolones and

to

β-Lactam Pudjiatmoko, Hideto Fukushi*, Yoshitsugu Ochiai, Tsuyoshi Yamaguchi, and Katsuya Hirai Department ofVeterinary Microbiology,Faculty ofAgriculture, GifuUniversity, Gifu,Gifu501-11,Japan Received August7, 1997.Accepted October 20,1997 Abstract:

The

romycin),

in

two

one ƒÀ-lactam

for

and

The

results

Key

words:

susceptibility

was

to

Chlamydia

and

1.0 ƒÊg/ml

pecorum,

pecorum

to

minocycline), The

were

0.063

ciprofloxacin. doxycycline

MIC,

Macrolide,

macrolides

0.004

are

MIC the

two

Quinolone,

to

for

for

most

effective

ithromycin pon

(Dynabott,

Japan)

quinolones,

Seika,

and

Japan); and

Japan);

Japan).

The

solubilized

for

macrolides,

tetracyclines,

and

antibiotics use

according

were

was

(Dai

Ichi

supplied to the

also

used

pecorum

strains

were

cell

centrifuged

then

aspirated

at

drugs

was

After

the

cultures

including

(MIC)

was

deter-

Japan

Society

were

done

in

a 24-well

with

g for

C.

tra-

cell-culture ml

incubation

of

fixed

10

of

wells

methanol

FITC-conjugated

the

as

the

(Denka lowest

in

medium were

dilution

cell at 37

C for

and

stained

for

72

hr,

inclu-

genus-spe-

Seiken,

the

of

monolayers.

cells

Japan).

antibiotic

inclusions

ml was

Drugs

anti-Chlamydia

antibody

per

culture ml.

Each of

organism

inoculum

of

per

inoculated

with

chlamydial

The

1 ml

of

HeLa-229

units

dilutions.

onto

defined

with

0.2

1 hr.

with

serial

overlaid

The

concentration cell

culture

at were

inhibited. MICs in

of

the

Table

1.

with

an

effective with

erythromycin

Abbreviation: 61

(16),

the

cycloheximide

was

cline,

*Address correspondence to Dr . Hideto Fukushi, Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, 1-1 Yanagido, Gifu, Gifu 501-11, Japan. E-mail: [email protected]

1969

tests

overlaid

monoclonal

most

in

of

at 900 •~

MIC

shown

and

A

isolate

inoculated

cific

The

(11),

(12),

(13).

104 inclusion-forming

two-fold

completely

1968

1954

1954

method

coverslips

was

and

with

which

powders

on

were

in an

Drug

1 ƒÊg of

tested

in in

concentration

standard

well

containing

Phar-

lamb

containing

and

lung brain

comparison.

(8).

Each

calf calf

L2/454/Bu, for

the

suspension

sions

from

from

monolayers

plate.

manufacturer's

tested

C. pecorum.

from

inhibitory

by

antibiotic, C.

isolate

Chemotherapy

instruction. Three

for

1,024 ƒÊg/ml.

against

isolate

strain,

mined

(Meiji as

an

Minimum

Japan);

ampicillin

0.008

to 0.5 ƒÊg/ml

than

isolate

an

chomatis

doxycycline

ofloxacin

and ƒÀ-lactam,

0.25 greater

an

Ov/IPA,

(Dainip-

(Lederle,

and

clarithromycin,

drugs

Bo/Shizuoka,

clar-

erythromycin

minocycline

ciprofloxacin

maceutical,

and

Japan)

Pharmaceutical,

(Pfizer,

included:

eryth-

ciprofloxacin)

Tetracycline

cultures. tested

for

was

and

and

erythromycin,

ampicillin

Bo/Maeda,

antibiotics

(ofloxacin

0.008 ƒÊg/ml

Chlamydiapecorum, a newly described chlamydial species, causes enteritis, pneumonia, encephalitis and conjunctivitis in cattle (2, 7, 10-12, 15), polyarthritis and intestinal infection in sheep (13, 15), polyarthritis, abortion and pneumoniain swine (15), and a severe reproductivetract disease in koala bears (3, 9). Data on the treatment of C. pecorum infection in animals are limited,and little is known about the susceptibilityof C. pecorum to antimicrobial agents. Some antibioticshave been used for treating chlamydiosis. Tetracyclines,macrolides,and lately,quinolones have been used for treating human chlamydial infections (5, 6). Tetracyclinesare the drug of choicefor treating avian chlamydiosis(18). In this study, we evaluated the susceptibilityof C. pecorum in vitro to macrolides, tetracyclines,quinolonesand plactam antibioticsin cell The

(clarithromycin

quinolones

to 0.125 ƒÊg/ml The

and

two

two

MICs

minocycline,

for

clarithromycin

Chlamydia

and

determined.

doxycycline

0.25

show

of

(doxycycline

(ampicillin)

to 0.031 ƒÊg/ml ofloxacin

vitro

tetracyclines

antibiotics

Clarithromycin MIC

of

antibiotics MICs

against

of with

MIC,

was

0.004

0.008 MIC

minimum

to

the

pecorum most

doxycycline

0.063

inhibitory

the and

0.031 ƒÊg/ml, of

are

effective

to 0.008 ƒÊg/ml;

were

an

C.

followed to

0.125 ƒÊg/ml,

concentration.

next

minocyby

62

PUDJIATMOKO

Table

1. Activity

and

of seven

ofloxacin

and

0.5 ƒÊg/ml

and

cillin,

at

tion.

The

MICs

The

than

drug

to be

pecorum.

has

tration,

most

been

used

for

psittacine

tested

by

LCR

or

vivo

In

and of

and

determination

crobial

activity

showed

no

against

activity.

conducted

to

advantages

had C.

in

(14). be

Therethe

drugs of

in

treatment

animals.

doxycycline,

antimi-

while

clinical

8)

ampicillin

studies

antibiotics

1) Fraschini, F., Scaglione, F., Pintucci, G., Maccarinelli, G., Dugnani, S., and Demartini, G. 1991. The diffusion of clarithromycin and roxithromycin into nasal mucosa, tonsil and lung in humans. J. Antimicrob. Chemother. 27 (Suppl. A): 61-65. 2) Fukushi, H., and Hirai, K. 1992. Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants. Int. J. Syst. Bacteriol. 42: 306-308. 3) Girjes, A.A., Hugall, A.F., Timms, P., and Lavin, M.F. 1988. Two distinct forms of Chlamydia psittaci associated with disease and infertility in Phascolarctos cinereus (koala). Infect. Immun. 56: 1897-1900. 4) Gylstorff, I. 1984. The treatment of chlamydiosis in psittacine birds. Isr. J. Vet. Med. 43: 11-19. 5) Hammerschlang, M.R., Hyman, C.L., and Roblin, P.M. 1992. In vitro activities of five quinolones against Chlamydia pneumoniae. Antimicrob. Agents Chemother. 36: 682683. 6) Hammerschlang, M.R., Qumei, K.K., and Roblin, P.M. 1992. In vitro activities of azithromycin, clarithromycin, 1ofloxacin, and other antibiotics against Chlamydia pneumoniae. Antimicrob. Agents Chemother. 36: 1573-1574. 7) Kawagami, Y., Omori, Y., Fukuhara, S., Tokuda,G., Ishii, S., and Matumoto, M. 1956. Studies on the disease of cattle caused by a psittacosis-lymphogranulomagroup virus (Miya-

minocy-

excellent

pecorum,

for

animals

evaluation

optimal

showed

which

chemotherapy

to

in

stain

in

Prospective

determine

shown

been

the

has of

should

of

infection

most

a variety

agent

for

clarithromycin,

next

in has

applicable

erythromycin

the

alveolar

Doxycycline

doxycycline

C. pecorum

conclusion,

cline

be

C. Clarpene-

and

was

C.

8).

intracellular

epithelium

immunoperoxidase

may

efficacy

regimens

and

to

against (6,

chlamydial

and

and

active

Doxycycline

clarithromycin

choice,

also

chlamydiosis

eradicating

similar

against

trachomatis

C. pecorum.

avian

(4).

in

is

clarithromycin

antibiotic

Doxycycline

against

birds

study,

tissue

treating

effective

pecorum

bronchial

for

ciprofloxacin,

ampicillin.

C.

is

17).

antibiotic

to

0.031 ƒÊg/ml

this

C.

References

forma-

comparable

and

effective

and

into (1,

effective

of

of

excellent

especially

macrophages

fore,

the

to

Ampi-

0.125 ƒÊg/ml

for

In

C. pneumoniae

ithromycin

were

species

0.25

inclusion

ofloxacin

Clarithromycin

psittaci,

be

for

species.

of

clarithromycin,

susceptibility

shown

inhibit

minocycline,

1,024 ƒÊg/ml

chlamydial

was

to

for

and

MICs

respectively.

trachomatis

0.25 ƒÊg/ml

greater

other

failed C.

four Chlamydia

with

1.0 ƒÊg/ml,

0.008 ƒÊg/ml

doxycycline

and

to

for

erythromycin,

against

ciprofloxacin

0.25

1,024 ƒÊg/ml,

C. pecorum: for

antibiotics

ET AL

should

offer

be

potential

animals.

9) We script.

thank This

Research Sports

Dr. study

No. and

C.-C.

Kuo

was

supported

10156763

Culture,

from

Japan.

for

the

critical by

reading

Grants-in-Aid

Ministry

of for

of Education,

the

manu-

Scientific Science,

10)

11)

gawanella). VII. Isolation of a virus belonging to this group from feces of cattle. Jpn. J. Exp. Med. 25: 51-63. Kumamoto, Y., Matsumoto, A., Nagayama, A., Soejima, R., Hirai, K., Hashizume, S., and Hagiwara, T. 1992. Method for in vitro determinationof chlamydial susceptibility (Minimum inhibitory concentration; MIC) to antimicrobial agents. Chemotherapy 40: 308-314. McColl, K.A., Martin, R.W., Gleeson, L.J., Handasyde, K.A., and Lee, A.K. 1984. Chlamydia infection and infertility in the female koala (Phascolarctos cinereus). Vet. Rec. 115: 655. McNutt, S.H., and Waller, E.F. 1940. Sporadic bovine encephalomyelitis (Buss disease). Cornell Vet. 30: 437448. Omori, T., Ishii, S., and Harada, K. 1954. Studies on the dis-

NOTES

ease of cattle caused by a psittacosis- lymphogranuloma group virus (Miyagawanella). I. Immunology and pathogenicity of Kyushu virus. Bull. Natl. Inst. Animal Health 27: 45-54. 12) Omori, T., Ishii, S., and Matumoto, M. 1954. Studies on the disease of cattle caused by a psittacosis-lymphogranuloma group virus (Miyagawanella). IV. Identification of a virus "Shizuoka strain" as a causative agent of bovine encephalomyelitis. Jpn. J. Exp. Med. 24: 257-273. 13) Page, L.A., and Cutlip, R.C. 1968. Chlamydial polyarthritis in Iowa lambs. Iowa Vet. 39: 10-11 and 14-18. 14) Patton, D.L., Sweeney, Y.T.C., Clark, A.M., and Stamm, W.E. 1996. Antibiotics/antiinflammatory treatment of acute chlamydial upper genetical tract infection in female macaques, p. 103. In Stary, A. (ed), Proceedings third meet-

63

ing of the European

society for Chlamydia research, Study

group for STD and dermatological microbiology of the Austrian Society for dermatology and venerology, Vienna, Austria. 15) Perez-Martinez, J.A., and Storz, J. 1985. Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmunofluorescence. Infect. Immun. 50: 905-910. 16) Schachter, J., and Meyer, K.F. 1969. Lymphogranuloma venerum. II. Characterization of some recently isolated strains. J. Bacteriol. 99: 636-638. 17) Schentag, J.J., and Ballow, C.H. 1991. Tissue-directed pharmacokinetics. Am. J. Med. 91 (Suppl. 3A): 5S-115. 18) Vanrompay, D., Ducatelle, R., and Haesebrouck, F. 1995. Chlamydia psittaci infection: a review with emphasis on avian chlamydiosis. Vet. Microbiol. 45: 93-119.