Bull Vet Inst Pulawy 52, 223-226, 2008
ANTIBIOTIC SUSCEPTIBILITY OF LACTOCOCCUS GARVIEAE
IN RAINBOW TROUT (ONCORHYNCUS MYKISS) FARMS
KURSAT KAV AND OSMAN ERGANIS
Department of Microbiology, Faculty of Veterinary Medicine,
Selcuk University, 42075 Konya, Turkey
[email protected]
Received for publication February 21, 2008
Abstract
The aim of the study was to describe bilateral
exophthalmos ‘‘pop eye syndrome’’ in rainbow trout
(Onchorynchus mykiss) fisheries in the Konya region and to
determine effective antibiotic treatments. Between June 2002
and August 2004, 180 ill fish were obtained from 6 rainbow
trout fisheries where the disease had been observed.
Lactococcus garvieae strains were isolated from fish tissues
during different periods. All the isolates were found
susceptible to penicillin-G, ampicillin, amoxycillin,
ampicillin+sulbactam,
amoxicillin/clavulanic
acid,
vancomycin, ciprofloxacin, marbofloxacin, chloramphenicol,
florfenicol, erythromycin, oxytetracycline, cefoperazone,
sulbactam/cefoperazone, and novobiocin. It seems that βlactam antibiotics are preferred in the treatment of L. garvieae
infection in the rainbow trout farms.
Key words: rainbow trout, Lactococcus
garvieae, antibiotic susceptibility.
Lactococcus garvieae is one of the most
important Gram-positive cocci pathogenic for fish. It is
responsible for a serious problem in cultured marine and
freshwater fish species, such as yellowtail (Seriola
quinqueradiata) in Japan and rainbow trout
(Onchorynchus mykiss) in Europe and Australia (2, 5, 6,
13). In Turkey, lactococcosis has appeared regularly in
rainbow trout farms since 2001 (5), becoming one of the
most important risk factors in the trout industry during
the summer months. Despite the increased clinical
significance of L. garvieae, studies on the
characterisation and epidemiological relationship of this
microorganism isolated from different species and/or
clinical samples are very limited (17).
Besides fish, L. garvieae has been isolated from
other homoeothermic and poikilothermic animals, such
as cows, buffalos, and freshwater shrimp (3, 15). In
addition, it has been also recovered from humans (7, 9)
and, therefore, the possibility of zoonosis should be
considered. Taking into account all of these facts, it is
clear that effective control measures to avoid the spread
of this important disease are required. Outbreaks of the
disease are usually associated with changes in
environmental conditions, such as stress, overcrowding,
sudden change of temperature, transfer of fish,
mishandling, poor water quality, and high water
temperature.
The aim of this study was to determine an
effective antibiotic treatment of lactococcosis.
Material and Methods
Sampling and microbiological examination.
A total of 180 fish were obtained from 6 rainbow trout
farms between June 2002 and August 2004. The samples
of the liver, kidneys, heart, and eyes were collected. The
samples were placed on a 5% sheep blood agar (Oxoid)
with 1% yeast extract agar (Merck) plates and then
incubated at 24°C and 37°C for 2-3 d under aerobic
conditions. Wet mounts of skin, fin, and gill smears
were also examined microscopically as well as
macroscopically for parasites.
Antibiotic susceptibility test. An antibiotic
susceptibility test was performed using the disk
diffusion method on Mueller-Hinton agar (Oxoid)
according to the National Committee of Clinical
Laboratory Standards (4, 12). A minimum of three
colonies from the Columbia blood agar medium,
incubated at 25°C for 24 h, were suspended in 2 ml of
sterile saline to a density approximately equal to the
McFarland Opacity Standard No. 0.5. A dry sterile
cotton wool swab was placed in the suspension and
excess liquid was expressed into the tube. The bacterial
suspension was inoculated onto Mueller-Hinton agar
with the swab in such a way that the whole surface of
the agar was covered.
Disks containing the following antibiotics were
used: penicillin G (10 U, Oxoid), ampicillin (10 µg,
Oxoid), amoxycillin (25 µg, Oxoid), ampicillin/
sulbactam (20 µg, Oxoid), amoxicillin/clavulanic acid
(30 µg, Oxoid), methicillin (5 µg, Oxoid), oxacillin
(5 µg, Oxoid), cloxacillin (25 µg, Oxoid), cefoperazone
(30 µg, Oxoid), sulbactam/cefoperazone (105 µg,
224
Oxoid), erythromycin (15 µg, Oxoid), neomycin (30 µg,
Bioanalyse), clindamycin (2 µg, BBL), lincomycin
(10 µg, Oxoid), gentamicin (10 µg, Oxoid), neomycin
(30 µg, Bioanalyse), enrofloxacin (5 µg, Oxoid),
ciprofloxacin (5 µg, Oxoid), danofloxacin (5 µg, Pfizer),
marbofloxacin (5 µg, Marbocyl®, Laboratoire
Vétoquinol), chloramphenicol (30 µg, Bioanalyse),
florfenicol (30 µg, Oxoid), oxytetracycline (30 µg,
Oxoid), bacitracin (10 U, Oxoid), novobiocin (30 µg,
Oxoid), spiramycin (100 µg, Oxoid), sulphamethoxazole/trimethoprim (25 µg, Oxoid), and vancomycin (5
µg, Oxoid). These disks were dispensed on the surface
of the medium and incubated aerobically at 25°C for 24
h. The media, in which a susceptibility to methicillin and
oxacillin was tested, were incubated at 25°C and 35°C,
and then evaluated at 24 h and 48 h, respectively.
The results were recorded as resistant or
susceptible by measurement of the inhibition of the zone
diameter according to the interpretive standards of
NCCLS (12, 14). The reference strains used for
antibiotic susceptibility assay were Enterococcus
seriolocida (junior synonym of L. garvieae) ATCC
49156, and L. garvieae NCDO 2155 (=ATCC 43921).
Results
In the macroscopic examinations, bilateral
exophthalmos, darkening of skin, congestion of the
intestine, liver, kidney, spleen, and brain, and
characteristic haemorrhagic enteritis were observed (Fig.
1). Gram-positive cocci were seen in Gram-stained
smears taken from the eyes, liver, and kidney. No
parasites in the skin, fin, gills, and internal organs were
found.
The growth of pure α-haemolytic grey colonies
was observed after culturing the material from the eyes,
brain, and internal organs. The bacteria were identified
as L. garvieae on the basis of colony morphology,
Gram-staining, and biochemical characteristics (15, 17).
All the isolates gave the expected 1100-bp PCR
amplification product, which is specific for this
microorganism (18), confirming the preliminary
biochemical identification.
Antibiotic susceptibilities of L. garvieae
isolates were given in Table 2. The isolates were found
to be susceptible to penicillin-G, ampicillin,
amoxycillin, amoxicillin/clavulanic acid, ampicillin
sulbactam,
cefoperazone,
sulbactam/cefoperazone,
vancomycin,
ciprofloxacin,
marbofloxacin,
chloramphenicol,
florfenicol,
erythromycin,
oxytetracycline, and novobiocin and to be resistant to
methicillin,
oxacillin,
cloxacillin,
spiramycin,
clindamycin, lincomycin, gentamicin, neomycin,
bacitracin and sulphamethoxazole/trimethoprim. On the
basis of the results of antibiotic susceptibility test,
erythromycin was chosen for the treatment of L.
garvieae infection.
Fig. 1. The appearance of the exophtalmus on the eyes (A) and haemorrhagic lesions on the liver (B) and eye (A), L.
garvieae colonies on the 5 % sheep blood agar (C) and microscopic appearance of L. garvieae (1) (D).
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Table 1
Antibiotic susceptibility of L. garvieae
Antibiotic disc
Penicillin-G
Ampicillin
Amoxycillin
Ampicillin/sulbactam
Amoxicillin/clavulanic acid
Methicillin
Oxacillin
Cloxacillin
Cefoperazone
Sulbactam/Cefoperazone
Erythromycin
Spiramycin
Clindamycin
Lincomycin
R1
S
S
S
S
S
R
R
R
S
S
S
S
S
S
R2
S
S
S
S
S
R
R
R
MS
S
S
R
R
R
ST1-30
S
S
S
S
S
R
R
R
S
S
S
R
R
R
Antibiotic disc
Gentamicin
Neomycin
Enrofloxacin
Ciprofloxacin
Danofloxacin
Marbofloxacin
Chloramphenicol
Florfenicol
Oxytetracycline
Bacitracin
Novobiocin
Sulphamethoxazole/Trimethoprim
Vancomycin
R1
R
R
S
R
MS
MS
S
S
S
R
S
S
S
R2
R
R
S
S
S
S
S
S
S
R
S
S
S
ST1-30
R
R
MS
S
MS
S
S
S
S
R
S
R
S
R1 - L. garvieae ATCC 49156, R2 - L. garvieae NCDO 2155, ST1-30 - L. garvieae isolates, S - susceptible, MS moderately susceptible, R - resistant.
Discussion
Lactococcosis is one of the infectious diseases
with significant economic and sanitary repercussions for
trout farms in Mediterranean countries during the
summer months. Bacteriologic and molecular studies
confirmed L. garvieae as the aetiological agent of
haemorrhagic septicaemia in farmed trout.
In this study, L. garvieae was found to be
susceptible to β-lactam antibiotics except for
penicillinase-resistant penicillin antibiotics. It is well
known that β-lactams have a high effect on
Streptococcus strains (16). Diler et al., (5) reported that
L. garvieae was resistant to penicilin-G and ceftriaxon
while it was sensitive to ampicillin. However, in our
study, L. garvieae was resistant to penicillinase-resistant
penicillins such as methicillin, oxacillin, and cloxacillin.
It is stated that penicillinase-resistant penicillins,
generally are less active in vitro than natural penicillins
against susceptible Gram-positive cocci, apart from
penicillinase producing Staphylococcus sp. (11).
L. garvieae was sensitive to erythromycin but it
was resistant to clindamycin, lincomycin, and
spiramycin in our study. It is reported that L. garvieae
resistance to clindamycin is accepted as an identification
marker (8). Similar results were reported by Diler et al.,
(5), while Alves d’Azevedo et al., (1) found that L.
garvieae was resistant to erythromycin. These
controversial results may be due to differences of L.
garvieae isolate and antibiotics usage in the area.
When the aminoglycoside antibiotic was
evaluated, no susceptibility was observed. In fact,
aminoglycoside antibiotics have no effect on
Streptococcus sp. (7). L. garvieae was susceptible to
ciprofloxacin and marbofloxacin, but it was moderately
susceptible to enrofloxacin and danofloxacin.
When the other antibiotics were evaluated, it
was determined that L. garvieae was susceptible to
amphenicoles, oxytetracycline, novobiocin, and
vancomycin.
Tetracycline,
vancomycin,
and
chloramphenicol susceptibilities were also reported (5,
8, 10).
As it was demonstrated, β-lactam antibiotics,
except for penicillinase-resistant penicillin, were the
most effective antibiotics in the inhibition of L. garvieae
growth. These groups of antibiotics may be preferred for
the treatment of lactococcosis in fish.
Acknowledgments:
This
work
was
supported by SUBAPK (No. 2002/003) Konya, Turkey.
This article is a part of doctoral thesis. We thank Dr
Jesus L. Romalde from the Departamento de
Microbiología y Parasitología, Facultad de Biología,
Universidad de Santiago, Spain, for providing L.
garvieae NCDO 2155, and Dr Deok-Chan Lee from the
Department of Aquatic Life Medicine, Pukyong
National University, Busan, Korea, for providing L.
garvieae ATCC 49156.
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