Download Antibiotic Susceptibility of Aeromonas hydrophila and A. sobria

Bull Vet Inst Pulawy 48, 391-395, 2004
ANTIBIOTIC SUSCEPTIBILITY
OF AEROMONAS HYDROPHILA AND A. SOBRIA
ISOLATED FROM FARMED CARP (CYPRINUS CARPIO L.)
LESZEK GUZ AND ALICJA KOZIŃSKA1
Institute of Infectious and Invasive Diseases, Subdepartment of Fish Diseases and Biology,
University of Agriculture, 20-033 Lublin, Poland
1
Department of Fish Diseases, National Research Veterinary Institute,
24-100 Puławy, Poland
e-mail: [email protected]
Received for publication April 22, 2004.
Abstract
Twenty one Aeromonas isolates pathogenic for carp
were tested for susceptibility to 22 antimicrobial agents. Of the
all isolates examined, 100% were resistant to ampicillin and
penicillin, and sensitive to trimethoprim-sulphamides, oxolinic
acid, flumequine, chloramphenicol, norfloxacin, linkomycin,
pefloxacin. Most isolates were resistant to cephalothin (57%)
and erythromycin (52%). The minimal inhibitory
concentrations (MICs) of seven antimicrobials agents
(chloramphenicol, enrofloxacin, flumequine, nalidixic acid,
norfloxacin, oxolinic acid and oxytetracycline) were
determined for A. hydrophila (n=18) and A. sobria (n=3).
MICs were determined using an agar dilution technique in
Mueller-Hinton medium. The MICs of each antimicrobial for
each isolate examined, together with the minimum
concentrations of each antimicrobial required to inhibit 50%
(MIC50) and 90% (MIC90) of the isolates examined, were also
determined. The more recently synthetized 4-quinolones
showed very good activity against all isolates examined
compared with lower activity of oxytetracycline. The
enrofloxacin was the most active (MIC90 = 0.25 mg L-1).
Key words: carp, Aeromonas, resistance to
antibiotics.
Aeromonas sp. are commonly found in a wide
range of aquatic environments including fish ponds and
it is the causative agent of motile aeromonad infection
(MAI), which occur in a wide variety of freshwater fish
species (1, 2, 18, 19). The disease caused by A.
hydrophila complex is the major disease problem for
commercial carp farming. At present, the most widely
used method of controlling MAI in cultivated fish is the
use of antimicrobial drugs. Because there is no suitable
vaccine available to control such an economically
important disease, the use of the correct antimicrobial
therapy should be taken into consideration.
Although there are certain alternatives to the
use of antimicrobial agents, such as vaccination,
immunostimulants
or
probiotics,
antimicrobial
chemotherapy still represents the method of choice for
control of most bacterial infections in both human and
veterinary medicine (3, 26, 29, 30). Intensive fish
farming has resulted in growing problems of bacterial
diseases, which have lead to a widespread antibiotic use
for their treatment, and has been associated with
increased antibiotic resistance in aquatic bacteria (17,
19, 29, 35). The aim of this study was to determine the
antimicrobial resistance rates among Aeromonas sp.
pathogenic for carp.
Material and Methods
All Aeromonas strains were recovered from
MAI diseased carp cultured in Poland. The colonies of
Aeromonas sp. strains were identified as Gram-negative,
oxidase positive, glucose fermenting and O/129 resistant
motile rods (12). Further identification of these bacteria
was performed by the API 20E assay (Bio Mérieux,
France) and the isolates were classified
as A.
hydrophila: J4N/95, 15s/94, 1N/95, 2s/94, 1s/95, F6/95,
F9/95, J4N, F11s/94, F14N/93, F15N/93, F10s/94,
F13J/92, 1N, 1s, 15s, F8/95, F12s/94 and A. sobria:
R8s/94, R6s/95, R7s/94.
All isolates were tested for the sensitivity to
antimicrobials by the disc diffusion method (5) using
antibiotic impregnated discs with the following
antibacterial concentrations: ampicillin (AM) 10 µg,
tetracycline (TE) 30 UI, kanamycin (K) 30 UI,
gentamicin (GM) 10 µg, chloramphenicol (C) 30 µg,
nalidixic acid (NA) 30 µg, tobramycin (NN) 10 µg,
amikacin (AN) 30 µg, streptomycin (S) 10 UI, penicillin
G (P) 10 UI, erythromycin (E) 15 UI, neomycin (N) 30
UI, colistin (CL) 50 µg, trimethoprim-sulfamides (ST)
1.25 µg + 23.75 µg, flumequine (AR) 30 µg, norfloxacin
(NR) 10 µg, lincomycin (L) 15 µg, pefloxacin (PF) 5 µg,
furazolidone (FM) 100 µg, oxolinic acid (OX) 2 µg,
cephalothin (CF) 30 µg, cefixime (CF) 5 µg,
oxytetracycline (O) 30 µg, enrofloxacin (EF) 5µg. Zones
392
of inhibition were read after incubation at 27°C for 24 h
and sensitivity was assessed.
Following identification, minimum inhibitory
concentrations (MICs) of selected antimicrobial agents
were determined for all isolates, using an agar dilution
method as described by Schmidt et al. (27). MuellerHinton agar (Difco) was the basic medium. Double
dilutions of antibacterial agent stock solutions were
incorporated into the agar plates, with final
concentrations ranging from 0.06 to 1024 mg L-1. The
Aeromonas isolates were cultured overnight in tryptic
soy broth (Sigma) at 28°C, and cultures were adjusted to
an optical density of a 0.5 McFarland standard, diluted
1:10 in PBS, and applied as 1 µl droplets to the plates.
Every test was run in duplicate on freshly prepared agar
plates. The first and last agar plates did not contain any
antibacterial agents in order to detect possible
contamination of the isolates or antibiotic carry-over.
After 2 d of incubation at 20°C the MIC for each isolate
was determined as the lowest concentration of the
antimicrobial agent able to inhibit bacterial growth.
Results
The resistance to antibiotics of the isolated
Aeromonas strains are presented in Table 1. All the
isolates were resistant to ampicillin and penicillin, and
sensitive to trimethoprim-sulphamides, oxolinic acid,
flumequine,
chloramphenicol,
norfloxacine,
enrofloxacin, linkomycin, pefloxacin. The isolates
revealed variable results of tests with the use of
remaining drugs and were resistant to oxytetracycline
(38%), cephalothin (57%), erythromycin (52%),
tetracycline (38%), oxytetracycline (38%), kanamycin
(29%) and colistin (24%).
The MIC value for 90% isolates was the lowest
(0.25 mg L-1) for enrofloxacin and the highest (64 mg L1
) for oxytetracycline (Table 2).
Table 2
In vitro activity, analysed by MIC, of 6 antimicrobial agents against 18 strains of A. hydrophila
Antimicrobial
agent
Minimum inhibitory concentration (mg L-1)
Test range
50%
90%
Chloramphenicol
Enrofloxacin
Flumequine
Nalidixic acid
Norfloxacin
Oxolinic acid
Oxytetracycline
>0.06-2.0
>0.06-0.4
>0.06-16.0
>0.06-256.0
>0.06-4.0
>0.06-32.0
>0.06-256.0
0.06
0.12
0.25
0.06
0.12
0.06
2.0
0.50
0.25
2.0
0.5
2.0
1.0
64.0
Discussion
In 2000, the World Health Organization Report
on Infectious Diseases declared that antibiotic resistance
possess a severe threat to human health, and that the
problem is growing and global (14, 36). Resistance to
the antimicrobial agents used in aquaculture has
increased in many countries in recent years (13, 15, 34).
MAI can still be controlled for the time being with
correct use of the oxytetracycline, flumequine, furanes
and trimethoprim-sulfonamides. The apparent resistance
of A. hydrophila to antibiotics may be a result of the
uncontrolled or subtherapeutic use of antimicrobials. In
order to control the diseases caused by viruses, extensive
use of antibacterials was considered necessary for
control of bacterial complications.
The use of antibiotics in aquaculture is the
important factor in amplifying the resistance in a given
reservoir. Multiple antibiotic resistance among
Aeromonas sp. has been reported from many parts of the
world (14, 17, 35, 19, 30). Radu et al. (23) showed the
%
susceptibility
100
100
100
95.2
100
100
61.9
frequent occurrence of multiple antimicrobial resistance
and the presence of similar resistance patterns in some
A. hydrophila, A. veronii biovar sobria, and A. caviae
strains isolated from fish. Most of the A. salmonicida
strains isolated by Kirkan et al. (16) were resistant to
penicillin, erythromycin, amoxycillin + clavulanic acid,
cefuroxime sodium, gentamicin, oxytetracycline and
sulphamethoxazole + trimethoprim. These results
confirment the statement saying that the use of
antimicrobial agents increased the problem of the
development of drug-resistant strains (11, 16). In the
antibiotic era, an increase in the resistance of strains of
Aeromonas sp. to commonly used antibacterial agents
has been observed (25, 35). Antibacterial agents are
mainly administered as supplementary feed for the
treatment of diseased fish. Initially sulfonamides were
used successfully as food additives, then the usefulness
of oxytetracycline and 4-quinolones was reported, and
antibiotics are still used extensively for the control of
Aeromonas infections (1, 31).
393
Table 1
Sensitivity / resistance patterns to 24 antibacterial agents of high degree of virulence of Aeromonas strains isolated from carp suffering from MAI
Isolate
NA
AM
TE
K
GM
C
FM
TS
NN
AN
S
E CP
J4N/95
15s/94
1N/95
2s/94
1s/95
F6/95
F9/95
J4N
F11s/94
F14N/93
F15N/93
R8s/94
F10s/94
F13J/92
1N
1s
R6s/95
15s
F8/95
R7s/95
F12s/94
HS
S
HS
HS
S
S
HS
HS
R
HS
HS
HS
HS
HS
S
S
S
HS
S
S
HS
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
HS
S
HS
HS
HS
R
HS
R
HS
HS
R
R
HS
R
R
S
S
S
HS
R
HS
HS
S
HS
HS
HS
R
HS
R
HS
HS
R
R
HS
HS
S
R
R
S
S
S
HS
HS
S
HS
S
HS
HS
S
S
S
HS
S
S
S
S
S
HS
S
S
HS
HS
S
HS
S
HS
S
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
S
HS
HS
HS
HS
HS
HS
HS
HS
HS
R
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
S
S
S
S
S
S
S
S
R
HS
S
S
S
S
S
S
S
S
S
S
S
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
S
R
S
S
S
S
S
S
S
HS
S
S
S
S
S
S
S
HS
HS
S
S
R
R
R
R
R
S
R
R
S
S
S
S
S
R
R
R
S
S
S
R
S
R
R
R
R
R
HS
R
HS
R
R
HS
HS
HS
HS
HS
S
R
R
R
S
R
CF
N CL
HS
S
HS
HS
HS
HS
HS
HS
R
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
R
S
R
S
S
S
R
R
S
S
S
S
S
S
S
S
S
S
S
S
P OX
AR
NR
L
PF
EF
O
HS
HS
HS
HS
HS
S
HS
HS
S
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
S
S
S
S
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
S
S
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
HS
S
HS
S
HS
S
S
S
HS
HS
S
HS
HS
S
S
S
HS
S
HS
S
S
S
S
S
HS
S
S
R
S
R
HS
HS
R
R
S
R
R
S
S
R
S
R
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R - resistance; S - sensitivity; HS - high sensitivity; AM, ampicillin; TE, tetracycline; K, kanamycin; GM, gentamicin; C, chloramphenicol; FM, furanes; NA, nalidixic acid; NN, tobramycin; AN,
amikacin; S, streptomycin, P, penicilline G, E, erythromycin, CP, cephalothin; CF, cefixime; N, neomycin; CL, colistin; TS, trimethoprim-sulphamides; OX, oxolinic acid; AR, flumequine; NR,
norfloxacin; PF, pefloxacin; L, linkomycin; EF, enrofloxacin; O, oxytetracycline.
393
394
Quinolones are widely used in Europe, Japan and other
countries in Asia and Latin America. The first
generation of 4-quinolones include nalidixic acid,
oxolinic acid, and flumequine. The second generation of
4-quinolones/fluoroquinolones, notably enrofloxacin
and sarofloxacin are effective at inhibiting the
Aeromonas sp., and offer promise for the future (2).
However, mutational resistance to this class of
antibacterial compounds can develop in A. salmonicida
(4, 18, 22, 37). In our study all strains were inhibited by
oxolinic acid (MIC90 1.0 mg L-1), flumequine (MIC90 2.0
mg L-1), enrofloxacine (MIC90 0.25 mg L-1), norfloxacin
(MIC90 2.0 mg L-1), and 95.2% of strains by nalidixic
acid (MIC90 0.5 mg L-1).
Resistance to chloramphenicol is extremely rare
in Aeromonas sp. Michel et al. (18) study demonstrated
that chloramphenicol MICs in A. salmonicida strains
displayed a bimodal distribution and revealed the
existence of a large and well-delineated resistant
population. Distribution of MIC values of
chloramphenicol in A. salmonicida strains were 0.25-2
µg ml-1 and 16->256 µg ml-1, whereas in motile
aeromonads were from 0.06 µg ml-1 to >256 µg ml-1.
Chang and Bolton (7) found 8% of strains resistant to
chloramphenicol (MIC 10 mg L-1) and Goni-Urreza et
al. (9) - 2% of resistant strains with value of MIC >0.132 mg L-1. However, Montoya et al. (20) reported a
single strain resistant to chloramphenicol (MIC 128 mg
L-1). Our results confirm that Aeromonas sp. are
susceptible to chloramphenicol (MIC > 0.06-2 mg L-1,
MIC90 0.5 mg L-1). Chloramphenicol is hazardous to
humans, causing an idiosyncratic, aplastic anaemia, and
at present, it is highly illegal to use in food animals.
Tetracyclines are the most frequently used
antimicrobial agents in veterinary medicine in many
parts of the word. Oxytetracycline is frequently used
antimicrobial in freshwater fish farming. Tetracycline
resistance is most commonly mediated either by active
efflux of tetracycline from the cell or by ribosomal
protection from the action of tetracycline, and, in rare
cases, through direct inactivation of the antibiotic or by
mutations in the 16S rRNA that prevent biding
tetracycline to the ribosome (6, 8, 24, 28, 32, 33). As
regards oxytetracycline and tetracycline, the strains of
Aeromonas sp. tested have been divided into three
populations: highly susceptible (14.3% and 47.6%
strains, respectively), moderately susceptible (47.6%
and 19.1% strains) and resistant (38.1% and 33.3%
strains). The Aeromonas sp. strains exhibited MICs of
oxytetracycline >0.06-256 mg L-1 (MIC90 64 mg L-1).
Resistance to β-lactam antibiotics is due to the
production of multiple inducible, chromosomally
encoded β-lactames (9). Resistance to the thirdgeneration cephalosporins is known to be associated
with the derepression of the chromosomal enzymes (9,
10). In this study, all Aeromonas strains were resistant to
ampicillin and penicillin. Surprisingly, 42.8% of the
strains were susceptible to cephalothin.
Widespread antibiotic use has resulted in the
rapid spread of multidrug-resistant pathogens. It must be
remembered that widespread use of antimicrobials is not
a substitute for efficient management or good husbandry
practice. If possible, alternative methods of disease
control (vaccination) should be use to reduce
antimicrobial use. Moreover, since the A. hydrophila
complex and A. sobria complex are potential pathogens
of fish and humans, characteristics of aeromonads have
public health significance and should be assessed.
Acknowledgments: This study was granted
by the Polish State Committee for Scientific Research
(grant No. 5 P06K 013 19 and WCZ/BW-5).
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