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					Bull Vet Inst Pulawy 48, 391-395, 2004

                                         LESZEK GUZ AND ALICJA KOZIŃSKA1

                Institute of Infectious and Invasive Diseases, Subdepartment of Fish Diseases and Biology,
                                      University of Agriculture, 20-033 Lublin, Poland
                              Department of Fish Diseases, National Research Veterinary Institute,
                                                   24-100 Puławy, Poland

                                             Received for publication April 22, 2004.

                          Abstract                                  chemotherapy still represents the method of choice for
                                                                    control of most bacterial infections in both human and
           Twenty one Aeromonas isolates pathogenic for carp        veterinary medicine (3, 26, 29, 30). Intensive fish
were tested for susceptibility to 22 antimicrobial agents. Of the   farming has resulted in growing problems of bacterial
all isolates examined, 100% were resistant to ampicillin and        diseases, which have lead to a widespread antibiotic use
penicillin, and sensitive to trimethoprim-sulphamides, oxolinic     for their treatment, and has been associated with
acid, flumequine, chloramphenicol, norfloxacin, linkomycin,         increased antibiotic resistance in aquatic bacteria (17,
pefloxacin. Most isolates were resistant to cephalothin (57%)
and erythromycin (52%). The minimal inhibitory
                                                                    19, 29, 35). The aim of this study was to determine the
concentrations (MICs) of seven antimicrobials agents                antimicrobial resistance rates among Aeromonas sp.
(chloramphenicol, enrofloxacin, flumequine, nalidixic acid,         pathogenic for carp.
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                           Material and Methods
Mueller-Hinton medium. The MICs of each antimicrobial for
each isolate examined, together with the minimum                             All Aeromonas strains were recovered from
concentrations of each antimicrobial required to inhibit 50%
(MIC50) and 90% (MIC90) of the isolates examined, were also
                                                                    MAI diseased carp cultured in Poland. The colonies of
determined. The more recently synthetized 4-quinolones              Aeromonas sp. strains were identified as Gram-negative,
showed very good activity against all isolates examined             oxidase positive, glucose fermenting and O/129 resistant
compared with lower activity of oxytetracycline. The                motile rods (12). Further identification of these bacteria
enrofloxacin was the most active (MIC90 = 0.25 mg L-1).             was performed by the API 20E assay (Bio Mérieux,
                                                                    France) and the isolates were classified           as A.
          Key words: carp, Aeromonas, resistance to                 hydrophila: J4N/95, 15s/94, 1N/95, 2s/94, 1s/95, F6/95,
antibiotics.                                                        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.
          Aeromonas sp. are commonly found in a wide                         All isolates were tested for the sensitivity to
range of aquatic environments including fish ponds and              antimicrobials by the disc diffusion method (5) using
it is the causative agent of motile aeromonad infection             antibiotic impregnated discs with the following
(MAI), which occur in a wide variety of freshwater fish             antibacterial concentrations: ampicillin (AM) 10 µg,
species (1, 2, 18, 19). The disease caused by A.                    tetracycline (TE) 30 UI, kanamycin (K) 30 UI,
hydrophila complex is the major disease problem for                 gentamicin (GM) 10 µg, chloramphenicol (C) 30 µg,
commercial carp farming. At present, the most widely                nalidixic acid (NA) 30 µg, tobramycin (NN) 10 µg,
used method of controlling MAI in cultivated fish is the            amikacin (AN) 30 µg, streptomycin (S) 10 UI, penicillin
use of antimicrobial drugs. Because there is no suitable            G (P) 10 UI, erythromycin (E) 15 UI, neomycin (N) 30
vaccine available to control such an economically                   UI, colistin (CL) 50 µg, trimethoprim-sulfamides (ST)
important disease, the use of the correct antimicrobial             1.25 µg + 23.75 µg, flumequine (AR) 30 µg, norfloxacin
therapy should be taken into consideration.                         (NR) 10 µg, lincomycin (L) 15 µg, pefloxacin (PF) 5 µg,
          Although there are certain alternatives to the            furazolidone (FM) 100 µg, oxolinic acid (OX) 2 µg,
use of antimicrobial agents, such as vaccination,                   cephalothin (CF) 30 µg, cefixime (CF) 5 µg,
immunostimulants       or    probiotics,    antimicrobial           oxytetracycline (O) 30 µg, enrofloxacin (EF) 5µg. Zones

of inhibition were read after incubation at 27°C for 24 h       was determined as the lowest concentration of the
and sensitivity was assessed.                                   antimicrobial agent able to inhibit bacterial growth.
         Following identification, minimum inhibitory
concentrations (MICs) of selected antimicrobial agents
were determined for all isolates, using an agar dilution                                 Results
method as described by Schmidt et al. (27). Mueller-
Hinton agar (Difco) was the basic medium. Double                         The resistance to antibiotics of the isolated
dilutions of antibacterial agent stock solutions were           Aeromonas strains are presented in Table 1. All the
incorporated into the agar plates, with final                   isolates were resistant to ampicillin and penicillin, and
concentrations ranging from 0.06 to 1024 mg L-1. The            sensitive to trimethoprim-sulphamides, oxolinic acid,
Aeromonas isolates were cultured overnight in tryptic           flumequine,         chloramphenicol,       norfloxacine,
soy broth (Sigma) at 28°C, and cultures were adjusted to        enrofloxacin, linkomycin, pefloxacin. The isolates
an optical density of a 0.5 McFarland standard, diluted         revealed variable results of tests with the use of
1:10 in PBS, and applied as 1 µl droplets to the plates.        remaining drugs and were resistant to oxytetracycline
Every test was run in duplicate on freshly prepared agar        (38%), cephalothin (57%), erythromycin (52%),
plates. The first and last agar plates did not contain any      tetracycline (38%), oxytetracycline (38%), kanamycin
antibacterial agents in order to detect possible                (29%) and colistin (24%).
contamination of the isolates or antibiotic carry-over.                  The MIC value for 90% isolates was the lowest
After 2 d of incubation at 20°C the MIC for each isolate        (0.25 mg L-1) for enrofloxacin and the highest (64 mg L-
                                                                 ) for oxytetracycline (Table 2).

                                                         Table 2
            In vitro activity, analysed by MIC, of 6 antimicrobial agents against 18 strains of A. hydrophila

                   Antimicrobial         Minimum inhibitory concentration (mg L-1)           %
                   agent                                                                susceptibility
                                         Test range          50%            90%

                   Chloramphenicol       >0.06-2.0           0.06           0.50             100
                   Enrofloxacin          >0.06-0.4           0.12           0.25             100
                   Flumequine           >0.06-16.0           0.25            2.0             100
                   Nalidixic acid       >0.06-256.0          0.06            0.5             95.2
                   Norfloxacin           >0.06-4.0           0.12            2.0             100
                   Oxolinic acid        >0.06-32.0           0.06            1.0             100
                   Oxytetracycline      >0.06-256.0           2.0           64.0             61.9

                      Discussion                                frequent occurrence of multiple antimicrobial resistance
                                                                and the presence of similar resistance patterns in some
         In 2000, the World Health Organization Report          A. hydrophila, A. veronii biovar sobria, and A. caviae
on Infectious Diseases declared that antibiotic resistance      strains isolated from fish. Most of the A. salmonicida
possess a severe threat to human health, and that the           strains isolated by Kirkan et al. (16) were resistant to
problem is growing and global (14, 36). Resistance to           penicillin, erythromycin, amoxycillin + clavulanic acid,
the antimicrobial agents used in aquaculture has                cefuroxime sodium, gentamicin, oxytetracycline and
increased in many countries in recent years (13, 15, 34).       sulphamethoxazole + trimethoprim. These results
MAI can still be controlled for the time being with             confirment the statement saying that the use of
correct use of the oxytetracycline, flumequine, furanes         antimicrobial agents increased the problem of the
and trimethoprim-sulfonamides. The apparent resistance          development of drug-resistant strains (11, 16). In the
of A. hydrophila to antibiotics may be a result of the          antibiotic era, an increase in the resistance of strains of
uncontrolled or subtherapeutic use of antimicrobials. In        Aeromonas sp. to commonly used antibacterial agents
order to control the diseases caused by viruses, extensive      has been observed (25, 35). Antibacterial agents are
use of antibacterials was considered necessary for              mainly administered as supplementary feed for the
control of bacterial complications.                             treatment of diseased fish. Initially sulfonamides were
         The use of antibiotics in aquaculture is the           used successfully as food additives, then the usefulness
important factor in amplifying the resistance in a given        of oxytetracycline and 4-quinolones was reported, and
reservoir. Multiple antibiotic resistance among                 antibiotics are still used extensively for the control of
Aeromonas sp. has been reported from many parts of the          Aeromonas infections (1, 31).
world (14, 17, 35, 19, 30). Radu et al. (23) showed the

                                                                                               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        CF      N CL        P OX       AR     NR      L      PF      EF      O

      J4N/95        HS      R     HS     HS      HS     HS     HS      HS     S      HS     S       R   R       HS      S   R       R   HS     HS     HS      HS     HS       S      S
      15s/94        S       R     S      S       S      S      HS      HS     S      HS     R       R   R       S       R   R       R   HS     HS     HS      HS     HS      HS      S
      1N/95         HS      R     HS     HS      HS     HS     HS      HS     S      HS     S       R   R       HS      S   S       R   HS     HS     HS      HS     HS       S     HS
      2s/94         HS      R     HS     HS      S      S      HS      HS     S      HS     S       R   R       HS      S   R       R   HS     HS     HS      HS     HS      HS      S
      1s/95         S       R     HS     HS      HS     HS     HS      HS     S      HS     S       R   R       HS      S   S       R   HS     HS     HS      HS     HS       S      S
      F6/95         S       R     R      R       HS     HS     HS      HS     S      HS     S       S   HS      HS      S   S       R   S      S      HS      HS     HS       S     R
      F9/95         HS      R     HS     HS      S      HS     HS      HS     S      HS     S       R   R       HS      S   S       R   HS     S      HS      HS     HS       S      S
      J4N           HS      R     R      R       S      HS     HS      HS     S      HS     S       R   HS      HS      S   R       R   HS     S      HS      S      HS      HS     R
      F11s/94       R       R     HS     HS      S      HS     R       HS     R      HS     S       S   R       R       S   R       R   S      S      HS      S      HS      HS     HS
      F14N/93       HS      R     HS     HS      HS     HS     HS      HS     HS     HS     HS      S   R       HS      S   S       R   HS     HS     HS      HS     HS       S     HS
      F15N/93       HS      R     R      R       S      HS     HS      HS     S      HS     S       S   HS      HS      S   S       R   HS     HS     HS      HS     HS      HS     R
      R8s/94        HS      R     R      R       S      HS     HS      HS     S      HS     S       S   HS      HS      S   S       R   HS     HS     HS      HS     HS      HS     R
      F10s/94       HS      R     HS     HS      S      HS     HS      HS     S      HS     S       S   HS      HS      S   S       R   HS     HS     HS      HS     HS       S      S
      F13J/92       HS      R     R      HS      S      HS     HS      HS     S      HS     S       R   HS      HS      S   S       R   HS     HS     HS      HS     HS       S     R
      1N            S       R     R      S       S      HS     HS      HS     S      HS     S       R   HS      HS      S   S       R   HS     HS     HS      HS     HS       S     R
      1s            S       R     S      R       HS     HS     HS      HS     S      HS     S       R   S       HS      S   S       R   HS     HS     HS      HS     HS      HS      S
      R6s/95        S       R     S      R       S      HS     HS      HS     S      HS     S       S   R       HS      S   S       R   HS     HS     HS      HS     HS       S      S
      15s           HS      R     S      S       S      HS     HS      HS     S      HS     HS      S   R       HS      S   S       R   HS     HS     HS      HS     HS      HS     R
      F8/95         S       R     HS     S       HS     HS     HS      HS     S      HS     HS      S   R       HS      S   S       R   HS     HS     HS      HS     HS       S      S
      R7s/95        S       R     R      S       HS     S      HS      HS     S      HS     S       R   S       HS      S   S       R   HS     HS     HS      HS     HS       S     R
      F12s/94       HS      R     HS     HS      S      HS     HS      HS     S      HS     S       S   R       HS      S   S       R   HS     HS     HS      HS     HS       S      S

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.


Quinolones are widely used in Europe, Japan and other         a substitute for efficient management or good husbandry
countries in Asia and Latin America. The first                practice. If possible, alternative methods of disease
generation of 4-quinolones include nalidixic acid,            control (vaccination) should be use to reduce
oxolinic acid, and flumequine. The second generation of       antimicrobial use. Moreover, since the A. hydrophila
4-quinolones/fluoroquinolones, notably enrofloxacin           complex and A. sobria complex are potential pathogens
and sarofloxacin are effective at inhibiting the              of fish and humans, characteristics of aeromonads have
Aeromonas sp., and offer promise for the future (2).          public health significance and should be assessed.
However, mutational resistance to this class of
antibacterial compounds can develop in A. salmonicida                 Acknowledgments: This study was granted
(4, 18, 22, 37). In our study all strains were inhibited by   by the Polish State Committee for Scientific Research
oxolinic acid (MIC90 1.0 mg L-1), flumequine (MIC90 2.0       (grant No. 5 P06K 013 19 and WCZ/BW-5).
mg L-1), enrofloxacine (MIC90 0.25 mg L-1), norfloxacin
(MIC90 2.0 mg L-1), and 95.2% of strains by nalidixic
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