魚 病 研 究 Gyobyo Kenkyu,27(3),131-142,1992.9
InVitro Activity of Quinolone Antibacterials against Selected Fish Pathogens
Roy Palmer, Kenji Kawai and Riichi Kusuda
Fish Disease Laboratory, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783, Japan
(Received January 7, 1992)
A comparison was made of the in vitro activities of five quinolone antibacterials (nalidixic acid, piromidic acid, oxolinic acid, flumiquine and miloxacin) currently used in aquaculture in Japan and five newer quinolones (ofloxacin, norfloxacin, enoxacin, ciprofloxacin and tosufloxacin), against selected fish bacterial pathogens. Of the earlier quinolones, flumiquine and oxolinic acid showed the highest bacteriostatic and bactericidal activities against Gram-negatives. The newer quinolones, however, showed considerably higher activities, with ciprofloxacin and tosufloxacin most active. The newer quinolones were also active against strains resistant to the earlier quinolones. Gram positive bacteria were resistant to all the earlier quinolones, but some strains were sensitive to the newer quinolones, particularly to ofloxacin, ciprofloxacin and tosufloxacin. The activities of the newer quinolones were found to be less affected by serum, pH or the addition of magnesium, than the earlier quinolones. The newer quinolones were considered to have potential for the treatment of Gram-negative, and possibly Gram-positive, bacterial fish pathogens.
Quinolone antibacterials are a large group of Mandell, 1988; Smith and Lewin, 1988). drugs, derived from 4-oxo-1,4-dihydro quinoline. These newer quinolones may have potential for An important property of the quinolones is that the treatment of fish bacterial diseases. Nakano development of plasmid mediated resistance is rare et al. (1989) found that the minimum inhibitory and is therefore non-transferable (Smith and Lewin, concentrations (MICs) of newer quinolones were 1988). Some of these drugs have been valuable in lower compared to earlier quinolones, against the treatment of Gram-negative bacterial diseases Listonella anguillarum (formerly Vibrio anguil of fish (Alderman, 1988). Resistance of bacteria larum), Pasteurella piscicida, Edwardsiella tarda, to these drugs has, however, become a problem in and Enterococcus seriorlicida (formerly Streptococ aquaculture, with cross resistance between these cus sp.). The newer quinolone, A-56620 (sara quinolones (Hastings and McKay, 1987 ; Nakano floxacin) has shown efficacy as a bath treatment et al., 1987, O'Grady et al., 1987; Kusuda et al., against experimental Aeromonas salmonicida infec 1988 ; Tsoumas et al., 1989). tion in chinook salmon Oncorhynchus tshawytscha In the 1980's a new group of quinolones (7-substituted(Markwardt and Klontz, 1989). Resistance de fluoroquinolones) was developed, which velopment to newer quinolones has been shown has improved activities against Gram-negatives to be lower than to earlier quinolones in some fish and Gram-positives, and has improved pharma pathogens (Nakano et al., 1989 ; Stamm, 1989 ; cokinetics in humans. The frequencies of resistance Tsoumas et al., 1989). development are lower for these drugs than for the In the present study, a comparison was made of earlier quinolones, and cross resistance between MICs and minimum bactericidal concentrations newer' and 'earlier' quinolones does not ' always (MBCs) of five earlier quinolones, licenced in occur (Crumplin and Odell, 1987 ; Norris and Japan for use in aquaculture*, and five newer * Japanese Fisheries Agency (1989): Use of drugs quinolones, against Gram-negative and Gram licenced for fish (8th ed.). Ministry of Agricul t positive fish pathogens. Rates of antibacterial ure, Forestry an Fisheries, Tokyo, 6 p. (in activity, and the effects of serum, pH and mag Japanese.) nesium on the activities against L. anguillarum, 132 R. Palmer, K. Kawai and R. Kusuda were also examined. all drugs were approximately 100 % active com pound, except ENX (92.2 %) and CPFX (85.8 %). Materials and Methods Initial solutions of 2 mg/ml were prepared in 0.1 M NaOH; subsequent dilutions were made in Antibacterial Agents distilled water or culture medium. The earlier quinolones tested were: nalidixic acid (NA), piromidic acid (PA), oxolinic acid (OA), Bacterial Strains miloxacin (MLX) and flumiquine (FLU). The Test strains were: 49 strains of Gram-negative newer quinolones tested were: ofloxacin (OFLX), bacteria (seven species: A. salmonicida, Aeromonas norfloxacin (NFLX), enoxacin (ENX), cipro hydrophila, Pseudomonas fluorescens, Psudomonas floxacin (CPFX) and tosufloxacin (TFLX, T-3262 putida, E. tarda, L. anguillarum and P. piscicida) base). Tests were based on active compound: and 20 strains of Gram-positive bacteria (three
Table 1. Bacterial strains used in this study
Activity of quinolone antibacterials 133
species : Streptococcus iniae, Staphylococcus epider over. Effects of drug carry over were determined
midis and E. seriolicida) isolated from fish disease by placing 5 ƒÊl volumes of drug dilutions onto an
occurrences in Japan (Table 1). Stocks were agar plate previously spread-inoculated with the
maintained as agar stabs at 15•Ž. After storage, test species (0.1 ml of a 105 CFU/ml broth), fol
strains were tested for viability and purity, and lowed by incubation for 48 h and examination for
were subcultured on agar at least twice before any growth inhibition (modified from Barry et al.,
using. 1987).
Culture Media and Conditions Effects of Serum
Mueller Hinton Agar (Eiken) and Sensitivity MICs and MBCs for OA, FLU, OFLX and
Test Broth (Eihen) were used, with an additional CPFX against L. anguillarum V-1037 were deter
1.5 % NaCl for culture of L. anguillarum and P. mined in broth, and in 50 % horse serum (heat piscicida. Cultures of Gram-positive species and deactivated) in broth. The media were adjusted E. tarda were incubated at 37•Ž, and other species to pH 7.0 with 1 M HCl. Other test conditions
at 25•Ž. Optical density versus plate count were as described above
curves were determined for each species, to
estimate cell concentration (colony forming units : Effects of pH and Magnesium
CFU/m/) in the MIC and MBC tests. MICs and MBCs for OA, FLU, OFLX and CPFX against L. anguillarum V-1037 were deter Agar Dilution MIC Assays mined in broth, with or without added magnesium Agar dilution MICs were determined according (2.49 g/l MgCl2, 26.2 mM Mg). The media were to the procedure of the Japanese Society of adjusted to pHs 6.0, 7.0 or 8.0, with 1 M NaOH Chemotherapy (1981). Agar plates containing or 1 M HC1. The adjusted media were determined serial dilutions of antibacterials were loop-inoculat to have buffeting capacities approximately equal ed with approximately 5 ƒÊl volumes of 18-20 h to 25 mm phoshate buffer. Other test conditions broth cultures, diluted to approximately 106 CFU/ were as described above. ml. After 20 h incubation, MICs were measured
as the lowest drug concentrations inhibiting growth Rate of Antibacterial Activity to less than six colonies (99.9 % inhibition). Determinations of antibacterial activity over a 24 h incubation period were made for OA, FLU, Broth Dilution MIC and MBC Assays OFLX and CPFX against L. anguillarum V-1037. Broth MIC and MBC determinations were con An 18 h broth culture was diluted to approximate ducted in microtiter wells (modified from Barry et ly 105 CFU/ml, and 4.5 ml aliquots were dispensed al., 1987). Doubling dilutions of the drugs in to culture tubes. Drug solutions in broth were broth were inoculated with 10 ƒÊl volumes of added (0.5 ml) to each tube, to give final concent
diluted broth cultures (18-20 h), to give appro rations of 0, 1/4, 1 and 4 times their respective ximately 105 CFU/ml final inocula. After 18 h broth MICs against V-1037. Samples (0.2 ml) incubation, MICs were determined as the lowest were taken from each tube, at 0, 1, 3, 6 and 24 h, drug concentrations at which no growth was for viable cell concentration (CFU/ml) determina visible. Cells were resuspended, and cultures at tions by serial dilution plate counts. Plates were drug concentrations of the MIC and above were incubated for 48 h before reading. Drug carry inoculated (5 ƒÊl) onto agar plates for MBC deter over in culture samples was not considered to be minations. Samples of cultures without drug significant, due to the subsequent high dilutions were included as controls. After incubation for in the viable counts.
48 h, MBCs were taken as the lowest drug con centrations at which less than two colonies grew Results from the sample inoculum (99.8 % kill). A small sub-culture inoculum size, deep agar plates (0.75 Agar Dilution MICs cm), five inocula per plate, and plate incubation for Agar dilution MICs for the 10 antibacterials 48 h were used to reduce any effects of drug carry against tested strains are shown in Fig. 1. MICs Fig. 1. Minimum inhibitory concentrations (MICs) of ten quinolones against fish pathogens determined by agar dilution assays. Axes of ordinate express number of strains. Former names of Listonella anguillarum and Enterococcus seriolicida are Vibrio anguillarum and Streptococcus sp., respectively. NA, nalidixic acid; PA, piromidic acid; OA, oxolinic acid; MLX, miloxacin; FLU, flumiquine; OFLX, ofloxacin; NFLX, norfloxacin; ENX, enoxacin; CPFX, ciprofloxacin; TFLX, tosufloxacin.
134 R. Palmer, K. Kawai and R. Kusuda Activity of quinolone antibacterials 135 Table 2. Bactericidal activity of ten quinolones against selected strains of fish pathogens 136 R. Palmer, K. Kawai and R. Kusuda
of •…0.39 ƒÊg/m/ were provisionally taken to in the respective plate MICs by one dilution, which
dicate full susceptibility, whereas, MICs of 0.78 was considered due to different culture conditions
1.56 ƒÊg/ml were taken as indicating intermediate - and inocula sizes. Strains were provisionally cate
susceptibility, and MICs of •†3.13 ƒÊg/ml were gorised as bactericidal (MBC•… 0.39 ƒÊg/ml, MBC/ taken to indicate resistance. MIC•…4), intermediate (MBC 0.78-1.56 ƒÊg/ml,
In the Gram-negative bacteria, all strains of A. MBC/MIC =8) and non-bactericidal (MBC•†3.13
hydrophila, E. tarda and L. anguillarum, plus the g/ml, MBC/MIC •† 16). ƒÊ majority of A. salmonicida and P. piscicida strains, For NA susceptible strains, the MBC to MIC
showed susceptibility or intermediate susceptibility ratios for all drugs were within one to four, with to NA. However, P. fluorescens, P. putida, one the exceptions of: ratios of eight for E. tarda with
strain of A. salmonicida (A-1083), and four strains FLU and P. piscicida (P-2833) with PA and OA, of P. piscicida (P-6000, P-6023, P-6024 and P-6038 and a ratio of 64 for A. hydrophila with NA. NA
) were resistant. was bactericidal against A. salmonicida (A-1006),
Among the NA sensitive bacteria, A. salmonicida and intermediate to E. tarda and L. anguillarum.
and A. hydrophila were the most susceptible PA was bactericidal against A. salmonicida (A-1006
species to all drugs. Newer quinolones were very ), A. hydrophila and intermediate against L. active, while PA was the least active against E. anguillarum. MLX was intermediately bacter tarda strains. L. anguillarum showed the least icidal against P. piscicida (P-2833) and bactericidal
differences between the earlier and newer quin against other species. Other drugs were fully
olones, with only a 2-4 •~ difference between OA bactericidal against all strains, with CPFX having
and the most active drug, CPFX. NA, PA and the highest activity or being equally high with
MLX were the least active drugs against P. TFLX. piscicida, with most MICs in the intermediate Ratios of MBCs to MICs of NA resistant strains range. were very variable, depending on strains and All NA resistant strains showed resistance to drugs. With A. salmonicida (A-1083), the ratios
PA and intermediate susceptibility or resistance were one to two for all drugs except for NA (eight to OA, FLU and MLX (except P. putida susceptible or higher). FLU and the newer quinolones show to OA). However, these strains were fully sus ed bactericidal activity, and OA was intermediate. ceptible to the newer quinolnes (except P. piscicida With P. piscicida (P-6000), NFLX and CPFX intermediate to ENX), although they showed showed bactericidal activity (MBC: MIC ratios
higher MICs than NA susceptible strains. The of two to four), TSFX was intermediate (MBC: newer quinolones were active against P. fluorescens, MIC ratio of eight), and other drugs were not
P. putida and NA resistant A. salmonicida and P. bactericidal (MBC: MIC ratios 16-•†64). The piscicida. earlier quinolones were not bactericidal against In the Gram-positive bacteria, all strains showed P. fluorescens, P. putida, S. iniae and S. epidermidis, resistance to NA, PA, OA, MLX, and FLU, ex whereas, the newer quinolones showed bactericidal
cept some strains of S. epidermidis which showed or intermediate bactericidal activity, except for intermediate susceptibility to FLU. Most strains ENX which was not bactericidal against S. iniae.
of S. iniae and E. seriolicida were susceptible to None of the drugs was bactericidal against E. TFLX, intermediate to OFLX and CPFX, and seriolicida.
resistant to NFLX and ENX. S. epidermidis In examination of drug carry over effects, 5 ƒÊl was the most susceptible of the 3 species to the volumes of drug dilutions were found to inhibit
newer quinolones, with all strains susceptible to growth on agar at concentrations of •†32 •~ TSFX, CPFX and OFLX, and most strains inter the MICs, depending on the drugs and species.
mediate to NFLX and ENX. Therefore, MBC: MIC ratios of •†32 may have been underestimated. MBC to MIC ratio
MBC to MIC ratio obtained from MBC and Effects of Serum
MIC values by broth dilution assay are shown in MICs and MBCs of OA, FLU, OFLX and Table 2. Some of the broth MICs differed from CPFX against V-1037 in the presence and absence Activity of quinolone antibacterials 137
Table 3. Effect of 50 % horse serum on the activity of four quinolone antibacterials against Listonella anguillarum* V-1037
Fig. 2. Effect of pH and magnesium on the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of four quinolones, against Listonella anguillarum (formerly, Vibrio anguillarum) V-1037 (broth dilution assays). A, oxolinc acid;
B, flumiquine; C, ofloxacin; D, ciprofloxacin. •œ--•œ MIC; •œ--•œ, MBC; •›--•›, MIC at added magnesium concentration of 26.2 mM ;•›--•› MBC at added magnesium concentra tion of 26.2 mM. Each point is the geometric mean of 3 determinations. of serum are shown in Table 3. The MICs and Effects of pH and Magnesium MBCs increased in the presence of serum by 4 •~ The MICs of OA and FLU against V-1037 for OA, approximately 6 •~ for FLU, and ap increased with a pH change from 6.0 to 8.0 by proximately 2 •~ for OFLX and CPFX. approximately 10 •~ and 16 •~ respectively. The 138 R. Palmer, K. Kawai and R. Kusuda
Fig. 3. Effect of four quinolones on the viability of Listonella anguillarum (formerly Vibrio an
guillarum) V-1037. Test drug concentrations were relative to previously determined minimum inhibitory concentrations (MICs, broth dilution assays) against V-1037. A,
oxolinic acid (MIC=0.025 ƒÊg/ml); B, flumiquine (MIC=0.05 ƒÊg/ml); C, ofloxacin
(MIC=0.05ƒÊg/ml); D, ciprofloxacin (MIC =0.012 ƒÊg/ml). •¥, no drug; •¤, drug con centration at 1/4 of the MIC; •œ, drug concentration at the MIC; •›, drug concentration at
4 times of the MIC.
MBCs of these drugs were affected similarly by 3. The bactericidal activity of OA and FLU pH. The MICs and MBCs of OFLX and CPFX were similar : at 1/4 •~ MIC there was some inhibi showed no major changes with pH (Fig. 2). tion of growth; at 1 •~ the MIC there was bac
The MICs of OA and FLU showed approxi tericidal activity but regrowth occurred by 24 h; mately 16 •~ and 12-26 •~ increases with added whereas, at 4 •~ MIC no regrowth occurred. magnesium, respectively. Addition of magnesium The activity of OFLX and CPFX were similar to gave increases of approximately 8 •~ and 8-10 •~ OA and FLU at 1/4 •~ their MICs, but at 1 •~ in OFLX and CPFX MICs, respectively. The their MICs no regrowth occurred within 24 h,
MBCs of each drug showed increases similar and at 4 •~ MICs, the viability fell below the to the MICs, except the MBC of OFLX which assay limit within 3 or 6 h. showed a relatively greater increase at lower pHs
(Fig. 2). Discussion
Rate of Antibacterial Activity Most of the Gram-negative strains were sus
Effects of quinolones on the viability of L. ceptible to all the quinolones tested, except P. anguillarum V-1037, with time, are shown in Fig. fluorescens, P. putida, one strain of A. salmonicida Activity of quinolone antibacterials 139
and four P. piscicida strains, which showed resis olicida, whereas NFLX and ENX activitives were tance to NA and some other earlier quinolones. intermediate or low. Some newer quinolones,
Pseudomonas spp. have innate low susceptibilities therefore, may be useful for treating Gram
to the early quinolones (Phillips et al., 1988), positive infections. These drugs may be par whereas resistance in A. salmonicida and P. ticularly useful where there is resistance to the piscicida strains would be acquired during drug more commonly used drugs such as the macrolides, use. Of the earlier quinolones, OA and FLU or in the case of mixed Gram-negative and Gram
were the most active drugs tested against the positive infections. Gram-negatives, showing good bacteriostatic and The newer quinolones, CPFX and OFLX, were bactericidal activity against all NA susceptible found to have more rapid and complete bactericidal strains, with low MBC: MIC ratios. NA and activity than OA and FLU, against L. anguillarum PA were the least active drugs, but showed good V-1037, at 1 •~ and 4 •~ their MICs. Similar dif bacteriostatic and bactericidal activity against ferences between these drugs have been found strains. However, where resistance did occur against other bacterial species (Smith and Lewin, to these drugs, it was generally at a higher level 1988). The bactericidal activities of the quin than against OA and FLU. MLX generally olones, particularly the earlier quinolones, have showed activities higher than NA and PA but been shown to have a biphasic dose response, with lower than OA and FLU. activity decreasing at concentrations above optim The bacteriostatic and bactericidal activities of al, although, the significance of this is unclear for the newer quinolones against Gram-negatives activity in vivo (Smith and Lewin, 1988; Tsoumas were generally higher than those of the earlier et al., 1989). This aspect was not examined in quinolones, with CPFX, TFLX and OFLX par the present study. ticularly active, and NFLX and ENX inter Activities of quinolones show reductions in the
mediate between these and the earlier quinolones. presence of serum, mainly due to protein binding, P. fluorescens and P. putida were also fully sus although this is generally less with the newer
ceptible to these drugs. Although some reduction quinolones (Hastings and McKay, 1987; O'Grady in activity against NA resistant A. salmonicida et al., 1987; Norris and Mandell, 1988). In the
and P. piscicida strains occurred, these drugs present study with L. anguillarum V-1037, OA and remained fully bacteriostatic, and were bactericidal FLU showed MIC increases of approximately 4 •~
except for OFLX and ENX against P. piscicida. and 6 •~ , respectively, whereas OFLX and CPFX
Therefore, these drugs are potentially usefull showed increases of approximately 2 •~ . The
against Gram-negative infections of fish, including MBCs showed increases similar to those of the cases showing low susceptibility to earlier quin MICs.
olones. Earlier quinolones generally show decreasing
Use of quinolones against strains with acquired activity with increasing pH, and also show decre resistance may lead to step-wise further increases ased serum uptake and lower antibacterial action in resistance (Norris and Mandell, 1988 ; Stamm, in the presence of multivalent cations such as 1989). Bimodal resistances to earlier quinolones magnesium and calcium (Monk and Campoli have been found in fish pathogens from farm Richards, 1987; Smith and Lewin, 1988). - In sea situations (Aoki et al., 1981; O'Grady et al., 1987). water fish, magnesium and calcium from ingested However, the frequency of resistance development water are concentrated in residual intestinal is lower in newer quinolones than in the earlier material, and magnesium concentrations of 50 quinolones, even where some decrease in sus 180 mM have been recorded. In fresh water,- ceptibility has previously occurred (Crumplin and however, there is active absorption of calcium and
Odell, 1987). magnesium and concentrations of 2 mM and None of the earlier quinolones showed noticeable 0 mM, respectively, have been recorded (Conte, activity against the Gram-positives tested. Of the 1969). In fresh water and sea water fish, stomach newer quinolones, TFLX, CPFX and OFLX were and intestinal pHs may vary from alkaline when bacteriostatic and bactericidal against S. iniae and fasting to acidic during feeding, but these pHs are
S. epidermidis and bacteriostatic against E. seri generally higher in sea water fish (Barrington, 140 R. Palmer, K. Kawai and R. Kusuda
1957). O'Grady et al. (1986) found that serum O'Grady et al., 1986; Ueno et al., 1988a, 1988b). uptake of orally administered FLU, and the ef With administraption of FLU to Atlantic salmon ficacies of OA and FLU were lower in Atlantic S. salar at 36 mg/kg, a peak serum level of 2.5 ƒÊg/ salmon Salmo salar held in sea water than those ml was obtained in seawater conditions, whereas held in fresh water. In bath treatments of fish in 11.4 ƒÊg/ml was obtained in fresh water (O'Grady fresh water and sea water, increased pH, calcium et al., 1986). Serum levels obtained with PA in and magnesium gave decreased uptake of OA and freshwater species have been recorded at: 1.1-6.8
FLU (Endo and Onozawa, 1987a, 1987b; O'Grady g/ml for 5-10 mg/kg doses, and 7.7-13.5 ƒÊg/mlƒÊ et al., 1988). Additionally, possible differences for 40 mg/kg doses (Katae et al., 1979a, 1979b, in the metabolism of OA have been found between c; Oida et al., 1982). Stamm 1979 (1989) con marine and fresh water fish (Ishida, 1990). The sidered that in vivo antibacterial concentrations newer quinolones are also affected by multivalent of at least four times the MIC were required to cations, but generally show greater activity with minimize the selection of resistant organisms. increasing pH (Nishio et al., 1988; Smith and The MIC guidelines for resistance, used in this Lewin, 1988). In the present study, using a L. study for the earlier quinolones, largely cor anguillarum strain as a test organism, the MICs responded to these levels, when effects of serum of OA and FLU showed large increases with pH, on activity were considered. However, drug tis whereas the MICs and MBCs of OFLX and sue distributions, clearance rates and other par CPFX showed no major changes. With added ameters also require consideration ; some of these magnesium (26.2 mM), corresponding to 50 % of aspects were examined in previous studies on the level in a standard sea water (Lyman and earlier quinolones in fish. Pharmokinetic studies Fleming, 1940), there were large increases in the of the newer quinolones in fish will be required for MICs for all drugs, but the increases in MICs of assessment of their applicability. OA and FLU were higher than those for OFLX and CPFX. Magnesium has been reported to Acknowledgement inhibit bactericidal activity more than bacteri ostatic activity (Smith and Lewin, 1988). How This work was supported by a scholarship from ever, with the present test organism and conditions, the Ministry of Education, Science and Culture of this was found only to some degree with OFLX. Japan. Provisional categories were used in this study to describe bacteriostatic and bactericidal suscepti References bility. In humans, a guide level of MIC •…2 ƒÊg/ Aoki, T., T. Kitao and K. Kawano (1981): Changes ml has been used to indicate susceptibility for in drug resistance of Vibrio anguillarum in cultured quinolones, although, with the improved phar ayu, Plecoglossus altivelis Temmink and Schlegel, macokinetics of newer quinolones, strains with in Japan. J. Fish Dis., 4, 223-230. MICs up to 4 ƒÊg/ml may be intermediately sus Alderman, D. J. (1988): Fisheries chemotherapy : ceptible (Grimm, 1987; Monk and Campoli a review. In "Recent advances in aquaculture" Richards, 1987; Phillips et al., 1988). - Higher (ed. by J. M. Muir and R. J. Roberts.) Croom values (MIC •…16 ƒÊg/ml) have been used for NA Helm, London, Vol. 3, pp. 1-61. against urinary infections, although serum levels Barrington, E. J. W. (1957): The alimentary canal are usually low (Norris and Mandell, 1988; and digestion. In "The physiology of fishes" (ed. Phillips et al., 1988). Various MIC test conditions by M. G. Brown). Academic Press, London, Vol. 1, pp. 109-161. and susceptibility guide lines have been used in Barry, A. L., R. V. Gardiner and R. R. Packer (1987): studies with fish pathogens (Hastings and McCay, Bactericidal activities of ten different fluoroquinol 1987; Kusuda et al., 1988; Nakano et al., 1989; ones against selected Enterobacteriaceae. Diag. O'Grady et al., 1987; Stamm, 1989; Tsoumas et Microbiol. Infect. Dis., 6, 81-83. al., 1989). With oral administration of OA, Conte, F. P. (1969): Salt secretion. In "Fish physio peak serum levels have been recorded between logy" (ed. by W. S. Hoar and D. J. Randall). Aca 1.4 and 5.1 ƒÊg/ml, for single or multiple doses of demic Press, New York, Vol. 1, pp. 241-292. 30-50 mg/kg (Endo et al., 1973, 1987a, 1987b; Crumplin, G. C. and M. Odell (1987): Development Activity of quinolone antibacterials 141
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