Strains of Aeromonas salmonicida have recently been isolated in lreland which have a reduced zone of inhibition when tested against discs containing ...
Bull. Eur. Ass. Fish parhol. Z (2),43, tggT
ISOLATION oF AeromonassalmonicidaSTRAINS RESISTANTTO THE QUINOLINE ANTIBIOTICS By P. O'GneDy,R. PALMER, H. RoocEnexn p. Svrrn Strains of Aeromonas salmonicida have recently been isolated in lreland which have a reduced zone of inhibition when tested against discs containing quinoline antibiotics.This note describesan investigation into these strains and a similar strain isolated in Scotland in 1985. The strains studied are shown in Table l. All strains were autoagglutinating. Strain SPU was isolated from a production hatchery from fish which had received a ten day courseof oxolinic acid therapy at lOmg/kg fish/day in fresh water. All strainsin the 600 serieswere isolatedfrom fish which had receivedthis treatment in fresh water followed by ten days of oxolinic acid therapy at 30mg/kg fish/day after their arrival at sea. Three methods of assessing the levels of antibiotic resistance were used. Two semiquantitative disc methods were used- a rapid method and the standardisedKirbyBauer method recommended by the USFDA. The results of these tests are shown in Tables 2 and 3. 1. Rapid method 0.lml of a stationary phaseculture was spread on a tryptone soya agar (Oxoid) plate. The plate was dried and antibiotic discs were pressed on the surface. Zones of inhibition were read after 36hr incubation at 22"C. 2. Kirby-Bauer method - this was performed as described by Brown and Blowers (1978). The plates were again read after a minimum of 36hr incubation at 22" C . Attempts to read zone sizesprior
to 36hr lead to significant underestimates of their size. 3. Quantitative measurementsof resistance to oxolinic acid was made by determining minimum inhibitory concectrations (MIC) and minimum bactericidal concentrations (MBC) in antibiotic media Nos. 2 and 3 (Oxoid) by the method recommended by Waterworth
(1e78).
The strains studied here clearly fall into three groups with respect to their resistanceto quinoline antibiotics. Group I strains, which were highly sensitiveto the quinoline antibiotics, include two Irish strains isolated prior to the introduction of the quinoline antibiotics and one strain, 640Va, isolated at a sea farm from fish suppliedby the smolt producrion unit involved in this outbreak. Strains MT206, MOWI, SpU, 644Ra. 646T, 652Da, and 657V1 can be placed into Group II. Strains, representativeof this group, that were tested show an approximately forty-fold increase in the resistanceto oxolinic acid in broth MIC tests.Two strains,644Rb and 652Db, can be placed in Group IIL Strain 644Rb shows a 200-fold increasein oxolinic acid broth MIC valuescompared with Group I strains. It is interesting to note both of thesestrainswere isolatedfrom individual fish that also yielded strains 644Ra and 652Da, members of Group II. Preliminary investigations have shown that the frequency of occurenceof strains with a Group III phenotype in stationary broth
Bull.Eur.Ass.FishParhol.Z (Z),44,lg11' cultures of Group II strains (644Ra and S P U ) i s i n t h e r e g i o no f 5 x l 0 - 6 t o 5 x l 0 - 7 . Thus it is possiblethat the srrains 644Rb and 652Db may have emerged during laboratory testing. However, we have recentlyisolateda strain with a Group III phenotype in pure culture from fish involved in this outbreak. It is highly probable that Group III strainsare clinically significant. Of major concern, however, is the question of whether the level of resistance in the Group II strains is sufficient to compromiseoral quinoline therapy. Field observationswould suggest that in one situation this may have been the case.The strain MOWI, a typical Group II strain, was isolatedfrom an outbreak of chronic furunculosisat the SRTI researchfacility which was reported not to respondto four ten day courses of oral oxolinic acid therapy at l0mg/kg fish/day (D. Piggins p e r s .c o m m . ) . Comparisons of in vitro M'IC or MBC valueswith levelsof quinoline antibiotics achieved in fish during oral therapy may be expectedto provide information on the likely significance of the Group II resistancephenotype. However such a comparison is complicated by the effects
on the MIC and MBC values of various parameters such as innoculum size and the presence or absence of serum. We have found that a 103-fold increasein the innoculum size will increase the determined broth MIC value by approximately five-fold. Further the presenceof fish serum in MIC teststends also to increasethe MIC value obtained two-fold relative to those obtained in broth. Also it is not clear which tissue antibiotic levels (serum, muscle, or internal organs) is the most significant to measure. These factors are presently under investigation. In conclusion we recommend that the semi-quantitativeKirby-Bauer method be used for the determination of the resistance phenotype of strains of Aeromonas salmonicida against the quinoline antibiotics as this is a rapid, easily reproducible and accurate method that requiresa minimum of lab. work. Summary Strainsof Aeromonassalmonicido have been isolated that are resistant to the ouinoline antibiotics. These strains have been divided into three groups on the basis of their level of resistance to the antibiotics. The resistant strains are likely to be found to be clinically significant in the treatment o f f u r u n c u l o s i sb y o r a l q u i n o l i n e t h e r a p y .
Bull. Eur. Ass. Fish Pathol. 7 (2), 45, 1987 Table l: Source of Aeromonas salmonicida strains. STRAIN
SOURCE
DATE OF ISOLATION
Strains isolated prior to the introduction of the quinoline antibiotics. A47S LP4IS LP44R
This Lab. This Lab. This Lab.
I 978 I 980 1981
Strains isolated from outbreaks reoorted not to be respondingto standardoral oxolinic acid theranv.
MT206 MOWI
DAFS Lab. (Aberdeen) SRTI Research Facility
I 985 May 1986
Strainsisolatedat the salmon Researchrrust of Ireland(sRTI) smolt production unit or from smoltssuppliedto seafarms from this unit. SPU SRTI 2/s/1986 640Va Sea Site V 2r/4/t986 644Ra Sea Site R 23/4/1986 644Rb Sea Site R 23/4/1986 652Da Sea Site D 5/s/1986 652Db Sea Site D 5/5/1986 657Vl SEa Site V 9/5/ 1986 Table 2: Diameter of zones of inhibition produced by quinoline antibiotics.
METHOD ANTIBIOTIC CONCENTRATION $e/disc)
RAPID METHOD OX 1
KIRBY.BAUER
FLU NAL 0
2
nt nt 43 2 9 2 8 29 27 nr 3 0 19 1 8
nr nt 43 2 2 20 20 22 2 ND B
3
0
OX 1
0
FLU
2
1
0
NAL 2
3
0
1
0
STRAIN A47S LP44R 640Va M't206 MOWI SPU 644Ra 652Da 6 5 7 VI 644Rb 652Db n t - n o t t e s t e d ,N D - n o NAL-naladixic acid.
nr 50 4l 48 nt 42 36 39 50 54 43 4'7 0 8 2 5 1 9 25 0 8 2 2 1 6 22 t4 29 20 2'7 ND 28 t9 25 ND 23 19 25 0 8 2 5 1 9 25 ND 19 IO 12 N D 1 8 N D 14
z o n e d e t e c t a b l e ,O X - o x o l i n i c
36 39 36 18 13 12 12 15 14 ND ND
47 39 47 ND ND ND ND ND ND ND ND
acid, FLU-flumequine,
Zone sizesare given in mm. and are the average of five determinations.
36 33 36 ND ND ND ND ND ND ND ND
Bull. Eur. Ass. Fish Pathol. 7 (2),46,1987 Table 3: Broth MIC/MBC
values of Aeromonas sqlmonicida.
STRAIN
MIC'(yglml)
MBC (yglml)
LP44R LP4IS 644Ra SPU 644Rb
o.0122'3 0.012 0.5 0 . 75 2.O
0.0174'5
0.017 0.75 1.0 3.0
l
lnoculum was 50ul of a l0-3 dilution of a 48hr brain heart infusion (Oxoid) broth grown at 22'C.
2.
Assaybroth was Antibiotic Medium No. 3 (Oxoid).
3.
R e s u l t sr e a d a f t e r 4 8 h r a t 2 2 " C .
4.
Assay medium was Antibiotic Medium No. 2 (Oxoid).
5.
R e s u l t sr e a d a f t e r 4 8 h r a t 2 2 " C .
References Brown, D., and Blowers, R. (1978).Disc methods of sensitivity testing and other semi-quantitative methods. In "Loboratory Methods in Antimicrobial Chemotherapy." (ed. Reeves, D.S., Phillips, I., Williams, J.D., and Wise, R.), p. 8-30, Livingstone Churchill, Edinburgh, London, New York. Waterworth, P.M. (1978). Quantitative methods for bacterial sensitivity testing. ln "Lqboralor! Methods in Antimiuobial Chemotherapy!' (ed, R e e v e s ,D . S . , P h i l l i p s , I . , W i l l i a m s , J . D . , a n d Livingstone Churchill, Wise, R.), p.3l-40, E d i n b u r g h ,L o n d o n , N e w Y o r k . Authors'address: F i s h D i s e a s eR e s e a r c hG r o u p , D e p t . o f M i c r o b i o l o g y , U . C . C . , G a l w a y , l r e l a n d
