inhibitory activity of antibiotic-produc - c. p. dopazo, m. l. lemos

8/12/2019 Inhibitory Activity of Antibiotic-produc - C. P. Dopazo, M. L. Lemos

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Journal o Applied Bacteriology 1988 65 97-101 2728/01/88

Inhibitory activity of antibiotic-producing marine bacteriaagainst fish pathogensC . P . D O P A Z O * , . L . L E M O S , . L O D E I R O S ,. B O L I N C H E S ,. L . B A R J AA L I C I AE . T O R A N Z Oepartamento de M icrobiologia y Parasitologia, Facultad deBiologia, Universidad de Santiago, Santiago de Co mp ostela 15706, SpainReceived 20 January 1988 revised 17 March 1988 and accepted 5 March 1988

D O P A Z O , .P. L E M O S ,M . L . , L O D E I R O S ,. B O L I N C H E S ,. BARIA, J. L.TORANZO . E . 1988. Inhibitory activity of antibiotic-producing marine bacteriaagainst fish pathogens. Journal of Applied Bacteriology 65 97-101.The activity of antibiotic-producing marine bacteria was assayed against bacterialfish pathogens belonging to the genera Vibrio, A eromonas, Pasteurella, Edwardsiella,Yersinia and Pseudomonas with the aim of evaluating the possible use of thesemarine strains for controlling epizootics in aquaculture. Inhibition tests on solidmedium showed that in general the majority of fish bacteria were strongly sensitiveto the marine bacteria. Only two strains Edwardsiella tarda and Pseudomonasaeruginosa), were resistant to all the antibiotic-producing strains. The results ofantagonism assays in sea water however varied according to the fish pathogensexamined. Experiments conducted using cell-free supernatant fluids of marine bac-teria demonstrated the involvement of antibiotic substances in the inhibition of fishpathogens.

During this century, investigations in the field ofmicrobial interactions have been focused mainlyon the study of competition for resources(Fred rickson 1977; Fredrickson Step-hanopoulos 1981). However, some authors(Alexander 1971; De Freitas . Fredrickson1978; Fredrickson Steph anopo ulos 1981)have emphasized the importance of the pro-duction of antibiotics in microbial ecosystems.Although the existence of antibiotic pro-duction by marine bacteria has been demon-strated (Krassilnikova 19 61; Dogget 1968;Gauthier Flatau 1976; Do pazo et al. 1986),there is a paucity of information about theantagonistic relationships in their environment(Toranzo et al. 1982; Lemos et al. 1985a).In a previous report (Lemos et a . 1985 a) westudied the antibiotic production by chromo-genic marine strains isolated from intertidalgreen and brown algae in the north-west ofSpain. Each producer strain displayed a broadinhibitory spectrum, including other similar* Corresponding author.

producer bacteria, some non-producer marinestrains also isolated from seaweeds, and terres-trial and clinical strains. In the present work wehave extended this study to the antagonisticeffects of 18 selected antibiotic-prod ucing strainsagainst different bacterial pathogens whichcause important diseases in aquaculture, inorder to explore the potential use of thesemarine bacteria for controlling epizootics in fishcultures.Materials and MethodsThe fish pathogenic strains (Table 1) wereobtained from culture collections, other workersor isolated from epizootics in o ur a rea (To ranzoet al. 1987), and belonged t o the followingspecies : Vibrio anquillarum (four strains), V uul-nificus (one), V . tubiashii (one), Aeromonashydrophila (four), A . sobria (one), A . salmonicida(five), Pasteurella piscicida (two), Edwardsiellatarda (three), Yersinia ruckeri (one) and Pseudo-rnonas aeruginosa (one). As reported previously(Lemos et al. 1985b) our antibiotic-producing


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Antagonism o marine bacteria t fish pathogens 99marine bacteria belong to the Pseudomonas-Alteromonas group.The inhibition test in solid medium wascarried out using a double-layer method. Platesof Marine Agar (MA: Difco Laboratories) wereinoculated with 18-24 h broth cultures of theproducer strains using an automatic multipointinoculator (Denley, England). After incubationat 20C for 4 d the cells were killed with chloro-form vapour (1 min) and an overlay containingthe pathogenic strain was poured on the MAplate. This overlay was made with tryptonesoya agar (TSA ) supplemented with 2 NaC lfor A . hydrophila, P. piscicida and the Vibriospecies, and TSA for the other fish pathogens.After a diffusion period (15-30 min) at 2WC,plates were incubated for 2 4 d at the optimumtemperature for each species. A clear zone ofinhibition around the growth of marine bacteriaindicated antibacterial activity.

For the antagonism assays in batch culture,250 ml E rlenm eyer flasks with 100 ml of agedand autoclaved sea water were simultaneouslyinocu lated with b roth culture s (0.2 ml) of bothantibiotic-producers and pathogenic strains.Flasks inoculated with only one strain wereused as controls. All cultures were shake-incubated (100 rev/min) at room temperature(18-22C). Samples were taken at regular inter-vals immediately after incubation. The bacterialdensity was measured by the standard platecount method and by the Miles Misramethod (Corry 1982).To rule out possible nutrient competition theantagonistic effects were also evaluated usingculture filtrates of marine strains which wereobtained as previously described (Lemos et al.1985a). Briefly, after incubation for 2 d in seawater, cells were removed by centrifugation at5000 rev/min for 15 min, an d supern atant fluidswere sterilized by filtration through 0.22 pmmembranes (Sartorius). Peptone (0.5 ) andyeast extract (0.1 ) were added to the filtrates,and cultures of each fish strain to be tested wereseeded in these cell-free supernatant fluids. Incu-bation and sampling were performed asdescribed above.Results and iscussionThe inhibition tests in solid medium (Table 1)revealed that from a total of 23 pathogenic bac-teria assayed, only two strains E . tarda KGE-

7901 and Ps. aeruginosa JC-9006) were resistantto all the antibiotic-producing strains. Theremaining fish pathogenic bacteria were strong-ly sensitive. In fact, three A . salmonicida, two P .piscicida and V . tubiashii were inhibited by allthe marine bacteria.

The majority of antibiotic-producing strainsshowed activity against mo re tha n 75 ofpathogenic strains. Only one marine strain(PA 2) inhibited less than 50 of fish pathogenicstrains.Our intention was to study the behaviour ofmarine bacteria producing antibiotics againstfish pathogenic bacteria. Because our marinestrains ar e obligate halophilic bacteria, it is pos-sible only to assay their antagonistic effect in asaline environment. We used three marine bac-teria and selected fish pathogenic bacteriacausing the most serious problems in marineaquaculture. V . anguillarum, a well-known fishpathogenic bacteria in marine cultures; A.hydrophila, ubiquitous micro-organisms of bothfresh and marine waters (Hazen et al. 1978), andA . salmonicida. Although A . salmonicida was for-merly considered to be a pathogen of freshwaterfish it has now been implicated in epizootics insea water (Paterson et al. 1980; Cornick et al.1984; Bruno 1986).The results in liquid media were slightly dif-ferent from those obtained in solid media. Aero-monas salmonicida was rapidly inhibited, in lessthan 2h, by the three marine bacteria and noviable cells were detected after 1 h (this rep-resented a reduction in count of more than fourlogs . In contrast, the population of A . salmoni-cida declined only one-half log in 12 h whengrowing alone in the same medium. Vibrioanguillarum and A . hydrophila, however, showedresistance to some of the strains that had dis-played inhibitory activity in solid medium.Figure 1 shows the distinct antagonistic pat-terns exhibited by the antibiotic producer FP6strain against a, A . hydrophila B-32; b, A . sal-monicida ATCC 14174 and c, V . anguillarum

The experiments conducted with cell-freeculture filtrates of FP6 (results not shown)revealed similar inhibitory kinetics of A . salmon-icida and A. hydrophila to those found usingmixed cultures of antibiotic producer and fishpathogen. These results indicate the presence ofantibiotic compounds released to the mediumand, hence, rule out nutrient competition.

R-82.


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C . P . Dopazo et al.00A preliminary characterization of these anti-biotic substances, purified by chromatographicand electrophoretic procedures (Lemos et al.1985a), demonstrated that they are anionic andlow molecular weight (< 1000 daltons) com-pounds, thermolabile and resistant to proteo-

lytic enzymes.T o date, few studies have dealt with the inter-actions between environmental strains andpathogenic fish bacteria in aqu atic microcosms.Harris Singleton (1986) reported the isolationof heterotrophic chromogenic bacteria whichcaused i i reduction of the numbers of Vibrio sp.in mixed culture. These authors did not con-clude that the basis for this populationreduction was competition for a limitingresourcr: or prod uction of non-specific inhibitoryextracellular products. Recently, Imada et al.(1985a, b) characterized an active substancefrom a marine Alteromonas sp. which inhibitedproteases released by strains of A . hydrophilaand V . anguillarurn.Our findings indicated that in general, theproduction of antibiotics give these marinestrains ;in antagonistic capacity against most ofthe fish pathogenic bacteria. We consider that,as stated by Imada et al (1985a, b), these strainscould be of interest for the biological control ofpathogenic bacteria in fish culture systems.Therefore, further work will be focused ondetermining if the results reported here can besuccessfully extended to the fish rearing systems.This work was supported by a grant from theComisibn Asesora de Investigacion Cientifica yTecnica (CAICYT) AQ-018/84, Ministerio deEducacion y Ciencia (MEC), Spain. C.P.Dopazo and M.L. Lemos also acknowledge theMEC and the Rectorado of the University ofSantiago, respectively, for Research fellowships.

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F i g 1. Effect of the marine strain FP6 against a,Aeromonas hydrophila B-32; b, A. salmonicida ATCC14174 and c, Vibrio anguillarum R-82, in the antago-nism assays in sea water. a Fish pathogen in mixedculture with FP6; 0 FP6 in mixed culture; U fishbacteria in pure culture con trol ).

ReferencesALEXANDER . 1971 Microbial Ecology, pp. 511. New

York ohn Wiley.BRUNO,D.W. 1986 Furunculosis in sea-reared atlanticsalmon, Salmo salar L. colonization of the gill epi-thelium. Bulletin of the European Association of FishPathologists 6,16-19.CORNICK,.W., MORRISON, .M., ZWICKER, .SHUM,G 1984 A typical Aeromonas salmonicidainfection in atlantic cod, adus morhua L. Journalof Fish Diseases 7,495-499.CORRY, .E.L. 1982 Quality assessment of culturemedia by the Miles Misra method . In Pro-


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Antagonism o marine bacteria to f i sh pathogens 101ceedings of the Symposium Quality Assurance andQuality Control of Microbiological Culture Mediaed. Corry, J.E.L. pp. 21-37. Dar ms tadt: V erlagErnst Giebeler.

DE FREITAS,M.J. FREDRICKSON,.G. 1978 Inhibi-tion as a factor in the maintenance of microbialecosystems. Journal of General Microbiology 106,DOGGET, .G. 1968 New anti-Pseudomonas agent iso-lated from a marine Vibrio . Journal of BacteriologyDOPAZO, .P., LEMOS, .L., BOLINCHES,., BARJA,.L.TO RA NZ O, .E. 1986 Inhibition of bacterial fishpathogens by antibiotic-producing marine bacteria.Abstracts of the X l V International Congress ofMicrobiology, Manchester. No. PG2-23, p. 232.FREDRICKSON,.G. 1977 Behav iour of mixed culturesof microorganisms. Annual Reueiw of MicrobiologyFREDRICKSON,.G. STEPHANOPOULOS,. 1981Microbial Competition. Science 213,972-979.GAU THIE R, .J. FL AT AU , .N. 1976 Antibacterialactivity of marine violet-pigmented Alteromonaswith special reference to the production of bromin-ated compounds. Canadian Journal of MicrobiologyHAZEN,T.C., FLIERMANS,.B., HIRSCH,R.P. ESCH,G.W. 1978 Prevalence and distribution of Aero-monas hydrophila in the United States. Applied andEnvironmental Microbiology 36,731-738.HARRIS,.L. SINGLETON,.L. 1 986 Studies on inter-action between pathogenic Vibrio species andchromogenic aqu atic bacteria. Abstracts of the 86thAnnual Meeting of the American Society for micro-biology, Wasing ton D .C. No. 1-45, pp. 172.

307-320.

95 1972-1973.

31,63-87.

22,1612-1619.

IMADA, ., SIMIDU, . TAGA,N. 1985a Isolation andcharacterization of marine bacteria producing alka-line protease inhibitor. Bulletin o the JapaneseSociety o ScientiJic Fisheries 51 799-803.IMADA, . MAEDA,M. TA GA , . 1985b Purificationand characterization of protease inhibitor monas-tatin from a marine Alteromonas sp. with referenceto inhibition of the protease produced by a bacte-rium pathogenic to fish. Canadian Journal of Micro-biology 31, 1089-1094.KRASSILNIKOVA,.N. 1961 Antibiotic properties ofmicroorganisms isolated from various depths ofworld oceans. Mikrobiologiya 30 45-550.LEMOS,M.L., TOR ANZ O, .E. BA RJA , .L. 1985aAntibiotic activity of epiphytic bacteria isolatedfrom intertidal seaweeds. Microbial Ecology 11149-1 63.LEMOS,M.L., TO RA NZ O, .E. BARIA, J.L. 1985 bModified medium for the oxidation-fermentationtest in the identification of marine bacteria. Appliedand Environmental Microbiology 49 1541-1543.PATERSON, .D., DOUEY, . DFSAUTELS, . 1980Isolation and identification of an atypical Aero-monas salmonicida strain causing epizootic lossesamong atlantic salmon Salmo salar) reared in aNova Scotian hatchery. Canadian Journal of Fish-eries and Aquatic Sciences 37,22362241.Antiviral activity of antibiotic-producing marinebacteria. Canadian Journal of Microbiology 28, 231-238.TORA NZO, .E., BAY A,A.M., ROBERSON,.S. BARJA,J.L., GRIMES,D.J. HETR ICK, .M. 1987 Specificityo slide agglutination test for detecting bacterial fishpathogens. Aquaculture 61,81-97.

TORANZO, .E. BARIA, J.L. HETR ICK , .M. 1982

inhibitory activity of antibiotic-produc - c. p. dopazo, m. l. lemos

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