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Cytophaga Aquatilis Sp INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY, Apr. 1978, p. 293-303 Vol. 28, No. 2 0020-7713/78/0028-0293$02.00/0 Copyright 0 1978 International Association of Microbiological Societies Printed in U.S.A. Cytophaga aquatilis sp. nov., a Facultative Anaerobe Isolated from the Gills of Freshwater Fish WILLIAM R. STROHLT AND LARRY R. TAITtt Department of Biology, Central Michigan University, Mt. Pleasant, Michigan 48859 A facultatively anaerobic, gram-negative, gliding bacterium was isolated from the gills of freshwater fish. Its deoxyribonucleic acid base composition (33.7 mol% guanine plus cytosine), lack of microcysts or fruiting bodies, cell size (0.5 by 8.0 pm), and hydrolysis of carboxymethylcellulose and chitin place it in the genus Cytophaga. This aquatic cytophaga is differentiated from other cytophagas by its fermentation of carbohydrates, proteolytic capabilities, and a number of addi- tional physiological and biochemical tests. The organism was compared to other similar isolates reported from fish, and it appears to belong to a new species, for which the name Cytophaga aquatilis is proposed. The type strain of C. aquatilis, N, has been deposited with the American Type Culture Collection under the accession number 29551. Borg (3) described four strains of facultatively isolates are similar to those strains of faculta- anaerobic cytophagas isolated from salmon at tively anaerobic cytophagas described by Borg the Skagit Fish Hatchery near Seattle, Wash. (3), Pacha and Porter (14), Anderson and Con- He associated those strains with bacterial gill roy (l),and Reichardt (16). disease although their pathogenicity was not MATERIALS AND METHODS proven. Bacterial strains. Thirteen strains of cytophagas While studying “myxobacterial” diseases of were isolated from diseased salmon or trout at the salmonbids, Anderson and Ordal(2) isolated and Platte River Fish Hatchery, Honor, Mich.; the Wolf studied a saprophytic fermentative cytophaga Lake Fish Hatchery, Wolf Lake, Mich.; and from that required COZ for the fermentation of glu- diseased suckers obtained from the holding tanks of cose. They named the organism Cytophaga suc- an Honor, Mich., baitshop. The strains and their cinicans. Pacha and Porter (14) isolated and sources are listed in Table 1. Methods. Three isolation methods were used to characterized a number of facultatively anaero- obtain the cytophagas: (i) salmon gills or fins were bic strains of saprophytic cytophagas from swabbed with sterile cotton swabs and streaked onto salmon, and Anderson and Conroy (I) compared skim milk medium (6), CP medium (5), and Pate and five strains of fermentative cytophagas to Borg’s Ordal medium (15); (ii) aseptically dissected gills or Skagit strains and again associated them with fins were placed onto CP or skim milk medium; and bacterial gill disease of salmon. (iii) aseptically dissected gills were blended with 100 A recent study by Bullock (4) showed that ml of sterile distilled water and plated onto CP and certain cytophaga-like strains, including a group skim milk media. Isolates from the plates were ob- of facultatively anaerobic cytophagas, could be tained by picking the edges of spreading colonies and placing them onto plated Pate and Ordal medium associated with bacterial gill disease as oppor- containing 0.9 or 1.5% agar. Spreading colonies from tunistic invaders, but no single pathogenic spe- these plates were checked for gliding motility. Gliding cies was found. of cells was determined by the use of hanging-drop Trust (22) showed that cytophagas are a part and wet-mount preparations and by observing plate or of the normal gill flora in a number of both wild slide agar mounts under phase and light microscopy. and hatchery-raised salmon, and he indicated A medium of 2% Casitone and Chu no. 10 basal salts that these organisms, along with pseudomonads, (20) was used as a basal medium and a maintenance are the predominant species of bacteria on the medium for the cytophagas throughout this investi- gills of healthy salmon. gation. In this report, we describe a new facultatively The temperature for optimal growth was deter- anaerobic species of Cytophaga which was iso- mined by growing the cultures in liquid basal medium at various temperatures from 0 to 37°C and comparing lated from the gills of diseased salmon at the the relative optical densities over a 2-week period. Platte River Fish Hatchery in Michigan. Our Methods for the determination of lauryl sulfate toler- ance, carboxymethylcellulose digestion, alginate liq- t Present address: Dept. of Microbiology, Louisiana State University, Baton Rouge, LA 70803. uefaction, and tyrosine degradation were those of -ftPresent address: Dept. of Microbiology, Wayne State Lewin and Lounsbery (12). The methods of Pacha and University, Detroit, MI 48202. Porter (14) were employed for the determination of 293 294 STROHL AND TAIT INT. J. SYST. BACTERIOL. TABLE1. Sources and strains of cytophagas isolated from fish in March 1974 Location on fish Strain desig- Source Type of fish nation where obtained ~~~ T I Platte R. Hatchery Salmonb Gills R I Platte R. Hatchery Salmon Gills 0 I Honor, Mich., baitshop' Sucker Gills Q I Platte R. Hatchery Salmon Gills N I Platte R. Hatchery Salmon Gills A I1 Wolf Lake Hatchery Atlantic salmon Fins B I1 Wolf Lake Hatchery Atlantic salmon Fins D I11 Platte R. Hatchery Trout Fins G I11 Platte R. Hatchery Trout Fins P IV Wolf Lake Hatchery Atlantic salmon Fins J IV Platte R. Hatchery Salmon Gills K IV Wolf Lake Hatchery Atlantic salmon Fins S IV Wolf Lake Hatchery Atlantic salmon Fins The group I strains are the strains which are discussed in detail in this paper. The species of the salmon or trout were not recorded. ' From holding tanks outside the baitshop. gelatin liquefaction, catalase production, casein, mined by the methods of Dworkin and Gibson (9) and starch, and esculin hydrolysis, digestion of chitin, and by placing the culture onto yeast streaks and observing cytochrome oxidase production. Oxidation or fermen- for fruiting structures over a 6-week period. The Cy- tation of carbohydrates was determined by the pro- tophaga pigments were extracted with 100% acetone duction of acid in phenol red basal agar medium or 95% ethanol, and absorption curves were run on a (Difco; 8) with 1%carbohydrate added. For the oxi- Gilford 2400 recording spectrophotometer. Analysis dation of carbohydrates, the acid reaction on plates of and gross characterization of the pigments were ac- aerobically incubated cultures was recorded as posi- complished by the methods of Reichenbach et al. (18). tive. For the fermentation tests, sterile Vaspar was For electron microscope observations of the colo- overlaid on agar deeps, and an acid reaction was nies, frozen-surface replicas were prepared by the considered as positive. Aerobic and anaerobic growth method of Steere (21) with cultures that were fixed for of the cultures was tested on 1%glucose supplemented 1h on plates of basal medium with 3% glutaraldehyde. with Chu no. 10 basal salts alone, basal salts plus 0.5% The agar was sliced approximately 3 mm below the yeast extract, basal salts plus 0.5% KN03, and basal surface and placed onto stubs for insertion into the salts plus 0.1% NH4H2P04.The degradation of native modified Denton DFE-2 freeze-etch unit. Replicas cellulose was tested by placing sterile Whatman no. 1 were shadowed at a 45" angle with platinum-carbon filter paper disks onto plates containing Chu no. 10 and were carbon coated. Electron micrographs were basal salts and 0.1% yeast extract. The cellulose plates taken, using a JEM lOOB electron microscope were incubated aerobically for a 4-week period, and equipped with a 60" tilt goniometer stage. Thin sec- positive results were recorded if dissolution of the tions were prepared by a modified Ryter-Kellenberger paper occurred. Salinity tolerance was tested by plac- technique (19). Plates of cells grown on basal medium ing 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0% NaCl into for 48 h were flooded with 0.02% oso4 for 20 to 30 min, the basal medium and observing growth over 2 weeks. and the clumped bacteria were scraped off the agar All other physiological tests were done with standard and transferred to 1%Os04 for 18 h. Acetone dehydra- procedures found in the Difco Manual (€9, except tion (using, in a series, 50, 70, 85, 95, 100, and 100% those done with the Enterotube apparatus (BBL Prod- acetone) and Mollenhauer no. 1 plastic embedding ucts, Inc., Baltimore, Md.). procedure (13) were followed, and thin sections of the The ability of the cytophagas to lyse bacterial cells cytophaga were obtained on a Sorvall MT-2 Porter- was examined by swabbing live cultures of various Blum ultamicrotome with glass knives. Electron mi- bacteria across non-nutrient agar plates and then stab- crographs (magnification approximately x40,OOO) of bing the cytophaga test culture into the center. Posi- thin sections of the cytophaga were taken by using a tive results were indicated by dissolution of the bac- Phillips 300 electron microscope. Measurements of cell terial colony with spreading of the cytophaga colony size were obtained with a Filar micrometer under beyond the swabbed cultures of test bacteria. Lysis of phase microscopy using 24-h-old liquid cultures. cyanobacteria was tested by the methods described by Shilo (20) and by Gromov et al. (10); cultures of RESULTS Plectonema boryanum, Anacystis nidulans, and Nos- The aquatic cytophaga reported here is a fac- toc sp. were used as test cultures. ultatively anaerobic bacterium that uses fer- Antibiotic susceptibilities were determined by plac- ing susceptibility disks of various antibiotics onto basal mentable carbohydrates as suitable substrates medium plates streaked with the Cytophaga cultures; for anaerobic growth. Growth factors in yeast positive results were indicated by zones of inhibition.
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