APPLIED MICROBIOLOGY, Jan. 1968, p. 97-101Copyright ( 1968 American Society for Microbiology

Vol. 16, No. 1Printed in U.S.A.

Characteristics of Cytophaga psychrophila (Borg)

Isolated During Outbreaks of BacterialCold-Water Disease

R. E. PACHA

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331

Received for publication 18 August 1967

Characteristics of 10 strains of Cytophaga psychrophila isolated during a numberof epizootics of bacterial cold-water disease were compared. Morphological, cul-tural, biochemical, and serological data showed that the organisms were very closelyrelated. The isolates exhibited gliding motility, formed neither fruiting bodies nor

microcysts, were actively proteolytic, and grew only at low temperatures. Data pre-

sented extend the description of C. psychrophila.

The etiological agent of bacterial cold-waterdisease was originally isolated by Borg (Ph.D.Thesis, University of Washington, Seattle, 1948).This organism was found to be a nonfruitingmyxobacterium which was unable to grow attemperatures above 25 C. On the basis of pitho-genicity and low optimal growth temperature,the organism was considered to be a new speciesof myxobacterium. The name Cytophaga psychro-phila was proposed. The description of this or-ganism is somewhat incomplete, and it i6 notincluded in Bergey's Manual of DeterminativeBacteriology, 7th ed.

Bacterial cold-water disease is generally foundin young silver salmon in early spring when watertemperatures are low. In some hatcheries, up to30% of the fry have been lost as a result of thisdisease. Generally, when water temperatures in-crease to 13 C the disease abates.

Because of the importance and widespread dis-tribution of this organism among the hatcherypopulations of fish in the Pacific Northwest, itwas of interest to compare cultures isolatedfrom different outbreaks of disease to determinewhether one or several strains of the organismwere involved. This report describes the results ofthese studies and extends the description of C.psychrophila.

MATERIALS AND METHODS

Source of strains. A total of 10 strains of C. psy-chrophila, isolated from silver and chinook salmontaken from various hatcheries in the Pacific Northwest,were studied. The code numbers and source of eachare shown in Table 1. The strains isolated at variouslocations in the State of Washington were provided

from the culture collection at the University ofWashington through the courtesy of E. J. Ordal.Strains 143a and 144a are two of the original culturesisolated by Borg.The cultures were maintained on Cytophaga agar

deeps. This medium is composed of tryptone, 0.05%;yeast extract, 0.05%; beef extract, 0.02%; sodiumacetate, 0.02%; and agar (Difco), 0.4%. The stockcultures were incubated at 18 C for about 1 week andthen stored at 4 C.

Liquid cultures used as inocula for the various testsperformed were grown in Cytophaga broth. Thecomposition of this medium is the same as thatdescribed above except that the agar is omitted. Allculture media were cooled to refrigeration tempera-tures before use.

Morphological characteristics. After 24 hr of incuba-tion in Cytophaga broth at 18 C, the organisms wereGram-stained. Cell morphology and motility weredetermined by examination of wet mounts with aphase-contrast microscope. Colony appearances onCytophaga agar were noted after 36 hr of incubation.In an attempt to induce fruiting-body and microcystformation, the organisms were placed on sterile fishtissues submerged in tap water. This procedure wasfirst described by Ordal (6).

Environmental characteristics. The ability of strainsto grow in Cytophaga broth at 37, 30, 23, 18, and 4 Cwas recorded after incubation periods ranging from 1day to 2 weeks, depending on the temperature em-ployed. Growth was determined by examining tubesfor turbidity.The NaCl tolerance of the organisms was deter-

mined by use of Cytophaga broth containing variousamounts of NaCl. Cultures were examined for growthwithin 2 weeks. The NaCl concentrations employedincluded 0, 0.1, 0.2, 0.4, 0.8, 1.0, and 2.0%.

Susceptibility to antibacterial agents. Susceptibilityto antibiotics was determined by placing Sensi-discs ofthe antibiotics (BBL) on agar plates streaked with the

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TABLE 1. Source of isolates

Strain Source Fish host

Simp 2 Simpson Fish Hatchery, Washington State Department of Fish- Silver salmoneries

L. Kal. 1 Kalama Fish Hatchery, Washington State Department of Fish- Chinook salmoneries

LMC 6 Lower Minter Creek Fish Hatchery, Washington State Depart- Silver salmonment of Fisheries

Si-5 Siletz Salmon Hatchery, Oregon Fish Commission Silver salmonA1-2 Alsea Salmon Hatchery, Oregon Fish Commission Silver salmonA1-3 Alsea Salmon Hatchery, Oregon Fish Commission Silver salmonDun 1 Dungeness Fish Hatchery, Washington State Department of Silver salmon

Fisheries Silver salmon143a Minter Creek Fish Hatchery, Washington State Department of Silver salmon

Fisheries144a Minter Creek Fish Hatchery, Washington State Department of Silver salmon

FisheriesIs-9 Issaquah Fish Hatchery, Washington State Department of Silver salmon

Fisheries

test organism. The strains were tested for susceptibil-ity to chlortetracycline, 30 ,ug; bacitracin, 10 units;chloramphenicol, 30 Mg; dihydrostreptomycin, 10,g;erythromycin, 2,g; neomycin, 30 ,g; penicillin, 10units; polymyxin B, 30 Mg; sulfadiazine, 1 mg; andtetracycline, 30 Mlg.

Physiological tests. The basal medium used to testthe ability of the isolates to degrade gelatin, starch,casein, tributyrin, tyrosine, xanthine, and albuminconsisted of tryptone, 0.1%; yeast extract, 0.05%; beefextract, 0.05%; and agar (Difco), 1.1%.

Gelatinase production was tested by use of thebasal medium supplemented with 0.5% gelatin. After1 week of incubation at 18 C, the plates were floodedwith acid mercuric chloride to show areas of gelatinliquefaction (3).

Starch hydrolysis was detected by use of a mediumcontaining 0.2% soluble starch. Plates of this mediumwere inoculated, incubated for 1 week, and thenflooded with an iodine solution. Colonies with theability to hydrolyze starch produced clear areas in anotherwise blue medium.The ability to hydrolyze casein was tested by inocu-

lating the cultures onto the basal medium containing10% skim milk. After 1 week, the plates were exam-ined for clear areas of hydrolysis in the opaquemedium.

Tributyrin hydrolysis was determined by clearingproduced on 0.05% tributyrin incorporated in thebasal medium. Plates were read after 2 weeks ofincubation.

Tyrosine breakdown was measured by the abilityof cultures to dissolve 0.5% tyrosine suspended inbasal medium within 2 weeks.The ability to decompose xanthine was tested by use

of basal medium supplemented with 0.4% xanthine.The cultures were examined for periods up to 1 monthfor the disappearance of crystals around the areas ofgrowth.

Albumin hydrolysis was detected by use of thebasal medium containing 0.25% egg albumin. Evi-

dence of hydrolysis was detected after 1 week incuba-tion by the addition of acid mercuric chloride to theplates.The method of Emerson and Weiser (2) was used to

detect cellulose digestion. The mineral salt mediumdevised by Stanier (8) was supplemented with 1.5%agar and used as a basal medium. The plates wereexamined every 2 days for 1 month for depressions inthe overlay surrounding the colonies.

Chitin decomposition was tested with a suspensionof chitin prepared according to the procedure de-scribed by Stanier (9). A 5-ml amount of agar (con-taining 0.1% peptone, 0.5% chitin, and 0.7% Difcoagar) was overlaid on non-nutrient agar plates andallowed to dry for 1 day. Cultures to be tested werespotted onto the surface of the overlay agar, andhydrolysis was detected by the dissolution of chitinaround the areas of growth.

Non-nutrient agar (1%), containing 1% (wetweight) autoclaved, washed cells of Escherichia coli,was used to determine the ability of the strains to lysebacterial cells. Evidence of lysis was indicated by theappearance of clear zones around the areas of growth.A slight modification of the Hugh-Leifson pro-

cedure (5) was used to test for the production of acidfrom carbohydrates. The basal medium consisted ofpeptone, 0.2%; sodium chloride, 0.2%; K2HPO4,0.03%; bromothymol blue, 0.0015%; and agar(Difco), 0.3%. The carbohydrate solution was filter-sterilized and added to the basal medium at a finalconcentration of 1.0%.A modified litmus milk medium was used to test

the action of the organisms on milk. The mediumconsisted of 5.0% skim milk and 0.04% litmus.

Hydrogen sulfide production was tested by use oflead acetate strips over tryptone broth cultures of thetest organisms.The medium used for the Voges-Proskauer and

methyl red tests consisted of 0.7% peptone and 0.5%glucose. Tests for acid production with the use of

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methyl red indicator and for acetylmethylcarbinolproduction with 40% KOH and a-naphthol reagentwere carried out.

Tests for the reduction of nitrate to nitrite, forindole formation, and for catalase were carried outaccording to the procedures described in Manual ofMicrobiological Methods (7).The presence of cytochrome oxidase was deter-

mined by the method of Gaby and Hadley (4). Theappearance of a blue color within 2 min after additionof the reagent was considered a positive reaction.

Nutritional requirements of the organisms weretested by use of a casein hydrolysate medium con-taining 0.1% (NH4)2HP04, 0.02% KCl, 0.02%MgSO4* 7H20, and 0.2% vitamin-free acid-hydrolyzedcasein. The ability to utilize citrate as a sole source ofcarbon was tested by use of a mineral base mediumcontaining 0.02% MgSO4*7H20, 0.1% NH4H2PO4,0.1% K2HPO4, 0.2% NaCl, 0.2% sodium citrate,1.5% agar (Difco), and 0.001% bromothymol blue.

Serological methods. Immune sera were obtainedfrom rabbits injected intravenously with saline-washedsuspensions of cells in successive doses of 0.2, 0.4,0.8, 1.5, and 2.0 ml on every 3rd day. The rabbitswere bled from the marginal ear vein 2 weeks afterthe last injection. The sera were stored in the frozenstate.A tube agglutination procedure was used to titrate

the antisera. The antigen suspension consisted ofdoubly washed saline suspension of cells adjusted toan optical density of 0.22 at a wavelength of 525 m,u.The tubes were incubated at 52 C for 2 hr and forapproximately 18 hr at 4 C before being read. A slideagglutination procedure was used to determine therelationships between the test isolates. The antigensuspension employed was the same as that used in thetube agglutination studies.

RESULTS

Morphological characteristics. All of the iso-lates were weakly refractile, slender, gram-nega-tive, flexible rods (Fig. 1). Actively growing cellsof the organisms were about 0.75 ,u in diameterand 1.5 to 7.5 .1i long. As the cultures aged, thecells tended to become somewhat shorter thanthose found in log-phase cultures.On Cytophaga agar, the colonies of the strains

of C. psychrophila studied were bright yellow incolor and generally showed thin, spreading mar-gins. No diffusible pigment was apparent. Aphotograph of an edge of a typical colony is shownin Fig. 2. Although the colony shown in Fig. 2 ischaracteristic for this organism, variations mayoccur. In some cases, deeply colored colonies withentire edges were formed. The fact that severalcolony types appeared on the same plate indi-cates that colony morphology is a variable char-acteristic for this organism. No attempt was madeto determine the effect of various environmentalfactors on colony appearance.

Neither fruiting-body formation nor rmicrocyst

production occurred on any of the culture media.Attempts to induce fruiting bodies on bits of fishtissue also were unsuccessful. Thus, these or-ganisms appear to be typical representatives ofthe genus Cytophaga as defined by Stanier (8).

Environmental characteristics. All of the testisolates grew readily at temperatures between 4and 23 C. As would be expected, growth at lower

FIG. 1. Phase-contrast micrograph of a culture ofCytophaga psychrophila. X 1,100.

FIG. 2. Phase-contrast micrograph of the edge of acolon0y of Cytophaga psychrophila on1 Cytophaga agar.X 140.

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TABLE 2. Selected physiological characteristics ofCytophaga psychrophila

Characteristic Percentage ofstrains positive

Gelatin degradation ................. 100Casein hydrolysis ................... 100Albumin digested................... 100Tributyrin hydrolyzed ........ ....... 100Tyrosine decomposed................ 20Digestion of autoclaved cells of Esch-

erichia coli..................... 100Starch hydrolyzed ........ .......... 0Chitin degraded .................... 0Cellulose decomposed. 0Xanthine hydrolyzed ........ ........ 0Litmus milk peptonized. 100Carbohydrate utilization:

Glucose oxidized.............. 0Glucose fermented ........... 0Cellobiose oxidized.............. 0Cellobiose fermented ............ 0

Hydrogen sulfide produced .......... 0Indole produced..................... 0Voges-Proskauer positive . 0Nitrate reduced to nitrite .0.......0Catalase produced................... 100Cytochrome oxidase produced ....... 0Growth in casein hydrolysate me-

dium ........... 100Growth on citrate medium .......... 0

temperatures was much slower than that occur-ring in the vicinity of 20 C. None of the organismswas capable of growing at 30 or 37 C.

All of the cultures grew in the presence of 0.8%NaCl. In 1% NaCl medium, only six of the or-ganisms were capable of growing, and 2% NaClinhibited growth of all of the isolates tested. Nodecrease in growth was noted in the basal me-dium alone without the addition of NaCI.

Antibiotic susceptibility. Antibiotics whichproved to be inhibitory to the 10 strains of C.psychrophila were chlortetracycline, bacitracin,chloramphenicol, dihydrostreptomycin, eryth-romycin, neomycin, penicillin, and tetracy-cline. In addition, 4 of the 10 isolates were sus-ceptible to polymyxin B.

Physiological properties. Results of 24 physio-logical tests carried out on the isolates are givenin Table 2. These data indicate that C. psychro-phila is actively proteolytic, but shows no abilityto degradeeither simple orcomplex carbohydrates.Tyrosine degradation appears to be a variablecharacteristic of this organism, since only 2 of the0isolates were able to decompose this compound.

The nutritional requirements of the organismwere not complex as indicated by the fact thatthe isolates were able to grow on a vitamin-freecasein hydrolysate medium.

Serological analyses. Three strains of C. psy-chrophila isolated during different outbreaks ofbacterial cold-water disease were selected andused to immunize rabbits. The three strainschosen were 144a, Dun 1, and LMC 6. Thehomologous titers of these antisera were 1:640.To test the serological homogeneity of the iso-

lates of C. psychrophila, slide agglutination testswere carried out on the organisms. It was foundthat each of the 10 cultures was agglutinated byeach of the three antisera. Thus, the isolates aresimilar serologically.The specificity of the antisera for C. psychro-

phila was tested by carrying out slide agglutina-tion studies with 24 nonpathogenic cytophagasisolated from fish. None of these isolates wasagglutinated by the three antisera available; hence,no serological evidence was found supporting arelationship between C. psychrophila and othermyxobacteria associated with fish.

DISCUSSION

On the basis of a limited number of tests, Borg(1) described C. psychrophila as a gram-negative,flexible rod which exhibited creeping motility.The organism produced neither fruiting bodiesnor microcysts, was strictly aerobic, and grewonly at low temperatures. In addition, casein washydrolyzed and catalase was produced by thisbacterium. Two of the cultures originally isolatedby Borg were available for comparison in thepresent investigation. These two isolates differedfrom the other organisms studied in that theywere able to degrade tyrosine. No other signi-ficant characteristics were noted which wouldallow Borg's isolates to be distinguished fromthe other cultures studied.The 10 strains of C. psychrophila studied in this

investigation were found to represent a veryhomogeneous group of microorganisms. Thisfinding is of interest since the organisms wereisolated over a period of years in widely separatedgeographic areas and from different species offish. The only cultural and physiological differ-ences noted were with regard to sodium chloridetolerance, sensitivity to sulfadiazine, and theability to decompose tyrosine. Morphologicallyand serologically the cultures were also veryclosely related.The fact that antiserum prepared against C.

psychrophila appears to be highly specific for thisorganism would suggest that somatic antigensmight be used to distinguish C. psychrophila fromother myxobacteria occurring on fish. Therefore,it may be possible to use serological proceduresas a means for rapid identification of the or-ganism. Before reliance can be placed on such a

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test, however, a greater number of myxobacteriawill have to be studied.

It is of interest to note that all of the isolatesof C. psychrophila examined in the present studywere actively proteolytic. Perhaps this activityplays an important role in the pathogenicity ofthe organisms.On the basis of the results of this investigation,

the description of C. psychrophila is extended asfollows:The cells are gram negative, rod-shaped and

vary in length depending on the age of the culture.The average size of young cells is about 0.75 by3.5 A. Gliding motility is exhibited on solid sur-faces.

Colonies on Cytophaga agar are yellow in colorand generally exhibit a thin, spreading edge. Non-spreading variants occur. No diffusible pigmentwas produced.

Gelatin, casein, albumin, tributyrin, and auto-claved cells of Escherichia coli hydrolyzed. Starch,chitin, cellulose, xanthine not degraded. Tyrosinedegradation is variable.Litmus milk peptonized.Glucose and cellobiose neither fermented nor

oxidized.Indole negative. Voges-Proskauer and methyl

red negative.Nitrate not reduced to nitrite.Hydrogen sulfide not produced.Catalase produced. Cytochrome oxidase not

produced.Growth on casein hydrolysate broth. No

growth on citrate medium.No growth in the presence of 2% NaCl.

Optimal temperature about 20 C. No growthat 30 C.

Source: Isolated from fish with bacterial cold-water disease.

Habitat: Freshwater. A pathogen of fish.

ACKNOwLEDGMENTThis investigation was supported by grant 5-R01-

WP00925-02 from the Department of Interior.

L1TERATuRE CirD1. BORG, A. F. 1960. Studies on myxobacteria associ-

ated with diseases in salmonid fishes. WildlifeDisease 8:1-85, 2 microcards.

2. EMERSON, J. E., Am 0. L. WEISER. 1963. Detectingcellulose-digesting bacteria. J. Bacteriol. 86:891-892.

3. FRAZER, W. C. 1926. A method for the detectionof changes in gelatin due to bacteria. J. Infect.Diseases 39:302-309.

4. GABY, W. L., AND C. HADLEY. 1957. Practicallaboratory test for the identification of Pseu-domonas aeruginosa. J. Bacteriol. 74:356-358.

5. HUGH, R., AND E. LEiFSON. 1953. The taxonomicsignificance of fermentative versus oxidativemetabolism of carbohydrates by various gramnegative bacteria. J. Bacteriol. 66:24-26.

6. ORDAL, E. J. 1946. Studies on myxobacteria. J.Bacteriol. 51:579.

7. SOCIETY OF AMERICAN BAcEERIoLoGisTs. 1957.Manual of microbiological methods. McGraw-Hill Book Co., Inc., New York.

8. STANIER, R. Y. 1942. The Cytophaga group: acontribution to the biology of myxobacteria.Bacteriol. Rev. 6:143-196.

9. STANIER, R. Y. 1947. Studies on nonfruitingmyxobacteria. I. Cytophaga johnsonae, n. sp., achitin-decomposing myxobacterium. J. Bacteriol.53:297-315.

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