Hemolysins of Edwardsiella tarda J. J. Watson and F. H. White*

ABSTRACT Isolates of Edwardsiella tarda from four sources produced nonfilterable hemolysin in trypticase soy broth. The cell-associated hemolysin was partially heat labile, destroyed by formalin and sensitive to treatment with trypsin. These characteristics, and the observation that Ca"+ or Mg+ + ions enhanced activity, suggest that a proteinaceous, enzymic component may be responsible for the hemolytic activity.

The most notable lesion associated with the isolation of Edwardsiella tarda from wildlife species has been a hemorrhagictype enteritis. It was also noted that E. tarda characteristically produces a betatype hemolysis on sheep blood agar plates at 37°C. In the present study, isolates of E. tarda from several sources were examined for hemolysin production, and the hemolysin was characterized.

MATERIALS AND METHODS RESUME Des souches d'Edwardsiella tarda, provenant de quatre sources distinctes, produisirent une hemolysine non filtrable, lorsqu'on les ensemenqa dans un bouillon 'a base de trypticase et de soya. Cette hemolysine, liee aux cellules bacteriennes, se revela partiellement thermolabile, destructible par la formaline et vulnerable a l'action de la trypsine. Ces caracteristiques et le fait que les ions Ca++ ou Mg+ accrurent l'activite de l'hemolysine, suggerent qu'une substance enzymatique de nature proteique pourrait etre responsable de l'activite hemolytique de cette hemolysine.

ISOLATES The following isolates of E. tarda were studied: 1) No. 10, isolated from the small intestine of a Brown pelican (Pelacannus occidentalis carolinensis), 2) No. 26, isolated from the liver of a largemouth bass (Micropterus salmoides), 3) No. 41, isolated from the small intestine of an alligator (Alligator mississippiensis) and 4) No. 43, isolated from the spleen of an armadillo (Dasypus novemcintus Linn). HEMOLYSIN PRODUCTION AND ASSAY

INTRODUCTION Edwardsiella tarda, a member of the Enterobacteriaceae, was described as a new genus and species by Ewing et al (4). This organism has a wide geographical distribution and infects numerous animal species, including man (2,6,7,13,14). *College of Veterinary Medicine, University of Florida, Gainesville, Florida 32611. Submitted February 20, 1978.

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Hemolysin was produced by propagation of the organisms in 100 ml amounts of trypticase soy broth (TSB)l in nephloflasks. The seed inoculum for each trial was prepared by growing the organisms in 10 ml of TSB for 18 hours. One ml of this fresh culture was inoculated into 99 ml of TSB. Duplicate nephloflasks were inoculated. Nephloflasks containing uninoculated TSB were incubated along with the test flasks as controls. Flasks were incubated in a shaking water bath2 at lBaltimor,e Biological Laboratory, Cockeysville, Maryland. 2Precision Scientific, Chicago, Illinois.

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40°C and 25°C. Optical density was re- density readings were made at a wavecorded at 525 nm on a Spectronic 20 spec- length of 540 nm. trophotometer3 every hour. At the end of the second hour, 4 ml of culture was assayed for hemolysin (11) . One and one-half STABILITY OF THE HEMOLYSIN ml were used to determine hemolysin actiHemolysin susceptibility to inactivation vity in unfiltered culture while the remaining sample was filtered through a by heat, formalin and trypsin was tested 0.22 ,um membrane filter.4 An additional according to the methods of Synder and assay on 1.5 ml of the filtered culture fil- Kock (11). trate was run to determine any filterable hemolysin activity. After finding that hemolytic activity was lost with filtration EFFECTS OF CALCIUM AND MAGNESIUM of whole culture through 0.22 ,um and 0.45 IONS ON HEMOLYTIC ACTIVITY ,um filters, the following procedure was The effect of adding calcium and maadopted. Four ml of culture were removed at the time of assay with whole culture gnesium ions on the hemolytic activity was activity tested as before. The remaining determined by employing physiological saportion of the sample was centrifuged at line containing 0.01 M CaC12 or 0.01 M 14,000 g for 30 minutes at 4 °C and the MgCl2 as test solutions. These solutions supernatant fluid was assayed for hemo- were used for the dilution of the hemolysin lytic activity. Also, the pelleted cells re- as well as erythrocyte suspensions. maining after centrifugation were suspended in 1.5 ml of the hemolysin diluent EFFECT OF PH ON HEMOLYTIC ACTIVITY and tested for hemolytic activity. The ability of the four strains of EdThe hemolysins were diluted in isotonic wardsiella tarda to lyse the erythrocytes from sheep, pelican, man and alligator was Tris-HCl buffered saline (0.2 M) at pH tested at both 400C and 25°C. The blood values of 9.0, 7.5, 7.0, 6.0 and 5.5. Washed was collected by cardiac puncture or vena- sheep erythrocytes (1%) were also suspuncture into acid citrate dextrose solution. pended in the same buffer at the various The whole blood was centrifuged at 4°C pH values. Samples of the culture incuat 3000 g to collect the erythrocytes which bated at 400C were removed at hourly inwere washed three times with physiological tervals and the hemolytic activity of whole saline (0.85%) containing 0.01M CaCl2 and culture and centrifuged culture was deter100 ,ug/ml streptomycin. The same solution mined at these pH values. In the above trials on stability and the also was used for hemolysin dilution and suspension of the washed erythrocytes. A effects of ionic supplementation and pH final suspension of 1 % washed erythrocy- on hemolytic activity the isolate from the Brown pelican was grown and assayed at tes was used in the hemolysin assay. A hemoglobin curve for each species was 40°C using sheep erythrocytes as the inprepared by making a series of dilutions of dicator. 1% erythrocytes in distilled water. The straight lines of these plots showed a direct relationship between optical density and RESULTS percent hemolysis. One hemolytic unit (HU) was arbitrarily defined as that amount of hemolysin that would produce 1 % hemolysis in a 1 % suspension of eryThe four isolates of Edwardsiella tarda throcytes. The hemolytic units of a parti- produced hemolysin in TSB at growth temcular sample were calculated by multiply- peratures of 40°C and 250C (Table I). ing the optical density of the hemolysin There was no hemolytic activity in the fildilution showing 50% hemolysis by the trate of cultures filtered through 0.22 ,um reciprocal of that same dilution. All optical or 0.45 ,um membrane filters. The supernatant fluid of cultures centrifuged at 14,000 g for 30 minutes showed a trace amount of 3Bausch and Lomb, Rochester, New York. hemolytic activity. The pellet of cells re4Millipore Corporation, Bedford, Massachusetts. sulting from the centrifugation, when re-

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TABLE I Comparative Hemolytic Activity of Edwardsiella tarda Isolates-

Sheep Temp C

Erythrocytes Pelican Man C Temp C Temp 25 40 25 40 90 31 44 355

25 40 Isolated from Brown Pelican Max. HU/1.5 ml 305 620 hemolysin Armadilio " 520 580 153 70 " 92 Largemouth bass 75 180 79 " 145 500 39 Alligator 90 aAfter growth and assay of the cultures at the temperature given

suspended in diluent and titrated, produced hemolytic activity equivalent to uncentrifuged cultures. Irrespective of the isolate or type of erythrocyte used as the indicator, hemolysin production occurred during the logarithmic phase of growth. During the growth trials at 40°C, maximum production appeared during the first quarter or middle log phase of growth. In all cases, maximum hemolytic activity occurred when the pH of the growth medium was lowest. Also, hemolytic activity showed a sharp and continuous decline after optimum activity was reached. There was no potentiation of hemolytic activity in samples incubated at 40C for extended periods. Figure 1 illustrates the typical kinetics of growth and hemolysin production in relation to pH. Attempts to culture E. tarda in the chemically defined medium of D'Empaire (3) were not successful. Additional growth media including nutrient broth, 1% peptone water, casein hydrolysate medium (11) and a chemically defined medium for Escherichia coli (11) were tested for their ability to support the production of a filterable hemolysin by E. tarda. However, these trials were unsuccessful. Unfiltered cultures of the four isolates of E. tarda lysed the erythrocytes from sheep, man, pelican and alligator. There was a variable susceptibility to hemolysis related to the species from which the erythrocytes were obtained. Sheep erythrocytes were the most susceptible, while alligator erythrocytes were the least sus-

ceptible (Table I). In regard to differential hemolytic activity of the four isolates tested, the isolates from the Brown pelican and the armadillo had greater overall hemolytic capability against the erythrocytes of all species at both 250C and 400C. The addi80

33 16 18

44 92 20

Alligator Temp C 25 40 17 16

9 8 6

17 31 32

tion of either calcium or magnesium ions enhanced the hemolytic activity of E. tarda hemolysin in whole culture (Table II). TABLE II. Effects of Calcium and Magnesium Ions on the Activity of Edwardsiella tarda Isolate 10 (Brown pelican) Hemolysin Hemolytic Activity in HU/1.5 ml 79 317 159 -Physiological saline solution (0.85% NaCi)

Substance added PSS PSS with 0.01M CaC12 PSS with 0.01M MgCl2

There was an immediate effect upon mixing trypsin and whole culture of E. tarda as evidenced by a sixfold loss in activity in the trypsin treated culture compared to the control culture tested at time zero. The trypsin treated culture continued to lose hemolytic activity upon further incubation, reaching 1:2 at 30 minutes and remaining at this level throughout the experiment. Incubation at 380C caused some loss in activity. A threefold loss in activity was demonstrated during the first fifteen minutes incubation, decreasing to 1:128 at 30 minutes and remaining at this level. The cell-associated hemolysin was partially heat labile. Compared to the control sample, a heated sample of whole culture was reduced fourfold in hemolytic activity (Table III). There was complete loss of hemolytic activity when the culture was treated with formalin. The whole culture hemolysin appeared active over a broad range of pH but the titer was highest at pH 5.5 (Table IV). The hemolytic activity of the supernatant fluid of centrifuged samples was much

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Growth *--e4-Hemolysin in whole culture o-o-H-o-OHemolysin in filtered supernatant fluid

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Hemolysins of Edwardsiella tarda.

Hemolysins of Edwardsiella tarda J. J. Watson and F. H. White* ABSTRACT Isolates of Edwardsiella tarda from four sources produced nonfilterable hemol...
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