F L O C C U L A T I O N P H E N O M E N O N OF C A N D I D A A L B I C A N S BY L Y S O Z Y M E
by T. KAMAYA
Dept. o/ Clinical Pathology Deer Lodge Hospital, D.V.A. Winnipeg, Manitoba, Canada ABSTRACT Young colonies of Sabouraud's glucose agar room temperature culture of Candida species from human isolation were suspended in distilled water. The suspension was mixed with a solution of lysozyme and incubated in a 37 ° C water bath. Within 3 - - 5 hours, various species of Candida ceils showed flocculation to varying degrees which occurred at varying periods of onset. Among seven Candida species, Candida albicans and Candida stetlatoidea showed the strongest flocculation, earliest onset and most solution clarity than did any other species. Candida stellatoidea was indistinguishable from Candida albicans in its degree of flocculation, and in the clarity of solution. Candida species may be arranged in the following order according to their decreasing positivity in flocculation: 1 2 3 4 5 6 7 8
Candida albicans Candida stellatoidea Candida tropicalis Candida krusei Candida pseudotropicatis Candida parapsilosis Candida guilliermondii Saecharomyces species may be placed after Candida guilliermondii.
t t seems possible to separate t!le Candida species into 3 groups by the rate of flocculation, and clarity of solution. Group I. Candida albicans and Candida stellatoidea. Group II. Candida tropicalis, C. krusei axtd Candida pseudotropicalis. Group III. Candida parapsilosis and Candida guiltiermondii. Saccbaromyces specimens (S. cerevisiae and others) were placed after group III. INTRODUCTION
In 1932 FRIEDBERGER • HODER found that many isolates of air borne saprophytic microorganisms, although resistant to the lytic action of lysozyme, were nevertheless, agglutinated by it. They noted that the flocculating capacity of lysozyme appeared to run parallel to its lyric activity and considered these two activities to be the manifestations of a single substance (Lysozyme). Accepted for publication: 1.I.1969
F L O C G U L A T I O N O F C. A L B , B Y L Y S O Z Y M E
321
KLEMPARSKAYA (1939) r@orted that lysozyme has the ability to dissolve and flocculate bacteria, and used such flocculation as a means of dividing bacteria into three groups. He speculated that th.e flocculation of microorganisms by lysozyme was dependant on its concentration, and the pH at which time it was used, and concluded that different types of behaviour shown by various organisms regarding flocculation by lysozyme are explained by differences in their colloidal structure. THOMPSON (1940) also observed occasional agglutination of very susceptible micrococci or sarcinas S.P.P. by purified egg white lysozyme, prior to its lytic activity, and the agglutination effect was attributed to adsorption of the enzyme b y the bacteria which, for some reason, were not dissolved. In 1948 WEBB noticed that lysozyme caused fioceulation to occur in suspensions of streptococcus faecalis and staphylococcus aureus. SALTON (1953) observed that the addition of lysozyme (1 mg/ml) to bacterial suspensions resulted in agglutination of the cells. In the present study human isolates of pathogenic and non-pathogenic members of the Candida species were tested for flocculation by lysozyme. M A T E R I A L AND ~/[ETHOD
Material The surface of a young 1) room temperature e) culture (2--5 days) of Candida species in Sabouraud agar 3) was gently scraped, using a loop. The material obtained was suspended in 10 mI of distilled water in a 100 × 16 mm size cuvette with a screw or rubber cap. The mixture was agitated to make an even suspension, and its turbidity was measured at 520 M# using a Coleman Junior Spectrophotometer. Each suspension of Candida was adjusted to 50 % transmittance either b y further dilution, or by the addition of Candida culture. A 10 ml distilled water blank was used to represent 100 % transmittance. Method Fresh lysozyme solution was prepared by dilution of lysozyme 4) in distilled water to a concentration of 10 mg/5 ml. 0.5 ml of the diluted lysozyme solution was placed in each of several clean sterile 1) Mainly consists of y e a s t cells. The use of young cultures is i m p e r a t i v e for definitive results. e) Not 37 ° C incubation. 3) N o t blood agar, nor corn meal agax. 4) Lysozyme (egg white) salt free, L y s o z y m e egg white 3 × crystalline, Nutritional Biochemical Company. N.B. L y s o z y m e chloride 3 × crystals (Nutritional Biochemical Co.) was tried a n d found to be unsatisfactory.
322
T,K~MAYA
tubes ~) to which was added 0.5 ml of the diluted 50 % T Candida suspension. The tubes were shaken gently and incubated in a 37 ° C water bath. Results were checked at 2 hour, 3 hour and 5 hour intervals. Before each reading the tubes were mixed by gently inverting each once or twice. Gentle agitation of the tube contents before each reading is an essential step, since the organism usually sediments to the bottom within two hours. I N T E R P R E T A T I O N OF T E S T S
There were two factors important for interpretation of the positive flocculation tests. One - - the degree of flocculation seen and the other - - the clarity (or decreasing turbidity) of the solution. 1 Flocculation was divided into three grades, as follows: i Strong flocculation + + + ii Moderate flocculation + + iii Slight flocculation + 2 Decreasing turbidity or "clarity" was arbitrarily divided into three grades. i Completely clear showing no turbidity + + + ii Moderately clear, moderately turbid + + iii Marked turbidity (little clarity) + Final readings were made by consideration of both factors mentioned. 1 Turbid milky solution with no flocculation indicated negative results. (see Fig. 1) 2 Numerous large heavy clumps with complete clarity (no turbidity) of solution indicated a strongly positive test. (see Fig. 1) 3 Increased flocculation with increased clarity (decreasing turbidity) of the solution indicated the grade of positivity. 4 A fine granular agglutination accompanied by high turbidity of the solution may be a precursor of true agglutination or m a y indicate pseudoflocculation. Pseudoflocculation m a y result from introducing tiny clumps of pseudomycelia of the original culture, as from an excess of Candida suspension. RESULTS
Twenty-five standard strains of seven Candida species isolated from humans were obtained from the National Institute of Health, Bethesda, Maryland, U.S.A.; The Communicable Disease Center, 1) ]3-]3 p r o d u c t 100 × 13 m m in size, factory fresh t u b e s were used. Trace aanounts of glucose in used t u b e s m a y interfere w i t h t e s t results. Sterility preferable, b u t n o t essential, t t u b b e r s t o p p e r or screw caps were used to seal the tubes.
trLOCCIJLATION OF C. ALB. BY LYSOZYME
323
Atlanta, Georgia, U.S.A.; The Department of Mycology, McGill University, Montreal, P.Q., Canada; and Temple University, Philadelphia, U.S.A. All twenty-five strains of seven Ca~dida species from four institu-
Fig. 1. F l o c c u l a t i o n reaction: P o s i t i v e flocculation (left tube} - - Candida albicans N e g a t i v e flocculation (right tube) ........Candida guilliermondii
Fig. 2. A g g l u t i n a t i o n Mass of Candida albicans s e e n microscopically (10 × 10)
324
:r. KAMAYA TABLE ~[ C o m m u n i c a b l e Disease C e n t e r s t r a i n s (after 3 h o u r s in 87 ° C w a t e r b a t h )
C. alb
C. stella
C. trop
C. pseudotrop
C. krusei
C. parapsilosis
C. guilliermondii
+++
+++
+
+
+
±
i
±++
±+±
±
±
±
--
_
(0)*
Floecilla-
tion Clarity of solution Final Reading
(6)
(6)
(2)
(2)
(2)
(0)*
+++
+++
+
+
+
(±)
(±)
± + +
+ + +
4-
+
±
--
__
C. guilliermondii
TASLE II
McGill University strains (after 3 h o u r s in 37 ° C w a t e r b a t h )
C. aZb
C. stella
C. trop
C. pseudotrop
C. krusei
C. parepsilosis
Flo eculation
+++
+++
±
+
+
±
C l a r i t y of solution
+++
+++
+
+
+
--
__
Final Reading
(6)
(6)
(~)
(2)
(2)
(o)*
(o)*
+-~+
+++
+
+
+
(:5)
+++
+++
+
+
±
(±)
--
__
TABLE I I I N a t i o n a l I n s t i t u t e of H e a l t h s t r a i n s (after 3 h o u r s in 37 ° C w a t e r b a t h )
C. alb
C. sfella
C. leop
C. pseudotrop
C. krusei
C. parapsilosis
C. gvtilliefmondii
Flocculatio n
+++
+++
++
+
+
+
dz
C l a r i t y of solution
+++
+++
++
+
+
.
(a) +++ +±+
(6)
(4)
(2)
(2)
(0)*
(0)*
+++ ±+±
++ -t-+
+ +
+ +
(±) ~--
(±) --
Final Reading
.
.
.
FLOCCULATION OF C.
ALB.
BY
325
LYSOZYME
TABLE I V
Temple University strains (after 3 h o u r s in 37 ° C w a t e r b a t h ) C. alb
C. stella
C. pseudotrop
C, krusei
C. guilliermondii
Flocculation
+++
+++
+
+
=~
Clarity of solution
+ + +
+++
+
+
--
(6) + + + + + +
(6) +++
(2) +
(2) +
(0)*
Final Reading
+++
+
+
--
(±)
L e g e n d of D i a g r a m 1, 2, 3, 4: R e s u l t s a f t e r 3 hour, 5 h o u r in 37 ° C w a t e r b a t h . P o s i t i v e c o n t r o l u s i n g k n o w n C. atbieans culture. O p t i m a l r e s u l t s were o b t a i n e d u s i n g y o u n g r o o m t e m p e r a t u r e c u l t u r e ( 2 - 4 d a y s culture). * (0) as long as t h e r e w a s no clarity (no d e c r e a s i n g t u r b i d i t y ) final r e a d i n g w a s considered n e g a t i v e e v e n if t h e r e w a s little flocculation (see w e a k flocculation in section of m e t h o d ) .
TABLE V
S a c c h a r o m y c e s species H o r t i c u l t u r a l Reseea'ch I n s t i t u t e of O n t a r i o S t r a i n (after 3 h o u r s ill 37 ° C w a t e r b a t h ) Saecharomyees cerevisiae
Saccharomyces ellipsoideus
Saecharomyces steineri
Saecharomyees ovi[ormis
LODDER t:~ I
Saccharomyces florentinus (CASTELLI)
VAN RIJ Flocculation
.
.
Clarity of solution
.
Final Reading
(0) . .
.
.
. .
.
.
.
. .
(0) . .
.
.
.
.
.
.
.
.
(0) .
.
.
.
(0) .
.
.
.
(0) .
.
tions were treated as described previously and the results obtained tabulated (Table I, II, III, IV). Young cultures of Saccharomyces species were treated in the same manner as Candida species (Table
V).
Various species of Candida cells showed flocculation to varying degrees, and at varying periods of onset. Results shown in tables I, II, III, IV, V are obtained within 3--5 hours incubation.
326
T. KAMAYA
I. C. albicans and C. steltatoidea Of all 25 strains of human isolation, Candida albicans showed the most striking flocculation and solution clarity (see Fig. 1). This reaction was discernable within 2 hours of incubation, earlier than any other species. Candida stdlatoidea reacted similarly to Candida albicans in its degree of flocculation and its early onset of reaction. II. Candida tr@icalis, Candida pseudotropicalis, Candida krusei Candida tr@icalis Results obtained using Candida tr@icalis were not always consistent, however the majority of cases showed a finely granular flocculation with slight to occasionally moderate clarity of the solution. This species, however, showed less flocculation as compared to C. albicans and C. stellatoidea. Also, the onset of agglutination was much slower except in the very rare case, in which strong Ilocculation comparable to Candida albicans and stellatoidea was observed. Reasons for this phenomenon are not postulated at the present time.
Candida pseudotropicalis and Candida krusei Both species showed considerably less flocculation as compared to Candida albicans and Candida stdlatoidea. In addition, the turbid solution showed no clarity or only slight clarity in 3--6 hours of incubation. In most cases a fine granular dust-like flocculation was observed. III. Candida parapsilosis and Candida guilliermondii Both species showed no clarity of solution nor agglutination from 3-5 hours incubation, however when subjected to more prolonged incubation, showed a very slight agglutination. IV. Saccharomyces species (S. cervisiae gr. 11; S. dlipsoideus; S. steineri. LOI)I)ER & VAN RIJ; S. oviformis; S./lorentinus). Young cultures of these species showed diffuse milky solution with no floccnlation after g hours incubation1). DISCUSSION
The previously described study demonstrates that cells of young cultures of Candida species were agglutinated b y lysozyme solution within 3--6 hours incubation in a 37 ° C water bath. 1) Some s t r a i n s u s e d f r o m v e r y old c u l t u r e s slxowed a slight a g g l u t i n a t i o n . H o w ever, as p r e v i o u s I y m e n t i o n e d , o n l y y o u n g c u l t u r e s s h o u l d be u s e d for t e s t i n g .
FLOCCULATION OF C. ALB. B Y LYSOZYM~
32~
Candida species of human isolation showed different rates of flocculation by different species. Candida albicans and Candida stellatoidea showed the strongest degree of flocculation, as compared to other species of Candida. The degree of flocculation appeared to be progressive with prolonged incubation. However, optimum times were established and should not be exceeded, to ensure reliable consistent results. There are certainly varying maximum rates of flocculation, and varying times of onset of the reaction, by various species. The cells of young cultures of saccharomyces species showed negative flocculation or only faint flocculation. However, old cultures of saccharomyces species showed some agglutination of similar grade as group III. A number of workers (FLEmNG & ALLISON (1922), GOLDWORTHY & FLOREY (1930), GOHAR (1930), THOMPSON (1940) and WEBB (1948)) reported that heat killed cocci were definitely less susceptible than the living organism to the lyric action of egg white lysozyme. In our study strongly positive agglutination and flocculation of C. albicans in lysozyme solution was obtained using the living organism. Heat or formaldehyde killed Candida albicans showed no flocculation or only faint flocculation. The influence of pH on the positive flocculation of C. albicans, C. stellatoidea, and on tile negative flocculation of C. ~ara~silosis and C. guilliermondii was compared before and after incubation at 37 ° C. Both positive and negative species showed a very similar pH range, around 5 to 5.5 before incubation and around 5.5 to 6 after incubation in both positive and negative tests. The determinant causing flocculation was considered to be a factor other than pH. SALTON (1957) speculated that in vitro lysozyme may be attracted by negatively charged groups on the bacterial surface and neutralization would account for the observed agglutination. SKARNIES & WATSON (1955) showed that acidic base complexes by acidic polymers were susceptible to ionic substitution by minute amounts of neutral salts or low ionic strength buffers. BOASSON (1938) suggested that the importance of electrostatic forces in absorption of lysozyme onto the organism, and linked the lysozyme reaction with other immunological reactions. In the present study, the dilution of both lysozyme, and the suspension of Candida cells was made using only distilled water and not physiologic saline solution. In fact, the presence of physiologic saline, inhibited the reaction. As has been shown previously, the various species of Candida showed flocculation to varying degrees and the reaction occurred at varying periods of onset. Results shown in tables I, II, III, IV, V are obtained within 3--5 hours from the start of incubation in the water bath. From the results, the degree of flocculation, and the turbidity of solution enables Candida species of human isolation to be grouped into 3 main groups.
328
T. KAMAYA
GROUP A
Candida Species
Group I Group II Group III
C. C. C. C. C. C. C.
albicans stettatoidea tropicalis krusei pseudotropicalis parapsilosis guilliermo~dii
Decreasing Flocculation
*
In the above grouping a question arises considering the two species C. tropicalis and C. parapsilosis. Candida tropicalis occasionally shows strong flocculation ( + + + ) and clarity of solution ( + + + ) to the same extent as C. albicans and C. stellatoidea. This result cannot be explained. If one considers the factor of strong flocculation, C. tropicalis should belong to group I instead of group II. From the occasional weak agglutination of C. flarapsilosis, perhaps this species should belong to group II. Considering above two points, the following grouping may be possible: GROUP B Group I Group II Group I I I
C. C. C. C. C. C. C.
labicans stellatoidea tropicalis krusei pseudotropicalis parapsilosis guilliermondii
Decreasing Flocculation
,
At the moment the author prefers Group A, however, decreasing positivity in fiocculation phenomenon in these 7 species always occurs in the same order, regardless of how the organisms are grouped. (1) C. albicans (3) C. tropicalis (5) C. pseudotropicatis (7) C. guilliermondii
(2) C. stellatoidea (4) C. krusei (6) C. parapsilosis
Saccharomyces species may be placed after these seven species. STANLEY & HURLEY (1967) separated the Candida species of human isolation into three groups according to the difference in their rate of destruction of epithelial cells. Group I - - c. albicans, C. stellatoidea and C. tr@icalis destroyed the culture faster and produced more mycelia than did Group II - - C. krusei, C. parapsilosis, C. pseudotropicalis. Group I I I - - c. guilliermondii was the least toxic to the cultured epithelial cells and rarely produced mycelium. The authors suggested that a correlation existed between (1) the rate at which the Candidas kill the cultured cell in vitro, (2) their frequency of isolation from disease in man and (3) their pathogenicity in laboratory animals. A parallel relationship exists between the grouping according to the destruction of the epithelial cells by various Candida species and
FLOCCULATION OF C. ALB. BY LYSOZYME
329
the grouping of the present study, by flocculation in lysozyme distilled water solution. Since lysozyme is considered to be involved in one of the most important defence mechanisms of the human body (FLEMING 1922, DUBOS 1946, AMANO et al. 1954, SKARNES & WATSON 1955). The close correlation between the two previously mentioned phenomena may have some significance in understanding the host-parasite relationship in Candidiasis.
Zusammenfassung J u n g e Kolonien y o n Candida Arten, auf Sabouraud's Glucose Agar bei Zimmertemperatur, isoliert v o m Menschen, w u r d e n in destilliertem Wasser verteilt. Die Suspension wurde m i t einer L6sung y o n L y s o z y m gemiseht u n d bei 3 7 ° C im ~Vasserbad inkubiert. Innerhalb y o n 3 - - 5 S t u n d e n h a b e n verschiedene Arten VOlt Candida-Zellen in verschiedenem Grade Flocculation gezeigt, dereI Beginn zu verschiedenen Z e i t p u n k t e n erfolgte. Unter sieben Candida Arten zeigten Candida albicans u n d Candida stellatoidea die st~rkste Floeeulation, den frfihesten Begilm u n d gr613te Durchsichtigkeit der L6sung, d e n n irgendwelche andere Arten, C. stellatoidea konnte m a n y o n C. albicans in der R a t e der Flocculation u n d in d e m Grade der Durchsichtigkeit der L6sung nicht unterscheiden. Candida Arten k 6 n n e n in der folgenden O r d n u n g gem~B der a b n e h m e n d e n Positivit~t eingereiht werden: C. albicans, C. stellatoidea, C. tropicalis, C. hrusei, C. pseudotropicalis, C. parapsilosis, C. guilliermondii, Saccharomyces Arten. Es ist m6glich, die Candida Arten gem~B der R a t e der Flocculation u n d der Durchsiehtigkeit der L 6 s u n g in drei Gruppen einzuordnen. Gruppe 1: C. albicans and C. stellatoidea; Gruppe 2: C. tropicalis, C. krusei und C. pseudotropicalis; Gruppe 3: C. parapsilosis u n d C. guillier~nondii. Saccharomyces Arten (S. cerevisiae u n d andere) w a r d e n n a c h der Gruppe 3 eingeordnet.
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