toxin and hemolysin, there is now ample evidence to show be distinguished from the ...

18 Pages·2007·1.16 MB·English

OBSERVATIONS ON HEMOLYSIN PRODUCTION BY THE
STREPTOCOCCI.*
HAROLD W. LYALL.
(From theDepartmentofBacteriology, Hoagland Laboratory, Brooklyn, N.Y.,
andtheBacteriologicalLaboratoryofBrown University,Providence, R.Z.)
Despite extensive study, our understanding of the factors
responsible for the grave toxic conditions arising from strep-
tococcal infections is stillfar from satisfactory. The toxemia
has generally been attributed to: (i) the elaboration of a
true soluble toxin, and (2) the production of an hemolysin.
The feeble action of the soluble toxin produced in vitro has
failed to explain the severe toxic effects so often manifested
in what appears to be only a mild and purely local process.
Although no sharp distinction was made formerly between
toxin and hemolysin, there is now ample evidence to show
that these are two separate and distinct substances. In their
behavior toward heat the relatively thermostabile toxin may
be distinguished from the thermolabile hemolysin, which is
readily destroyed at a temperature of 560 C. for thirty
minutes. The relation of the hemolysin to the general
toxemia and the extent to which this factor is responsible for
the pathogenesis of streptococcic infections, has attracted
little attention from either bacteriologists or clinicians. The
recent work of M'Leod and M'Nee represents the most care-
ful study ofthe pathological processes produced by strepto-
coccushemolysin. Workingwith actively hemolyticfiltrates,
they have been able toshow that such filtrates, in rabbits, are
capable of producing a marked hemoglobinemia and hemo-
globinuria, and, in some cases, even death. They were also
able to show that the disappearance of the hemolytic power
ofthe filtrate is accompanied by the loss of its toxic action.
Inasmuch as this is accomplished after forty-eight hours at
370 C., a period and temperature not sufficient to materially
*ReceivedforpublicationJune8,I914.
(515)
SI6 LYALL.
impair the potency of any true toxin, it seems to emphasize
the importance of hemolysin in producing pathological
changes.
With aviewto discovering any possible correlation between
hemolysin production and pathogenicity, determinations of
the hemolytic titerwere made with several strains. Thistitra-
tion consisted in adding decreasing amounts of an eighteen-
hour, two per centpeptone, ascitic broth culture to a constant
quantity ofwashed sheep's red blood cells (one cubic centi-
meter of a five per cent suspension). These tubes were
incubated in a water-bath at 37.50 C. for one hour and read-
ings made at the endofthattime. Itispossiblein this wayto
determine accurately the minimum amount ofany given cult-
ure capable of producingcomplete solution of the blood cells.
This amount may be considered as representing one hemo-
lytic unit. The results do not afford any very marked evi-
dence of distinct differences according to the source of the
strain. Table I. presents these results:
OBSERVATIONS ON HEMOLYSIN PRODUCTION. 517
TABLE I.
Culture. Source. HemolyticTiter.
1II./2 ................. Sputum -bronchiectasis. 0.05
A 38 .................. Gland - septic sore throat. 0.2
A39 .................. Ear " " " 0.I
A40 .................. 0.1
A 33 .................. Mastoiditis. 0.1
A35 .................. Blood- post-partemsepsis.' 0.1
A42 .................. Mastoiditis. 0.1
A 67 .................. Throat- acutetonsilitis. 0.2
A68 .................. Spinal fluid- meningitis. 0.I
A 73 .... .......... Cellulitis. 0.05
BV. .................. Uterus- post-partem sepsis. 0.03
B VI.................. Blood " " " 0.02
B VIII................. Blood " " " 0.I
BX. .................. Ear-otitis media. 0.02
BXIII................. Spinal fluid-meningitis. 0.2
B XIV................. Abscess -knee. 0.4
BXV.................. Multiple furunculosis. 0.3
A83 .................. Blood - septic sore throat. 0.4
BXXX................ Mastoiditis. 0.2
B XXVII.............. Nose -sinusitis. 0.4
B XXVIII.............. Ear-mastoiditis. 0.I
B XXXII............... Gland -suppurative adenitis. 0.2
It does not appear justifiable, from the above titrations, to
draw any distinctive conclusions as to the relationship
between hemolytic titer, virulence, and the severity of the
disease evoked by of the strains. It be
process any can
pointed out that, in general, the strains isolated from the
blood show a relatively high titer. Strain A 83, with the
5I8 LYALL.
comparatively low titer of .4, was isolated from the blood of
a patient during a severe attack of septic sore throat. A
subsequent culture taken within a week failed to show the
presence of bacteria in the blood, and the patient made a
rapid recovery. It is reasonable to infer, in this case, that
the presence in the blood represented merely a temporary
invasion of the organisms responsible for the throat condi-
tion. This may account for the low titer. Strain III/2 from
bronchiectatic sputum and Strain A 73, which was respon-
sible for a very swift cellulitis with subsequent bacteremia
and death, would clinically be considered marked opposites.
The fact that they possess the same hemolytic titer is against
using the hemolytic titer as a positive criterion for virulence.
Although the nature of the hemolysin produced by several
other bacterial species has been investigated in detail, in the
case of the streptococci, this phenomenon has been accu-
rately studied by only a few investigators. Marmorek was
the first to observe this characteristic for streptococci. He
was able, as previously described,to detect hemolysis in vivo
as well as in vitro. Attempts to produce anti-hemolysin in
animals failed. This is true of the results of all subsequent
endeavors to produce, artificially, a specific antibody against
streptococcic hemolysin. M'Leod and M'Nee have shown
that no immunity is produced by repeated injections of
streptolysin, but that, on the contrary, an increased suscep-
tibility may result. The greater part of the work on this
subject, up to the last few years, has not been to any degree
quantitative, and therefore is open to criticism. This, how-
ever, does not apply to the work of Braun, von Hellens, and
M'Leod and M'Nee. The work of these authors has added
much to our knowledge of the hemolysin produced by
streptococci. The observations of Braun and M'Leod
appear to demonstrate conclusively the filterability of the
streptolysin, thereby establishing it as a true secretion prod-
uct. This has been a mooted question and as late as I908
Pribram and Russ, in their review of the bacterial hemoly-
sins, conclude that the streptococci belong to that class
"which does not produce a true hemotoxin, but which,
OBSERVATIONS ON HEMOLYSIN PRODUCTION. 519
under certain conditions, is able to dissolve red blood cells."
The above authors did much to further our knowledge of
the nature of streptolysin, the conditions of optimum pro-
duction, and other factors necessary for a more complete
understanding of this relatively little understood phenome-
non. Previous to the appearance of their publication, the
writer was engaged in a study of some of the features of
hemolysin production bystreptococci. The results presented
below confirm, in part, the earlier results and, in addition,
afford new data.
The successful production ofa potent hemolysin in broth
cultures ofstreptococci is dependent on several factors:
I. Culture media.- (a) Enrichment: The original
observations of Marmorek showed that some enriching sub-
stance was necessary for hemolysin production in order to
insure constant and reliable results. According to M'Leod,
nutrient bouillon with the addition of fifteen per cent sterile
horse serum is the best medium for securing a potent hem-
olysin. Owing to the difficulty, in the present investigation,
of securing horse serum, tests were carried out with human
blood serum, ascitic, and hydrocele fluid, and also with
rabbit, sheep, and guinea-pig serum, to determine which
would offer the best medium. Of these, human ascitic or
hydrocele fluid yielded the best results. Optimum produc-
tion was found to occur in broth containing from fifteen to
twenty per cent of the enriching fluid. The enriching prop-
erty of this material was found to deteriorate very little with
age, and separate specimens showed practically no variation
in their ability to furnish substances suitable for maximum
hemolysin production. Media containing two per cent
peptone possesses an advantage over ordinary nutrient
bouillon containing only one per cent ofthis material. (b)
Reaction: The optimum reaction was found to be at a
point distinctly alkaline to litmus, or about + .3 to phenol-
phthalein. To test the effect of acids or alkalis onhemolysin
production, varying amounts of normal hydrochloric acid
5:20 LYALL.
and sodium hydroxide were added to two per cent peptone
broth and the amounts of hemolysin produced in each
accurately titrated. Very little difference in the titrations
were noted between +2 and -2. Beyond these points the
content of acid or alkali had a marked inhibitory effect on
hemolysin production. From some experiments, to be cited
later, it would appear that acid formed as a result of the
growth ofthe streptococci has a different effect. However,
in order to minimize this factor, it was considered advisable
to add a piece of marble to each culture tube in order to
neutralize any excess offree acid formed during incubation.
Testing the effect ofthe salt content it was found that over
two per cent inhibits hemolysis. Certain sugars were also
found to cause inhibition. Their action will be discussed
later. Hemolysin production was unaffected by anaerobic
cultivation.
2. Incubation.- The length of incubation was found to
constitute an important factor. Hemolysin production could
be demonstrated in cultures three hours after inoculation, and
increased progressively until the twelfth hour when a maxi-
mum was obtained. From the twelfth to the eighteenth hour
the curve remains fairly level and then begins to drop, dis-
appearing rapidly until it entirely disappears after thirty-six
to forty-eight hours at 370C. The amountof culture used for
inoculation presumably influences the rapidity with which
the initial appearance occurs, and possibly also the height
ofthe curve attained. The disappearance of the hemolysin
is not, however, correlated with the loss of viability of the
culture. Cultures in calcium carbonate broth will remain
alive for at least six days at incubator temperature, although
all hemolytic activity has disappeared. Streptococci grown
on solid media are able to retain this property for a con-
siderable time. Some strains studied in the present investi-
gation not only showed no diminution in their hemolytic
activity when cultured for a period of over two years but
actually exhibited an increase.
OBSERVATIONS ON HEMOLYSIN PRODUCTION. 521
The discussion concerning the filterability ofthe hemolysin
produced by streptococci has already been referred to.
Because oftheir inability to secure a hemolytic filtrate, Pri-
bram and Russ are disinclined to classify the streptococci
among those organisms capable, in vitro, ofproducing a true
hemotoxin. Recently, M'Leod and Braun have been able
to secure, with great regularity, potent hemolytic filtrates.
They offer, as an explanation of previous failures, the fine
grade offilter used and the necessityof using a medium con-
taining a relatively low percentage ofserum. In the present
investigation, filtration through the coarse grade (N) of
Berkefeld filters was attempted with negative results. Unfor-
tunately, it has not been possible totest the Maasen filter as
recommended by M'Leod. If, however, the hemolytic prod-
uct will not pass through a coarse grade Berkefeld candle,
this would appear to argue for the close association of this
substance to thebacterial cells, and against its beingin a true
and permanent solution as we commonly understand this
term. To further test this point, the following experiment
was carried out with a strain capable of producing a very
potent hemolysin. The surface growth of four tubes of an
eighteen-hour culture onplainagar (+.3 tophenolphthalein),
after pipetting off any water ofcondensation, was emulsified
in five cubic centimeters of isotonic salt solution (i). This
was then centrifugalized for twelve minutes at a speed of
three thousand revolutions per minute and the supernatant
liquid carefully withdrawn (2); the sediment was then
thoroughly emulsified in 4.5 cubic centimeters of isotonicsalt
solution (3); whirled again (supernatant No. 4) and the
resulting sediment emulsified in fourcubic centimeters ofsalt
solution (S). One-half cubic centimeter of eachofthe above
designated fluids was then added to one cubic centimeter of
a five per cent suspension of sheep's cells and incubated for
one hourin a water bath at 37.50 C. withthe followingresults:
522 LYALL.
Tube. OneHourat37.50C. OverNightonIce.
I.................... + + + + + +
2.................... ± i
3.................... 0 +
4.................... 0 0
5.0 ±........
It will be seen that, although the original emulsion was
actively hemolytic, the supernatant fluid resulting after the
first centrifugalization was only weakly hemolytic. The sus-
pension ofthe sediment resulting from the first whirling was
also weakly hemolytic and the subsequent supernatant failed
entirely to hemolyze. Thethird emulsion showed hemolysis
over night on ice. Centrifugalization ofa broth culturegave
comparable results, whereas .02 cubic centimeter,before cen-
trifugalization, produced hemolysis in one cubic centimeter
of cells; it required .3 cubiccentimeterto producethe faintest
trace afterward.
Regarding the exact nature of the streptolysin little is
known. Von Lingelsheim, in Kolle-Wassermann, inclines
to the view that it most nearly approaches the nature
of an enzyme, but this has not been definitely proved.
In order to study the possible enzymatic nature of this sub-
stance, observations were made on the effect of chloroform,
toluol and formalin on streptolysin. Chloroform and toluol,
although capable of destroying bacterial life, should not, in
the proportions used, affect the activity of any enzyme
present. To tubes of actively hemolytic cultures, the above
chemicals were added in the following proportions: formal-
dehyde one to two thousand, chloroform one to ten and
toluol one to twenty. The tubes were thoroughly shaken
and allowed to stand in the ice-box, together with control
tubes. After contact with the above substances for as short
a period as three hours, the hemolysin had completely dis-
appeared, whereas the control tube gave approximately its
OBSERVATIONS ON HEMOLYSIN PRODUCTION. 523
original titer. Subcultures from the tubes containing the
added chemicals proved sterile. From this it is reasonable
to assume that the disappearance of hemolysin is to some
extent associated with the loss ofviability ofthe organism.
Distinguished from the true soluble toxin of the strepto-
coccus, the hemolysin is distinctly thermolabile. It is
completely destroyed by thirty minutes' exposure to a tem-
perature of 560 C. In order to determine the length of time
which the hemolysin persists at various temperatures, a series
of tubes of an actively hemolytic strain, after a preliminary
incubation period, was divided into three lots and kept
respectively at 37.50 C., at room temperature, and in the ice-
box. The following chart shows the effect ofthese tempera-
tures on the persistency ofhemolysin:
524 LYALL.
CHART 1.
o6/' ___\_\
_ ___ ___
6.3 I_
NJo.oldays 53
1 2. i Is
Therapidisappar __f_hemol
perature has been discussed previously. The curves for
room and ice-box temperature, it will be seen, compare
closely except for the fact that the hemolytic titer of the
cultures kept in the ice-box does not suffer as sudden loss as
it does in those at room temperature. Tubes kept in the