and its Abolition by Iron Compounds

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Immunology, 1971, 20, 391.
The Bacteriostatic Effect of Serum on Pasteurella septica
and its Abolition by Iron Compounds
J. J. BULLEN, HENRYJ. ROGERS ANDJ. E. LEWIN
National Institutefor Medical Research, Mill Hill, London, N.W.7
(Received 11th June 1970)
Summary. Methods are described for controlling the oxygen tension and pH of
bacterial cultures in vitro. In fresh normal rabbit serum or plasma at a Po2 of
80-90 mmHg and a pH of 7-5, the presence ofspecific antibody has a powerful
bacteriostatic effect against Pasteurella septica. It is suggested that the serum com-
ponents involved in this reaction consist of transferrin, specific antibody, and
complement. Bacteriostasis can be abolished by a variety ofiron compounds. This
evidence together with that obtained in vivo (Bullen, Wilson, Cushnie and Rogers,
1968a) strongly suggests that an interference with bacterial iron metabolism plays
an important role in resistance to infection against P. septica.
INTRODUCTION
There is now good evidence to suggest that interference with bacterial iron metabolism
by the host may play an essential role in resistance to infection. Rogers (1967) showed that
the bacteriostatic effect ofserum against Clostridium welchii type A could be attributed to
the iron binding protein transferrin acting in concert with /32 or y-globulin. Bullen and
Rogers (1969), in a preliminary report, suggested that transferrin, specific antibody, and
complement were involved in the bacteriostatic reaction ofserum against P. septica and
that transferrin and other components of serum were also involved in the bactericidal
and bacteriostatic effects of sera on Escherichia coli. The significance of these results lies
in thefactthattheycan becorrelated with experiments invivowhereithas beenshown that
a variety ofiron compounds can abolish passive immunity to Cl. welchii (Bullen, Cushnie
and Rogers, 1967) and P. septica (Bullen et al., 1968a) and greatly enhance the viru-
lence ofE. coli (Bullen, Leigh and Rogers, 1968b).
In the present paper we describe the methods used for carrying out the bacteriostatic
tests in vitro, together with further observations on the effect ofiron compounds on bac-
teriostatic reactions ofserum against P. septica.
MATERIALS AND METHODS
Pasteurella septica
P. septica strain CN3449 (Wellcome Research Laboratories) previously used for experi-
ments in vivo (Bullen et al., 1968a) was also used for all the experiments in vitro. The
methods used for passage and storage have been described previously (Bullen et al.,
1968a). For experiments in vitro the culture was sown by an automatic inoculator at
6 a.m. into papain digest broth containing 1 per cent of yeast extract (Difco). Three
391
392 J. J. Bullen, H. J. Rogers andJ. E. Lewin
hours later 2 ml ofthe culture was sown into 20 ml ofyeast broth and incubated at 370
for a further 1l hours. This gave viable counts of approximately 1 x 109/ml. Suitable
dilutions were made in 10 per cent broth saline and viable counts on fresh blood agar
plates made in the usual way (Bullen, Wilson and Cordiner, 1961).
Rabbit serum
Normal rabbits were bled from an ear vein into a glass container lined with paraffin
wax. The clotted blood was incubated for 2j hours at 370 and thenkept atroomtempera-
ture for a further 1-1 hours. The serum was separated by centrifugation, sterilized by
filtration through an Oxoid cellulose acetate membrane, and stored at -700. This
technique gave serum containing a minimum ofhaemoglobin.
Rabbitplasma
Normal rabbits were anaesthetized with fluothane and nitrous oxide. The right carotid
artery was exposed and ligated. The artery was severedjust proximal to the ligature and
the blood collected in siliconized glass vessels containing heparin to give a final concen-
tration of0a15 mg/ml ofblood. The plasma was separated bycentrifugation, sterilized by
filtration through an Oxoid cellulose acetate membrane and stored at -700. A pooled
sample ofplasma which contained 1-3 mg ofhaemoglobin/litre was used for a number of
experiments. Haemoglobin was estimated by a modification of the method of Hanks,
Cassel, Rayand Chaplin (1960). o-Dianisidine (0l percent) wasusedinsteadofbenzidine
and the optical density was measured at 450 nm, 20 minutes after the addition ofH202.
Rabbitplasma serum
Platelet free plasma serum was prepared by the method ofHirsch (1960). Siliconized
centrifuge tubes (15 ml) were placed in 50 ml centrifuge buckets containing water which
was then frozen. Blood was collected from an ear vein directly into the cold tubes. After
centrifugation at 4000 rev/min for 20 minute at 00 the unclotted plasma was removed and
incubated at 370 for 1-1 hours. After the plasma had clotted the clear plasma serum was
separated by centrifugation and stored at -700.
Normal human serum
A pooled sample ofnormal human serum was sterilized by filtration through an Oxoid
cellulose acetate membrane and kept at -700.
Human transferrin
Human transferrin was prepared by the method ofRogers (1967).
Normal goat serum
A normal goat was bled from thejugular vein and the serum separated by the same
technique used for rabbit serum. This was kept at -700.
Normal horse serum
Normal horse serum was obtained from the Wellcome Research Laboratories and kept
at -700.
Bacteriostatic Effect ofSerum on Pasteurella septica 393
P. septica horse antiserum
Horse antiserum Pony PS1 was the same as that used previously for experiments in vivo
(Bullen et al., 1968a).
Clostridium welchii horse antiserum
This contained Cl. welchii alpha antitoxin 770 units/ml, theta antitoxin 146 units/ml,
and kappa antitoxin 1100 units/ml.
Ferric ammonium citrate
This contained 20percentw/wiron estimated colorimetrically bythemethodofCollins,
Diehl and Smith (1959): 0-625 g was dissolved in 100 ml of 0-85 per cent NaCl and
sterilized by autoclaving at 15 lb for 15 minutes.
Haematin hydrochloride
Haematin hydrochloride (British Drug Houses Ltd) was ground in a ball mill in water
(25 ml) for 3 hours. After centrifugation the water was removed and the haematin placed
in ethyl alcoholfor 30 minutes. The mixture was again centrifuged, the alcohol discarded,
and the haematin dried in a desiccator over CaCl2.
Crystalline human haemoglobin
Crystalline human haemoglobin was prepared by the method ofDrabkin (1949). The
crystals were dissolved in 0-85 per cent NaCl and sterilized by filtration through a Seitz
EK pad.
Technique in vitro
The glass culture vesselillustrated in Fig. 1 contained 8 ml ofserumin theinner conical
container. The water circulating in the outer jacket was pumped from a water bath
(Grant Instruments Ltd) maintained at 37-3+00010. This gave a temperature of37*0 in
the serum. The oxygen electrode (Oxygen macro electrode, Beckman Instruments Inc.)
was inserted through a silicone rubber bung (Esco Rubber TC156). Before insertion the
electrode tip was cleaned, moistened with electrolyte (Beckman Instrument Inc) and
covered with a polypropylene membrane previously sterilized by U.V. irradiation. The
membrane was held in position by an 0 ring cut from Esco silicone rubber tubing and
was of sufficient size to completely cover the electrode barrel, the edge of the mem-
brane being squeezed between the top ofthe barrel and the silicone bung.
The silicone bung also accommodated three 14 gauge stainless steel tubes, one for the
admittance of5 per cent CO2 in air, or 5 per cent CO2 in nitrogen, the second for 5 per
cent CO2 in oxygen, and the third for the exit of gas and for sampling. Samples were
removed with a 1 ml tuberculin syringe fitted with a 9 5 cm 20 gauge needle. The serum
was stirred with a plastic coated magnetic follower. The culture vessel was clamped on to
a magnetic stirrer, the clamp being arranged to hold the silicone bung in the neck ofthe
vessel to form a gas tight seal.
Control ofoxygen tension
The electronic system for controlling the oxygen tension in the culture vessel is shown
diagramatically in Fig. 2.
I
394 J. J. Bullen, H. J. Rogers andJ. E. Lewin
6
3 JJ1 4 5
2
I
water
*inlet
FIG. 1. Culturevessel. (1) Glasscontainer; (2) siliconerubberbung; (3) C02/N2 inlet; (4) C02/02
inlet; (5) gasexit; (6) oxygenelectrode; (7) magneticstirrer; (8) serum8-9ml; (9) circulatingwater
at 370.
02+Co2
-* TOVESSEL
DILUENT_ N2+CO
FIG. 2. Diagramofsystemforcontrolof02tension. The electrode (02) is situated in culture vessel.
(SeeFig. 1 and text.)
The oxygen concentration was monitored as a current flowing through a polarographic
electrode (Oxygen macro electrode, Beckman Instruments Inc.) mounted in the culture
vessel (Fig. 1). This electrode required a polarizingpotential of750 mVwhichwas derived
from a long-life battery and was measured on a front panel meter. The current flowing
was measured as a voltage drop across a series resistance which could be switched to
Bacteriostatic Efect ofSerum on Pasteurella septica 395
produce some 3 mV, at the input ofan electrometer amplifier (Vibron 33B.2, Electronic
Instruments Ltd). Itwas important that this voltage drop was kept small so that the effec-
tive polarizing potential did not change significantly over the range of oxygen concen-
trations to be measured. The amplifier output was recorded continuously on a potentio-
metric strip chart recorder (Electronic Mark 3, Kent Instruments Ltd) fitted with a slave
cam carrying control contacts, which could be set to open at any position on the scale
corresponding to the required upper limit ofoxygen concentration.
The oxygen flow rate was controlled by adjusting the proportion oftime during which
a solenoid valve in the flow line from the oxygen balloon was held open. The rate of
operation ofthisvalvewas controlled byaunijunction transistoroscillatorwhichproduced
pulses at a rate ofbetween 5 and 100 per minute. The pulses were used to trigger a mono-
stable vibrator which controlled the interval during which the valve was held open;
this varied between 100 milli-seconds and 1 second. These pulses were fedvia the recorder
contacts to the base ofa switching transistor which energized the solenoid valve.
By selecting a rapid succession of relatively short bursts of oxygen it was possible to
prevent overshoot caused by flushing the vessel with a high concentration of oxygen.
Theresolutionofthesystemwaslimitedonly bydiffusiontimeconstantswithintheculture
medium. In practice the Po2 could be controlled within 10 mmHg provided the rate of
drift ofthe oxygen electrode did not exceed 0-1 mV during the course ofan experiment
(usually 6 hours).
For each experiment the oxygen electrode was calibrated by passing 5 per cent CO2
in air over the surface ofthe serum which was stirred continuously. A steady reading was
obtained in 15-30 minutes. The slave cam on the recorder was then set to open at the
required upper limit of oxygen concentration with a resistance selected to give a con-
venient recording on the 10 mV scale on the Vibron electrometer. The serum was inocu-
lated with the bacteria as soon as the oxygen tension was controlled at the required figure.
Control ofpH
The pH was controlled by the presence of 5 per cent CO2 in the gasses (air, 02 or
N2) passing over the surface ofthe serum. The serum was exposed to 5 per cent CO2 for
l-Il hours before inoculation. With rabbit serum a concentration of5 per cent CO2 gave
apH of7'50. Thiswasmeasured bythetechnique described byDobson and Bullen (1964).
In some experiments a concentration of25 per cent CO2 was used giving a pH of6-85.
RESULTS
GROWTH OF P. septica IN NORMAL RABBIT SERUM OR PLASMA
At a Po2 of80-90 mmHg, pH 7-5, P. septica grew rapidly in fresh normal rabbit serum
or plasma with a generation time ofapproximately 25 minutes (Fig. 3).
THE EFFECT OF P. septica ANTISERUM
AtaPo2 of80-90mmHg, pH 7*5, thepresenceof15percentP. septicaantiseruminfresh
normal rabbit serum, heparinized plasma, or platelet free plasma serum had a well-
marked bacteriostatic effect. Immediately after inoculation there was a sharp fall in the
viable count. This was followed by some regrowth between the 1st and 4th hour but
396 36. 3. Bullen, H. 3. Rogers andj. E. Lewin
after the 4th hour the growth of the organism was inhibited and this persisted for at
least 10 hours (Fig. 3). A concentration of5 per cent antiserum did not produce bacterio-
stasis, butthe growth ofthe bacteriawas much slower than normal with ageneration time
ofapproximately 1 hour (Fig. 3).
9
8
7
0'
0'0 5
0
4
3
0 2 4 6 8 10
Hours
FIG. 3. The effect ofantibody on thegrowth ofP. septica in normal rabbit serum. A, No antiserum.
A, 15percentCl.welchiiantiserum. 0,5percentP.septicaantiserum.0, 15percentP.septicaantiserum
(Po2 80-90 mmHg,pH7.5).
THE EFFECT OF HETEROLOGOUS ANTISERUM
The presence of15 per cent Cl. welchii antiserum had no inhibitory effect on the growth
ofP. septica; the rate ofgrowth being identical to that seen in normal rabbit serum (Po2
80-90 mmHg, pH 7.5) (Fig. 3).
THE EFFECT OF PH ON BACTERIOSTASIS
In the presence of 15 per cent P. septica antiserum well-marked bacteriostasis occurred
when the concentration ofCO2 in the gas phase was 5 per cent (Po2 80-90 mmHg). This
gave a pH of7-5 in the serum. When the concentration ofCO2 was increased to 25 per
cent, giving a pH of6-85 in the serum, the bacteriostatic effect was lost (Fig. 4).
Bacteriostatic Effect ofSerum on Pasteurella septica 397
6
-5
-o
0
2 4 6 8 10
0
Hours
FIG. 4. The effect ofpH on bacteriostasis. 155 per cent P. septica antiserum in normal rabbit serum
(Po2 80-90 mmHg). 0, pH7.5; 0, pH6-85.
6
-5
E
004
._Q
9
31
- 0 2 4 6 8 10
Hours
FIG. 5.Theeffectofheatingnormalrabbitserum. 0, 15percentP.septicaantiseruminnormalrabbit
serum. *, 15 percentP.septica antiserum inheatedrabbitserum (Po2 80-90 mmHg,pH 7 5).
THE EFFECT OF HEATING RABBIT SERUM
Thepresence of15percentP. septicaantiseruminrabbitserumthathadbeenpreviously
heated to 560 for 30 minutes had no bacteriostatic effect (Po2 80-90 mmHg, pH 7.5)
(Fig. 5). This showed that a heat labile factor in rabbit serum, whichwas assumed to be
complement, was essential for bacteriostasis.
398 3. J. Bullen, H. J. Rogers andJ. E. Lewin
THE EFFECT OF IRON COMPOUNDS
(1) Ferric ammonium citrate. At a Po2 of80-90 mmHg, pH 7 5, the addition ofsufficient
ferric ammonium citrate to provide a concentration ofFe in the serum twice that ofthe
total iron binding capacity ofthe transferrin (200 per cent saturation) resulted in a rapid
bacterial growth, but only after a delay of 6 hours (Fig. 6). The response to iron was
strongly influenced by the oxygen tension of the medium. In the experiment shown in
Fig. 7 the oxygen tension ofthe medium was reduced to zero; the pH was kept at 7-5. The
oxygen was removed by passing 02 free gas (5 per cent CO2 in N2) over the surface ofthe
serum. When the 02 tension had fallen to approximately 10 mmHg the last traces of02
wereremoved byaddingascorbic acidtoafinalconcentrationof0-25percent. Underthese
7
6
05-D
0~~~~~~~~~
40
0 2 4 6 8 10
Hours
FIG.6. TheeffectofsaturatingserumtransferrinwithFe3+. 15percentP.septicaantiseruminnormal
rabbitserum(Po280-90mmHg,pH7.5). o,NoFeadded; *,serumtransferrin200percentsaturated
with Fe3+.
circumstances the bacteriostatic power of 15 per cent P. septica antiserum was unaffected,
but the addition ofsufficient ferric ammonium citrate to saturate the transferrin to either
90 per cent or 200 per cent ofits total iron binding capacity resulted in rapid bacterial
growth after a delay ofonly 1 hour (Fig. 7).
(2) Haem compounds. The addition ofwashed lysed rabbit red cells to give a final con-
centration of 1-2 mg ofhaemoglobin/ml rapidly abolished the bacteriostatic effect of 15
per cent P. septica antiserum in fresh normal rabbit plasma (Po2 80-90 mmHg, pH 7.5)
(Fig. 8).
Finely powdered haematin (1 mg/ml) and crystalline human haemoglobin (3 mg/ml)
were also effective in abolishing bacteriostasis although the bacterial growth was not quite
so fast as with lysed red cells (Fig. 8). The haem compounds behaved differently from
ferric ammonium citrate in that bacterial growth occurred without delay at the relatively
high PO2 80-90 mmHg.
Bacteriostatic Effect ofSerum on Pasteurella septica 399
THE EFFECT OF DELAY IN ADDING IRON COMPOUNDS
Rogers (1967) found that saturation ofthe serum transferrin with iron at 3 hours after
inoculation with Cl. welchii type A failed to abolish bacteriostasis, although it hadjust this
effect ifadded with the bacteria at 0 hours. This suggested that during the first 3 hours in
serumthe bacteria underwentsomeirreversible changewhichprevented themfromtaking
up ferric iron after this period. Similar experiments were done with P. septica but in view
ofthe long delay in the response to iron added with the bacteria at 0 hours that occurs at
a Po2 of 80-90 mmHg, pH 7-5 (see Fig. 6) it was essential to allow the experiments
A
6-
5-
0.
-D
0
03-
.23
0 2 4 6 8
Hours
FIG. 7. Theeffectofsaturatingserumtransferrinwith Featzero02tension (Po2OmmHg;pH7-5).
15percentP.septicaantiseruminnormalrabbitserum.0,noFeadded;*,serumtransferrin200percent
saturatedwithFe; A,serumtransferrin90percentsaturatedwithFe.
to run for a long period. In the control experiment without additional Fe, normal rabbit
plasma with 15 per cent P. septica antiserum was inoculated with 2-5x 103 organisms/ml
and a viable count made after 20 hours incubation at a Po2 of80-90 mmHg, pH 7.5. By
this time the viable count had reached 1-5x 105/ml. In a second experiment underidenti-
cal conditions sufficient ferric ammonium citrate was added after 3 hours to provide a
concentration ofFe twice that ofthetotal iron binding capacityoftheserum (200percent
saturation). At 3 hours the viable count was 4-5x 102/ml but had risen to 7-3x 109/ml at
20 hours. Thus P. septica was apparently capable ofacquiring Fe from the environment
when it was present in large excess even though the bacteriostatic reaction had been
allowed to proceed for 3 hours beforehand. In the case ofhaem compounds there was no
delayintheresponsewhenlysedredcellswereaddedafter3hourstogiveafinalconcentra-
tion of 1-2 mg haemoglobin/ml (Po2 80-90 mmHg, pH 7.5) (Fig. 9).
400 J. J. Bullen, H. J. Rogers andj. E. Lewin
EXPERIMENTS WITH SERA FROM OTHER SPECIES
In pooled normal human serum there was an initial period of2-3 hours during which
the viable count fell by 10-50-fold. Thereafter growth occurred slowly with a generation
time ofapproximately 1 hour. P. septica grew rapidly in fresh normal horse or goat serum.
In sera from both these species the presence of 15 per cent P. septica antiserum (Po2
80-90 mmHg, pH 7-5) had a powerful bactericidal effect, the viable count falling from
8
7
6
E5
-40Dc0)4
3
2
0 2 4 6 8 10
Hours
FIG.8.Theeffectofhaemcompoundsonbacteriostasis. 15percentP.septicaantiseruminnormalrabbit
plasma(Po280-90mmHg,pH7.5). o,Nohaemcompoundsadded; *,lysedredcells 1-2mgHb/ml;
0, crystallinehumanhaemoglobin 3mg/ml; A,haematin 1 mg/ml.
1.6-5.3x 103 organisms tozero in 2 hours. Five per centP. septica antiserum in goat serum
produced a bacteriostatic reaction resembling that seen in rabbit serum containing 15 per
cent antiserum (Fig. 10). Bacteriostasis produced with 5 per cent P. septica antiserum was
fairly rapidly abolished by the presence ofhaematin (1 mg/ml) (Fig. 10).
THE EFFECT OF TRANSFERRIN ON THE GROWTH OF P. septica
Human transferrin stabilized bythe additionofasmall amount ofalbumin inhibits the
growth of Cl. welchii (Rogers, Bullen and Cushnie, 1970). This does not occur with P.
septica. The organism grew well in a semi-synthetic medium (Rogers, 1967) containing