Note: Descriptions are shown in the official language in which they were submitted.
1;~738~7
The invention relates to a method of submersely
growing Pseudomonas aeruginosa bacterial strains of the
taxonomic familiy of the Pseudomonadaceae.
Bacterium Pseudomonas aeruginosa is opportunistic
pathogen which often occurs with hospital infections mainly
patients having a weakened immune defense, such as patients
suffering from burns, persons suffering from cystic
fibrosis or having defective organic functions, and pa-
tients suffering from tumours. Due to the occurrence of
resistances, antibiotics are active against Pseudomonas
infections to a limited extent only, and therefore attempts
are made to fight infections caused by Pseudomonas aerugi-
nosa by immunological methods.
Infections may be triggered by a plurality of strains
producing 0-group antigens and H-antigens. According to the
H-antigen pattern according to Ansorg (ZBl. Bakt. Hyg. I.
Abt. Orig. A 242, 228-238 (1978)), by using the indirect
immunofluorescence technique, with Pseudomonas aeruginosa
there is differentiated between a complex flagellar antigen
a having the partial antigens ao, al, a2~ a3/ a4, and a
uniform flagellar antigen _. The partial factors ao - a4
are independent determinants, a flagellar antigen pattern
having several H-types resulting 0-groups and H-type show
free combinations.
It is already known to produce Pseudomonas vaccines
for the prophylaxis against Pseudomonas infections, wherein
as starting materials either Pseudomonas aeruginosa bac-
terial masses themselves and/or culture filtrates which
were obtained by growing the micro-organisms on surface
cultures or submersely in complex nutrient media are used.
-- 1
~;~73&~7
With these complex nutrient media beside a carbon and
energy source (mostly carbohydrates) and essential nutri-
tive salts, various extracts and/or hydrolysates of animal,
microbial or vegetable proteins (so-called peptones) were
used. Such nutritive solution supplements are not defined
as to their exact composition, and furthermore they are
variable from lot to lot. Beside amino acids, they also
contain incompletely degraded protein fragments and un-
defined complexes of the same and serve substantially for
meeting the amino acid and growth promoting substance de-
mands. Culture supernatants, therefore, are always rich in
substances of non-bacterial origin, which has the disadvan-
tage that for preparing a flagellar antigen of Pseudomonas
aeruginosa always several separating steps, which are to
follow the cultivation step, are necessary in order to free
an immunogenically effective flagellar antigen as far as
possible from impurities stemming from the nutrient medium.
A further disadvantage of the hitherto used growing
methods in lots or batches consists in that the bacteria
are subjected to a condition of continuous physiological
change from the time of their inoculation to the time of
their separation from the culture. The trigger moment for
this physiological and thus functional differentiation of
the bacterial biomass is caused by the cellular growth
itself and by the temporal change of the composition of the
nutrient medium caused in turn by the cellular growth. The
visible result of this spontaneous physiological differen-
tiation is already recognizable by the various phases of
the growth curve of a batch culture. As a consequence of
the described direct relationship between the physiological
-- 2
' .
~3~387
activity of the cellular mass on the one hand and its
surrounding environment on the other hand, a preferred
enrichment in various intra and extra cellular metabolic
products depending on the growth phase and differentiation
phase is connected therewith. Therefore, the culture su-
pernatants of a batch culture contain an integrated mixture
of all the metabolic products formed temporally over indi-
vidual differentiation phases.
The invention aims at avoiding these difficulties and
disadvantages and has as its object to provide a method of
submersely growing strains of Pseudomonas aeruginosa bac-
teria which gives, in much higher yields than has hitherto
been the case, a bacterial biomass for a recovery of anti-
gens whose physiological state remains constant during the
total period of cultivation; in which a synthetic nutrient
medium is used from which there result no additional
impurities caused by autoclaving; and which enables an
easier chemical working up of the bacterial biomass to
flagellar antigen.
According to the invention, this object is achieved in
that the following H-type antigen producing Pseudomonas
aeruginosa bacterial strains
strain H-type
1 170001 - b
M-2 - b
2 5940 - aol a2
3 5939 - ao, a3
4 5933 _ ao, al, a2
1210 - ao~ al, a2
16990 - ao~ al, a2
-- 3 --
~3~87
170018 - ao, a3, a4
are grown in an aqueous nutrient medium, the aqueous
nutrient medium being free from antigens and free from
proteins and containing a nitrogen source and mineral
salts as well as succinate or urea as carbon source.
The carbon source mentioned has proved to be particu-
larly growth-promoting.
For the strains 170001, 5940, 5939, 5933 and 170018
the classification according too Ansorg is used. The allo-
cation of the strains M-2, 1210 and 16990, not classified
in types, to the corresponding H-serotype is effected on
the basis of comparative examinations of the molecular
weights and serological cross reactions by Montie et al.
Preferably, the Pseudomonas aeruginosa bacterial
strains are grown continuously.
According to a further preferred working method, the
Pseudomonas aeruginosa bacterial strains may be grown tur-
bidostatically, the nutrient medium having an oxygen con-
tent of from 5 to 20 %, preferably approximately 10 %, a
cellular density of 2-3 x 109 cells/ml being maintained and
a substrate dilution rate of from 0.1 ~ to 0.3 ,u being
maintained by supplying fresh nutrient medium.
The growing of Pseudomonas aeruginosa bacterial
strains according to the invention is effected under agi-
tation and airing of an "open c~llture" in a manner that
they are caused to grow maximally and form the flagellas.
The nutritive substrate is supplied in dependence on the
growth rate of the culture or on the state of equilibrium
reached by the culture. The regulation or control of the
fresh nutrient medium supply is effected in dependence on a
.
1~3~7
parameter that is proportionate to the growth of the cul-
ture, preferably the temporal change of the cellular densi-
ty. However, also other parameters which are proportionate
to the development of the culture, such as the breathing
activity, C02 production, change in the hydrogen ion con-
centration, change in the carbon source concentration,
respiration quotient, nitrogen consumption, oxygen con-
sumption and heat tone may be used. Furthermore, the supply
of fresh nutrient medium may be effected in dependence on
the amount of the antigen formed.
With the continuous growing method according to the
invention, culture liquid is withdrawn from the growing
vessel simultaneously with the supply of fresh nutrient
medium, so that between the growth and the physiologic
activity, in particular the forming of cells on the one
hand and the surrounding environment of the cells on the
other hand, there adjusts a flow equilibrium state that is
precisely definable and reproducible at any time and which
can be maintained practically for an unlimited period of
time.
As nutrient medium, a synthetic medium free from pro-
teins is used, which exclusively contains chemically as
well as physico-chemically qualitatively and quantitatively
determinable components. An inorganic nutrient solution
containing all essential elements is suitable.
With the preferred embodiment according to the inven-
tion, for the fermentative production of the germs, the
chemostatic principle of the continuous culture, in which
the physiological activity of the culture is controlled by
a limiting substrate, preferably the carbon and energy
-- 5 --
~2'7~8~7
source, as well as the turbidostatic principle of the
continuous culture, with which the dilution rate (micron)
and thus the physiological activity of the culture is
externally controlled via a growth parameter, preferably
via the instantaneous cellular density of the culture, are
applicable.
Preferably, the turbidostatic continuous cultivation
is used.
In order to promote a maximal bacterial growth, also
the temperature and the pH value are of importance; thus,
suitably, while growing of the Pseudomonas aeruginosa bac-
terial strains takes place, a temperature of from 20 to
35C, preferably 30C, and a pH of from 6.6 to 7.S, pref-
erably 7.0, are to be maintained.
In detail, the method according to the invention,
with the turbidostatic working manner, is carried out in a
fermentor, in that at first the nutrient medium is inoccu-
lated with a bacterial strain and it is proceeded under
maintenance of the conditions stated, until the cellular
density stated, of 2 - 3 x 109 cells/ml is achieved in the
logarithmic final phase. As soon as this is the case,
bacterial suspension is continuously withdrawn from the
fermentor and substituted by fresh nutrient solution.
Suitably, the dilution rate shall not be more than 0.3
preferably of from 0.1 to 0.2 ~u.
Under the conditions stated, after achieving the flow
equilibrium state, 1.5 to 2.0 g/l wet cells are obtained,
which corresponds to a turbidity value of the suspension of
from 0.5 to 0.7, prior to centrifugation at 590 nm, ac-
cording to the method by Tyndall.
1~3~
The method according to the invention shall now beexplained in more detail by way of the following example:
Example:
A nutrient solution of the following composition was
used:
Disodium succinate 4.05 g/l,
Dipotassium monohydrogen phosphate 7 g/l,
Potassium dihydrogen phosphate 3 g/l,
Ammonium hydrogen phosphate 1 g/l,
Magnesium sulfate . 7 H2O 0 05 g/l,
Ferric chloride 0.0025 g/l,
the pH was adjusted to 7.0 and the temperature was brought
to 30C. The nutrient solution was inocculated with the
strain Pseudomonas aeruginosa M-2 and introduced into a
15 1 fermentor. During a fermentation period of 96 hours,
140 1 of the nutrient medium were passed through, the air
flow rate amounting to 10 % P02 (oxygen partial pressure).
The dilution rate was 0.1 tU. The cellular density amounted
to 2-3 x 109 cells/ml after the flow equilibrium state had
been reached.
Under the same conditions, the strains 170001 and
1210 were cultivated, the following table giving the cul-
ture conditions, dilution rate, oxygen partial pressure in
% and cell yield in g/l wet weight after centrifuging twice
at 15,000 x g. Furthermore, the turbidity values of the
suspension prior to centrifuging are given.
~73~
Culture
Strain conditions Cell yield
Dilution Oxygen Turbidity value g/l wet
rate content at 590 nm;d=lcm cells
_ ~
170001 0.1 ~ 10 % PO2 0.6 - 0.67 1.7
M-2 0.1 y 10 % PO2 0.6 - 0.7 1.6 - 1.7
1210 0.1 ~ 10 % PO2 0.55 - 0.65 1.5 - 1.6
1210 0.2 ~ 10 % PO2 0.6 1.5 - 1.6
