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7/25/2019 Changes in Preservative Sensitivity for The
1/8
Journal of i ippl ied Bacteriology 1980,
49
119-1
26
6941121/79
Changes in Preservative Sensi tiv ity fo r t h e
USP Antimic rob ial Agents Effectiveness
Test Micro -org anisms
M. M.
A. AL HITI
AND P.
GILBERT
Department of Pharmacy, Universi ty
of
Manchester, Manchester M I 3 9PL, U K
Received
24
December 19 79 an d accepted 19 February 19 80
Chemically defined and semi-defined media were designed for the preservative-effi
cacy testing micro-organisms designated by the United States Pharmacopoeia, in
which the organisms went into the stationary phase of growth at an optical density
E470) of 1.0, because of depletion of a single carbon, nitrogen or phosphate source.
Aspergillus niger
was grown on solid media containing concentrations of these
nutrients which limited the rates of mycelial development and sporulation density. The
ability of the micro-organisms to survive and grow in the presence of chlorhexidine
diacetate, benzalkonium chloride and thiomersal varied markedly with the nutrient-
depletion of the inocula.
No
universal pattern of sensitivity emerged among micro-
organisms. Only A niger showed little overall change in preservative sensitivity. These
results highlight the need to define more adequately growth media and conditions for
the production of inocula for antimicrobial challenge tests.
MICROBIALH A L L E N G E S form a useful basis for evaluating the biological availability
of preservatives in pharmaceutical and cosmetic products that are liable to microbial
spoilage (Anderson Crompton 1967; Eriksen 1970; Nortonet al. 1974). The United
States Pharmacopoeia1 (XIX) (USP) Antimicrobial Agents Effectiveness Test pro-
vides an officially recommended test for evaluating the effectivenessof preservatives in
medicinal products (Anon. 1975). The test was designed for use with ophthalmic, aural
and nasal preparations but is often used as a guideline for other products including
non-pharmaceutical materials. In many instances, however, products complying with
this test are subsequently found to be inadequately preserved (Moore 1978). Results of
the USP Test indicate whether the product will contend adequately with a defined
challenge by five designated strains of micro-organisms. The conditions specified by
the Pharmacopoeia, however, leave ample room for variation; indeed conflicting
results may often be obtained within a single laboratory (Moore 1978).
The bacterial envelope is remarkably flexible in its structure and composition. It is
highly responsive to changes in the nutritional environment, resulting in differences in
the sensitivity of cells towards drugs, through variation in permeability of the cell
envelope (Brown 1975). Thus Gram negative bacteria (Finch Brown 1975; Gilbert
Brown 1978a, b ) and Gram positive bacteria (Gilbert Brown 1980) and yeasts
(Johnson et al. 1978), have all been reported to vary in drug sensitivity according to
conditions of vegetative growth. It has been suggested that the organisms used in the
USP Test for antimicrobial effectiveness might also vary in a similiar fashion (Brown
1977; Hobbs et al. 1979). In addition a preserved system is unlikely to be challenged
during normal use with typical laboratory cultures grown in chemically rich media
designed to give optimal conditions for growth. Instead the micro-organisms would
have adapted to their own particular environments (Yablonski 1972). Thus the nutri-
0021
-8847/80/040119+08 01.00/0
[ 191
980 The Society
for
Applied Bacteriology
7/25/2019 Changes in Preservative Sensitivity for The
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120 M. M . A. AL-HIT1 AND P. GILBERT
tional status of the challenge inocula might influence the decision about the adequate
preservation of a product. As the USP Test is essentially a biological assay of
preservative availability, it is not absolutely necessary for
it
to mimic the in-use
situation; it must, however, be reproducible. Growth conditions of the challenge
inocula are inadequately specified in the USP and might therefore lead to results that
vary between laboratories.
This study therefore investigates the effectsof depletion of carbon (C-dep), nitrogen
(N-dep) and phosphate (P-dep) during the growth of the USP Test organisms upon
their ability to grow subsequently in the presence of three typical preservatives. The
object
was
to improve the reliability of the test. A preliminary report of some of these
results has already been published (Al-Hiti Gilbert 1979).
Materials and Methods
Organ sms
Stuphylococcus aureus,
ATCC 6538;
Escherichia coli,
ATCC 8739;
Pseudomonas ueru-
ginosa,
ATCC 9027;
Candida ulbicuns,
ATCC 8739, and
Aspergillus niger,
ATCC
16404 were used throughout. Cultures were maintained on slopes of Nutrient Agar
(Oxoid, CM3) at room temperature after incubation overnight at 35C for the bacteria
and yeast and 7
d
at 30C for the fungus. Slopes were replaced at 28 d intervals.
Chemicals
Chlorhexidine diacetate was obtained as Hibitane from ICI Ltd. (Macclesfield, Chesh-
ire) and benzalkonium chloride and morpholinopropane-sulphonic acid (MOPS) from
Sigma Chemicals (Poole, Dorset). Microbiological media were supplied by Oxoid
except for the yeast extract which was purchased from Difco Laboratories. All other
reagents were obtained from BDH and were of the purest available grade.
Liquid media
Chemically defined or semi-defined simple-salts media were designed for the growth of
the micro-organisms, based initially on those of Gilbert Brown
(1
9786) for Esch. coli,
Vogel Bonner
( 1956)
for
Ps. aeruginosa,
Kobayashi
t al. (1
964) for
C. albicans
and
supplemented with yeast extract and thiamine-HC1 for Staph. uureus. Although media
for the different organisms must inevitably be different quantitatively, reflecting their
different growth requirements, it was desirable that qualitatively they were as similar as
possible. Thus the constituents of these established simple salts media were rational-
ized as much as possible with respect to carbon, nitrogen and phosphate sources, buffer
systems and salts. The media were sterilized by autoclaving at 1 15Cfor 30 min. Biotin,
thiamine-HC1 and FeNH4(S0 were sterilized separately as concentrated solutions
by membrane filtration.
Cleated Erlenmeyer flasks (250 ml) containing 100 ml of the various media were
inoculated from overnight cultures grown in simple-salts liquid media and incubated in
an orbital shaker (Gallenkamp Ltd., London) at 100 osc./min and 35C. Growth was
monitored by optical density (Euo) using a Cecil CE303 spectrophotometer (Cecil
7/25/2019 Changes in Preservative Sensitivity for The
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NUTRIENT-DEPLETION AND PRESERVATIVE SENSITIVITY
121
Instruments Ltd., Cambridge) and 1 cm glass cuvettes. Initially the concentrations of
either the carbon, nitrogen or phosphate source (Table 1) were varied and the optical
density of the cultures determined when they reached the stationary phase of growth
(Fig.
1).
All remaining nutrients [Mg
S 0 4 ,
0.5 m~;FeNH~(S04)2,.03 mM, and for
Esch. coli and Staph . aureus KCl, 13.4 m ~ ]ere present in excess. The media for Staph.
aureus were also supplemented with yeast extract (1O g/l), thiamine-HCl(1 O mg/l) and
biotin (0.15 mg/l) and that for
C
albicans with biotin (0.15 mg/l). pH was measured
before and after growth of the cultures to check the buffering capacity of the media (pH
TABLE
Media composition
for
the growth of Escherichia coli, Pseudomonas aeru-
ginosa, Staphylococcus aureusand Candida albicans
Concentration
(mM)
to give stationary phase at an opti-
cal density
E470) of 1
O
Growth
limiting
Escherichia
nutrient coli
Glucose
Glycerol
8.5
Sodium citrate
K 2 H P 0 4 0.13
(NH4)zS04 1.2
Staphylococcus Psrudomonas Candida
aureus arruginosa alhicans Nutrient-depletion
3.0 6.0
12.5
Carbon-depleted*
9.0
t 0.05 0.15 0.05
Phosphate-depleted*
2.5 2.0 2.5
Nitrogen-depleted*
* If
non-limiting added at five times these concentrations.
t
No added phosphate for phosphate-
depleted, otherwise
2.0 mM. No
added
NH4)2S04
for nitrogen-depleted, otherwise
5.0 mM.
7.2). The P-dep and all staphylococcal cultures were buffered using MOPS (200 mM);
the remainder were buffered with phosphates (KH*P04,28mM; K*HP04,72 mM). The
concentrations of the carbon, nitrogen and phosphate sources within the media
causing the cultures to enter their stationary phase of growth at an optical density of
1.0 were used in the design of the simple-salts media. The nutrient to be depleted was
supplied at this concentration, and the remainder at 5 times this level.
Solid media design
For
A
niger the USP Test specifies that the challenge inocula is a spore suspension.
Solid media were therefore devised for the growth of this organism based on the
simple-salts liquid media of Kobayashi et
al. (1964)
and solidified with 1 (w/v)
bacteriological agar (Oxoid L1 1). Plates were inoculated centrally with an agar disc cut
from a 7 d simple-salts plate culture ofA niger, using a 4 mm flamed cork borer. Rates
of increase in colony size were determined directly by daily measurement over 7 d and
the density
of
spores within the colony was assessed daily for 7 d by flooding replicate
plates with water, agitating with a glass spreader and performing total spore counts on
the final suspension. Concentrations of carbon, nitrogen and phosphate sources were
varied as with the liquid media. Rates of increase in colony size altered linearly with
respect to limiting nutrient concentration. At very low phosphate concentrations,
however, although mycelial growth rate was limited by the phosphate concentration,
7/25/2019 Changes in Preservative Sensitivity for The
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122 M .
M .
A. AL-HIT1
AND
P. GILBERT
the density of sporulation within the colony was very much reduced. Choices of
limiting nutrient concentrations for the final media (Table 2) were therefore made
subjectively, selecting those that restricted the rate of colony development but allowed
a sufficient level of sporulation for harvesting and preparation of spore suspensions
after
7
d incubation. For C-dep and N-dep cultures these reduced the rate of increase in
0 5
0 2
0.4 0 6 0 8
poG3-
oncentration
CO MI
Fig. 1 . Effect of phos phate concentration upon the stationary phase optical density
E470)
of (a)
Psrudomonas aeruginosa grown in simple-salts media with glycerol 0 ) and sodium citrate as
carbon source 0 ) ; (b) Staphylococcus aureus grown in simple-salts media including yeast
extr act (I g/l.) as a source of vitamins and amin o acids.
colony diameter by 50 from that on complete media. For P-dep cultures, however,
where sporulation density varied with phosphate concentration, this was not possible
and that concentration giving a 50 reduction in spore density of the colony was
selected.
Sensitivity to w ar d preservatives
Liquid cultures of the bacteria and yeast, depleted either in carbon, nitrogen or
phosphate source, were prepared in 250 ml cleated Erlenmeyer flasks containing 100ml
of the appropriate media (Table l), grown overnight (16 h) at 35C in an orbital shaker
(100 osc./min). These had been inoculated from similarly grown cultures in identical
7/25/2019 Changes in Preservative Sensitivity for The
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NUTRIENT-DEPLETION AND PRESERVATIVE SENSITIVITY 123
media. Aspergillus niger was grown on solid media (Table 2) for
7
d at 35C and the
spores harvested by flooding the plates with water and agitating using glass spreaders.
Cultures of micro-organisms
so
obtained were serially diluted in distilled water and 0.1
ml amounts, containing 1 x lo2,1 x lo3and
1
x lo4viable cells, spread on to predried
nutrient agar plates containing varying concentrations of thiomersal, benzalkonium
chloride or chlorhexidine diacetate. The number of colony forming units (c.f.u.) was
TABLE
Media composition
f o r
the growth of
Aspergillus niger
Concentrations
of
growth limiting
nutrients
(mM)
for
Aspergillus niger
in Bacteriological Agar
( 1
wiv)
wiv)
Carbon- Phosphate- Nitrogen-
Nutrient limited limited limited
Glucose 3.0
45.0 45.0
K2HP04
72.0
0 05
72.0
(NH4)2S04
15.0
15.0 0.0
Additives MgS04 , 0.5 mM; FeS04, 0.03 mM; biotin,
0.15
mg/l; KH2P04,
30
mM (carbon and nitrogen-
limiting media only); MOPS buffer,
200
mM (phos-
phate-limiting media
only).
determined after incubation of the plates at 35C for 24 h for the bacteria and 48 h for
the yeast and fungi. Experiments were done in triplicate and results were expressed as
percentage reduction in c.f.u. relative to the controls.
Results and
Discussion
M edia design
Chemically defined and semi-defined liquid media were designed for the bacteria and
yeast in which logarithmic growth ceased at an optical density of 1.0 because of
depletion of one key nutrient, either a carbon, nitrogen or phosphate source (Table 1).
All non-limiting nutrients were available in excess. For the growth of Staph. aureus it
was necessary to supplement the simple-salts media with a source of vitamins and
amino acids. These also served to some extent as nitrogen and phosphate sources. Thus
no added nitrogen or phosphate were included in the media when these were the
required nutrients for depletion (Fig. lb).
For
Ps.
aeruginosa
two carbon sources were used (sodium citrate or glycerol). This
organism utilizes citrate in preference to other carbon sources (Hamilton Dawes
1959), it was therefore of interest to see whether the nature of the carbon source
influenced drug sensitivity. Curiously, not only were the molar requirements of the
organism different for the two carbon sources, but their phosphate requirement
increased threefold when sodium citrate replaced glycerol as the sole carbon source
7/25/2019 Changes in Preservative Sensitivity for The
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124 M . M . A .
AL-HIT1
A N D
P . GI LBERT
(Table
1,
Fig. 1). This o bviously reflects a m ajor ch ange in the physiological status of
the cells.
Preservative sensitirity
The ability of the USP Test organisms, grown under C -dep, N -d ep and P-dep condi-
tions, to survive and grow in the presence of varying concentrations of preservatives
was assessed. Typical results are illustrated in Fig.
2,
and collected results for all the
Benzoihonium chloride
concentration
( % w/v
lo4
Fig. 2. Effect of depletion of carbon m), nitrogen (o), or phosphate 0 ) dur ing the growth of
Staphylococcus aureus, upon
its
ability
to
survive and g row on agar plates containing various
concentrations of benzalkonium chloride.
micro-organisms, as tha t concen tration of preservative reducing the c.f.u. by
90 ,
in
Table 3. With the exception of
A
niger the preservative sensitivity of the micro-
organisms varied markedly with nutrient-depletion. Least variation in sensitivity was
observed towards thiomersal, and the greatest towards benzalkonium chloride. Nota -
bly Ps
aeruginosa
was most resistant to all the agents, justifying its notoriety a s an
organism p articularly resistant
to
chemical inactivation (Brown
1975).
Greatest varia-
tion in preservative sensitivity with nutrient-depletion was also observed for this
organism; the iso-effective concentrations for benzalkonium chloride, for example,
varied fro m
2.5 x f>
N-dep
3
P-dep; for
Ps . aeruginosa,
P-dep
>
N-dep C-dep and for
Stap h. aureus,
N-dep
>
P-dep C-dep.
From the results of this study and others (Hobbs et al. 1979) there appears to be no
rationale for the choice of a single nutrient-depletion, minimizing preservative sensiti-
vity, for the growth of challenge test inocula. The results do, however, indicate that the
use of different media within different laboratories could be a primary cause of
interlaboratory variation, and influence the results of a challenge test for preservative
TABLE
Ef fec t of various nutrient-depletions upon the ab ility of micro-organism s to grow in
the presence of preservative
Preservative concentration required
to
reduce the number
of
colony
forming units
by
907,; (7 wjv
x
lo4)
Benzalkonium
Thiomersal Chlorhexidine chloride
Organ ism N-dep C-dep P-dep N-dep C-dep P-dep N-dep C-dep P-dep
Escherichiu coli 0.53 0.37 0.60 24.0 13.0 20.0 100.0 65.0 82.0
Pseudomonus
ueruginosu
(grown o n citrate)
1.90
1.40 0.25 28.0 24.0 30.0
100.0
450.0 25.0
(grown o n glycerol) 5.00
5.00 3.70
62.0
45.0 41.0
500.0 500.0 180.0
Staphylococcus
uureus 0.12
0.54 0.14 8. 0 10.5 10.5 0.5 0.6 0 .6
Cundidu albicuns
0.009
0.009 0 013
10.5
11 0
10.0 52.0
45.0 35.0
Aspergillus
niger
0.025 0.025
0.025
2.3
2.3 2.3
2.6 2.5 2.6
N-dep , nitrogen depleted; C-dep, carbo n depleted; P-dep, pho sphate depleted.
efficacy, especially when the concentrations of preservative employed are just ade-
quate. Growth conditions are inadequately specified within the USP Antimicrobial
Agents Effectiveness Test and allow such a situation to occur. There would appear,
therefore, to be justification in designating a single medium for the growth of each
organism to be used in this and similar tests. Insufficient data are available to decide
whether this medium shall be chemically-defined and of low complexity or undefined,
such as nutrient broth. There is, however, one major advantage in using chemically-
defined media in that they are unlikely to vary significantly between manufacturers.
Adoption of these measures would increase test reproducibility, but to increase
relevance to the
in viuo
state, additional testing must be done with organisms from an
unpreserved or inadequately preserved product (Yablonski 1972), and possibly also
with organisms isolated from the manufacturing environment and grown in the
unpreserved product.
References
AL-HITI,M . M .
A . GILBERT,. 1979
Effect
of
nutrient-depletion upon the sensitivity
of
the
US
Pharmacopoeia1 preservative testing strains towards thiomersal, benzalkonium chloride
and chlorhexidine diacetate.
Society for General Microbiology Quarterly
7 3435.
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AND
P. GILBERT
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Recommended