changes in preservative sensitivity for the

7/25/2019 Changes in Preservative Sensitivity for The

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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


2/8

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


3/8

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,


4/8

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


5/8

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


6/8

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.


8/8

126 M . M. A. AL-HIT1

AND

P. GILBERT

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