Journal of Clinical Pharmacy and Therapeutics, 2012
Evaluation of preservative efficacy in pharmaceutical products: the use of
psychrotolerant, low-nutrient preferring microbes in challenge tests
C. Charnock PhD and E. Otterholt MSc
Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, St. Olavs plass, Oslo, Norway
Received 8 July 2010, Accepted 5 April 2012
Keywords: preservative efficacy, survival characteristics, wild-type challenge test strain
SUMMARY
What is known and Objective: Preservative efficacy in medi-
cines is typically investigated using challenge tests. In such
tests, the product is artificially contaminated with a high con-
centration of standard bacterial and fungal test strains such as
Pseudomonas aeruginosa and Candida albicans. The rate and
extent of reductions in inoculum viability over a specified per-
iod forms the basis for acceptance/rejection of preservative
efficacy. None of the strains named for inclusion in the chal-
lenge test outlined in the European Pharmacopoeia are associ-
ated with the contamination of high-quality water used in
pharmaceutical production. Alpha- and Betaproteobacteria are
easily the most common microbes in waters intended for
pharmaceutical production. In addition, none of the standard
test strain panel prefer low-nutrient, dilute conditions or grow
at or around refrigeration temperatures. This is important
because the water activity and nutrient content of medicines
can vary greatly and medicines are often stored cold. We
investigate the relevance of these factors when testing preser-
vative efficacy by including other strains in challenge tests.
Methods: Psychrotolerant, low-nutrient preferring strains
(Beta- and Alphaproteobacteria and a yeast) were isolated
from pristine waters. These were compared in challenge tests
with C. albicans and P. aeruginosa using different storage
temperatures. Pharmaceutical products differing widely in
water-content, pH and preservative systems were included in
the study.
Results and Discussion: Regardless of the type of medicine
tested C. albicans always showed superior survival character-
istics to the yeast isolate (Cryptococcus terricola). One of the
three screened bacterial strains (a Sphingomonas sp.) survived
significantly better than P. aeruginosa in all but one product
tested. However, the results for all products taken together
cannot easily be explained by reference to this strain’s psy-
chrotolerancy or its preference for dilute, low-nutrient envi-
ronments. This study supports previous work indicating that
the inclusion of wild-type test strains, in this instance strains
that are suited to survival in high-quality waters, improves
preservative efficacy tests.
Correspondence: Colin Charnock, Department of Health Sciences,
Oslo and Akershus University College, Pilestredet 46, 0167 Oslo,
Norway. Tel.: +47 22 452348; fax: +47 22 452335; e-mail: colin.char-
nock@hf.hio.no
ª 2012 Blackwell Publishing Ltd
doi: 10.1111/j.1365-2710.2012.01354.
What is new and Conclusion: Use of a Sphingomonas sp. iso-
lated from a pristine water as a challenge test strain, gave a
more stringent indication of preservative efficacy in a wide
range of pharmaceuticals than did P. aeruginosa.
WHAT IS KNOWN AND OBJECTIVE
The antimicrobial efficacy of medicines and allied products con-
taining preservatives can be investigated using the challenge
test outlined in monograph 5.1.3 of the European Pharmaco-
poeia (EP).1 The test involves inoculating containers with the
specified strains of Pseudomonas aeruginosa, Staphylococcus aureus,
Aspergillus niger and Candida albicans. Changes in the start con-
centration with time are followed by plating onto high-nutrient
growth media and are interpreted to give a measure of the pre-
servative efficacy by reference to criteria given in the mono-
graph. The test strains should be supplemented with others if
these are relevant as potential contaminants of the product in
question.1 According to Cowen and Steiger,2 challenge test
strains should be chosen for their known association with prod-
uct contamination, high level of resistance to preservatives,
opportunistic contamination capacity, low-nutrient demand and
adaptability. Even though many medicines have a high water
content and are stored refrigerated, none of the EP strains are
specifically associated with dilute environments or are psychro-
tolerant. For example, P. aeruginosa is ubiquitous in nature, and
the particular strain (ATCC 9027/DSM1128) used in challenge
tests was originally isolated from an outer ear infection.
This study will use the term ‘low-nutrient preferring’ opera-
tionally to denote a strain that grows better on media with a
lower nutrient content than tryptone soya agar (TSA) and Sabou-
raud dextrose agar (SAB) that are specified for inoculum produc-
tion and cell recovery in the EP challenge test.1 A
psychrotolerant microbe is one that can grow at 7 C or less irre-
spective of the optimum growth temperature.3 Growth or sur-
vival at low temperatures requires general physiological
adaptations that might also be expected to effect preservative
efficacy. For example, adaptation to low temperatures in fungi
can involve increases in the fraction of unsaturated membrane
lipids to maintain fluidity (see Ref. 4 for a review of cold adapta-
tion in yeasts). All of the named indicator strains in the EP mono-
graph grow well or best at 37 C on nutrient-rich media. The
standard preservative test may then be insufficient to describe
the survival capacity in pharmaceuticals of strains adapted to
low-nutrient, dilute environments and/or low temperatures.
Bacterial diversity in partially purified water used in pharma-
ceutical manufacturing has been investigated using non-cultiva-
tion-based techniques.5,6 It was found that the dominant strains
1
Evaluation of preservative efficacy in pharmaceutical products C. Charnock and E. Otterholt

were Alpha- and Betaproteobacteria. An examination of the bac-

teria contaminating ultrapure water in industrial systems con-
cluded that certain bacterial populations appear common to
many industrial ultrapure waters. The majority of these were fac-
ultative oligotrophs and Sphingomonas spp. were represented in
several systems.7 The presence of P. aeruginosa (Gammaproteo-
bacteria) was not reported in any of the above-mentioned studies.
By virtue of their universality and presumably good survival
characteristics in high-quality waters, it is pertinent to investigate
whether low-nutrient preferring, Alpha- and or/Betaproteobacte-
ria species show fundamentally different survival characteristics
to P. aeruginosa in high water-content medicines. Furthermore,
the Alpha- and Betaproteobacteria include such members as
Sphingomonas and Methylotrophs that have also been associated
with opportunistic infections.8–12 On the basis of the guidelines of
Cowen and Steiger2 for choosing relevant challenge test strains,
we have isolated and identified psychrotolerant, low-nutrient
preferring environmental strains of Alpha- and Betaproteobacte-
ria and a yeast. The survival characteristics of these strains are
compared with those of the standard test organisms P. aeruginosa
and C. albicans in a wide range of pharmaceutical products.
MATERIALS AND METHODS
Pharmaceutical products included in the study
Table 1 provides an overview of the products tested and their
compositions, including preservative system.
Determination of total dissolved substances
The proportion (w/w) of dissolved substances in products for
testing was estimated by weighing sample portions (2 mL)
before and after drying at 200 C for 12 h.
Isolation of microbial strains from cold, low-nutrient environ-
ments for use in challenge tests
Three bacterial strains were chosen (see criteria below) from
those isolated from bottled Norwegian mineral waters originat-
ing from cold, pristine sources. In addition, a yeast was isolated
from tap water at Oslo and Akershus University College. The
techniques used were as follows:
Bacterial isolates: Samples of 0Æ1 mL from bottles were spread
onto the relatively low-nutrient R2A agar (Oxoid, Basingstoke,
UK). Plates were incubated in the dark at 22 ± 2 C for 2 weeks.
Well-isolated colonies were inoculated onto R2A and TSA. Sets
of plates were incubated at 4 ± 1 C, 8 ± 1 C, 22 ± 2 C and
37 ± 1 C and read for growth over a period of 14 days. Three
easily distinguishable (i.e. different coloured) colony types that
grew at 4–8 C and grew on R2A agar and poorly or not at all
TSA were selected for identification and use in challenge tests.
Fungal isolate: Five litres of tap water were filtered through a
membrane filter of nominal pore size 0Æ45 lm. The filter was
placed into 200 mL of 2% w/v malt extract broth (Oxoid) con-
taining 0Æ5 mg/mL chloramphenicol to prevent bacterial growth.
The bottle was incubated at 8 C. After 4 weeks incubation, a
sample (0Æ1 mL) was transferred to bottles containing 200 mL of
0Æ00002% w/v malt extract with chloramphenicol and incubated
further for 7 days. At this point, samples spread onto low-nutri-
ent agar (20 g bactoagar (Oxoid); 0Æ002g malt extract in 100 mL
Milli-Q water) showed a pure yeast culture at about
5 · 102 CFU/mL. Colonies were visible 2–3 days after incuba-
tion at 8 C.
Growth characteristics of environmental isolates and EP test
strains
Yeasts. Yeasts were tested for growth characteristics by inoculat-
ing water agar (15 g bactoagar in 1000 mL MilliQ water), R2A,

Table 1. Salient information on the medicines and allied products tested for efficacy of antimicrobial preservation

Percentage (w/w)
of total dissolved
substances and pH
Brand name Produced by Declared contents (measured)

Atropine 1mg/mL Norwegian Pharmacy 1 mL contains: atropine sulphate 1 mg; NaCL 9 mg; HCL 1 m, 0Æ03 0Æ46 ± 0Æ11; 5Æ9
(for injection) Association, Oslo micromoles; 1 mg methylparaben, water to 1 mL

Bricanyl (syrup) AstraZeneca AS, Oslo 1 mL contains: terbutaline sulphate 0Æ3 mg; sorbitol 105 mg; ethanol 12Æ39 ± 2Æ0; 3Æ8
96% 2 mg; sodium benzoate 1 mg; aroma; water to 1 mL
Ephedrine 2 mg/mL Nycomed Pharma AS 1 mL contains: ephedrine hydrochloride 2 mg; 96% ethanol, 65 mg; amonium 2Æ52 ± 0Æ02; 7Æ4
(syrup) chloride 30 mg; anise oil, liquorice extract, water to 1 mL

Ephedrine 1 mg/mL Norwegian Pharmacy 1 mL contains: ephedrine hydrochloride 2 mg; unsugared blackcurrant juice 22Æ8 ± 0Æ1; 3Æ1
(syrup) Association, Oslo 150 mg, sorbitol 250 mg, 0Æ8 mg methylparaben; propyl- paraben 0Æ2 mg;
96% ethanol 9 mg
Fluoride 2 mg/mL Norwegian Pharmacy 1 mL contains: sodium fluoride 2 mg: 0Æ8 mg methylparaben; 0Æ2 mg 0Æ05 ± 0Æ02; 5Æ9
(mouth wash) Association, Oslo propyl-paraben; permaseal peppermint 84137-51
0Æ8 mg; water to 1 mL
Camphor (syrup) Norwegian Pharmacy 1 g contains: camphor racemic 5 mg, 96% ethanol 10 mg, mucilago gummi 2Æ81 ± 0Æ08; 5Æ2
Association, Oslo acaciae 100mg, potassium sorbate 1 mg, water to 1 mL
Paracetamol (liquid) Weifa AS, Oslo 1 mL contains: paracetamol 24 mg, sodium cyclamate (ND), saccharin 11Æ43 ± 0Æ51; 4Æ6
sodium (ND), glycerol (ND), forest berries taste (ND), macrogol
1540 (ND), 1Æ33 mg methylparaben, 0Æ67 mg propylparaben

ND, not declared.

ª 2012 Blackwell Publishing Ltd Journal of Clinical Pharmacy and Therapeutics, 2012
2
Evaluation of preservative efficacy in pharmaceutical products
TSA, SAB and blood agars. Plates were incubated at 4 ± 1,
8 ± 1, 22 ± 2 and 37 ± 1 C.
Bacteria. Strains were inoculated onto water agar, R2A and TSA
and incubated as for yeasts.
Identification of bacteria based on colony-polymerase chain
reaction (PCR) of the 16S rRNA gene
PCR amplification and sequencing of approximately 500 bp of a
segment of the 16S rRNA gene were performed exactly as previ-
ously described.13 Sequence similarity searches were conducted
in April 2012 using the blast network service and the GenBank
database.
Identification of yeast based on colony-PCR of the fungal 5.8S
gene and the non-coding Internal Transcribed Spacer regions
ITS1 and ITS2
PCR amplification and sequencing of the 5.8S gene and the non-
coding Internal Transcribed Spacer regions ITS1 and ITS2 were
performed exactly as previously described.13 Similarity searches
were performed as described above for bacteria.
Test for efficacy of antimicrobial preservation
Strain combinations and inoculum preparation. Challenge testing
was initially performed using two microbial combinations. One
combination was selected from standard test strains listed in
section 5.1.3 of the EP1 and consisted of P. aeruginosa
(DSM1128 = ATCC9027) and C. albicans (DSM1386 = ATCC10231).
The other combination consisted of the yeast and bacterial iso-
lates. By virtue of its superior survival characteristics, one bacte-
rial strain, Ssp.1 (see strain identifications below), was chosen
along with the yeast isolate for further investigation. Inoculae
for challenge tests were prepared in physiological saline from
colonies grown at 22 ± 2 C for 72 h on R2A. In investigations
of strain survival characteristics at low temperatures, strains
were grown at 8 ± 1 C for 4 days prior to inoculum produc-
tion. Basic initial studies were performed to (i) determine the
relationship between optical density and viable cell numbers
such that the correct inoculum size could be chosen (ii) test
whether a mixed inoculum affected the viability of its constitu-
ent species.
Preservative efficacy test (challenge test). Cells were added to bot-
tles to give start concentrations of 1Æ0 · 105 CFU/mL of each
individual strain whether used alone or in a combined inocu-
lum. The bottles were held without shaking in the dark at
22 ± 2 C and sampled immediately after inoculation and at
intervals over a 28-day test period as follows: samples (0Æ1 mL)
taken directly from the container and from serial dilutions in
casein peptone lecithin broth amended with 4% v/v Tween 20
(Sigma-Aldrich, St. Louis, MO, USA) were plated in duplicate
on R2A to estimate the viable cell population. Results are
expressed as the surviving fraction at each test time as a per-
centage of the initial inoculum concentration.
RESULTS
Environmental isolates were identified based on partial sequenc-
ing av rDNA genes. The putative identifications given below
ª 2012 Blackwell Publishing Ltd
3
C. Charnock and E. Otterholt
are based on 100% similarity to published sequences. The iden-
tification is given to the genus level, unless 100% similarity
across the entire sequence length was also found with a single
named species. The fungal isolate was putatively identified as
Cryptococcus terricola. Abbreviated to C. t. The closest identities
for the three bacterial isolates chosen for challenge tests were as
of April 2012: Curvibacter lanceolatus (grey-white colonies).
Abbreviated to C. l Sphingomonas sp. 1. (yellow-orange colonies
becoming brown). Abbreviated to Ssp.1. Sphingomonas sp.2. (yel-
low colonies). Abbreviated to Ssp. 2 The Sphingomonadaceae (Al-
phaproteobacteria) form part of a recently published large-scale
identification of the bacterial flora of Norwegian drinking
waters13 and have accession numbers HQ601937 (Ssp.1) and
HQ601940 (Ssp.2). Strains C. t and C. l (a Betaproteobacterium)
are new to this study and have the accession numbers (JF766377
and JF766378). These sequences are also provided as Supple-
mentary material.
Growth characteristics of the test strains
Yeasts. Cryptococcus terricola grew in the temperature range 4–
8 C, but not at 37 C. The cardinal temperature for growth was
22 C. At 8 C colonies of diameter 2 mm were obtained within
1 week on R2A agar. Under the same conditions, C. albicans pro-
duced faint background growth, and no colonies. Cryptococcus
terricola grew on TSA, SAB and blood agars, but the fastest and
most copious growth was on R2A. The strain produced also 2-
mm colonies on water agar (22 C) within 1 week. C. albicans
grew best on SAB. C. albicans gave colonies on water agar
(22 C), but these appeared first after 2 weeks incubation and
were then <1 mm in diameter.
Bacteria. None of the environmental bacterial isolates grew on
TSA, but all grew on R2A. P. aeruginosa grew well on R2A, but
strongest growth was obtained with TSA. All of the environ-
mental isolates grew weakly in the temperature range 4–8 C,
but not at 37 C. The cardinal temperature for growth was
22 C. P. aeruginosa grew best at 37 C and gave only faint
background growth after 1 week at 8 C on R2A. Ssp.1 pro-
duced 1-mm colonies on water agar after incubation for 1 week
at 22 C. The other strains did not produce colonies under these
conditions.
pH and content of dissolved solids in the tested preparations
Table 1 presents the measured pH and content of dissolved sub-
stances as (% w/w) in the tested products. Salient product
details (including the preservative system) are also supplied.
Challenge tests
Mixed and single stain inoculums gave essentially the same
results. Preliminary work (results not shown) indicated that
there was no inter-strain inhibition in mixed inoculums.
Tables 1–3 show the outcome of challenge tests. Regardless of
the medicine type, the growth temperature (8, 22 C) used for
preparation of the inoculum or the storage temperature of the
products after inoculation (4, 22 C), C. t showed inferior sur-
vival characteristics to C. albicans (Table 2). C. albicans and
P. aeruginosa were not tested for survival characteristics at 4 C
as the growth rate of the strains on agar at low temperatures
excluded the possibility of making an inoculum from cells
Journal of Clinical Pharmacy and Therapeutics, 2012
Evaluation of preservative efficacy in pharmaceutical products C. Charnock and E. Otterholt

Table 2. Survival characteristics of two strain combinations (C.a.; P.a.) and (C.t.; Ssp.1, Ssp.2, C.l.) in medicines and allied products
containing preservatives

Viable fraction as percentage of the initial inoculum

Formulation Inoculum 0h 0Æ25 h 1h 3h 6h 18 h 1 day 3 day 7 day 14 day 28 day

Ephedrinea C.a 102 63 44 13 2Æ7 0Æ2 0Æ05 nd nd nd nd

P.a 30 0Æ03 nd nd nd nd nd nd nd nd nd
C.t 11 0Æ5 nd nd nd nd nd nd nd nd nd
Ssp.1 0Æ2 nd nd nd nd nd nd nd nd nd nd
C.l nd nd nd nd nd nd nd nd nd nd nd
Ssp.2 nd nd nd nd nd nd nd nd nd nd nd
Ephedrine C.a 79 83 58 50 43 29 48 3Æ2 nd nd nd
P.a 1Æ3 nd nd nd nd nd nd nd nd nd nd
C.t 1 1 2Æ9 1Æ3 1Æ5 nd nd nd nd nd nd
Ssp.1 48 0Æ1 nd nd nd nd nd nd nd nd nd
C.l 11 nd nd nd nd nd nd nd nd nd nd
Ssp.1 nd nd nd nd nd nd nd nd nd nd nd
Camphor C.a 78 62 79 59 70 33 70 6Æ2 0Æ05 nd 0Æ3
P.a 0Æ3 nd nd nd nd nd nd nd nd nd nd
C.t 14 1Æ8 0Æ7 0Æ4 0Æ2 nd nd nd nd nd nd
Ssp.1 37 0Æ1 nd nd nd nd nd nd nd nd nd
C.l 39 nd nd nd nd nd nd nd nd nd nd
Ssp.2 nd nd nd nd nd nd nd nd nd nd nd
Atropine C.a 92 112 93 100 96 51 23 4Æ2 nd nd nd
P.a 40 0Æ1 nd nd nd nd nd nd nd nd nd
C.t 85 87 13 0Æ1 nd nd nd nd nd nd nd
Ssp.1 96 4Æ8 0Æ4 nd nd nd nd nd nd nd nd
C.l 20 nd nd nd nd nd nd nd nd nd nd
Ssp.2 20 nd nd nd nd nd nd nd nd nd nd
Fluoride (mouthwash) C.a 66 85 82 65 58 4Æ4 1Æ8 0Æ01 nd nd nd
P.a 4Æ0 nd nd nd nd nd nd nd nd nd nd
C.t 51 72 44 0Æ8 0Æ03 nd nd nd nd nd nd
Ssp.1 86 1Æ8 0Æ01 nd nd nd nd nd nd nd nd
C.l 67 0Æ3 nd nd nd nd nd nd nd nd nd
Ssp.2 0Æ1 nd nd nd nd nd nd nd nd nd nd
Bricanyl C.ab 106 91 106 77 80 46 33 44 0Æ7 nd 0Æ03
P.a 0Æ15 nd nd nd nd nd nd nd nd nd nd
C.t 29 25 23 14 4Æ3 nd nd nd nd nd nd
Ssp.1 59 38 5Æ6 0Æ5 0Æ01 nd nd nd nd nd nd
C.l 25 nd nd nd nd nd nd nd nd nd nd
Ssp.2 19 nd nd nd nd nd nd nd nd nd nd

nd, no (cfu) detected; Ssp., Sphingomonas sp; P.a, Pseudomonas aeruginosa; C.t, Cryptococcus terricola; C.a, Candida albicans; C.l, Curvibacter lanceolatus.
a
Self-preserving.
b
Samples taken after 28 days over a total of 6 weeks showed cfu but not regrowth of the strain.

grown cold. Two of the tested environmental isolates (C. l and
Ssp. 2) showed similar survival characteristics to P. aeruginosa.
That is, viable cells were almost never found after 0Æ25 h in
bottles (Tables 2 and 3) and never found after 1 h. However,
strain Ssp.1 showed better survival characteristics than P. aeru-
ginosa in all but one (Ephedrine 2 mg/mL) of the seven prod-
ucts tested. In this instance, both strains became non-viable
within 1 h. The most striking example of the difference
between the strains was seen with the liquid Paracetamol
preparation (Table 3). The P. aeruginosa inoculum became non-
viable within 1–3 h, whereas Ssp.1 could still be grown from
bottles after 24 h (Table 3). Similarly, in the Bricanyl syrup,
Ssp. 1 could still be grown from bottles 6 h after inoculation,
whereas P. aeruginosa became non-viable within 0Æ25–1 h
(Tables 2 and 3). Table 4 presents the results of challenge tests
using a cold-grown inoculum (Ssp.1 and C. t) and pharmaceu-
tical products stored cold after inoculation. In most instances,
particularly in the case of C. t, cold storage led to longer sur-
vival times (Tables 2–4) and the opposite was never the case.
However in some cases (e.g. Ephedrine 1 mg/mL and Paracet-
amol), the survival characteristics of Ssp.1 varied only margin-
ally at the two test temperatures.
Table 5 ranks the survival times of Ssp.1 and P. aeruginosa for
each product. Details of the preservative system, dissolved sub-
stances, pH and the authors’ own evaluation of a possible nutri-
ent base are given. With the exception of Ephedrine 2 mg/mL,
the strains show similar rank orders. Pseudomonas aeruginosa
became non-viable in all of the 8 pharmaceutical products in the
course of 1 h, and the data are not sufficiently spread to draw
conclusions on the relative survival characteristics of this strain in

ª 2012 Blackwell Publishing Ltd Journal of Clinical Pharmacy and Therapeutics, 2012
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Evaluation of preservative efficacy in pharmaceutical products C. Charnock and E. Otterholt

Table 3. Survival characteristics of P. a. and Ssp.1 in medicines and allied products containing preservatives

Viable fraction as percentage of the initial inoculum

Formulation Inoculum 0h 0Æ08 h 0Æ25 h 1h 3h 6h 18 h 1 day 3 day 7 day 14 day 28 day

Ephedrinea Ssp.1 33 0Æ01 nda nd nd nd nd nd nd nd nd nd

P.a 54/140 28/21 1Æ2/21 nd/2Æ3 nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd
Ephedrine Ssp.1 74 38 1Æ9 nd nd nd nd nd nd nd nd nd
P.a 0Æ6/0Æ06 0Æ06/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd
Camphor Ssp.1 89 3Æ3 0Æ2 nd nd nd nd nd nd nd nd nd
P.a 4Æ2 nd nd nd nd nd nd nd nd nd nd nd
Atropine Ssp.1 72 36 23 0Æ5 nd nd nd nd nd nd nd nd
P.a 16/39 nd/0Æ6 nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd
Fluoride (mouthwash) Ssp.1 97 26 16 nd nd nd nd nd nd nd nd nd
P.a 120 31 nd nd nd nd nd nd nd nd nd nd
Bricanyl Ssp.1 58 53 36 14 0Æ6 0Æ04 nd nd nd nd nd nd
P.a 13 0Æ2 0Æ2 nd nd nd nd nd nd nd nd nd
Paracetomol Ssp.1 81/120 66/77 55/45 55/17 47/12 20/3Æ7 11/2Æ4 5Æ3/1Æ8 nd/nd nd/nd nd/nd nd/nd
P.a 2Æ9/23 5Æ1/20 4Æ6/7Æ2 nd/0Æ06 nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd nd/nd

nd, no (cfu) detected; Ssp. 1, Sphingomonas sp; P.a, Pseudomonas aeruginosa.

a
Self-preserving.

Table 4. Survival characteristics of cold-adapted C. t. and Ssp.1 in preservative-containing medicines and allied products at 4 ± 1 C

Viable fraction as percentage of the initial inoculum

Formulation Inoculum 0h 0Æ08h 0Æ25h 1h 3h 6h 18h 1d 3d 7d 14d 28d

Ephedrinea C.t 20 1Æ2 0Æ9 0Æ6 nd nd nd nd nd nd nd nd

Ssp.1 41 6Æ0 0Æ8 nd nd nd nd nd nd nd nd nd
Ephedrine C.t 14 2Æ5 0Æ6 2 2 2Æ1 2Æ5 1Æ8 nd nd nd nd
Ssp.1 11 0Æ2 0Æ03 nd nd nd nd nd nd nd nd nd
Camphor C.t 58 59 39 33 16 16 8Æ9 5Æ6 0Æ6 nd nd nd
Ssp.1 8Æ9 4Æ4 1Æ7 0Æ1 nd nd nd nd nd nd nd nd
Atropine C.t 15 4Æ2 3Æ7 3Æ2 1Æ6 1Æ2 nd nd nd nd nd nd
Pcr3 44 15 9Æ4 7Æ8 2Æ3 0Æ7 nd nd nd 0Æ02 nd nd
Fluoride (mouthwash) C.t 73 54 41 35 17 7Æ8 nd nd nd nd nd nd
Ssp.1 25 26 15 5Æ1 0Æ94 0Æ02 nd nd nd nd nd nd
Bricanyl C.t 21 5Æ4 5Æ3 3Æ2 2Æ0 1Æ0 1Æ8 1Æ5 nd nd nd nd
Ssp.1 18 12 12 6Æ8 7Æ5 0Æ9 0Æ3 0Æ9 nd nd nd nd
Paracetomol C.t 22 20 30 12 4Æ4 2Æ9 0Æ4 0Æ3 nd nd nd nd
Ssp.1 25 7Æ6 13 4Æ9 2Æ6 1Æ7 2Æ1 2Æ1 nd nd nd nd

nd, no (cfu) detected; Ssp. 1, Sphingomonas sp; C.t, Cryptococcus terricola.

a
Self-preserving.

the products tested. Ssp.1 showed a much greater spread (0Æ08–
24 h) in this parameter. However, no obvious trend in survival
times with respect to product variables (dissolved substances,
preservative system, pH, etc.) is discernible in the data set.
DISCUSSION
Environmental strains were isolated and chosen for challenge
tests based on their affiliation with high-quality waters, psy-
chrotolerancy, low-nutrient demand and, in the case of the bac-
teria their taxonomic affiliations (Alpha-/Betaproteobacteria).
These attributes were hypothesized to have relevance for bacte-
rial survival in pharmaceutical products (see What is known
and objective section). The attributes of the environmental
strains are in contrast to the more mesophilic, high-nutrient pre-
ferring status of the EP standard challenge test strains, C. albi-
cans and P. aeruginosa included for comparison.
The use of C. t and Ssp. 1 in challenge tests
Cryptococcus terricola isolated from drinking water was both
psychrotolerant and grew on water agar. A search of the liter-
ature does not implicate C. t in any infections or as a contam-
inant of medicines, and its potential value as a challenge test

ª 2012 Blackwell Publishing Ltd Journal of Clinical Pharmacy and Therapeutics, 2012
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Evaluation of preservative efficacy in pharmaceutical products C. Charnock and E. Otterholt

Table 5. Survival times of ssp. 1 and P.

Ranked based a in pharmaceutical products
on survival time
in each producta

Product details: description; percentage of

Ssp. l P.a Product dissolved substances; pH

1 2 Paracetamol Clear liquid, medium nutrientb, parabens; 11; 4Æ6

2 3 Bricanyl Syrup, high nutrientb, Na-benzoate; 12; 3Æ8
3 5 Atropine Clear liquid, low nutrientb, methylparaben; 0Æ5; 5Æ9
4 4 Fluoride Clear liquid, low nutrientb, methylparaben; 0Æ05; 5Æ9
5 6 Ephedrine 1mg/mL Syrup, very high nutrientb, parabens; 23; 3Æ1
6 7 Camphor Syrup, low nutrientb, K-sorbate, 3; 5Æ2
7 1 Ephedrine 2 mg/mL Syrup, medium nutrientb, self-preserving; 3; 7Æ4

Ssp. 1, Sphingomonas sp; P.a, Pseudomonas aeruginosa.

a
In cases of equal survival time in two products, the order is based on the initial rate of inactivation and
averaging the survival times from parallels (Tables 2–4).
b
Based on the authors’ own evaluation of each product’s declared content (Table 1).

strain would thus lie in its showing better survival character-
istics than the standard EP test yeast C. albicans. However,
regardless of the medicine type, the growth temperature used
for preparation of the inoculum or the storage temperature of
the medicines after inoculation C. t was inferior to C. albicans.
Thus, the use of this yeast in challenge tests would not seem
to offer any improvements on the standard EP C. albicans
strain.
Members of the genus Sphingomonas in general and Ssp.1 in
particular fulfil a number of the criteria for ideal challenge test
strains:2 Sphingomonas spp. are widely distributed in nature and
are resistant to many disinfecting and toxic chemicals.14 They
have been found in water intended for hospital, pharmaceutical
and industrial use5–7,15 and have also been isolated from medi-
cines containing preservatives.16,17 Furthermore, some species
have been associated with community-acquired and nosocomial
infections including bacteremia, meningitis, cather-related sepsis
and diarrhoeal disease.8,11,12 Ssp.1 was isolated from a pristine
water. In addition to its survival characteristics in preservative-
containing products, it shows a low-nutrient demand and grows
across a temperature range suitable for the storage of pharma-
ceuticals. The better survival characteristics of Ssp.1 relative to
P. aeruginosa in seven of eight pharmaceuticals mean it can pro-
vide a more stringent test of preservative efficacy for a wide
range of products.
The eight products tested represent not only a wide range
of different medicinal groups (e.g. parenteral products and
syrups), but also contain three fundamentally different preser-
vative systems and undergroups of these (self-preserving,
weak acids:benzoate/sorbate, methylparben alone, paraben
combinations). Given their preference for lower nutrient agars
and isolation from clean waters, it was hypothesized that the
putative superiority (i.e. longer survival times) of the environ-
mental isolates over C. albicans and P. aeruginosa might be
greatest for dilute, low-nutrient pharmaceuticals. Table 5 that
summarizes the findings for Ssp. 1 does not provide support
for this view. There were no discernible trends in the data
that would be consistent with better survival of this strain in
dilute, low-nutrient products. Nor were trends with respect to
the particular preservative system used and product pH
found. Ssp.1 survived best in the Paracetamol and Bricanyl
products (Tables 2 and 3). The Paracetamol preparation is
preserved with a paraben mixture, whereas Bricanyl contains
sodium benzoate (Table 1). Ssp. 1 quickly lost viability (<1 h)
in Ephedrine 1 mg/mL that contains the same two parabens
as Paracetamol, and in Camphor that contains a weak acid
preservative (sorbate).
It is most likely that the survival characteristics of Ssp.1 in
pharmaceuticals are a complex combination of factors. Notwith-
standing, the superiority of Ssp.1 over P. aeruginosa for a wide
range of medicines and their preservative systems makes it a
more stringent indicator of preservative efficacy.
Cold storage led, in most instances, to longer survival times
(Tables 2–4) and the opposite was never the case. However, pre-
servative efficacy is known to increase with temperature,2 and
in some cases (e.g. Ephedrine 1 mg/mL and Paracetamol), the
survival characteristics of Ssp.1 varied only marginally at the
two test temperatures. This aspect (i.e. psychrotolerancy vs. pre-
servative kinetics) is worthy of further examination.
A Sphingomonas sp. (Ssp.1) isolated from a pristine bottled
water survived better than the EP test strain P. aeruginosa DSM
1128 in all but one of eight pharmaceuticals tested. Although it
is unrealistic to expect that manufacturers always perform
experiments such as the one described here before choosing
strains for inclusion in challenge tests, it is surprising that the
four strains that form the core of the EP test all grow best at
temperatures greatly in excess of those used in the storage of
medicines. Furthermore, the EP strains prefer environments
with a high-nutrient content, whereas many pharmaceuticals
are dilute products, and none of them are common contami-
nants of waters used in the production of preservative-contain-
ing medicines. This study demonstrates that other strains that
meet these conditions can give a more stringent and perhaps
more relevant test of preservative efficacy.
WHAT IS NEW AND CONCLUSION
The use of environmental isolates has been recommended for
challenge tests, and the results of this study bear out the conten-
tion that these can offer more stringent tests of preservative effi-
cacy. Use of a Sphingomonas sp. isolated from a pristine water as
a challenge test strain gave a more stringent indication of pre-
servative efficacy in a wide range of pharmaceuticals than did
P. aeruginosa.

ª 2012 Blackwell Publishing Ltd Journal of Clinical Pharmacy and Therapeutics, 2012
6
Evaluation of preservative efficacy in pharmaceutical products
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