Short report

Resistance or adaptation? How susceptible is a
‘glutaraldehyde-resistant’ Pseudomonas aeruginosa
isolate in the absence of selection pressure?

G. Kampf

a

,

b

,

*

, C. Ostermeyer

c

, S. Tschudin-Sutter

d

, A.F. Widmer

d

a

Bode Science Center, Bode Chemie GmbH, Hamburg, Germany

b

Institute for Hygiene and Environmental Medicine, Ernst-Moritz-Arndt University, Greifswald, Germany

c

Microbiology, Bode Chemie GmbH, Hamburg, Germany

d

Division of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Switzerland

A R T I C L E

I N F O

Article history:
Received 12 March 2013
Accepted 13 May 2013
Available online 5 July 2013

Keywords:
Glutaraldehyde
Resistance
Adaptation
Pseudomonas aeruginosa

S U M M A R Y

The activities of glutaraldehyde solution and an instrument disinfectant based on
glutaraldehyde on a Pseudomonas aeruginosa isolate with reduced susceptibility to
glutaraldehyde after the first and fifth passages were determined using three concen-
trations and temperatures. No significant difference was found between the first and fifth
passages so phenotypic adaptation is unlikely.

ª 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

A pseudo-outbreak was reported recently with a glutaral-

dehyde (GA)-resistant Pseudomonas aeruginosa isolate that
was detected from the rinsing water and drains of washer-
disinfectors for flexible endoscopes.

1

GA resistance was

assumed because the isolates were not killed consistently
within 10 min at 55

C by a 1% dilution of a product based on 20%

GA (which the manufacturer’s state is bactericidal at 1% within
5 min between 50

C and 55

C

1

). An ATCC strain of

P. aeruginosa, however, was killed within 10 min at 55

C by a

product dilution of 0.5%. Such a resistance pattern has only
been described previously for mycobacteria.

2

Although

continuous exposure to a sublethal concentration of an active

ingredient may result in reduced susceptibility of bacterial
cells, phenotypic resistance subsides once bacteria are
removed from the disinfectant. It was hypothesized that
resistance of this P. aeruginosa strain is a phenotypic adapta-
tion, rather than persistent expression of resistance. As such,
the bactericidal activity of a commercially available disinfec-
tant based on GA was compared with the bactericidal activity
of a GA solution in suspension tests on isolates from the first
and fifth passages of the strain propagated without any selec-
tion pressure.

Methods

The pseudo-outbreak isolate of P. aeruginosa (I38) was ob-

tained directly after its first isolation, and passaged either once
or five times on tryptone soya agar (TSA) prior to inoculation in
the suspension test. For passaging, a colony was transferred to
a TSA plate which was incubated for 24 h at 37

C before the

next passaging, similar to the method used by Gradel et al.

3

* Corresponding author. Address: Bode Science Center, Bode Chemie

GmbH, Melanchthonstrasse 27, 22525 Hamburg, Germany. Tel.:

þ49 40

54006 0; fax:

þ49 40 54006 165.

E-mail address:

[e-mail]

(G. Kampf).

Available online at

www.sciencedirect.com

Journal of Hospital Infection

j o u r n a l h o m e p a g e : w w w . e l s e v i e r h e a l t h . c o m / j o u r n a l s / j h i n

0195-6701/$

e see front matter ª 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jhin.2013.05.010

Journal of Hospital Infection 84 (2013) 316

e318

The disinfectant Neodisher

Septo DN 2 (glutardialdehyde 20 g

per 100 g; Chemische Fabrik Dr. Weigert GmbH & Co. KG,
Hamburg, Germany) and a GA solution (Dow Advanced Mate-
rials, Buchs, Switzerland) in water (glutardialdehyde 20 g per
100 g) were tested at 1%. Both test solutions contained 0.2%
glutardialdehyde.

Bactericidal activity was determined using the European

standard method for chemical disinfectants EN 13727.

4

Briefly,

the test organism was cultured on TSA and transferred into the
diluent after 24 h of incubation at 37

C. The cell number was

adjusted to 1.5

e5 10

8

colony-forming units per mL. Eight

millilitres of disinfectant dilutions (0.5%, 1% and 2%) or the GA
solution were mixed with 1 mL of an organic load (‘clean con-
ditions’: 0.03% bovine albumin) and 1 mL of the bacterial test
suspension and held at 20

C, 50

C and 55

C. The higher tem-

peratures were chosen because products based on gluta-
rdialdehyde are often used at 50

C and 55

C for processing

endoscopes.

5

After 2.5 min, 5 min or 10 min, 1 mL of the mixture

of bacteria, organic load and product was transferred to a tube
containing 8 mL of neutralizer and 1 mL of water. The following
substances were used as neutralizers: polysorbate (3%), lecithin
(0.3%), histidine (0.1%), sodium thiosulphate (0.5%) and saponin
(3%). After neutralization for 5 min, serial dilutions were per-
formed; 1-mL aliquots were divided into two portions of
approximately equal size, and spread on TSA plates that were
incubated at 36

C for 48 h. Colonies were counted and the

viable count of the test mixture was calculated. The reduction
of bacteria was calculated as the difference in the logarithms of
viable counts before and after the application time. The mini-
mum requirement for bactericidal activity according to EN
13727 is a reduction of at least 5 log

10

steps. Three replicates of

each experiment were performed. The t-test for independent
samples was used to compare the mean log reduction of the first
and fifth passages for each time and temperature. A P-value
<0.05 was considered to indicate significance.

Results

At a concentration of 1%, the disinfectant produced a 5 log

10

reduction at 50

C (10 min) and at 55

C (10 min) for the first and

fifth passages (

Table I

). The GA solution with the same con-

centration produced similar results. There was no significant
difference between the mean log

10

reductions of the first and

fifth passages for both formulations at any temperature or
application time (p

> 0.05; t-test).

Discussion

The data indicate that the reduced susceptibility of the

P. aeruginosa isolate to GA cannot be explained by phenotypic
adaptation caused by selection pressure due to a sublethal
concentration of GA. Even after five passages, the suscepti-
bility pattern remained unchanged. The selected isolate seems
to have reduced susceptibility to GA, a well-established active
ingredient with a broad spectrum of bactericidal activity,

6

in

suspension tests and under practical conditions.

5

Some bacterial cells may reveal a cross-adaptive response

after exposure to oxygen-releasing compounds; pretreatment
with a sublethal dose of H

2

O

2

makes specific Escherichia coli

cells resistant to aldehydes including GA.

7

The stages during

the reprocessing were analysed to identify risk factors for the
emergence of resistance. Endoscopes in this hospital were
decontaminated with a compound containing peracetic acid
(PA) to ensure that they were safe to handle before manually
wiping the outer surface and brushing the inner channels. PA is
a known oxygen-releasing compound and causes surface fixa-
tion of organic loads such as blood.

8

Surface fixation is to be

avoided during cleaning.

9

However, the most likely explanation

for GA resistance was that the washer-disinfector was

>5 years

old, so biofilm formation had probably occurred, possibly
enhanced by the use of PA.

10

The use of PA during cleaning may

Table I
Mean log

10

-reduction

SD obtained with Neodisher Septo DN 2 (1%) and glutaraldehyde (1% of a solution containing 20% glutaraldehyde)

against the first and fifth passages of a Pseudomonas aeruginosa isolate formerly described as glutaraldehyde-resistant (N

¼ 3; t-test for

independent samples)

Product (concentration)

Temperature (

C)

Exposure time (min)

Passage

P-value

First

Fifth

Neodisher Septo DN 2 (1%)

20

2.5

0.70

0.19

0.89

0.32

0.428

5

0.86

0.17

0.97

0.22

0.531

10

1.74

0.89

1.67

0.31

0.904

50

2.5

2.51

0.38

2.93

0.42

0.268

5

3.90

0.76

4.80

0.75

0.216

10

6.00

0.25

5.46

0.73

0.290

55

2.5

4.20

0.61

3.90

0.68

0.606

5

4.66

0.25

4.67

0.56

0.986

10

5.70

0.34

5.59

0.79

0.830

Glutaraldehyde (1% of a solution

containing 20% glutaraldehyde)

20

2.5

0.70

0.19

0.89

0.32

0.428

5

0.77

0.13

0.89

0.32

0.559

10

1.60

0.90

1.48

0.45

0.850

50

2.5

3.03

0.86

3.02

1.05

0.990

5

5.08

1.39

5.34

0.51

0.773

10

5.63

1.07

6.12

0.13

0.513

55

2.5

4.77

0.34

4.44

0.82

0.554

5

6.32

0.24

5.49

0.85

0.179

10

6.22

0.19

5.89

0.68

0.460

G. Kampf et al. / Journal of Hospital Infection 84 (2013) 316

e318

317

have induced GA tolerance of bacterial cells, thus leading to
inadequate processing. However, as long as the cellular
mechanism for the reduced susceptibility of the P. aeruginosa
isolate is not understood, it remains unclear why the cells were
not killed as effectively.

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