Eissa 2016
May 2016, 2(4):1-5
International Journal of Microbiology and Allied Sciences (IJOMAS)
ISSN: 2382-5537
May 2016, 2(4):1-5
© IJOMAS, 2016
Perspective
Page: 1-5
Limitations of the current chemical neutralization tests for
assessment of microbial recovery from antimicrobials: A new
prospective approach
Mostafa Essam Eissa1*
Quality Control Department, Microbiology Laboratory Division, HIKMA Pharma
Pharmaceutical Company, 2nd Industrial Zone, Plot no.1, 6th of October City, Giza, Egypt
1
*Corresponding Author:
Mostafa Essam Eissa
Quality Control Department, Microbiology Laboratory Division, HIKMA Pharma
Pharmaceutical Company, 2nd Industrial Zone, Plot no.1, 6th of October City, Giza, Egypt
Mobile: 00201006154853, E-mail: [email protected]
Abstract
Neutralization of antimicrobial properties is considered as a crucial aspect when considering
an evaluation of efficacy of biocidal products and microbial recovery of microorganisms from
hostile chemical environment. Chemical neutralization method is considered one of these
main important techniques for neutralizing antimicrobial properties of the biocidal agents.
However, unless reaction time is nearly instantaneous the declining antimicrobial
concentration will take time that may affect the survival and recovery of the organism. In
general, the duration is uncontrolled and contingent which leads to biased and/or exaggerated
estimation of the true potency of the antimicrobial activity and possibly a low microbial
recovery from the environmental monitoring (EM) samples. A thorough review on the
preparation of protocols for the chemical neutralization is required in order to determine the
significance and influence of the reaction time for the diminishing antimicrobial compounds.
The consequences of stressed or injured microbial cells due to the exposure to harsh chemical
environment may lead to a viable but not culturable (VBNC) state in which no colonies can
be observed in a conventional culture medium. This may lead to overestimated potency of
the true biocidal activity. This fact highlighted the importance of the application of new
advanced technologies for the enumeration of the viable microbial particles using rapid
microbiological methods (RMMs). In healthcare providing facilities, proper disinfection
program must be ensured to face the ever growing list of objectionable microbes that affect
human health.
Key words: Biocidal, environmental monitoring, microbial recovery, neutralization, reaction
time.
International Journal of Microbiology and Allied Sciences, May 2016, Volume 2 Issue 4
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Eissa 2016
May 2016, 2(4):1-5
Introduction
The process of neutralization of a biocidal
effect is a pivotal step in many quality control
monitoring
and
testing
protocols.
Pharmacopeial methods of neutralization
include
filtration,
dilution,
chemical
inactivation or combinations of these methods
[1]. Chemical neutralization techniques have
been studied extensively by many
investigators and researchers [2]. A valid
protocol has been developed. Such protocol
involved the mixing of a neutralizing agent
with an antimicrobial product for a sufficient
time (ex. ten minutes) followed by the
addition of the test microorganism. After
adequate incubation time, such samples were
plated to examine the microbial recovery [2].
B. Toxicity of the neutralizing agent and/or
the byproduct(s) of its reaction with the
biocidal agent
Since chemical neutralization is based on
chemical reaction between a neutralizing
broth and the antimicrobial compound, the
formed byproduct(s) may possess intrinsic
adverse effect on the viability of the microbial
cells. Even the neutralizing compound itself
may show some toxicity against some types of
microorganisms. Table 1 demonstrates the
toxicity of some neutralizing agents – which
are dedicated for neutralizing some
antimicrobial chemical classes - against some
microorganisms [4]. On the other hand,
Figure 1 shows schematic diagram of how this
property can be assessed and determined in
the neutralization study [5].
Main challenges of the chemical
neutralization study of the biocidal agents
2. General challenge for the neutralization
process:
A. Viable but not culturable state of
microbial cells (General challenge)
Even for those tests that depend on a relatively
longer
contact
times
between
the
antimicrobial products and microorganisms
such as preservative efficacy test (PET), the
possibility of entering viable but not
culturable (VBNC) state cannot be excluded.
This may lead to an over estimation of the true
antimicrobial power of the biocidal product.
The estimated number of microbial particles
might be far below the true bioburden density,
even if the incubation conditions such as
temperature and time have been modified. [6].
Several factors affect the validity and integrity
of the results derived from chemical
neutralization studies. Some of these factors
are general for all types of neutralization
procedures (chemical, filtration, dilution).
While other factors influence the chemical
neutralization process only.
1. Specific challenge for chemical
neutralization
A. Chemical reaction time till complete
neutralization
The kinetics studies of the antimicrobial
efficacy test (AET) depends on the process of
stopping the action of a biocidal agent on the
exposed
test
microorganisms
at
predetermined intervals. If this action cannot
be stopped instantaneously, then there will be
a residual amount of antimicrobial
components that may affect the viability of
microbial particles [3]. In addition, the time of
microorganisms’ exposure to the decreasing
concentration of the biocidal components is
unknown and hence the true activity of an
antimicrobial product may be overestimated.
The reaction time impact on the results of a
neutralization study possesses a great effect
on the microbial recovery in case of short
contact time between microorganisms and
antimicrobial products.
B. Accuracy of microbial number
estimation
Specification of the optimum CFU range for
microbial enumeration of plated samples is
necessary to ensure the accuracy of data
derived from the neutralization study. There
are some regulatory authorities that suggest
25-250 CFU (Colony Forming Units)/plate as
a countable limit range [7]. The core of this
dilemma is that the yield of the test results
demonstrates extremely low accuracy in plate
count under 25 CFU (when the number is 25
CFU/plate, error as a percent of the average is
International Journal of Microbiology and Allied Sciences, May 2016, Volume 2 Issue 4
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Eissa 2016
May 2016, 2(4):1-5
Table 1: The antimicrobial compound class, the neutralization method of the biocidal
activity for the antimicrobial compound, and the possible toxicity (from the neutralizing
agent and/or the byproduct of the reaction between the neutralizing agent and the
antimicrobial compound) on specific class of microorganisms [5] are shown
Antimicrobial Compound
Neutralization Technique
Glutaraldehyde, Mercurials Bisulphate/Chemical
Phenolics,
Alcohol,
Dilution
Glutaraldehyde
Glutaraldehyde
Glycine/Chemical
Quaternary
Ammonium
Compounds
(QACs), Lecithin/Chemical
Parabens, Bis-biguanides
Magnesium and Calcium
EDTA
Ions/Chemical
QACs, Iodine, Parabens
Tween/Chemical
Mercurials
Thioglycollate/Chemical
Possible
Toxicity
on
Microorganism
Non-spore
forming
bacteria
n/a
Growing cells
Bacteria
n/a
n/a
Staphylococci and Spores
Figure 1: Diagram showing the basic process core of the evaluation of the neutralization
process which is valid for any type or combinations of the neutralization processes [6].
20 %). Theoretically, it can be assumed that
the microbial count expressed as CFU follows
Poisson distribution [8], and hence the
estimated error expressed as a percent can be
obtained from calculating the square root of
the mean [8]. Thus, the optimum countable
range is a balance between the error expressed
as a percent of the average and the standard
error. However, the error percent of the
average decreases
increasing the count.
exponentially
with
Importance of the application of new
technologies in microbial recovery
New technologies that are based on rapid
microbiological methods can be divided into
the following classes: Growth-based
techniques: e.g., carbon assimilation,
International Journal of Microbiology and Allied Sciences, May 2016, Volume 2 Issue 4
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Eissa 2016
May 2016, 2(4):1-5
validity of a chemical neutralization process,
either in case of sanitizer validation or in-situ
evaluation of disinfection program.
enzymatic
reactions,
carbon
dioxide
generation or ATP bioluminescence; Artifactbased methods, e.g., fatty acid analysis using
gas chromatography, ELISA and MALDITOF mass spectrometry of cell components
such as nucleic acids and proteins; Nucleic
acid-based methods: e.g., PCR and automated
Southern blotting; Viability-based methods:
e.g., fluorescent labelling methods, such as
flow
fluorescence
cytometry;
immunofluorescence and fluorescent nucleic
acid stains used as a viability marker along
with propidium iodide as a membranecompromised cell marker. The role of rapid
microbiological methods (RMMs) in such
situations becomes apparent where they can
detect VBNC microbes in fairly short time
compared to the conventional culture-based
methods [9, 10]. While conventional culturebased techniques are relatively inexpensive
and reproducible, yet they usually show a very
low level of microbial recovery (0.01–1 %)
from the true bioburden density for any given
specimen as shown by some authors [4].
References
1. US Pharmacopoeia <1072>. 2014.
Disinfectants
and
Antiseptics.
Baltimore, MD, USA: United States
Pharmacopeia.
2. Sutton SV and Proud DW, Rachui S,
Brannan DK. 2002. Validation of
microbial recovery from disinfectants.
PDA Journal of Pharmaceutical
Science and Technology. 56(5):25566.
3. Weerasinghe BN. 2008. Kinetics of
tap water dechlorination and aquatic
health
impacts
of
selected
dechlorination chemicals. Doctoral
dissertation, University of Ottawa.
4. Sutton S. 2016. Antimicrobial
Efficacy
Test
[Internet].
The
Microbiology Network. [cited 10
April 2016]. Available from:
http://www.microbiol.org/resources/
monographswhitepapers/antimicrobial-efficacy-test/
5. Sutton S. 2016. Method Suitability
Control Studies for Microbial Testing:
Quantitative Comparisons | IVT GMP - Microbiology [Internet].
Ivtnetwork.com. [cited 02 April
2016].
Available
from:
http://www.ivtnetwork.com/article/m
ethod-suitability-control-studiesmicrobial-testing-quantitativecomparisons
6. Watkins J and Jian X. 1997. Cultural
methods
of
detection
for
microorganisms: recent advances and
successes. Freshwater Biological
Association. 19-27.
7. Maturin LJ and Peeler JT. 2011.
Bacteriological Analysis Manual,
Lessons learned
It could be concluded that there are three
requirements for effective neutralization: 1Effective cessation of the antimicrobial
product by neutralizer. 2- Absence of intrinsic
toxicity of the neutralizer on the microbial
cells. 3- The reaction product(s) of the
neutralizing agent and the antimicrobial agent
must be non-toxic to the microbial cells.
Another three factors are required to be
considered when implementing successful
protocol of neutralization study: 1- Test
microorganisms counts should be in the valid
count range with minimal errors specified by
guidelines and/or verified using method
discussed pharmacopeially. 2- According to
the nature and purpose of the study, the
classical culture media may include specific
components that are required for the recovery
of the stressed and/or injured microorganisms
otherwise the search for adopting RMMs will
be crucial in such instances. 3- The
neutralization reaction time between the
biocidal agent and the neutralizing agent is an
important factor in the assessment of the
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Eissa 2016
May 2016, 2(4):1-5
Chapter 3: Aerobic Plate Count.
[Online]. Available from, URL:
http://www.fda.gov/Food/
Sd.ence
Research
Laboratory
Methods
Bacteriological Analytical Manual.
Htm
8. Sutton
S.
Counting
Colonies
[Internet]. 2016. The Microbiology
Network. [cited 01 February 2016].
Available
from:
http://www.microbiol.org/resources/
monographswhite-papers/countingcolonies/
9. Eissa M. 2015. RMM: In-Depth Focus
2015
[Internet].
European
Pharmaceutical Review. [cited 15
March 2016]. Available from:
http://www.europeanpharmaceuticalr
eview.com/35997/supplements/rmmin-depth-focus-2015/
10. RMM Tutorial - Rapid Microbiology
and Rapid Microbiological Methods
[Internet].
2016.
Rapidmicromethods.com. [cited 08
April 2016]. Available from:
http://rapidmicromethods.com/files/tu
torial.php.
For Citation:
Eissa ME. 2016. Limitations of the current chemical neutralization tests for assessment of
microbial recovery from antimicrobials: A new prospective approach. International Journal
of Microbiology and Allied Sciences. 2(4):1-5.
International Journal of Microbiology and Allied Sciences, May 2016, Volume 2 Issue 4
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