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Rapid Detection of Micro-Organisms using AK:
AKuScreen is a rapid microbiology release test for
non-sterile products, in-process samples, and raw
materials. These products are currently tested by
the USP Microbial Limit Test <61> or the equivalent.
During routine testing these products typically produce
a zero count and have an absence of objectionable
organisms, therefore they could be classified as “commercially
sterile”. A rapid screen of these products
to show which ones have any bioburden and which ones
do not allows 90-99% of products to be released faster.
Any products that are positive by AKuScreen need
further investigation to enumerate and identify any
contamination.
The AKuScreen protocol requires that the product
be sampled into Difco Letheen or TAT broth (or any
other type of broth that is validated for use with
the Celsis system) at typically 1%. The sample suspension
is then incubated at 30±2°C for 18 or
24 hours. Following incubation and any additional
sample preparation, a bioluminescence assay enhanced
with the linear amplification of ATP is carried out
automatically inside the Advance luminometer on a
small aliquot of the incubated product suspension.
AK Technology Explained:
ATP bioluminescence is now a commonly used method
for the microbial screening of a wide range of
products. It is applicable to a very wide range
of sample types and can detect the presence of
an unacceptable level of microbial contamination
within 1-2 working days.
A more sensitive method for the detection of microorganisms
has been developed by Celsis International: AK
technology.
All living organisms contain the compound ATP as
a vital part of their energy metabolism. It is this
ATP that is released and detected by a standard bioluminescence
test. ATP rapidly reacts, in the presence of the
luciferase enzyme, with luciferin and oxygen to give
a photon of yellow-green light:
A luminometer automatically adds the required reagents
to a sample and detects the emitted light with great
sensitivity. Although ATP bioluminescence is a very
sensitive technique it is limited by the fact that
it is detecting a metabolite (ATP), and an organism
can contain only a finite amount of ATP.
All living organisms also contain adenylate kinase
(AK), another vital part of energy metabolism. Because
this is an enzyme, rather than a metabolite, it is
possible to use AK to generate almost unlimited amounts
of its products.
The reaction catalysed by AK is:
Note that one of the products is ATP, which can
be measured with bioluminescence. The longer the
above reaction is allowed to proceed, the more ATP
is generated and the greater the bioluminescence
signal.
The amount of AK in a microorganism – like
the amount of ATP – varies from species to
species and is governed mainly by the size of the
cell: the larger the organism, the more AK it normally
contains. As a general guide, the AK enzyme in a
bacterium can be made to produce, in one minute,
approximately 40-fold more ATP than the organism
originally contained. If the reaction is allowed
25 minutes, the amount of ATP can be 1000-fold
more than the organism originally contained. In effect,
AK technology is based on the amplification
of microbial ATP.
Since the use of AK technology as the end-point
detection method gives a more sensitive measure of
microbial contamination than ATP bioluminescence,
the enrichment period can be shorter than with ATP
bioluminescence and the overall time to result will
be faster.
The General Procedure:
The Celsis system can be used to detect low levels
of contamination in non-sterile products. Any microorganisms
present in the sample may have low metabolic activity.
For this reason an incubation step is included
in the protocol before the assay is performed.
During incubation the sample itself is diluted
in growth medium so that any effects on the final
assay are minimized, and any microbes present in
the original sample are allowed to multiply and
generate adenylate kinase and ATP.
The AKuScreen protocol requires that the product
be sampled into Difco Letheen or TAT broth (or any
other type of broth that is validated for use with
the Celsis system) at typically 1%. The sample suspension
is then incubated at 30±2°C for 18 or
24 hours.
Two protocols can be performed with the AKuScreen
assay. The first protocol is the standard protocol
for detection of bacteria and yeasts in 18 hours.
The second is the mold protocol for detection of
bacteria, yeasts, and molds in 24 hours.
A different procedure is required for molds because
of the physiology of their growth. Whereas bacteria
and yeasts normally divide as they grow and disperse
evenly throughout the enrichment culture, molds tend – especially
during the first 24 hours after spore germination – to
grow as discrete units or mycelia. When a small sub-sample
is taken for the end-point assay it is quite likely
not to contain any mold biomass, and therefore there
is a strong possibility of failing to get a positive
signal.
Homogenization of large volumes (100 mL) is achieved
using a device called a Linear Shaker. Glass beads
(to aid the breakage of mold mycelia) and antifoam
(to make sampling easier at the end of the treatment)
are added to the enriched sample container. The container
is placed on the Linear Shaker which then rapidly
agitates for 30 minutes. At the end of the treatment,
mold adenylate kinase will be dispersed throughout
the broth and a sub-sample can be assayed in a cuvette
as normal. A description of the two protocols is
shown in Table 1:
| |
Standard Protocol
|
Mold Protocol |
| Prepare |
1 gram sample into 99mL sterile enrichment
broth |
1 gram sample into 99mL sterile enrichment
broth |
| Incubate |
Shake at 30°C for 18 hours |
Shake at 30°C for 24 hours |
| Disruption |
N/A |
Add 15g sterile beads and 100 μL of antifoam.
Place on linear shaker and homogenize for not
less than 30 minutes |
Pipette
|
Pipette 50µL into cuvette |
Pipette 50µL into cuvette |
Assay
|
Advance.im automatically controls all injections
and measurements |
Advance.im automatically controls all injections
and measurements |
Table 1: Adenylate Kinase Protocols
Following incubation and additional sample prep
(if necessary), a portion of the product in broth
is added to a cuvette that is placed in the luminometer.
Celsis LuminAMP is first injected automatically followed
by Celsis LuminEX. The Celsis LuminEX permeabilises the microbial
cell membranes (if present) to allow AK and ATP to
come into solution. The Celsis LuminAMP (purified ADP) is
converted to ATP by the microbial AK. This reaction
takes place for 40 minutes at 30°C. The Celsis
LuminATE is then injected and any light produced
is measured by the instrument in relative light units
(RLU). Results indicate the presence of microbial
AK and ATP if the product in broth gives a signal
significantly above that of the broth alone.
Signal to Noise Ratio
Studies demonstrate that AKuScreen exhibits a significantly
higher signal to noise ratio than the standard
ATP Bioluminescence assay. The Signal to Noise
(S/N) ratio – also called signal to blank
ratio – is an indicator of a kit’s
ability to distinguish between regular background
noise and contaminated samples and therefore is
a measure of assay sensitivity. As the S/N ratio
increases, so should the confidence of the user.
Any S/N value significantly above the control baseline
indicates a contamination. With AKuScreen, S/N
ratio is greatly increased compared to traditional
ATP bioluminescence. An internal Celsis study,
performed in February 2004, evaluated a number
of personal care products using both the AKuScreen
and RapiScreen reagents. The study demonstrated
that in those instances where contamination was
detected by both kits, the Signal to Noise ratio
for AKuScreen was substantially higher. In fact,
63% of the samples detected using AKuScreen provided
S/N readings in the overload category.
Figure 3: Signal to noise Ratio Distribution – RapiScreen
and AKuScreen
Limit of Detection
The limit of detection of Celsis AKuScreen is 1 organism
per sample. The sample size is generally 1g (equivalent
to 1 organism per gram of product) although this
can be increased or decreased as required.
Detection of very low numbers of different organisms,
in the presence of a 1% product suspension in 100
ml Letheen broth, has been demonstrated.
Growth
Media
Similar to broth selection for ATP Bioluminescence
assays, the broth used to incubate the sample must
be capable of supporting growth of viable organisms,
be able to neutralize any preservative systems,
contain relatively low levels of ATP and have good
batch to batch consistency. In addition, the broth
must have low levels of the enzyme adenylate kinase
in order to help ensure low background and therefore
good sensitivity.
A large number of broths were initially screened
for ATP content before testing some of the most promising
broths for AK content. Table 2 presents the data
for a range of broths, including those typically
used for testing pharmaceutical and personal care
products.
| Type of Broth |
ATP content (pM) |
AK content
(fmols ATP/min/50µl sample) |
Letheen Broth
TAT
Dey Engley
Eugon LT100
Nutrient Broth
Tryptic Soy Broth |
26
59
1432
700
453
2774 |
0.6
0.1
1
4
9
1 |
Table 2; Results of testing for ATP and AK content
of a range of growth media
Consistency in ATP content from batch to batch is
also important in the selection of an appropriate
broth for enrichment. Table 2 shows the results of
an assessment of ten batches of Letheen broth for
ATP content.
| Batch of Letheen Broth |
ATP content (pM) |
1
2
3
4
5
6
7
8
9
Mean ATP concentration |
33
25
26
19
19
27
27
35
16
36
26.4 pM
Standard deviation 7.3pM |
Table 3; Results showing ATP concentration in 9
batches of Letheen broth
