• MLAB_30_ 01.02.09.doc


  •   
  • FileName: MLAB_30.pdf [read-online]
    • Abstract: ATP detection and the detection of surface protein. residues as well as pathogen specific ... causing food poisoning or worse, present in the. environment, which may not have been ...

Download the ebook

Microgen Bioproducts Newsletter N o 30 2010
N o 7 2002
.
Dairy Farmers Confirm the Superior
Performance of Path-Chek Listeria
To ensure products are free from microbial the presence of specific food pathogens capable of
contamination food manufacturers are increasingly causing food poisoning or worse, present in the
adopting environmental sampling program as part of environment, which may not have been eliminated by
their HACCP (Hazard Analysis Critical Control Point) routine cleaning and sanitizing procedures (2).
plans. An environmental monitoring programme
focused on risk assessment and enables the The detection of Adenosine trisphosphate (ATP) is
detection of microbial contamination, particularly the an established method of hygiene monitoring within
detection of important food pathogens in a timely the food industry. ATP analysis is the detection of a
manner. If samples are taken in a planned manner to nucleotide which exists in all cells so it does not
reflect the differing working conditions and the results specifically detect pathogens but acts as a surrogate
are tabulated correctly, meaningful comparisons and marker of product residue. ATP analysis is not a
the analysis of trends can be examined easily. microbiological method as it is based on the
enzymatic conversion of Luciferin to Oxyluciferin.
The importance of microbial contamination and the ATP tests provide a rapid result but it does not
implications for consumer safety and commercial indicate if the ATP detected is from bacteria and
damage is highlighted by an outbreak of Listerosis whether the bacteria are important pathogens so it
caused by contaminated meat products in Canada. can be used a complementary tool to rapid and
The outbreak was linked to 22 deaths and cost the effective pathogen monitoring. Detection of specific
company $20 million in a product recall of 220 pathogens within the manufacturing and/or
products and $25-27 million to settle law suits (1). processing environment is vital to detect the
Listeriosis is caused by Listeria monocytogenes, the presence of important food pathogens introduced into
pathogenic species of the genus Listeria, a gram- the food handling environment and highlight the
positive, catalase positive and oxidase negative sources of these pathogens which may be resident in
group of organisms. The disease is a serious the environment.
problem as it has a high fatality rate (>25%). Listeria
is widespread in the environment so consequently Traditionally food manufacturers either have to send
there is considerable opportunity for food products environmental samples to commercial laboratories
and food handling environments to be contaminated. for analysis, which can be expensive and timely, or
The problem of Listeria contaminating food is a follow traditional microbiological methods of
concern for any food manufacturer as the organism environmental monitoring. The traditional
grows well in a wide range of salt concentrations, environmental microbiological testing methods for
pH's and temperature conditions giving it a Listeria involves sample swabs being incubated for
competitive advantage over other mesophilic flora. 48 hours in an appropriate broth medium followed by
subculture onto a suitable agar plate medium such as
Environmental Monitoring ALOA and/or Oxford agar. This is a laborious
process which can take up to 5 days to achieve a
A variety of methods can be employed for final result. Th e time to result and the 3 step process
environmental monitoring such as visual analysis, is a disadvantage for any food manufacturer
ATP detection and the detection of surface protein especially if they operate a positive release
residues as well as pathogen specific environmental programme and this is limited to those companies
monitoring. However, these methods do not and large enough to have suitable laboratory facilities or
cannot demonstrate the presence of specific food access to contract testing laboratories.
poisoning bacteria. They either detect the presence
of bacteria non specifically, or food residues on
surfaces that will most likely harbour bacteria. Only
specific environmental testing methods can detect
1
The Solution?
Method
Microgen Bioproducts has overcome problems
associated with these traditional methods of Environmental swabs were collected within one of
environmental monitoring by the introduction of Path- the Dairy Farmers manufacturing sites. In each area,
Chek Hygiene Pathogen system for the detection of 3 separate swabs were taken within a 5 minute
important food-borne pathogens (Listeria spp, period to minimise variability and each site was
Coliforms and Salmonella spp.) from work surfaces sampled 5 times over a 5 week period. One swab
and manufacturing equipment in food handling and from each site was tested for the presence of Listeria
manufacturing environments. The pathogen spp. using each of the systems under evaluation.
detection system consists of two units; a pre-
moistened swab, which has the benefit of neutralising
the effects of cleaning solutions and improving 3M PetrifilmTM
bacteria recovery from dry surfaces; and a highly
specific and sensitive detection media which gives
results by providing a clear visual colour change in 3M PetrifilmTM Listeria is a quantitative method which
18-24 hours for Coliforms and Salmonella spp. and involves sample ready plates, which are inoculated
30-48 hours for Listeria spp. if specific organisms are and incubated for 292 hours after an environmental
present on surfaces (Figure 1). The pathogen sample has been resuscitated (Figure 2).
detection system meets the requirements of ISO:
18395:2004(E) and is compliant with the
requirements of USDA, FSIS and BAM but unlike
similar methods does not require a pre-enrichment
step (3).
Salmonella spp. broth
Figure 2. 3M PetrifilmTM being inoculated.

bioMerieux's VIDAS
Coliform broth Listeria spp. broth

bioMerieux's VIDAS system is an automated
qualitative instrument which utilises ELFA (Enzyme
Linked Fluorescent ASSAY) technique.
Environmental Listeria samples for analysis via a
Figure 1. Path-Chek Hygiene Pathogen detection VIDAS system requires pre-enrichment in half fraser
broth. Positive reactions are on the left and negative broth for 24 hours followed by enrichment in fraser
are on the right. broth for 24 hours. After a sample has been pre-
enriched and enriched, 0.5 ml of the fraser broth

inoculates the VIDAS strip, which is then analysed
Evaluation automatically by the instrument and a test value
generated for each sample (Figure 3).
One of Australia's largest food manufacturing
companies (Dairy Farmers) evaluated Path-Chek
Hygiene Listeria to determine its effectiveness in the
isolation of Listeria from environmental samples.
This validation includes parallel testing using

bioMerieux VIDAS from a NATA accredited
laboratory (NATA is Australia's internationally
recognised laboratory accreditation authority) as well
as the 3M Listeria PetrifilmTM.

Figure 3. bioMerieux's VIDAS
2
Path-Chek
Test Date
Site Description
4/03/2009 9/03/2009 11/03/2009 16/03/2009 18/03/2009
Result Result Result Result Result
Drain, Line 9 Positive Positive Positive Positive Positive
Crack in floor, Line 7 Positive Positive Positive Positive Positive
Conveyor, Line 4 Positive Positive Positive Negative False Positive
Crate Conveyor, Line 9 Positive Negative Negative Negative Negative
Conveyor, Line 3 Negative Negative False Positive False Positive Positive
3M Petrifilm
Test Date
Site Description
4/03/2009 9/03/2009 11/03/2009 16/03/2009 16/03/2009
Result Result Result Result Result
Drain, Line 9 False Negative False Negative False Negative False Negative False Negative
Crack in floor, Line 7 Positive Positive Positive Positive Positive
Conveyor, Line 4 Positive False Negative False Negative Negative Negative
Crate Conveyor, Line 9 Positive Negative Negative Negative Negative
Conveyor, Line 3 Negative Negative Negative Negative False Negative
Siliker (NATA Approved Method)
Test Date
Site Description
4/03/2009 9/03/2009 11/03/2009 16/03/2009 18/03/2009
Result Result Result Result Result
Drain, Line 9 Positive Positive Positive Positive Positive
Crack in floor, Line 7 Positive Positive Positive Positive Positive
Conveyor, Line 4 Positive False Negative False Negative Negative Negative
Crate Conveyor, Line 9 Positive Negative Negative Negative Negative
Conveyor, Line 3 Negative Negative Negative Negative Negative
Table. 1 Summary of Listeria detection using 3 testing methods used on 5 sites on 5 separate occasions
3
Path-Chek and PetrifilmTM are both low cost in-house
methods used for environmental monitoring although
PetrifilmTM requires the expertise of a microbiologist
as there are two aseptic steps (resuscitation and
inoculation). Path-Chek is a simple one-step closed
Site Description Identification of system as the swab tip is snapped off into the
Confirmed Isolate detection tube, ensuring 100% of the sample is in the
detection system and this is the only time the
Drain, Line 9 L. monocytogenes detection medium is opened during the procedure.
Crack in floor, Line 7 L. monocytogenes The simple one-step procedure allows non-
microbiologists to use the system and any result can
Conveyor, Line 4 Listeria species
be confirmed by an external laboratory.
Crate Conveyor, Line 9 L. monocytogenes
Conveyor, Line 3 Listeria species Path-Chek is a more cost-effective test to PetrifilmTM
as it is a complete kit comprising of sanitising
neutraliser, pre-moistened swab which has the
benefit of neutralising any detergents or sanitizers
and chromogenic detection broth. PetrifilmTM
Table 2. Identification of confirmed positive
requires an additional purchase of a collection swab
isolates from each site
and resuscitation broth which adds to the cost.
Discussion
This study indicates Path-Chek Listeria as the most
sensitive, easy-to-use rapid (24-48hrs to result) in-
house method for the detection of Listeria. This
In this study PetrifilmTM demonstrated a lower
would result in a significant time and cost saving
sensitivity, with detection of only 47% of the
compared to an external laboratory, helping prevent
confirmed positive samples using other methods, in
a serious health problem and an expensive product
comparison to Path-Chek Listeria (100%) and
recall.
VIDAS (87%). Path-Chek did have a false positive
rate of 12% based on initial visual interpretation,
References:
followed by confirmation by subculture and the
identification of suspect colonies in their laboratories.
1. Canadian Food Inspection Agency
These false positive results are however still
(www.inspection.gc.ca)
considered valuable as they highlighted the fact that
2. A Guide to Environmental Microbiological
high background levels of certain organisms such as
Testing for the Food Industry. Philip Mugg.
Bacillus spp. and Enterococcus spp., which are good
3. ISO:18395:2004(E) Microbiology of food and
indicators of a poor hygiene level or poor sanitising
animal feeding stuffs Horizontal methods
practice still existed. A high false negative rate is not
for sampling techniques from surfaces using
acceptable as it may result in the release of food
contact plates and swabs.
products with high bacterial loads which may result in
rapid food spoilage. .
We wish to acknowledge and appreciate the
work of Mr David Wong, Scientific Services
The lack of sensitivity of the 3M PetrifilmTM is
Manager at Dairy Farmers, Australia, in
possibly due to fact that this method does not involve
production of this data.
an enrichment step that would increase the overall
sensitivity, particularly when a low number of cells
are involved or cells have been damaged or stressed
by temperature or detergents and sanitizers.
FOR MORE INFORMATION ON THE MICROGEN IDENTIFICATION PRODUCTS OR
ANY OF THE OTHER MICROGEN PRODUCT, PLEASE CONTACT YOUR LOCAL
MICROGEN DISTRIBUTOR
DISTRIBUTED BY:
2


Use: 0.4944