Strategic Diagnostics Inc Technical Bulletin Listeria #4
Comparison of Methods for the Detection of Listeria species in Foods
Version 1.0 01-01-04
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Strategic Diagnostics Inc Technical Bulletin Listeria #4
Introduction:
Listeria is a formidable organism that is widely distributed in nature, found commonly in plants, soil and decaying vegetation. Listeria monocytogenes is the primary species of concern to human health, and this pathogen has been linked to various foodborne outbreaks. The ubiquitous distribution of the organism in the environment, its ability to grow in adverse conditions including extremes of salt and temperature and its pathogenic nature make it of particular concern for RTE foods.
In general, the food industry tests primarily for Listeria species as an as an indicator of sanitation, since the organism can frequently persist in the food environment, recontaminating foods by forming biofilms and proving difficult to eliminate by cleaning solutions used for equipment etc. Therefore, routine testing for the absence of Listeria species in high risk products such as RTE foods and environmentals samples is wise to prevent illness, avoid costly product recalls and for general brand protection.
Strategic Diagnostics Inc. has developed an effective one- step enrichment system for the selective growth, isolation and detection of Listeria species in RTE foods and environmental samples. The system consists of proprietary enrichment media which facilitates the maximal recovery of both sub-lethally injured and healthy cells in a sample matrix, while selectively inhibiting closely related competitive flora, within a 40 h time period. No additions or amendments are needed and the broth also allows the option of autoclaving the media or adding the dehydrated media directly to pre-warmed sterile water just prior to use. After the enrichment process, 400ul of the sample is removed and boiled in a test tube for 5 minutes. A test strip is added directly to the test tube and a result is achieved after 10 minutes. During both development and application of the RapidChekÔ Listeria system, the assay was subjected to vigorous reactivity tests utilizing typed and field isolates from a variety of sources including clinical, environmental and food. The attached Technical Bulletin data characterizes and defines the attributes of the RapidChekÔ system for Listeria species compared to another immunoassay.
Aim:
The main objective of this study was to compare the performance of three enrichment/detection methods including two immunoassays and one cultural method for the isolation of Listeria species from various foods.
Experimental Protocol:
Various Listeria isolates including L. monocytogenes 4b (ATCC 19115, SDI M19) and L. monocytogenes 4c (ATCC 19116, SDI M20) were isolated from frozen stock and
grown in 10 ml TSB broth at 37°C for 18 h. Based on microbial plate counts, cheese and hot dog samples were batch inoculated with individual strains of Listeria at two levels, low (1- 2 cells/25g) and medium (2-5 cells/25g) inoculum. Inoculated samples were stored at 4°C for 72 h to allow bacteria to acclimatize to the food environment and more closely mimic what happens in real world samples.
For the cheese matrix, three strains were inoculated in three batches of cheese (ATTC 19115; SDI M19; ATCC 19116), at a low (5 replicates for each strain) and medium (5 replicates for each strain) inoculum. Twenty five gram samples were transferred to stomacher bags and 225ml of either RapidChekÔ one step Listeria enrichment broth (for SDI method) or buffered Listeria enrichment broth (BLEB for VIDAS and reference method). Broths were incubated at 30°C for 40 h (RapidChekÔ media) or 48 h (BLEB).
For the hot dog matrix, three strains were inoculated in three batches of hot dogs (ATTC 19115; SDI M20; ATCC 19116), at a low (5 replicates for each strain) and medium (5 replicates for each strain). Twenty five gram samples were transferred to stomacher bags and 225ml of either RapidChekÔ one step Listeria enrichment broth (for SDI method) or buffered Listeria enrichment broth (BLEB for VIDAS and reference method). Broths were incubated at 30°C for 40 h (RapidChek media) or 48 h (BLEB).
In addition to inoculated samples, 4 different brands of smoked salmon were purchased from a local supermarket and each brand was divided into three subsets of 25g. Nine to ten replicates of each brand were subsequently enriched using either RapidChekÔ enrichment broth or BLEB and incubated at 30°C for 40 h and 48 h respectively.
SmokeSalmon / Total # of Samples / RapidChek / Vidas / Reference Method
Brand Name 1 / 10 / 0 / 0 / 0
Brand Name 2 / 9 / 5 / 0 / 4
Brand Name 3 / 9 / 7 / 1 / 2
Brand Name 4 / 9 / 9 / 9 / 9
Total / 37 / 21 / 10 / 15
After enrichment, both inoculated and naturally contaminated materials were screened using either RapidChekÔ Listeria lateral flow strip, the semi automated VIDAS immunoassay or by plating onto selective agar (MOX).
All presumptive positive results for Listeria species on screening methods were further confirmed utilizing the FDA confirmation procedure.
Results :
Evaluation of RapidChek and Vidas with various Listeria strains in cheese and hot dog samples
Matrix / Strain / Cfu/25g / RapidChek / Vidas / Reference MethodCheese / L.mono 4b (19115) / 0.8 / 2 / 1 / 1
1.6 / 4 / 1 / 3
L.mono 4b
(SDIM19) / 2.6 / 3 / 2 / 4
5.2 / 3 / 3 / 3
L.mono 4c (19116) / 2.1 / 5 / 0 / 2
4.3 / 3 / 1 / 4
Hot Dog / L.mono 4b (19115) / 0.8 / 4 / 2 / 2
1.6 / 3 / 4 / 4
L.mono 4c (SDI M20) / 2.5 / 1 / 3 / 3
5.0 / 4 / 5 / 5
L.mono 4c (19116) / 2.1 / 1 / 1 / 1
4.3 / 3 / 3 / 3
Total / 36 / 26 / 35
Evaluation of Smoked Salmon for natural Listeria contamination
Discussion and Conclusions:
The results in all the tables represent confirmed positive results. These data indicate that the RapidChekÔ system facilitated better recovery of cells at both low and medium levels as compared to the other methods. This trend was evident in both inoculated and naturally contaminated foods where RapidChek detected Listeria in up to 40% more of the samples than either VIDAS or the conventional cultural method. This is important particularly if cells are present at low levels, a sensitive and specific method should be routinely utilized to ensure that all Listeria present is detected. It is concluded from this study that the RapidChekÔ system is more sensitive than either the comparative immunoassay or the cultural method for the detection of Listeria species in RTE foods.
For Technical & Customer Service call:
Strategic Diagnostics Inc
111 Pencader Drive
Newark, DE 19702
Phone: 800-544-8881
Fax: 302-456-6782
e-mail:
www.sdix.com
Version 1.0 01-01-04
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