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Temelli, Detection of Listeria in retail dairy products
Detection of Listeria spp. and Listeria monocytogenes in retail dairy products using the Vitek immunodiagnostic assay system LDUO method and ISO method 11290-1 1
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By Seran TEMELLI , Zafer ATA , Sahsene ANAR , Kamil Tayfun CARLI , Aysegul EYIGOR * 1
Department of Food Hygiene and Technology, Department of Microbiology, Faculty of Veterinary Medicine, Uludag University, 16059, Görükle Campus, * Bursa, Turkey. E-mail:
[email protected] 3 Military Veterinary School and Educational Central Commandership, Gemlik, Bursa, Turkey 2
This study aims to determine the presence of Listeria monocytogenes, an important infectious agent in humans, and other Listeria spp. in retail dairy products using the Vitek Immunodiagnostic Assay System LDUO (VIDAS LDUO) rapid detection method and ISO method 11290-1 (ISO 11290-1). One hundred dairy products (10 pasteurised milk, 10 kashar cheese, 20 white cheese, 18 mihalic cheese, 12 curd cheese, 20 ice cream, and 10 butter samples), collected randomly from food stores in Bursa, Turkey were analysed using the VIDAS LDUO and ISO 11290-1 methods. The results showed that one dairy sample (1%), a mihalic cheese, was positive for both Listeria spp. and L. monocytogenes. This finding highlights the risk of Listeria contamination of cheese manufactured from raw milk. Nachweis von Listeria spp. und Listeria monocytogenes in Milchprodukten des Einzelhandels mit dem Vitek immunodiagnostic assay system LDUO und der ISO-Methode 11290-1 In dieser Studie sollte das Vorkommen von Listeria monocytogenes, einem bedeutsamen Infektionserreger des Menschen, sowie anderer Listeria-Arten in Milchprodukten des Einzelhandels unter Einsatz des Vitek Immunodiagnostic Assay-Systems LDUO und der ISO-Methode 11290-1 ermittelt werden. 100 Milchprodukte (10 pasteurisierte Milchen, 10 Kashar- Käse, 20 Weichkäse, 18 Mihalic-Käse, 12 Lab-Käse, 20 Speiseeis- und 10 Butterproben) aus Lebensmittelgeschäften in Bursa, Türkei, wurden mit den genannten Methoden untersucht. Die Ergebnisse zeigten, dass in einer Probe (1%), einem Mihalic-Käse, sowohl Listeria spp. wie auch L. monocytogenes nachgewiesen werden konnten. Dieser Befund macht deutlich, dass das Risiko einer Listeria-Kontamination von aus Rohmilch hergestelltem Käse gegeben ist. 26 Listeria monocytogenes (Turkey, detection in dairy 26 Listeria monocytogenes (Türkei, Nachweis in products) Milchprodukten)
1. Introduction Listeria monocytogenes is a zoonotic pathogen that can contaminate dairy products and therefore cause food-borne listeriosis in humans. Listeria can be introduced to dairy products before, during, or after processing, and it can survive in the product for long periods. Cheese represents one of the most important potential carriers for this pathogen. Reports from various countries (1), including the United States (2), Japan (3), and Canada (4), have identified dairy products as important sources of listeriosis outbreaks. Compliance with hygienic standards during the processing and consumption of dairy products is critical to improving public health and minimising related economic losses. In Turkey, official controls of dairy products using rapid, reliable and cost-effective measures based on risk analysis using ‘internationally recognised methods’ have become mandatory. Accordingly, the Directive on Microbiological Criteria of the Turkish Food Codex (2001/19) (5) indicates the absence of L. monocytogenes in five randomly selected 25-g samples of a particular food. Currently, the most commonly applied standard guidelines for the detection of Listeria monocytogenes in foods in the United States and in the European Union are the detection method indicated in Chapter 10 of the United States Food and Drug Administration Bacteriological Analytical Manual Chapter 10: Detection and Enumeration of Listeria monocytogenes in Foods (6) and the International Organization for Standardization Method 11290-1 (ISO 11290-1) (7),
respectively. These bacteriological guidelines primarily cover conventional culture methods and are regarded as the ‘gold standard’ for the identification of this pathogen. However, isolation and confirmation using these methods may require up to 7 days to produce a positive result, and they are therefore not appropriate for the routine testing of larger numbers of samples. Alternative methods for the detection of Listeria spp. and L. monocytogenes, such as Enzyme Linked Fluorescent Immunoassay (ELFA) in a fully automated VIDAS system, offer performance equivalent (in sensitivity and specificity) to these standard methods but are more rapid (2 days), less complicated, and less labour-intensive (8-16). This study aims to determine the presence of Listeria spp. and L. monocytogenes in retail dairy products using the Vitek Immunodiagnostic Assay System LDUO (VIDAS LDUO) rapid detection method and the ISO 11290-1 method.
2. Materials and methods 2.1 Listeria monocytogenes standard strain Listeria monocytogenes ATCC 7677 obtained from the Refik Saydam National Public Health Agency in Ankara, Turkey, was used as a positive control.
2.2 Samples One hundred dairy product samples (10 pasteurised milk, 10 kashar cheese, 20 white cheese, 18 mihalic cheese, 12 curd cheese, 20 ice cream, and 10 butter samples) were randomly purchased from local Milchwissenschaft 67 (4) 2012
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food stores and transferred to the laboratory on ice. The samples in their original packages were individually repacked in sterile polyethylene bags to prevent cross-contamination during purchase and transfer. All of the samples were analysed immediately after transfer to the laboratory.
2.3 Analysis of samples by ISO and VIDAS The original packages of pasteurised milk, ice cream and butter were opened under aseptic conditions, and the whole sample was transferred into a sterile polyethylene bag. The sample was homogenised by hand from the outside of the bag, and 25-ml sub-samples were then taken for ISO and VIDAS. For all of the cheese samples, the whole cheese was placed onto a sterile polyethylene sheet and chopped 3 into 1-cm pieces under aseptic conditions. These pieces were mixed well on the sheet, and 25-g subsamples were then taken for ISO and VIDAS. For ISO 11290-1, each 25-ml (or g) sample was aseptically placed into a sterile stomacher bag with a filter and 225 ml of Half Fraser Broth (Oxoid CM 0895B). The samples were incubated at 30°C for 24 h for pre-enrichment after homogenisation by hand from the outside of the bag for 2 min. A 100-µl aliquot of this pre-enrichment culture was then transferred to 10 ml Fraser Broth (Oxoid CM0895B) for selective enrichment and incubated at 37°C for 48 h. Then, one loopful each of the pre-enrichment and selective enrichment cultures were streaked onto ALOA (Chromogenic Listeria Agar [ISO], Oxoid CM1084B) and Oxford (Listeria Selective Agar Base, Oxford, Oxoid CM856B) agar plates, which were incubated at 37°C and at 35°C for 24 h, respectively. Plates without typical colonies were incubated for a further 24 h. A maximum of 5 typical colonies were sub-cultured on plates of Blood Agar Base No. 2 mixed with 7% sterile sheep blood (Oxoid, CM0271). The plates were incubated at 37°C for 24 h, and selected colonies were subjected to Gram staining. Isolates with Gram-positive nonspore-forming small rods were then exposed to initial biochemical confirmation by catalase, oxidase and haemolysis assays, and further confirmation by API Listeria (Biomerieux 10300) (Fig. 1). All Listeria spp. isolates identified were stored at -20°C. For the VIDAS LDUO protocol, 25 ml/g of sample was homogenised in 225 ml Listeria XPress Broth (LX Broth, Biomerieux 42626) and incubated at 30°C for 24 h for pre-enrichment. A 100-µl aliquot of this preenrichment culture was then transferred to 6 ml LX Broth (Biomerieux 42117) for a 24 h primary enrichment at 30°C. Following this incubation, a 1-ml ali quot of the LX broth culture was heat treated at 100°C f or 5 min in a block heater (Techne, FBD02DD), and the remaining broth was stored at 4 °C until confirmati on. After cooling to room temperature, 500 µl of the heattreated LX broth culture was placed into a VIDAS LDUO strip (Biomerieux 30225), which was then loaded into a miniVIDAS (Biomerieux, Marcy L’Etoile, France) instrument for 127 min. Analysis was performed according to the manufacturer’s instructions for presumptive L. monocytogenes (DLMO) and for Listeria spp. (DLIS) positivity. Relative fluorescence values of < 0.05 in DLMO and < 0.1 in DLIS were considered negative for L. monocytogenes and Liseria Milchwissenschaft 67 (4) 2012
Fig. 1: Flowchart of the detection of Listeria spp. and L. monocytogenes in dairy products
spp., respectively. Samples with relative fluorescence values of < 0.05 in DLMO and of ≥ 0.1 in DLIS, indicating the absence of L. monocytogenes but the presence of Listeria spp., respectively, were further subjected to DLIS confirmation. Samples with relative fluorescence values of ≥ 0.05 in DLMO and positivity in DLIS indicating the presence of L. monocytogenes and thereby of Listeria spp., were confirmed by DLMO. For both DLIS and DLMO confirmation, a loopful of the LX broth culture that had been reserved at 4 °C was streaked on to Ottaviani Agosti Agar (OAA, Biomerieux 43641) and incubated at 37°C for 24 h. Confirmation was performed as indicated above in ISO 11290-1 (Fig. 1). L. monocytogenes ATCC 7677 was used as a positive control in ISO 11290-1 standard method applications and for each VIDAS LDUO sample test run.
3. Results Of the 100 dairy products collected randomly from food stores in Bursa, Turkey, only one dairy sample (1%), a mihalic cheese, was found to be positive for both Listeria spp. and Listeria monocytogenes by the VIDAS LDUO and ISO 11290-1 methods. None of the pasteurised milk, kashar cheese, white cheese, curd cheese, ice cream or butter samples were found to harbour this pathogen or any other Listeria spp.
4. Discussion and conclusions The most important finding in this study is the detection of L. monocytogenes in a mihalic cheese sample. This type of hard cheese, which is produced from raw milk, has 10-25% salt content. The presence of Listeria in this type of a cheese can be attributed not only to its ubiquitous distribution in the environment but also to its ability to survive at a wide range of pH values and in high salt concentrations. Our findings are consistent with those of ARDA et al. (17), JACOBSEN et al. (18), and LITTLE at al. (19), who reported the detection of L. monocytogenes in 2.7% of hard
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cheeses, 1.4% of cheeses produced from raw cow’s milk, and 1% of cheeses produced from raw milk, respectively. In this study, none of the pasteurised milk samples was found to harbour Listeria spp. or L. monocytogenes by either method. This is lower than the previous detection rates of 5.6% for Listeria spp. and 0% for L. monocytogenes reported by KELLS and GILMOUR (20). In their study, the authors linked the presence of Listeria spp. in pasteurised milk samples to contamination after pasteurisation. The absence of Listeria spp. and L. monocytogenes in the kashar cheese samples in our study are consistent with the results of JOHANSSON et al. (21), who did not find any L. monocytogenes in their hard or semi-hard cheese samples. In contrast, other studies conducted in Turkey by DÜMEN et al. (22) and OKTAY and HEPERKAN (10) reported L. monocytogenes in kashar cheese samples, with detection rates of 2.7% and 7.1%, respectively. The absence of Listeria spp. and L. monocytogenes in curd cheese samples in our study is consistent with the findings of UYSAL and ANĞ (23) but contrasts with the results of GEBRETSADIK et al. (24), who found 4% Listeria spp. and 1% L. monocytogenes in their cottage cheese samples. None of the white cheese samples in our study harboured Listeria spp. or L. monocytogenes. This is consistent with the findings of JOHANSSON et al. (21), who did not detect any L. monocytogenes in their fresh cheese samples. In contrast, a previous study on the detection of L. monocytogenes in cheese made from pasteurised milk (19) reported its presence in 0.2% of samples. Additionally, FRECE et al. (25), DÜMEN et al. (22), KARA et al. (26), and UYSAL and ANĞ (23) detected this pathogen in their fresh cheese samples at reported rates of 3%, 2%, 2.94%, and 4%, respectively. No Listeria spp. or L. monocytogenes were detected in any of the ice cream samples examined in our study, consistent with the findings of KESKIN et al. (27). However, GÖNÜLALAN and GÖNÜLALAN (28) and ARSLAN et al. (29) reported the detection of Listeria spp. in 18% and 1.36% of ice cream samples, respectively. No Listeria spp. or L. monocytogenes were detected in any of the butter samples in our study, similar to the results of a study by OKTAY and HEPERKAN (10). However, previous studies reported the detection of this pathogen in cream samples (12% by UYSAL and ANĞ (23), 1% by DÜMEN et al. (22), and 8.3% by FRECE et al. (25)). In conclusion, the differences between our results and those of other studies can be related to many factors related to the sample (type of product, production technology, hygiene-related processing conditions, conditions of the pre/post processing stages, number of samples tested, year/season and location of sampling) and the method used for analysis (sensitivity and specificity). The detection of Listeria spp. and L. monocytogenes in a mihalic cheese indicates that this type of cheese, which is produced from raw milk, may pose a risk of contamination with this pathogen. The production of dairy under hygienic conditions,
Temelli, Detection of Listeria in retail dairy products
prevention of the contamination of raw material and/or milk, sufficient pasteurisation and the avoidance of contamination after pasteurisation, particularly during packaging, storage, handling and in wholesale/retail settings, are the most effective methods to eliminate Listeria spp. and/or L. monocytogenes in the final product. Acknowledgements This work was funded by the Uludag University Scientific Research Unit Grant, Project No: 2008/67. The article has been edited for English by American Journal Experts. We thank Harald Sendelbach for summary in German and Özlem Zengin for technical assistance.
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