Before inoculation anatomical cuts were controlled for the absence of Listeria spp. following ISO 11290-1:1996/Amd 1:2004 (ISO, 2004) protocol intended for L. monocytogenes detection, excepting that suspected colonies were confirmed by biochemical miniaturized tests (API Listeria kit; BioMérieux, France). For L. innocua enumeration, samples were diluted 1/10 in Buffered Peptone Water (homemade) and homogenized in stomacher for 60 s. Ten-fold serial dilutions were plated onto ALOA agar (Biolife, Milan, Italy) and incubated at 37°C for 48 h. Suspected colonies were confirmed by biochemical miniaturized tests (API Listeria kit; BioMérieux, France). In samples below the quantification limit (10 CFU/g), the qualitative analysis was carried out as described before. Results of L. innocua counts were expressed in CFU/g and converted into Log10 CFU/g.
Api listeria kit
Manufactured by bioMérieux
Sourced in France
The API Listeria kit is a laboratory test kit used for the identification and confirmation of Listeria monocytogenes, a pathogenic bacterium. The kit provides a standardized biochemical identification method for isolates obtained from food and environmental samples.
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7 protocols using api listeria kit
1
Listeria spp. Detection and Enumeration
2
Listeria Monocytogenes Detection in Artisanal Cheese
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The presence of L. monocytogenes in the cheese samples was determined in accordance with the ISO standards and Food and Drug Administration guidelines [67 ,68 ]. Detection was performed with the VIDAS®Listeria monocytogenes II commercial kit (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Briefly, 25 g of artisanal fresh cheese was homogenized, selectively pre-enriched for Listeria spp. in 225 mL Half-Fraser broth (Biomerieux) and incubated at 30 °C for 24 h. Then, 1 mL of this suspension was transferred to 10 mL Fraser broth (Biomerieux) and incubated at 30 °C for 24 h. From this final suspension, 500 μL were used for the detection in the VIDAS® system. Those suspensions containing L. monocytogenes were cultured in Oxford and PALCAM (bioMérieux) selective media at 35 °C for 24 h. and the obtained colonies were confirmed through biochemical tests using the API® Listeria kit (bioMérieux) according to the manufacturer’s instructions. All assays were performed in triplicate, and at least two independent assays were performed for each cheese sample.
3
Listeria Detection in Food Samples
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Bacterial detection in food samples was performed using the VIDAS Listeria DUO kit (Biomerieux), according to the manufacturer's instructions. Briefly, analytical portions of 25 g of each sample were pre-enriched for Listeria in 225 ml of Listeria Xpress (LX) Broth and incubated at 30°C for 22–24 h. Then 0.1 ml of this broth was transferred to 6 ml of LX Broth and again incubated at 30°C for 22–24 h. Finally, 500 μl of each sample were tested in the VIDAS platform. Positive samples according to the VIDAS test were seeded in ALOA chromogenic agar (Agar Listeria Ottaviani and Agosti, Biomerieux), and further confirmed by the API Listeria kit (Biomerieux) and seeding in TSA agar + 5% sheep blood. Confirmed colonies were conserved in 20% non-fat milk at −20°C and 40% glycerol at −70°C. The clinical strains were kindly provided by the Instituto de Salud Pública de Chile. All strains were collected from patients with listeriosis in the years 2008 and 2009. The number of isolated strains from food and clinical samples are shown in Table 1 . The project protocols were approved by the Comite Asesor de Bioética de FONDECYT—CONICYT, Chile.
4
Biofilm Formation in Listeria monocytogenes
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Two L. monocytogenes strains (LM2 and LM9) with different ability to produce biofilm were selected. Both strains were obtained from a collection of strains of Microbiology Institute, Department of Public Health and Infectious Diseases, “Sapienza” University of Rome.
Strains were biochemically controlled by the API Listeria kit (Bio Mérieux, France), according to the manufacturer's instructions. Haemolysis on Muller Hinton agar (Oxoid) supplemented with 5% sheep blood was used as additional test. Bacteria were maintained as stock cultures in 15% glycerol-brain heart infusion broth (BHI) (Oxoid) at −80°C.
LM2 and LM9 strains were previously characterised for biofilm formation and classified as moderate and strong biofilm producers, respectively [28] .
Cells derived from a human colon carcinoma (CaCo-2) (ATCC® HTB-37) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco-modified minimum essential medium with Earle's salts (D-MEM, EuroClone), supplemented with 10% (v/v) heat-inactivated foetal calf serum (FCS, JRH Biosciences), and 2 mM glutamine. All incubations were carried out in a 5% CO2 atmosphere at 37°C. Cells were used 48 hrs after seeding.
Strains were biochemically controlled by the API Listeria kit (Bio Mérieux, France), according to the manufacturer's instructions. Haemolysis on Muller Hinton agar (Oxoid) supplemented with 5% sheep blood was used as additional test. Bacteria were maintained as stock cultures in 15% glycerol-brain heart infusion broth (BHI) (Oxoid) at −80°C.
LM2 and LM9 strains were previously characterised for biofilm formation and classified as moderate and strong biofilm producers, respectively [28] .
Cells derived from a human colon carcinoma (CaCo-2) (ATCC® HTB-37) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco-modified minimum essential medium with Earle's salts (D-MEM, EuroClone), supplemented with 10% (v/v) heat-inactivated foetal calf serum (FCS, JRH Biosciences), and 2 mM glutamine. All incubations were carried out in a 5% CO2 atmosphere at 37°C. Cells were used 48 hrs after seeding.
5
Isolation and Identification of Listeria monocytogenes
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Positive samples according to real-time PCR results were streaked onto two selective media plates: Oxford medium base with Modified Oxford Antimicrobic Supplement (BD – Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and BBL™ CHROMagar™Listeria (BD), both incubated for 24–48 h at 35–37 °C. Suspect colonies (small, convex and surrounded by a black halo; blue colonies less than 3 mm in diameter and surrounded by a white halo, respectively) were identified using API Listeria kit (bioMérieux, Marcy l’Etoile, France) and additional standard biochemical assays that included: β-Hemolysis halo production on Trypticase Soy Agar (BD) supplemented with sheep blood (5%) plates, catalase reaction, bile esculin; and Christie-Atkins-Munch-Petersen (CAMP) tests with control strains of Staphylococcus aureus and Rhodococcus equi.27
When food samples from retail shop or factory displayed positive results to L. monocytogenes, it was visited again and samples of the same food types were taken and analyzed according to the above described procedure.
Confirmed L. monocytogenes isolates were conserved in 10% reconstituted skim milk at −20 °C and also in 20% glycerol at −80 °C for further assays.
When food samples from retail shop or factory displayed positive results to L. monocytogenes, it was visited again and samples of the same food types were taken and analyzed according to the above described procedure.
Confirmed L. monocytogenes isolates were conserved in 10% reconstituted skim milk at −20 °C and also in 20% glycerol at −80 °C for further assays.
6
Listeria Strain Characterization Protocol
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Tables
1 ,
2 , and
3 list the panel of Listeria strains used in this study. Strains were obtained from the Food Microbiology Microbial Collection (University College Cork) and the Special Listeria Culture Collection (SLCC). All strains were cultured at 37°C for 16 h in Brain Heart Infusion (BHI) broth or agar (Oxoid, Hampshire, UK) unless otherwise stated. Where necessary, the characterisation of strains as L. innocua was confirmed biochemically by means of the API listeria kit (BioMérieux, Lyon, France) and 16S ribosomal DNA (rDNA) with CO1 and CO2 primer pairs previously described by Simpson et al.[14 (link)]. Escherichia coli EC101 was used as an intermediate vector host. Antibiotics were incorporated as follows
[8 (link)]: Erythromycin (Ery) 150 μg/ml E. coli, 5 μg/ml L. innocua. Chloroamphenicol (Cm) 10 μg/ml E. coli and L. innocua. Ampicillin (Amp) 100 μg/ml E. coli. 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) was incorporated at a concentration of 40 μg/ml.
[8 (link)]: Erythromycin (Ery) 150 μg/ml E. coli, 5 μg/ml L. innocua. Chloroamphenicol (Cm) 10 μg/ml E. coli and L. innocua. Ampicillin (Amp) 100 μg/ml E. coli. 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) was incorporated at a concentration of 40 μg/ml.
7
Listeria Species Identification Protocol
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Phenotypic characterization of the isolates to species level was done by subjecting them to Gram staining and various biochemical tests such as catalase and hemolysis tests, as well as arylamidase (3,3′-diindolylmethane test), esculin hydrolysis, presence of α-mannosidase, and fermentation of D-arabitol, D-xylose, L-rhamnose, α-methyl-D-glucoside, D-ribose, glucose-1-phosphate, and D-tagatose using analytic profile index (API) Listeria kit (Biomerieux, France) as per the manufacturer’s instructions.
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