Linkage of bacterial protein synthesis and presentation of MHC class I-restricted Listeria monocytogenes-derived antigenic peptides.

The processing and MHC class I-restricted presentation of antigenic peptides derived from the p60 protein of the facultative intracellular bacterium Listeria monocytogenes is tightly linked to bacterial protein synthesis. We used non-linear regression analysis to fit a mathematical model of bacterial antigen processing to a published experimental data set showing the accumulation and decay of p60-derived antigenic peptides in L. monocytogenes-infected cells. Two alternative models equally describe the experimental data. The simulation accounting for a stable and a hypothetical rapidly degraded form of antigen predicts that the antigenic peptides p60 217-225 and p60 449-457 are derived from a putative instable form of p60 with an average intracellular half-life of approximately 3 minutes accounting for approximately 31% of all p60 molecules synthesized. The alternative model predicts that both antigenic peptides are processed from p60 degraded intracellularly with a half-life of 109 min and that antigen processing only occurs as long as bacterial protein synthesis is not inhibited. In order to decide between both models the intracellular accumulation of p60 in infected cells was studied experimentally and compared with model predictions. Inhibition of p60 degradation by the proteasome inhibitor epoxomicin revealed that during the first 3 h post infection approximately 30% of synthesized p60 molecules were degraded. This value is significantly lower than the approximately 50% degradation of p60 that would be expected in the presence of the predicted putative short-lived state of p60 and also fits precisely with the predictions of the alternative model, indicating that the tight connection of bacterial protein biosynthesis and antigen processing and presentation of L. monocyctogenes-derived antigenic peptides is not caused by the presence of a highly instable antigenic substrate.

Foodproof Salmonella Detection Kit. Performance Tested Method 120301.

The foodproof Salmonella Detection Kit was previously validated in the Performance Tested Methods program for the detection of Salmonella species in a variety of foods, including milk powder, egg powder, coconut, cocoa powder, chicken breast, minced meat, sliced sausage, sausage, smoked fish, pasta, white pepper, cumin, dough, wet pet food, dry pet food, ice cream, watermelon, sliced cabbage, food dye, and milk chocolate. The method was shown to be equivalent to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) and the U.S. Department of Agriculture, Food Safety and Inspection Service's Microbiology Laboratory Guidebook reference culture procedures. In the first Emergency Response Validation (ERV) extension study, peanut butter was inoculated with S. enterica. ser Typhimurium. For the low inoculation level (1.08 CFU/25 g), a Chi-square value of 2.25 indicated that there was no significant performance difference between the foodproof Salmonella Detection Kit and the FDA-BAM reference method. For high-level inoculation (11.5 CFU/25 g) and uninoculated control, there was 100% agreement between the methods. In the second ERV extension study, peanut butter was inoculated with S. enterica. ser Typhimurium. For both inoculation levels (0.1 and 0.5 CFU/25 g by most probable number), Chi-square values of 0 indicated that there was no significant performance difference between foodproof Salmonella Detection Kit and the FDA-BAM reference method.

Singlepath Salmonella. Performance Tested Method 060401.

Singlepath Salmonella is an immunochromatographic (lateral flow) assay for the presumptive qualitative detection of Salmonella spp. in food. A previous AOAC Performance Tested Method study evaluated Singlepath Salmonella as an effective method for the detection of Salmonella spp. in the following selected foods: dried skimmed milk, black pepper, dried pet food, desiccated coconut, cooked peeled frozen prawns, raw ground beef, and raw ground turkey. In this Emergency Response Validation extension, creamy peanut butter was inoculated with S. enterica. ser. Typhimurium. For low contamination level (1.08 CFU/25 g), a Chi-square value of 0.5 indicated that there was no significant difference between Singlepath Salmonella and the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) reference method. For high-level and uninoculated control there was 100% agreement between the methods.

Evaluation of the Duopath Legionella lateral flow assay for identification of Legionella pneumophila and Legionella species culture isolates.

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries' regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.

Nucleic acid-based, cultivation-independent detection of Listeria spp and genotypes of L monocytogenes.

Based on comparative analysis of 16S rRNA gene sequences, two oligonucleotide probes for in situ detection of all members of the genus Listeria were designed. These probes allowed fast and reliable in situ detection of Listeria spp. even in complex samples like raw milk. Almost full-length iap (invasion-associated protein) gene sequences were determined for 69 Listeria monocytogenes strains of all 13 known serotypes. A comparison of these sequences revealed that the L. monocytogenes strains can be grouped into three distinct genotypes. These clusters correlate well with distinct serotypes. Thus, strains of serotypes b and d belong to genotype I, a and c to genotype II, and 4a and 4c, which are rarely isolated from humans, group together within genotype III. These results could be corroborated by further comparative sequence analysis of genes encoding two phospholipases - plcA and plcB. Based on the iap gene sequences, a highly specific and reproducible competitive PCR detection method was developed. Primer pairs targeting genotype-specific regions of the iap gene were designed. The amplification of non-specific PCR products from DNA of non-target strains was prevented by adding competitive primers. By applying this method, the rapid and reliable distinction of the three L. monocytogenes genotypes was possible.

Isolation and characterisation of a 13.8-kDa bacteriolytic enzyme from house dust mite extracts: homology with prokaryotic proteins suggests that the enzyme could be bacterially derived.

Bacteriolytic activity was detected in extracts of whole mite and spent growth medium (SGM) from the clinically important Dermatophagoides pteronyssinus and Dermatophagoides farinae mites and was most abundant in whole mite extract. Gram-positive organisms Micrococcus lysodeikticus, Bacillus megaterium and Listeria monocytogenes were preferentially lysed and the lytic activity was enhanced by thiols, destroyed by mite proteases, inhibited by HgCl2 and high concentrations of NaCl but was resistant to heat and acid treatment. Substrate SDS-PAGE analysis indicated the presence of several lytic enzymes, two of which were isolated from D. pteronyssinus spent growth medium extract by hydroxyapatite chromatography. The N-terminal amino acid sequence of one of them was then used in PCR-based cloning studies. The complete amino acid sequence of this protein was determined and cDNA found to encode a 130-amino acid residue mature protein with a 20-amino acid leader sequence. The deduced protein demonstrated sequence similarity with the C-terminal regions of a group of bacterial proteins belonging to the P60 superfamily. These data suggest that the enzyme is derived from bacteria within the mites rather than from mites per se.

Colony-blot assay with anti-p60 antibodies as a method for quick identification of Listeria in food.

The present study evaluated the ability to isolate Listeria from foods, using shortened procedure of sample enrichment followed by immunomagnetic separation or filtration methods, and serological identification of isolated bacteria by colony-blot and Western blot methods with anti-p60 antibodies. By these rapid methods, identification of Listeria was achieved in much shorter time (40-48 h) than with standard cultivation and biochemical identification procedures. The rapid methods used are easy to perform and, what is most important, their specificity is very high and fulfills the expectations. The possibility to select Listeria colonies growing on non-selective media by blotting with anti-p60 antiserum seems to be particularly valuable in examination of food samples containing/not too many Listeria (1-10 CFU/25 g). However, the blot method using anti-PepD mAb specific to unique region of L. monocytogenes p60 is necessary to distinguish L. monocytogenes from other Listeria species.