Virulence Pattern Analysis of Three Listeria monocytogenes Lineage I Epidemic Strains with Distinct Outbreak Histories.

Strains of the food-borne pathogen Listeria (L.) monocytogenes have diverse virulence potential. This study focused on the virulence of three outbreak strains: the CC1 strain PF49 (serovar 4b) from a cheese-associated outbreak in Switzerland, the clinical CC2 strain F80594 (serovar 4b), and strain G6006 (CC3, serovar 1/2a), responsible for a large gastroenteritis outbreak in the USA due to chocolate milk. We analysed the genomes and characterized the virulence in vitro and in vivo. Whole-genome sequencing revealed a high conservation of the major virulence genes. Minor deviations of the gene contents were found in the autolysins Ami, Auto, and IspC. Moreover, different ActA variants were present. Strain PF49 and F80594 showed prolonged survival in the liver of infected mice. Invasion and intracellular proliferation were similar for all strains, but the CC1 and CC2 strains showed increased spreading in intestinal epithelial Caco2 cells compared to strain G6006. Overall, this study revealed long-term survival of serovar 4b strains F80594 and PF49 in the liver of mice. Future work will be needed to determine the genes and molecular mechanism behind the long-term survival of L. monocytogenes strains in organs.

Rapid testing and quantification of Salmonella in ileocaecal lymph nodes of Austrian pigs slaughtered for consumption.

Traditionally, quantitative microbial risk assessment (QMRA) is based on culture-dependent technologies. However, molecular quantification could forge additional, detailed information. A prerequisite of quantitative real-time PCR in animal science is a tissue preparation method where large volumes of tissue material can be reduced and particularly target cells can be concentrated. An easy-to-use sample preparation method for food (Matrix-Lysis) was recently adapted to tissues and now permits quantification of target cells from up to 5 g of organic matrix. The aim of this study was to examine the suitability of Matrix-Lysis for quantification of Salmonella in porcine ileocaecal lymph nodes (ICLNs). After demonstrating constant recovery rates, ICLNs from 540 pigs were examined for Salmonella spp. with Matrix-Lysis. Samples were also analysed using ISO 6579:2002, a combined enrichment/qPCR method and a lateral flow test. It could be shown that qPCR coupled with Matrix-Lysis can contribute to QMRA in food safety by enabling reproducible quantitative data, even at low contamination rates.

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.

13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA.

The carbon metabolism of Listeria monocytogenes (Lm) EGD and the two isogenic mutant strains LmDeltaprfA and LmDeltaprfApPRFA* (showing no or enhanced expression, respectively, of the virulence factor PrfA) was determined by 13C isotopologue perturbation. After growth of the bacteria in a defined medium containing a mixture of [U-13C6]glucose and glucose with natural 13C abundance (1:25, wt/wt), 14 amino acids were isolated and analyzed by NMR spectroscopy. Multiply 13C-labeled isotopologues were determined quantitatively by signal deconvolution. The 13C enrichments and isotopologue patterns allowed the reconstruction of most amino acid biosynthesis pathways and illustrated that overproduced PrfA may strongly influence the synthesis of some amino acids, notably that of the branched amino acids (Val, Ile, and Leu). Retrobiosynthetic analysis of the isotopologue compositions showed that degradation of glucose occurs to a large extent via the pentose phosphate pathway and that the citrate cycle is incomplete because of the absence of 2-oxoglutarate dehydrogenase activity. The reconstructed labeling pattern of oxaloacetate indicated its formation by carboxylation of pyruvate. This metabolic reaction seems to have a strong impact on the growth requirement in defined minimal medium. Bioinformatical steady-state network analyses and flux distribution predictions confirmed the experimental data and predicted metabolite fluxes through the enzymes of the pathways under study.

Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening.

A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.

Inefficient replication of Listeria innocua in the cytosol of mammalian cells.

The efficiency of adherence to, internalization by, and replication in the cytosol of J774 macrophages and HEp-2 epithelial cells was compared between a nonspreading Listeria monocytogenes actA mutant and L. innocua. The studied L. innocua strains were equipped either with listeriolysin alone or with listeriolysin O (LLO) and the recently identified hexose-phosphate transporter of L. monocytogenes. All listerial strains expressed green fluorescent protein (GFP) under the control of the PrfA-dependent actA promoter. GFP expression was observed exclusively in the cytosol of host cells. Escape from the phagosome of LLO-expressing L. innocua strains was as efficient as that from L. monocytogenes. hpt-positive L. innocua showed significantly enhanced adherence to HEp-2 cells, but internalization was only slightly increased, compared with hpt-negative L. innocua. Subsequent replication of L. monocytogenes in the cytosol of the host cells proceeded within the next 6 h in most infected host cells, with a generation time <40 min. L. innocua prfA hly replicated more slowly (with a generation time of 60-90 min), and, in most host cells, bacterial replication stopped after 2-3 rounds of replication. In some cells, bacterial replication did not occur. Twenty-four hours after infection, the majority of J774 cells (>90%) infected with L. monocytogenes actA were dead, whereas most host cells infected with L. innocua were still alive. L. innocua equipped with the prfA, hly, and hpt genes of L. monocytogenes did not show significantly increased cytosolic replication, which indicates that expression of this sugar phosphate uptake system is not sufficient for extensive listerial replication in the cytosol of host cells.

Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation in Listeria.

Efficient replication in vivo is essential for a microparasite to colonize its host and the understanding of the molecular mechanisms by which microbial pathogens grow within host tissues can lead to the discovery of novel therapies to treat infection. Here we present evidence that the foodborne bacterial pathogen Listeria monocytogenes, a facultative intracellular parasite, exploits hexose phosphates (HP) from the host cell as a source of carbon and energy to fuel fast intracellular growth. HP uptake is mediated by Hpt, a bacterial homolog of the mammalian translocase that transports glucose-6-phosphate from the cytosol into the endoplasmic reticulum in the final step of gluconeogenesis and glycogenolysis. Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which upon entry into host cells induces a set of virulence factors required for listerial intracellular parasitism. Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice. Hpt is the first virulence factor to be identified as specifically involved in the replication phase of a facultative intracellular pathogen. It is also a clear example of how adaptation to intracellular parasitism by microbial pathogens involves mimicry of physiological mechanisms of their eukaryotic host cells.