An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions. This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping. The sigB sequence was resolved in four local L. monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined. The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages. Based on nucleotide sequences L. monocytogenes lineage F serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L. innocua strains clustered together as an outgroup. SNPs differentiating the three lineages were identified. Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L. monocytogenes isolates into the three lineages. (C) 2003 Elsevier B.V. All fights reserved.

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.

Monitoring changes in nisin susceptibility of Listeria monocytogenes Scott A as an indicator of growth phase using FACS

Listeria monocytogenes has previously been shown to adapt to a wide variety of environmental niches, principally those associated with low pH, and this compromises its control in food environments. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. The present Study aimed to gain a further understanding of the physiological basis for the differential effects of one control strategy, namely the use of the lantibiotic nisin. Using propidium iodide (PI) to probe membrane integrity it was shown that L. monocytogenes Scott A was sensitive to nisin (8 ng mL(-1)) but this was growth phase dependent with stationary phase cells (OD600=1.2) being much more resistant than exponential phase cells (OD600=0.38). We demonstrate that, using a combination of techniques including fluorescence activated cell sorting (FACS), the membrane adaptations underpinning nisin resistance are triggered much earlier (OD600 < 0.5) than the onset of stationary phase. The significance of these findings in terms of mechanism and application are discussed. (c) 2005 Elsevier B.V.All rights reserved.

Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Kunjin virus replicon vectors for human immunodeficiency virus vaccine development

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8(+) T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8(+) T-cell responses. Two immunizations with KUNgag replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of greater than or equal to1:10,000. Immunization with KUNgag replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8(+) T-cell responses, with one immunization of KUNgag VLPs inducing 4.5-fold-more CD8(+) T cells than the number induced after immunization with recombinant vaccinia virus carrying the gag gene (rVVgag). Two immunizations with KUNgag VLPs also provided significant protection against challenge with rVVgag. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8(+) T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.

CD40 and dendritic cell function

CD40 has emerged as a key signaling pathway for the function of B cells, monocytes, and dendritic cells (DC) in the immune system, and plays a major role in inflammatory pathways of nonhemopoletic cells. CD40 is expressed by monocytes and DC and is up-regulated when DC migrate from the periphery to draining lymph nodes (DLN) in response to microbial challenge. CD154 signaling by MHC-restricted, activated CD4* T cells induces differentiation of DC, as defined by an increased surface expression of MHC, costimulatory, and adhesion molecules. Thus, CD40 functions in the adaptive immune response as a trigger for the expression of costimulatory molecules for efficient T-cell activation. CD40 ligation of DC also has the capacity to induce high levels of the cytokine IL-12, which polarizes CD4(+) T cells toward a T helper 1 (Th1) type, enhances proliferation of CD8(+) T cells, and activates NK cells. CD40 may also play an important role in the decision between tolerance and immunity and the generation of regulatory CD4(+) T cells that are thought to maintain peripheral self-tolerance in vivo.

Quantification of Listeria spp. contamination on shell and flesh of cooked black tiger prawns (Penaeus monodon)

Aims: To quantify Listeria levels on the shell and flesh of artificially contaminated cooked prawns after peeling, and determine the efficacy of Listeria innocua as a model for L. monocytogenes in this system. Methods and Results: A L. monocytogenes and L. innocua strain were inoculated separately onto cooked black tiger prawns using two protocols ( immersion or swabbing with incubation). Prawns were peeled by two methods ( gloved hand or scalpel and forceps) and numbers of Listeria on shells, flesh and whole prawn controls were determined. Prawns were exposed to crystal violet dye to assess the penetration of liquids. Regardless of preparation method or bacterial strain there were ca 1log(10) CFU more Listeria per shell than per peeled prawn. Dye was able to penetrate to the flesh in all cases. Conclusions: Shell-on prawns may be only slightly safer than shell-off prawns. Listeria innocua is an acceptable model for L. monocytogenes in this system. Significance and Impact of the Study: Reduced risk from L. monocytogenes on prawns can only be assured by adequate hygiene or heating.

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Attachment of Shiga toxigenic Escherichia coli to beef muscle and adipose tissue

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (S-r) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. S-r values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and S-r values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.

Comparison of selected methods for measuring the virulence properties of Listeria spp.

The comparative ability of different methods to assess virulence of Listeria species was investigated in ten Listeria strains. All strains were initially subjected to pulsed-field gel electrophoresis analysis to determine their relatedness. Virulence characteristics were subsequently tested for by (i) determining the presence of six virulence genes by polymerase chain reaction; (ii) testing for the production of listeriolysin O, phosphatidylcholine phospholipase C, and phosphatidylinositol-specific phospholipase C; (iii) investigating the hydrophobicity of the strains; (iv) determining the strains ability to attach to, enter, and replicate within the Caco-2 cells. Variations in most of the virulence characteristics were obvious across the strains for the range of tests performed. A wide range of anomalous results among methods were apparent. In particular, the presence of virulence genes was found to be unrelated to the production of virulence-associated proteins in vitro, while virulence protein production and hydrophobicity in Listeria monocytogenes were found to be unrelated or marginally related, respectively, to the ability to invade the Caco-2 cell line. It was concluded that the methods investigated were unable to consistently and unequivocally measure the differences in the virulence properties of the strains.

In vitro antibacterial activity of Australian native herb extracts against food-related bacteria

The antibacterial activities of water, ethanol and hexane extracts of five Australian herbs (Backhousia citriodora, Anetholea anisata, Eucalyptus staigerana, Eu. olida and Prostanthera incisa) against seven food-related bacteria (Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Enteritidis, Sal. Typhimurium and Staphylococcus aureus) were determined by the microtitre broth microdilution assay. The water extracts of all the herbs displayed no or low antimicrobial activity against all of the bacteria tested with the exception of S. aureus. Relatively high levels of activity (minimum inhibitory concentrations of 125-15.6 mu g ml(-1)) against this pathogen were present in water extracts from all herbs except P. incisa. The ethanol and hexane extracts of all herbs displayed some activity against a number of the bacteria tested, with no one particular herb displaying an obviously higher level or range of activity. Staphylococcus aureus proved to be the most sensitive of the bacteria tested against the solvent extracts with all extracts displaying activity ranging from 125 to 7.8 mu g ml(-1), while E. coli and L. monocytogenes, on the other hand, proved the least sensitive with only five of 15 herb/extract combinations displaying any activity against these pathogens. The extracts of the Australian native herbs examined in this study have potential for application in foods to increase shelf-life or promote safety. (c) 2005 Elsevier Ltd. All rights reserved.