Use of Carnobacterium piscicola to limit the growth of Listeria monocytogenes in mussel products : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Bacteria were screened in order to find an organism antagonistic to Listeria monocytogenes which could be applied to mussel products and enhance their safety, especially when temperature-abused. A Listeria monocytogenes isolate from the seafood industry was selected as the target organism. Strains of Lactobacillus reuteri and Enterococcus fecium were screened on plates incubated at 35°C and 10°C for anti-listerial compounds, but none were found. A non-bacteriocinogenic strain of Carnobacterium piscicola, A9b- was selected as the antagonist for detailed examination of growth in broth, agar and mussel systems at 10°C. This temperature was chosen to represent temperature abuse of refrigerated products. To distinguish between the growth of the Carnobacterium piscicola strain and wild-type Listeria monocytogenes a "semi-selective" agar was developed using phenol-red indicator, and mannitol as the sole carbohydrate source. Growth rates of Carnobacterium piscicola and Listeria monocytogenes were compared when grown alone and as a co-culture in agar and broth. Growth rates of Listeria monocytogenes when grown alone, and in the presence of Carnobacterium piscicola, were determined on mussels. Regression analyses were done for the inhibition of Listeria monocytogenes by Carnobacterium piscicola. In all cases Carnobacterium piscicola significantly inhibited the growth of Listeria monocytogenes (P broth = 0.018, P agar <0.001, P mussels < 0.001). Growth of both organisms was faster in broth, than on mussels or agar. The greatest inhibition of Listeria monocytogenes was observed in broth reaching log₁₀4.8 at 41 hours of incubation, prior to decreasing after this time. In agar and mussels the inhibition lasted longer and had not decreased at the end of the trial. The log₁₀ reduction in growth of Listeria monocytogenes in agar was measured at 3.4 and in mussels measured at 1.6. These results were statistically significant (P<0.001 for all). Inhibition of wild type Listeria monocytogenes was also shown in broth when a much lower concentration of Carnobacterium piscicola was used. These results should be considered as preliminary and further confirmatory work should be done. However, Carnobacterium piscicola A9b- shows promise as an antagonistic organism to assist in the control of Listeria monocytogenes in mussel products along with industry-accepted good hygienic practices

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens

Development of a nucleic acid lateral flow immunoassay for simultaneous detection of Listeria spp. and Listeriamonocytogenes in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food sample

Development of ListeriaBase and comparative analysis of Listeria monocytogenes

Background: Listeria consists of both pathogenic and non-pathogenic species. Reports of similarities between the genomic content between some pathogenic and non-pathogenic species necessitates the investigation of these species at the genomic level to understand the evolution of virulence-associated genes. With Listeria genome data growing exponentially, comparative genomic analysis may give better insights into evolution, genetics and phylogeny of Listeria spp., leading to better management of the diseases caused by them. Description: With this motivation, we have developed ListeriaBase, a web Listeria genomic resource and analysis platform to facilitate comparative analysis of Listeria spp. ListeriaBase currently houses 850,402 protein-coding genes, 18,113 RNAs and 15,576 tRNAs from 285 genome sequences of different Listeria strains. An AJAX-based real time search system implemented in ListeriaBase facilitates searching of this huge genomic data. Our in-house designed comparative analysis tools such as Pairwise Genome Comparison (PGC) tool allowing comparison between two genomes, Pathogenomics Profiling Tool (PathoProT) for comparing the virulence genes, and ListeriaTree for phylogenic classification, were customized and incorporated in ListeriaBase facilitating comparative genomic analysis of Listeria spp. Interestingly, we identified a unique genomic feature in the L. monocytogenes genomes in our analysis. The Auto protein sequences of the serotype 4 and the non-serotype 4 strains of L. monocytogenes possessed unique sequence signatures that can differentiate the two groups. We propose that the aut gene may be a potential gene marker for differentiating the serotype 4 strains from other serotypes of L. monocytogenes. Conclusions: ListeriaBase is a useful resource and analysis platform that can facilitate comparative analysis of Listeria for the scientific communities. We have successfully demonstrated some key utilities of ListeriaBase. The knowledge that we obtained in the analyses of L. monocytogenes may be important for functional works of this human pathogen in future. ListeriaBase is currently available at http://listeria.um.edu.my

100K Pathogen Genome Project: 306 Listeria Draft Genome Sequences for Food Safety and Public Health.

Listeria monocytogenes is a food-associated bacterium that is responsible for food-related illnesses worldwide. This is the initial public release of 306 L. monocytogenes genome sequences as part of the 100K Pathogen Genome Project. These isolates represent global genomic diversity in L. monocytogenes

Production of antibodies for use in a biosensor-based assay for Listeria monocytogenes

The inclusion of L. monocytogenes in the list of organisms subject to HACCP has recently driven the search for detection methods suitable for on-line monitoring. The aim of the work presented in this thesis was the development of a biosensor-based immunoassay for the detection of Listeria monocytogenes using SPR. Two polyclonal antibodies were generated from Listeria monocytogenes MB extract and heat-treated Listeria monocytogenes cells. Both antibodies were purified and characterised by ELISA, SDS-PAGE and Western blotting. An inhibition ELISA-based immunoassay was developed with each antibody for the detection of Listeria monocytogenes. Intra- and interday studies were performed to evaluate the accuracy and intermediate precision of the assays. The feasibility of detecting Listeria monocytogenes cells in chocolate milk, a food matrix which has been reported to have been the cause of a well known Listeria monocytogenes-&ssoc\atQ<\ food poisoning outbreak, was also examined. To determine the potential cross reactivity of each antibody, inhibition ELISAs were performed with a number of bacterial strains. It was concluded that both antibodies can be used as valuable tools for the genus-specific detection of Listeria cells, but were severely limited for the species-specific detection of Listeria monocytogenes cells. Expressing Listeria monocytogenes invasion-associated proteins in E. coli allows the safe and efficient production of high quantities of pure protein for use in the generation of Listeria monocytogenes specific antibodies and immunoassay development. Two Listeria monocytogenes invasion-associated proteins, Intemalin B (InlB) and p60 (also known as iap), were cloned and expressed in E. coli XL-10 Gold cells. Expressed protein was purified by immobilised affinity chromatography (IMAC) and used for the selection of specific antibodies (Chapter 5) and for the development a biosensor-based immunoassay for the detection of Listeria monocytogenes (Chapter 6). The emergence of recombinant antibody phage display technology has transformed the way we generate antibodies for the specific detection of a chosen analyte. Chapter 5 describes the development of two combinatorial antibody libraries from mice immunised with Listeria monocytogenes cells and InlB protein extract. Phage scFv antibodies were selected from both murine antibody libraries and from a large naive human antibody library against Listeria monocytogenes cells and invasion associated protein. A number of the selected phage-scFv antibodies could not be expressed as soluble scFv and also showed tendencies to cross react with various bacterial strains tested. A soluble scFv antibody was selcctcd that recognised the Listeria monocytogenes invasion-associated protein, Internalin B, but did not recognise Listeria monocytogenes cells. An inhibition ELISA was developed with this antibody to indircctly detect Listeria monocytogenes. The two polyclonal antibodies and the selected anti-InlB scFv antibody were used in development of three biosensor-based immunoassays using a BIAcore 3000 instrument (chapter 6 ). Various assay formats and sensor chip surfaces were evaluated. Intra- and interday assay variability studies performed to determine the precision and reproducibility of each assay. The assay developed with the polyclonal anti-InlB polyclonal proved to be most sensitive and cost effective while the assay developed with the anti-InlB scFv antibody fragment proved most specific for the detection of Listeria monocytogenes

Activation of the Listeria monocytogenes Virulence Program by a Reducing Environment.

Upon entry into the host cell cytosol, the facultative intracellular pathogen Listeria monocytogenes coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator PrfA. Here, we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge than bacteria grown in conventional media. During cultivation in vitro, PrfA activation was completely dependent on the intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in a synthetic medium supplemented with oligopeptides, but the repression was relieved by stimulation of the stringent response. These data suggest that cytosolic L. monocytogenes interprets a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression in vivoIMPORTANCE Intracellular pathogens are responsible for much of the worldwide morbidity and mortality from infectious diseases. These pathogens have evolved various strategies to proliferate within individual cells of the host and avoid the host immune response. Through cellular invasion or the use of specialized secretion machinery, all intracellular pathogens must access the host cell cytosol to establish their replicative niches. Determining how these pathogens sense and respond to the intracellular compartment to establish a successful infection is critical to our basic understanding of the pathogenesis of each organism and for the rational design of therapeutic interventions. Listeria monocytogenes is a model intracellular pathogen with robust in vitro and in vivo infection models. Studies of the host-sensing and downstream signaling mechanisms evolved by L. monocytogenes often describe themes of pathogenesis that are broadly applicable to less tractable pathogens. Here, we describe how bacteria use external redox states as a cue to activate virulence

Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp. and Listeria monocytogenes from smoked fish processing chains

Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp. and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold-smoked fish. From all methods, Listeria spp. and L. monocytogenes were respectively present in 56 and 34 of 315 samples analysed. Fraser broth incubated for 48 h gave the fewest false negative Listeria spp. results [4/56; (7.1%)], but concurrently only 15/34 (44.1%) samples were correctly identified as containing L. monocytogenes. Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp. positive samples. Despite this lower isolation rate, LED identified 20/34 (58.8%) L. monocytogenes positive samples correctly and gave fewer false positive results. The overall conclusion was that more than one isolation method is needed to accurately estimate L. monocytogenes contamination rates

Using RAPD-PCR as molecular assessment on the performance of CHROMAgar™ Listeria and PALCAM agar on isolation of Listeria spp. and L. monocytogenes from foods

In this study, Listeria spp. were isolated from naturally contaminated samples of beef burger, minced beef and sliced cheese using PALCAM and CHROMagar™. Samples were enriched with FDA-BAM method and plated on PALCAM and CHROMagar™ Listeria before confirmation using PCR on hlyA and LLO toxin genes specific to L. monocytogenes. Identification of isolates showed a total of 45 isolates of Listeria spp. and two L. monocytogenes. All the 47 isolates were then subjected to RAPD analysis using two oligomers (OPA14 and OPA15) and fingerprint clustering was able to cluster the L. monocytogenes from Listeria spp. based on isolation from agar types as well as L. monocytogenes from Listeria spp. Studies showed that OPA14 and OPA15 are useful for rapid discrimination of Listeria spp. and L. monocytogenes . The differences observed on the isolation of Listeria spp. from PALCAM and CHROMagar™ Listeria that may have an impact on epidemiological studies