The seed germination process during sprout production provides suitable environmental conditions for the growth of pathogenic bacteria, such as Listeria monocytogenes. A potential way to control this bacterial growth is through the use of bacteriophages, which are naturally occurring viruses that specifically attack bacterial targets and have been shown to be effective antimicrobials in some foods. Therefore, the objective of this study was to evaluate the antimicrobial susceptibility of L. monocytogenes to bacteriophage on alfalfa sprouts during seed germination and subsequent refrigerated storage at 4 °C. Alfalfa sprout seeds were dip-inoculated with 5.5 x 105 CFU/ml L. monocytogenes serogroups 1 and 4. This was followed by treatment with the commercial bacteriophage LISTEX™ P100 at a concentration of 5.3 x 107 PFU/ml. The seeds were then soaked and germinated for 80 h using the glass jar method. The concentration of L. monocytogenes was determined every 24 h using PALCAM agar plated in triplicate. When compared to the spiked, untreated control, treatment of sprout seeds with LISTEX™ P100 resulted in a statistically significant (p < 0.05) reduction of 1.6 log10 CFU/g L. monocytogenes after the initial 24 h of germination. However, the bacteriophage did not show a lasting inhibitory effect, with no statistically significant reductions in L. monocytogenes growth as compared to the control at subsequent time points. The bacteriophage remained stable over the entire germination and storage period. Although biocontrol of Listeria with bacteriophages has high potential to serve as an alternative strategy to control foodborne illnesses, factors such as phage delivery and dose optimization in sprouts need to be further investigated.

Master of Science / Food Science Institute / Daniel Y.C. Fung / James L. Marsden / Listeria monocytogenes is known to be an environmental contaminant in food processing facilities. Floor drains in processing environments harbor Listeria spp. due to continuous presence of humidity and organic substrates. The cleaning and washing activities undertaken may translocate the bacterial cells from the drain to the surrounding environment, thus contaminating food products being produced. This study validates the effectiveness of Johnson Diversey ‘Eliminex’ Foaming Drain Cleaner and Johnson Diversey ‘Final Step’ 512 sanitizer for inhibition of Listeria monocytogenes in drain surfaces and evaluates the potential for translocation of L. monocytogenes from drains to food contact surfaces in the surrounding environment using Listeria innocua as a surrogate. A 7x 7 x 8 feet flexi glass chamber was built in which a 10 inch diameter drain mounted on an aluminum cabinet was placed. The drain was inoculated with the surrogate organism, L. innocua, at specific time intervals and then treated with the given chemicals. Sponge samples were taken and bacterial populations were recovered on Tryptic Soy Agar (TSA), Modified Oxford Medium (MOX) and Thin Agar Layer MOX (TALMOX). Stainless steel coupons (6.4 x 1.9 x 0.1 cm) were hung at 3 different heights 1, 3 and 5 feet inside the chamber and cell translocation from the drain on to the stainless steel coupons was studied. Reductions up to 4 Log CFU/area or ml were seen at the drain surface, drain crate, drain pipe and wash water for both free cells and cells entrapped in biofilms Treatment had a significant effect (p<0.05) on the reduction of bacterial cells. The wash water showed the greatest reduction from 8 Log CFU/ml to est. 0.23 Log CFU/ml. The given cleaner and sanitizer were found to be effective for reducing Listeria spp. on drain surfaces. Results for the second part indicated translocation at all three heights with percentage translocation ranging between 2-17%. Significantly higher translocation (p<0.05) was seen at 1 foot, followed by 3 feet and 5 feet indicating the closer the height to the drain, the greater the number of bacterial cells that are able to transfer from the drain to the surrounding environment.

Master of Science / Food Science Institute, Animal Science & Industry / Daniel Y.C. Fung / The objectives of these studies were to determine the antimicrobial effects of sodium lactate (SL) and acidified calcium sulfate (ACS) on frankfurters formulated with and without lactate in the frankfurter formulation. Two studies were performed, one which mimicked home storage, and the other evaluated the effectiveness of SL (12% v/v) and ACS (12% v/v) as antimicrobial dips when used prior to and after inoculation of Listeria monocytogenes on frankfurters formulated without lactate. In the first study, five peeled frankfurters with and without lactate in the formulation were either dipped in SL or in ACS, stabilized for 30 minutes, vacuum packaged and stored for 30 days at 4°C. Controls were also prepared by dipping in 0.1% peptone. After 30 days the packages were opened and frankfurters were dip inoculated, stabilized for 30 minutes, and one frankfurter from each treatment was sampled. All other frankfurters were then placed in storage at 7°C and sampled after an additional 7, 14, and 21 days. For the second study, treatments consisted of five frankfurters that were first inoculated with a five-strain cocktail of L. monocytogenes, stabilized for 30 minutes, then dipped in SL or acidified ACS; or were first dipped in SL or ACS, stabilized for 30 minutes then dip inoculated. Controls were prepared by dip inoculating frankfurters. One frankfurter from each treatment was sampled immediately. The remaining frankfurters were vacuum packaged, stored at 4°C and sampled after 30, 60, 90, and 120 days. For both studies, on sampling days one frankfurter from each treatment was pulsified and plated on Tryptic Soy Agar (TSA) for viable cell counts and Modified Oxford Medium (MOX) for L. monocytogenes counts. The results indicated that SL dipped frankfurters had lower total aerobic counts and L. monocytogenes counts compared with ACS treatments and the controls. Use of lactate formulation in frankfurters resulted in lower bacterial counts of both natural microflora and inoculated L. monocytogenes in frankfurters after prolonged storage at 4 °C. This research indicates that sodium lactate (12% v/v) may be effective as an antimicrobial dip on frankfurters for the reduction of natural microflora and L. monocytogenes.

Listeria monocytogenes is known to cause epidemic and sporadic cases of listeriosis. The present study investigated its occurrence, antibiotic sensitivity and serotyping of the organism in foods in various retail outlets in Gaborone, Botswana. Food samples were obtained randomly from selected supermarkets and street vendors from 5 geographical areas in Gaborone from May to September 2007. Listeria monocytogenes was isolated and positively identified by using morphological and biochemical tests. Furthermore, the organism was identified using multiplex PCR. From a total of 1324 food samples tested 57(4.3 %) were positive for Listeria monocytogenes. Out of the 57 isolates, 7 (12.3%), 3 (5.3%), 0 (0.0%), 27 (47.4%) and 20 (35.1%) were isolated from cheese, raw milk, meat (biltong), frozen cabbage and salad (coleslaw). From the 5 geographical areas selected for sampling in this study, Gaborone south recorded the most number 19 (33.3%) of L. monocytogenes isolates while Gaborone west recorded the least, 7 (12.3%). Most of the isolates (49%) belonged to serogroups 4a, 4b and 4c. These isolates were found mostly in cabbage. This was followed by serogroups 4b, 4d and 4e which comprised 30% of the isolates. This is in contrast to most studies that have found serotypes 1/2a and 1/2b to be the most common serotypes in food. That serotype 4b was detected in this study was a significant finding, because this is the number one serotype associated with human listeriosis. REP-PCR was used as a typing tool to characterize the L. monocytogenes strains. The method showed great promise as all of the L. monocytogenes strains were typable using this method, with good correlation between the REP-PCR profiles and the antibiotic resistant profiles. The findings reveal the presence of multi-drug resistant and virulent L. monocytogenes serotype 4b in ready to eat food in Gaborone, Botswana and highlight the need for education and training in food safety programmes. / Life and Consumer Sciences / M. Sc. (Microbiology (Life Sciences))

The primary aim of this investigation was to identify and assess the interactions (synergies and antagonisms) that exist between 20 minute detergent and 5 minute disinfectant treatments upon three factory isolated strains of surface adhered (1-hour attached) and surface adapted (24-hour biofilm) populations of Escherichia coli and Listeria monocytogenes, plus a comparison with vero-toxin producing strains of E. coli, when used as part of a cleaning and disinfection regime. The detergents chosen for assessment were two non-ionic (91/4 - Alcohol Ethoxylate and KCL5 - Polyethoxylated Alcohol), two anionic (LX28 - Sodium Lauryl Sulphate and Nec28 - Sodium Laurylether Sulphate) and two novel bismuth thiols (BisEDT - 1:1 Bismuth nitrate 1,2-ethanedithiol and BisTOL - 2:1 Bismuth nitrate 3,4-dimercaptotoluene), developed at Winthrop University Hospital, New York. The disinfectants chosen for assessment were a quaternary ammonium compound (BAC - Benzyl alkonium Chloride) and a chlorine releasing agent (NaDCC - Sodium Dichloroisocyanurate). The investigation showed that there were no specific cleaning and disinfection regimes that will adequately target both E. coli and L. monocytogenes strains. It was also concluded that to maximise the removal and disinfection of persistent strains of a given microorganism, it may be necessary to design a regime to specifically target not just the species, but the strain involved and where possible requires mechanical cleaning. The novel bismuth thiols were seen to be promising detergents to aid in the removal of E. coli strains and warrant further attention for future studies. Finally, an investigation to identify possible mechanisms of resistance to disinfectant treatments following detergent treatment, showed that different detergents can induce expression of the stress response proteins, HSP60 and HSP70, at differing levels of expression after the same contact time and against different states of adherent populations, i.e. 1-hour attached or 24-hour biofilm populations.

Le bois est un matériau traditionnellement utilisé pour l'affinage des fromages, en raison de ses propriétés hydriques spécifiques. Le caractère ubiquiste de L. monocytogenes, bactérie responsable de la listériose, rend la contamination des fromages possible à toutes les étapes de la fabrication. La maîtrise sanitaire de L. monocytogenes sur les surfaces en IAA est donc un objectif prioritaire, pour lequel les inhibitions microbiennes peuvent jouer un rôle. L'objectif de ce travail était de vérifier que les planches d'affinage en bois, en condition d'utilisation étaient capables de limiter l'implantation de Listeria monocytogenes. Après l'étude de l'environnement des planches dans les caves de l'affineur partenaire, l'étude de l'écologie microbienne de la surface des planches a mis en évidence la prédominance des flores d'intérêt technologique de surface du reblochon et les faibles dénombrements des flores indésirables. Après contact avec les fromages, le nettoyage séchage des planches entraîne des modifications du biotope de surface qui le rendent défavorable à la croissance microbienne, sauf pour les flores d'intérêt technologique. L'étude du comportement de L. monocytogenes inoculée sur des planches d'affinage a révélé un effet inhibiteur de celles-ci, lié à la présence du biofilm natif, naturellement implanté sur ces planches. Cet effet inhibiteur a été démontré pour 2 souches de L. monocytogenes et à 2 stades de prélèvement des planches, dont le stade après nettoyage, le plus exposé au risque d'introduction de L. monocytogenes dans les caves. La coexistence de plusieurs mécanismes est suggérée avec un mécanisme inhibiteur principal par compétition nutritionnelle.

L'objectif était de déterminer si la diversité des espèces microbiennes peut contribuer à la maîtrise de Listeria monocytogenes à la surface de fromage au lait cru. La stratégie reposait sur le criblage de communautés microbiennes de croûtes de fromage St Nectaire fermiers puis la reconstitution de communautés de composition plus simplifiée. Dix consortiums microbiens naturellement présents à la surface de ces fromages au lait cru sur trente quatre testés protégeaient contre L. monocytogenes. Le consortium de croûte le plus inhibiteur, composé de 8 espèces de bactéries lactiques dont Brochothrix thermosphacta, Marinilactobacillus psychrotolerans et Carnobacterium mobile peu fréquentes dans les produits laitiers, 12 espèces de bactéries à Gram positif et catalase positive, 10 espèces de bactéries à Gram négatif, 4 espèces de levures et 3 moisissures, était difficile à reconstituer. Par méthodes cultures dépendantes et indépendantes (Single Strand Conformation Polymorphism) il a été montré qu'au cours d'affinage les profils bactériens du consortium naturel était plus divers que ceux des consortiums reconstitués. Néanmoins, un consortium simplifié composés de flores cultivables sur un milieu "Brain Heart Infusion" et caractérisé par la présence de bactéries à Gram négatif et la dominance, notamment en fin d'affinage, d'espèces halophiles Brochotrix, Carnobacterium et Marinilactibacillus, était presque aussi inhibiteur que le consortium complexe. L'inhibition par ce consortium serait essentiellement associée à la production d'acide lactique en début d'affinage et d'acide acétique en fin d'affinage. Elle pourrait être contrecarrée par une consommation de lastate par les levures

La surveillance de la listériose en France s'est construite par étapes depuis les années 1980 sur deux piliers, la microbiologie et l'épidémiologie. Grâce à la création du Centre National de Référence des Listeria et à la mise au point de techniques de typage performantes, l'Institut Pasteur assure une surveillance microbiologique depuis 1987. Une surveillance épidémiologique initiée entre 1984 et 1992 par le Laboratoire National de la Santé, a été développée par le Réseau National de Santé Publique de 1993 à 1999, puis amplifiée par l'Institut de Veille Sanitaire à partir de 2000. Le premier objectif de cette thèse est de décrire les différentes phases de la construction de cette surveillance afin d'analyser leurs contributions respectives au cours de ces 30 dernières années. Cette construction s'est faite en 4 phases : 1. L'étape fondatrice de 1982 à 1992 a été la reconnaissance et la prise en compte du rôle des aliments dans la transmission de la maladie et dans la survenue d'épidémies. 2. La deuxième phase de 1993 à 2000 a été l'édification d'un système de surveillance opérationnel pour détecter et investiguer les épidémies en France. 3. La troisième phase de 2000 à 2005 a permis de consolider le système de surveillance et de le perfectionner en ajoutant un volet complémentaire avec des prélèvements alimentaires.4. Depuis 2005, nous sommes dans la quatrième phase avec comme objectif l'optimisation du système. Cette optimisation repose sur l'adaptation des outils de surveillance et d'alerte aux connaissances. Ainsi, après avoir montré que la durée d'incubation de la maladie varie selon la forme clinique de la maladie, nous avons proposé d'intégrer cette variation pour déterminer la période d'évaluation des expositions alimentaires à risque. L'analyse des performances du système a permis à deux reprises de proposer de nouveaux seuils de signalement plus spécifiques afin d'optimiser cette surveillance tout en réduisant son coût. Le deuxième objectif de cette thèse est de montrer la contribution des données de surveillance à une politique de santé publique. Un premier travail a consisté à mettre en perspective les variations temporelles d'incidence observées avec les différentes sources de données disponibles afin d'en analyser les déterminants. La phase de décroissance de 1987 à 1997 a été concomitante des mesures de contrôles prises par l'industrie agro-alimentaire et de la réduction de la contamination des aliments. La phase d'augmentation en 2006-2007 semble multifactorielle. L'augmentation de la prescription de traitements de réduction de l'acidité gastrique par des inhibiteurs de la pompe à protons pourrait être l'un des déterminants majeurs de cette augmentation.Dans une deuxième analyse, nous avons hiérarchisé les groupes à risque de listériose sur la base de l'estimation du taux d'incidence de listériose et de sa mortalité dans ces groupes. Cela a permis d'identifier les groupes les plus vulnérables : hémopathies, certains cancers (digestifs, cérébral et pulmonaire), maladie de Horton, cirrhose hépatique, les dialysés rénaux, les greffés, et les femmes enceintes. Une analyse épidémiologique des listérioses materno-néonatales (MN) a montré une association entre les régions avec une incidence plus faible de listérioses materno-néonatales et les régions où la séroprévalence toxoplasmique des femmes enceintes est la plus faible, ce qui suggère un effet positif des recommandations contre la toxoplasmose pour la prévention de la listériose MN.

The research within this thesis sought to determine the ability of various animal derived fats and plant derived oils to support the survival of several pathogenic cocktails over a multitude of storage times. The Salmonella study explored the survival rate of a four strain Salmonella cocktail in beef tallow, pig lard, duck fat, coconut oil, and extra virgin olive oil over seven days at 26˚C and 37˚C storage. The animal fats and the coconut oil supported the survival of the bacteria until the conclusion of the study. The Shiga-toxin producing Escherichia coli study explored the survival rate of a five strain STECs cocktail in extra virgin olive oil over seven days at 26˚C and 37˚C storage. The two Listeria studies explored the survival rate of a four strain Listeria monocytogenes cocktail in extra virgin olive oil over several time periods with different frequencies of sample mixing. In vitro, all genuses showed a 2.5-log cfu/mL to ≥ 7-log cfu/mL reduction in the extra virgin olive oil by the conclusion of the experiments. Extra virgin olive oil was then applied to cooked pork tenderloin, cheddar cheese snack squares, and turkey lunchmeat in hopes of inhibiting the L. monocytogenes cocktail. No reduction was observed.

Challenges encountered in pathogen identification and detection include the genetic heterogeneity of strains within species of some foodborne pathogens, isolation of injured cells, mixed strains or mixed species contamination of foods, and differentiation between viable and dead cells. The first objective of this research was to evaluate an isolation medium that was based on time-delayed release (5 to 6 h) of selective agents in tablet format to a modified Listeria recovery enrichment broth (mLRB) medium for enhanced and rapid recovery of injured Listeria. The second objective involved the use of Fourier transform infrared (FT-IR) spectroscopy and chemometric analysis for the differentiation of: Listeria monocytogenes epidemic clones (ECs); viable versus heat-killed populations; different mixed strains and mixed species of Listeria; and different injury treatments and repair in Listeria populations. Nitrite- or acid-injured Listeria at approximately 10 CFU/ml were recovered in mLRB medium, and cell populations enumerated at various times (12 to 48 h) of incubation at 37oC. Analysis of variance revealed that acid-injured Listeria populations in mLRBS6 (mLRB plus the selective agents at 6 h) were significantly higher (P < 0.05) than those in mLRBS0 (mLRB plus the selective agents at 0 h) at 24 h; however, the differences in populations on these two media were not significant for nitrite-injured Listeria. Cell populations of four strains of Listeria recovered in mLRBTD (mLRB plus the time-delayed release tablets of the selective agents) were significantly higher than when those strains were enriched in the U.S. Food and Drug Administration (FDA), International Organization for Standardization (ISO), and U.S. Department of Agriculture (USDA) broths at 24 h. Comparison between artificially contaminated milk and meat samples with a four-strain cocktail of Listeria resulted in cell populations that were significantly higher (P < 0.05) on mLRBTD for contaminated meat than on mLRBTD for contaminated milk at 24 h. FT-IR spectroscopy in the mid-infrared region (4000 to 600 cm-1) and chemometrics was successfully applied to discriminate L. monocytogenes strains belonging to the same EC (ECII or ECIV) (100% accurate spectral classification), intact and heat-killed populations of each EC strain (100% accurate spectral classification), and spectral wavenumbers 1650 to 1390 cm-1 were used to differentiate heat-killed from intact populations. FT-IR spectroscopy and chemometrics in the wavelength region 1800 to 900 cm-1 could successfully discriminate different mixed strains of L. monocytogenes (98.15% accurate spectral classification) and different mixed species of L. monocytogenes and L. innocua (92.06% accurate spectral classification) from individual strains; Wavelength range 1800 to 900 cm-1 was successfully used to discriminate between intact, acid-injured, and heat-injured Listeria, with repaired cells from acid and heat treatments clustering closer to intact cells (93.33% of spectra accurately classified). Delayed-addition of selective agents to broth medium improves recovery of injured Listeria by allowing repair time, could minimize contamination through manual addition of selective agents, and saves analyst time; FT-IR spectroscopy is a highly discriminatory and reproducible technique that can be used for the differentiation of strains and various physiological states of Listeria.