Simulación y control de biofilms portadores de "Listeria monocytogenes" en la industria alimentaria

1 OBJETIVOS Evaluar el impacto de las bajas temperaturas, la interacción entre P. fluorescens (Pf) y L. monocytogenes (Lm) en biofilms (BF) mixtos y la capacidad para persistir de Lm sobre (i) la capacidad de Lm para desarrollar BF; (ii) los parámetros estructurales de los BF formados; (iii) la respuesta de las células adheridas de Lm frente al tratamiento con quitosano; (iiii) y su capacidad para recuperarse tras el tratamiento, tanto a nivel de densidad celular como estructural. Además, se pretendió valorar el quitosano como agente de limpieza y desinfección de los BF en la industria alimentaria, profundizando en su impacto sobre la estructura. 2 METODOLOGÍA Para llevar a cabo estos objetivos se seleccionaron un total de 10 cepas de Lm (6 persistentes y 3 esporádicas aisladas por Ortiz y col. (2010) de una industria cárnica y la cepa Lm Scott A como referencia) y la cepa Pf ATCC 948TM para formar BF puros, de cada especie, y mixtos, de Pf y cada una de las cepas de Lm, iniciando los cultivos a un nivel de cada una de las bacterias de 104 UFC·ml-1. Para la formación de BF se empleó un sistema en batch, en el que cupones de vidrio desechables (22x22 mm) semisumergidos actuaron como soporte para la adhesión. Los BF se desarrollaron a 20°C (BF “templados”) y 4°C (BF “fríos”). Los tratamientos con quitosano al 1% (w/v) consistieron en la inmersión durante 1h. Para la recuperación, los cupones tratados se revitalizaron en medio fresco (Trytone Soya Broth, TSB) a 20°C/48h. La densidad celular adherida antes, después y durante la revitalización se determinó mediante siembra del BF residual recogido del cupón en medios generales, o selectivos en el caso de los BF mixtos. Para la determinación de la biomasa adherida mediante densitometría (λ=520-570 nm), los BF fueron teñidos con una solución al 1‰ de azul de coomassie. Para las observaciones de la estructura mediante microscopía confocal láser de barrido (CLSM) las muestras se tiñeron con diferentes fluorocromos, procesándose las imágenes obtenidas mediante el software Imaris 8.2. El tratamiento estadístico de los datos se hizo con el software Statgraphics Centurion...

Biochemical and Structural Studies on PrfA, the Transcriptional Regulator of Virulence in Listeria monocytogenes

Abstract

Listeria monocytogenes is a gram-positive soil saprophytic bacterium that is capable of causing fatal infection in humans. The main virulence regulator PrfA, a member of the Crp/FNR family of transcriptional regulators, activates the expression of essential proteins required for host cell invasion and cell-to-cell spread. The mechanism of PrfA activation and the identity of its small molecule coactivator have remained a mystery for more than 20 years, but it is hypothesized that PrfA shares mechanistic similarity to the E. coli cAMP binding protein, Crp. Crp activates gene expression by binding cAMP, increasing the DNA binding affinity of the protein and causing a significant DNA bend that facilitates RNA polymerase binding and downstream gene activation. Our data suggests PrfA activates virulence protein expression through a mechanism distinct from the canonical Crp activation mechanism that involves a combination of cysteine residue reduction and glutathione (GSH) binding.

Listeria lacking glutathione synthase (ΔgshF) is avirulent in mice; however virulence is rescued when the bacterium expresses the constitutively active PrfA mutant G145S. Interestingly, Listeria expressing a PrfA mutant in which its four cysteines are mutated to alanine (Quad PrfA), demonstrate a 30-fold decrease in virulence. The Quad and ΔgshF double mutant strains are avirulent. DNA-binding affinity, measured through fluorescence polarization assays, indicate reduction of the cysteine side chains is sufficient to allow PrfA to binds its physiological promoters Phly and PactA with low nanomolar affinity. Oxidized PrfA binds the promoters poorly.

Unexpectedly, Quad also binds promoter DNA with nanomolar affinity, suggesting that the cysteines play a role in transcription efficiency in addition to DNA binding. Both PrfA and Quad bind GSH at physiologically relevant and comparable affinities, however GSH did not affect DNA binding in either case. Thermal denaturation assays suggest that Quad and wild-type PrfA differ structurally upon binding GSH, which supports the in vivo difference in infection between the regulator and its mutant.

Structures of PrfA in complex with cognate DNA, determined through X-ray crystallography, further support the disparity between PrfA and Crp activation mechanisms as two structures of reduced PrfA bound to Phly (PrfA-Phly30 and PrfA-Phly24) suggest the DNA adopts a less bent DNA conformation when compared to Crp-cAMP- DNA. The structure of Quad-Phly30 confirms the DNA-binding data as the protein-DNA complex adopts the same overall conformation as PrfA-Phly.

From these results, we hypothesize a two-step activation mechanism wherein PrfA, oxidized upon cell entry and unable to bind DNA, is reduced upon its intracellular release and binds DNA, causing a slight bend in the promoter and small increase in transcription of PrfA-regulated genes. The structures of PrfA-Phly30 and PrfA-Phly24 likely visualize this intermediate complex. Increasing concentrations of GSH shift the protein to a (PrfA-GSH)-DNA complex which is fully active transcriptionally and is hypothesized to resemble closely the transcriptionally active structure of the cAMP-(Crp)-DNA complex. Thermal denaturation results suggest Quad PrfA is deficient in this second step, which explains the decrease in virulence and implicates the cysteine residues as critical for transcription efficiency. Further structural and biochemical studies are on-going to clarify this mechanism of activation.

Effetto di composti d'aroma sulla risposta fisiologica di Listeria monocytgenes

La citofluorimetria è una tecnica applicata per misurare le caratteristiche fisiche, morfologiche e fisiologiche di cellule microbiche ed ha il pregio di generare un dato per ogni singola particella (cellula) analizzata. Poiché è noto che l’assenza di sviluppo in piastra non implica necessariamente l’assenza di forme microbiche vitali, questa tecnica ha un grande potenziale nello studio dei trattamenti termici che mirano alla stabilizzazione ed alla sicurezza igienico-sanitaria dei prodotti alimentari. Infatti, nel contesto industriale la tendenza è quella di ridurre l’entità di questi trattamenti (tempi/temperature). Ciò può avvenire anche grazie all’utilizzo di composti d’aroma, la cui attività antimicrobica è ben documentata, poiché il trattamento termico, incrementando la tensione di vapore di queste sostanze, ne potenzia l’attività antimicrobica. Questa tesi è incentrata su due aspetti: da una parte, l’effetto dell’esposizione di L. monocytogenes a diverse concentrazioni di timolo e carvacrolo (terpenoidi prevalenti in oli essenziali di Labiatae tra cui timo e origano), dall’altra la valutazione degli effetti di trattamenti termici subletali (45, 50, 55°C), anche in presenza di composti d’aroma, sulla disattivazione e sul successivo recupero di L. monocytogenes. I risultati hanno confermato la forte sinergia tra trattamento termico e presenza di sostanze terpeniche. È stato inoltre dimostrato che la presenza di tali composti incide drasticamente sulle potenzialità di recupero degli eventuali sopravvissuti dopo il trattamento termico. I risultati a volte discordanti tra l’analisi citofluorimetrica (focalizzata sull’integrità della membrana) e i conteggi in piastra hanno evidenziato come i due approcci debbano essere utilizzanti in modo complementare. L’utilizzo di altri coloranti legati ad altre funzioni biologiche e metaboliche permetterà l’ottenimento di informazioni aggiuntive circa la risposta fisiologica di questo microrganismo.

Effetto di olio essenziale di Satureja Montana ed estratto di Cotinus coggygria sulle curve di morte termica di Listeria monocytogenes e Saccharomyces cerevisiae

Negli ultimi anni, è aumentato notevolmente l'interesse per piante e prodotti vegetali, e composti da essi derivati od estratti, in alternativa ai conservanti chimici per prevenire o ritardare lo sviluppo microbico negli alimenti. Questo deriva dalla percezione negativa, ormai diffusa a livello pubblico, nei confronti di sostanze di sintesi che sono ampiamente utilizzate come conservanti nell’industria alimentare. Sono stati effettuati diversi studi sull’attività antimicrobica di questi composti negli alimenti, anche se il loro utilizzo a livello industriale è limitato. Ciò dipende dalla difficile standardizzazione di queste sostanze, dovuta alla variabilità della matrice alimentare che ne può alterarne l’attività antimicrobica. In questa sperimentazione si sono utilizzati l’olio essenziale di Sateureja montana e l’estratto di Cotinus coggygria e sono state fatte delle prove preliminari, determinandone le componenti volatili tramite gas-cromatografia abbinata a microestrazione in fase solida. Sono stati selezionati un ceppo di Listeria monocytogenes (Scott A) e uno di Saccharomyces cerevisiae (SPA), e sono stati utilizzati per realizzare curve di morte termica in sistema modello e in sistema reale. Dai risultati ottenuti si può affermare che Satureja montana e Cotinus coggygria possono essere presi in considerazione come antimicrobici naturali da impiegare per la stabilizzazione di alimenti, nonché per ridurre l’entità dei trattamenti termici atti a salvaguardare le proprietà nutrizionali ed organolettiche di alimenti, come ad esempio succhi di frutta, garantendone la sicurezza e qualità microbiologica.

Studio propedeutico del potenziale di crescita di Listeria monocytogenes durante la shelf life di confezioni monoporzione di battuta di vitello

Scopo della tesi è la valutazione del potenziale di crescita (δ) di Listeria monocytogenes espressa come differenza tra il carico cellulare (log10 ufc/g) alla fine e all’inizio della prova, in monoporzioni di battuta di vitello (tartare) confezionate sottovuoto e conservate a temperatura di refrigerazione. Si tratta di un alimento ready to eat – RTE – prodotto dalla Ditta INALCA Spa e destinato ad una catena di ristorazione. I challenge test hanno lo scopo di fornire informazioni sul comportamento in determinate condizioni di conservazione, di L. monocytogenes inoculata artificialmente in un alimento. L. monocytogenes è un microrganismo patogeno ubiquitario nell’ambiente e resistente a diverse condizioni ambientali. E’ stato dimostrato che il batterio è responsabile della contaminazione post-processo degli alimenti, in quanto è stato isolato da impianti di trasformazione, macchine per l’imballaggio, nastri trasportatori, guanti, congelatori e guarnizioni. Sono state oggetto di studio: - i) 3 u.c. inoculate con soluzione fisiologica sterile su cui sono stati valutati Aw, pH, carica aerobia mesofila, batteri lattici, Pseudomonas, Enterobacteriaceae, muffe e lieviti; - ii) 1 u.c. non soggetta ad alcun inoculo per la ricerca qualitativa/quantitativa di L. monocytogenes; - iii) 9 u.c. contaminate con una miscela di 5 ceppi di L. monocytogenes (circa 100 ufc/g). Le confezioni inoculate sono state conservate per 21 giorni (pari alla shelf-life commerciale), di cui i primi 7 alla temperatura di +3°C, ed i successivi 14 giorni a +5°C. Il valore δ è risultato pari a 0.57: essendo superiore, seppure di poco, al valore soglia 0.5, le tartare in esame sono classificate come alimenti pronti che costituiscono terreno favorevole alla crescita di L. monocytogenes (categoria 1.2); in base al regolamento (CE) 2073/2005, il microrganismo deve essere assente in 25 g alla produzione e < 100 ufc/g durante la shelf–life (nota 5 e 7).

Meningitis por Listeria monocytogenes en paciente inmunocompetente

Listeria monocytogenes es un patógeno de origen alimentario que suele producir gastroenteritis, procesos febriles, sepsis y meningitis. Afecta característicamente a neonatos, embarazadas, ancianos e inmunocomprometidos, con una epidemiología controvertida y poco conocida. Se presenta un caso de meningitis y síndrome de secreción inadecuada de hormona antidiurética secundario en paciente inmunocompetente.

Determinación de Listeria monocytogenes en dos variedades de quesos artesanales, comeercializados en los mercados La Tiendona, Central y San Miguelito

Se realizó un análisis microbiológico a dos variedades de quesos (fresco y duro blando), comercializado en los mercados de la Tiendona, Central y San Miguelito. Se tomó en cuenta los parámetros utilizados dentro del Reglamento Técnico Centro americano (RTCA) 67.04.50:08 (Alimentos. Criterios microbiológicos para la inocuidad de alimentos) de quesos madurados y no madurados el cual dice que por cada 25 g de muestra, la Listeria monocytogenes debe ser “ausente”. Para determinar las dos variedades de quesos se utilizó una guía de observación, para determinar la presencia o ausencia de Listeria monocytogenes se analizó por medio de la metodología descrita por el BAM (Manual de Análisis Bacteriológico de la Administración de Drogas y Alimentos (FDA). Se realizó un conteo en placa para la determinación de la sobrevivencia de la L. monocytogenes; haciendo una comparación del día 1 y a los 5 días, sometiendo la bacteria a ciertas condiciones de temperatura. Todos los análisis respectivos fueron realizados en el Laboratorio de Microbiología de Alimentos del Centro de Investigación y Desarrollo en Salud (CENSALUD) de la Universidad de El Salvador. Los resultados obtenidos de los análisis, demostraron que el 25% de las dos variedades de quesos analizados no cumplen con los parámetros requeridos del RTCA (67.04.50:08), se demostró que la L. monocytogenes sobrevive a condiciones de temperatura de 4 °C. Se consideró que los quesos fresco y duro blando no son aptos para el consumo humano, se recomienda que el ministerio de salud aumente el monitoreo y de capacitaciones para mejorar las condiciones higiénicas en los puestos de los mercados, además de supervisar que empleen leche pasteurizada en la elaboración de queso así como prohibir el consumo de quesos de dudosa procedencia.

Eficácia de películas edíveis suplementadas com componentes de óleos essenciais no controlo de Listeria monocytogenes e Salmonella enterica Typhimurium

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Stress response and autolytic behaviour of Listeria monocytogenes isolated from portuguese cheese

Tese de dout. em Biologia, especialidade de Microbiologia, Unidade de Ciências e Tecnologias Agrárias, Univ. do Algarve, 2000

Prevalence of Listeria species in some foods and their rapid identification

Purpose: To investigate the occurrence of Listeria spp., (particularly L. monocytogenes ), in different foods and to compare diagnostic tools for their identification at species level. Methods: Samples of high protein foods such as raw meats and meat products and including beef products, chicken, fish and camel milk were analysed for the presence of Listeria spp. The isolates were characterised by morphological and cultural analyses, and confirmed isolates were identified by protein profiling and verified using API Listeria system. Protein profiling by SDS-PAGE was also used to identify Listeria spp. Results: Out of 40 meat samples, 14 (35 %) samples were contaminated with Listeria spp., with the highest incidence (50 %) occurring in raw beef products and raw chicken. Protein profiling by SDSPAGE was used to identify Listeria spp. The results were verified with API Listeria system. Approximately 25 % of the identified isolates were Listeria seeligeri , Listeria welshimeri , and Listeria grayi (three positive samples), while 16.66 % of the isolates were Listeria monocytogenes (two positive samples); 16.6 % of the isolates were Listeria innocua (two positive samples), while 8.3 % of the isolates were Listeria ivanovii (one positive sample). Conclusion: High protein foods contain different types of Listeria species; whole-cell protein profiles and API Listeria system can help in the identification of Listeria at the species level.

Caracterização molecular de cepas de Listeria monocytogenes oriundas de corte bovino e abatedouros frigoríficos de bovinos localizado no Distrito Federal

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Saúde Animal, 2016.

A bioengineered nisin derivative, M21A, in combination with food grade additives eradicates biofilms of Listeria monocytogenes

The burden of foodborne disease has large economic and social consequences worldwide. Despite strict regulations, a number of pathogens persist within the food environment, which is greatly contributed to by a build-up of resistance mechanisms and also through the formation of biofilms. Biofilms have been shown to be highly resistant to a number of antimicrobials and can be extremely difficult to remove once they are established. In parallel, the growing concern of consumers regarding the use of chemically derived antimicrobials within food has led to a drive toward more natural products. As a consequence, the use of naturally derived antimicrobials has become of particular interest. In this study we investigated the efficacy of nisin A and its bioengineered derivative M21A in combination with food grade additives to treat biofilms of a representative foodborne disease isolate of Listeria monocytogenes. Investigations revealed the enhanced antimicrobial effects, in liquid culture, of M21A in combination with citric acid or cinnamaldehyde over its wild type nisin A counterpart. Subsequently, an investigation was conducted into the effects of these combinations on an established biofilm of the same strain. Nisin M21A (0.1 μg/ml) alone or in combination with cinnamaldehyde (35 μg/ml) or citric acid (175 μg/ml) performed significantly better than combinations involving nisin A. All combinations of M21A with either citric acid or cinnamaldehyde eradicated the L. monocytogenes biofilm (in relation to a non-biofilm control). We conclude that M21A in combination with available food additives could further enhance the antimicrobial treatment of biofilms within the food industry, simply by substituting nisin A with M21A in current commercial products such as Nisaplin® (Danisco, DuPont).

Combining expert opinions in prior elicitation

A method of eliciting prior distributions for Bayesian models using expert knowledge is proposed. Elicitation is a widely studied problem, from a psychological perspective as well as from a statistical perspective. Here, we are interested in combining opinions from more than one expert using an explicitly model-based approach so that we may account for various sources of variation affecting elicited expert opinions. We use a hierarchical model to achieve this. We apply this approach to two problems. The first problem involves a food risk assessment problem involving modelling dose-response for Listeria monocytogenes contamination of mice. The second concerns the time taken by PhD students to submit their thesis in a particular school.

Combining expert opinions in prior elicitation

We consider the problem of combining opinions from different experts in an explicitly model-based way to construct a valid subjective prior in a Bayesian statistical approach. We propose a generic approach by considering a hierarchical model accounting for various sources of variation as well as accounting for potential dependence between experts. We apply this approach to two problems. The first problem deals with a food risk assessment problem involving modelling dose-response for Listeria monocytogenes contamination of mice. Two hierarchical levels of variation are considered (between and within experts) with a complex mathematical situation due to the use of an indirect probit regression. The second concerns the time taken by PhD students to submit their thesis in a particular school. It illustrates a complex situation where three hierarchical levels of variation are modelled but with a simpler underlying probability distribution (log-Normal).

Spontaneous fermentation of traditional sago starch in Papua New Guinea

Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago samples collected from PNG villages, lactic acid bacteria (LAB) were present (>= 3.6 x 10(4) cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all samples, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 degrees C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with similar to 10(4)/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to <30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days (<36/g), but Escherichia coli was still detectable after three weeks (>10(2)/g). Fermentation appeared to increase the storability and safety of the product.