No description available.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular. / La industria chilena hortofrutícola enfrenta grandes desafíos en el área de inocuidad alimentaria y bioseguridad. Garantizar el cumplimiento de los más altos estándares internacionales, le permite a empresas exportadoras, mantener su liderazgo en los más exigentes y demandantes mercados extranjeros. Para validar la inocuidad de sus productos, una empresa chilena líder en la exportación de fruta congelada, solicitó a Ciencia Pura SpA, una compañía enfocada en I+D+i (investigación, desarrollo e innovación), evaluar algunos de sus protocolos de bioseguridad, los cuales deben garantizar la ausencia de patógenos posiblemente letales y ubicuos, como las bacterias Listeria monocytogenes y ciertas cepas de Escherichia coli. Este seminario de título se desarrolla en el marco de la solución propuesta por Ciencia Pura SpA para satisfacer la solicitud de la empresa solicitante. La investigación desarrollada consta de dos series de experimentos con objetivos distintos: la primera, determinar la efectividad del proceso de lavado de la fruta con una solución de hipoclorito de sodio a diferentes concentraciones, ensayando distintos tiempos de exposición al agente desinfectante, sobre la superficie de fruta previamente inoculada; y la segunda serie, analizar la supervivencia bacteriana en la superficie de fruta inoculada, en condiciones de almacenamiento previas y posteriores a su congelación. Los estudios se realizaron sobre cuatro especies de berries cultivadas con gran éxito económico en territorio nacional: frutillas, frambuesas, moras y arándanos. La disminución de la carga bacteriana, y por ende el éxito del tratamiento o almacenaje, fue evaluada comparando el número de Unidades Formadoras de Colonias (CFU) sobre una placa de agar, al ensayar muestras de la superficie de los frutos, luego de inocularlos, y de someterlos a ensayo de lavado o almacenaje. Las características estudiadas de los protocolos utilizados por la empresa cumplen con las regulaciones demandadas por la industria, sin embargo, la relación entre concentración de cloro y tiempo de exposición al desinfectante podrían no ser las óptimas en todos los casos ensayados. Respecto a los ensayos de supervivencia bacteriana bajo diferentes condiciones de almacenamiento y transporte, un efecto bactericida fue observado en las cuatro especies estudiadas, siendo especialmente destacable en frambuesas. / Food exporting companies are always struggling to ensure their biosafety protocols are up to satisfy the most demanding international markets, focusing on high reliability to guarantee operations overseas. To validate innocuity of their exported goods, an important Chilean food export company, asked Ciencia Pura SpA, a company focused on R&D in the agricultural sector, to evaluate that in their frozen berries production chain the risk of contamination from potentially dangerous microbiological risks, such as the health-threatening bacteria Listeria monocytogenes and Escherichia coli, is nil. This seminar is the result of the research proposed and conducted by Ciencia Pura SpA. The upcoming investigation is divided as two sets of experiments with different objectives: the first one, to determine the disinfection effectiveness of household bleach (sodium hypochlorite dissolution), at different concentrations and exposure times, in the surface of bacteria-inoculated berries; the second set, to analyze bacterial growth in fruit surface at different storage conditions (pre-freezing and freezing temperatures). The research was carried out on four commercially successful berry fruits grown in Chilean territory: straw-, rasp-, black- and blue-berries. Bacterial load of inoculated berries was assessed to determine disinfection efficiency and bacterial survival, comparing Colony Forming Unit (CFU) occurrence in an agar plate culture before and after the treatments. / Agosto 2019

<div><div><div><p>Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, and biofilms are known to be the frequent source for infection and outbreaks of foodborne illness. Therefore, it is essential to understand the pathogenicity of bacteria in biofilms and methods to inactivate biofilm-forming microbes from food processing environments, including school cafeteria or other community-based food production facilities, and to prevent foodborne outbreaks. Pathogen transmissions occur primarily through raw or under cooked foods and by cross contamination during unsanitary food preparation practices. Then, pathogens can form biofilms on the surface and become persistent in food production facilities and can be a source for recurrent contamination and foodborne outbreaks. In this study, our first aim was to use L. monocytogenes as a model pathogen to study how an enteric infectious pathogen isolated from biofilm modifies its pathogenesis compared to its planktonic counterpart. Both clinical and food isolates with different serotypes and biofilm-forming abilities were selected and tested using cell culture and mouse models. L. monocytogenes sessile cells isolated from biofilms express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains revealed that at 12 and 24 h post-infection (hpi), L. monocytogenes burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile L. monocytogenes from intestinal content are about 6.0- and 280-fold higher than the sessile inoculum, respectively, suggesting sessile L. monocytogenes can still upregulate virulence genes shortly after ingestion (12 h).</p><p>After learning biofilm isolated L. monocytogenes cells have similar virulence potential as the planktonic counterparts, our next goal was to effectively prevent or inactivate biofilms using food-grade natural microbials. Since L. monocytogenes cells are usually found in multi-pathogen biofilm in nature, I combined two food-grade broad-spectrum natural antimicrobials, chitosan nanoparticles (ChNP) and ε-poly-L-lysine (PL), as ChNP-PL nanoconjugates and tested its function on single or mixed culture biofilms of L. monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica serovar Enteritidis, and Pseudomonas aeruginosa. ChNP- PL not only was able to significantly (P<0.05) prevent the biofilm formation but also inactivate pre-formed biofilms when analyzed by crystal violet staining and plate counting. In vitro cytotoxicity analysis (LDH and WST-based assays) using an intestinal cell line, indicated ChNP- PL to be non-toxic. In conclusion, our results showed ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens in food processing environment. Application of ChNP-PL could inhibit the colonization of foodborne pathogens, minimize cross-contamination during food production, and eventually reduce foodborne outbreaks.</p></div></div></div>

La presencia de L. monocytogenes en canales bovinas la convierte en un riesgo potencial de enfermedad para los consumidores por ser causante de la Listeriosis, enfermedad a la que son más susceptibles las mujeres embarazadas y aquellas personas con un sistema inmune deprimido. Debido a ello el objetivo del presente estudio fue evaluar el efecto del ácido láctico a una concentración del 2.5% como descontaminante de canales sobre la presencia de L. monocytogenes presentes en la superficie de las canales bovinas, así como evaluar el comportamiento de esta bacteria en refrigeración y bajo la acción del ácido láctico. El estudio se desarrolló en un centro de beneficio de la ciudad de Lima donde se muestrearon un total 58 canales bovinas al azar divididas en dos grupos. Las muestras tomadas fueron delimitadas por marcos estériles y correspondieron a un área total de 400 cm2 por canal bovina, tomadas de 4 zonas diferentes (cadera, falda, pecho y cuello) mediante método no destructivo de hisopado. Estas muestras fueron enriquecidas en medios de cultivo especificos y sometidas a incubación para determinar finalmente la presencia de L. monocytogenes mediante el empleo de un Kit diagnóstico. En los resultados obtenidos se observaron canales que tuvieron cambios respecto a la presencia de la bacteria sobre la superficie de las canales antes y después de la refrigeración y de la acción del ácido láctico, sin embargo al evaluar los resultados mediante la prueba estadística de McNemar se observa que estos cambios se debieron al azar (p>0.05) para el grupo 1 y para el grupo 2, el cual fue sometido a la acción del ácido láctico, se observa que hay diferencia estadistica significativa (p<0.05) por lo que se demuestra la acción bactericida del ácido láctico al 2.5% contra de L. monocytogenes presentes en la superficie de las canales. Se concluye que el tratamiento con ácido láctico al 2.5% es útil como descontaminante pero su uso no reemplazaría las buenas prácticas ni a la higiene durante el proceso de beneficio o el almacenamiento en las cámaras frigoríficas debido a que el tratamiento tuvo un efecto bactericida en más del 50% pero no en la totalidad de canales bovinas evaluadas. / --- The presence of L. monocytogenes in beef carcasses makes it a potential disease risk to consumers because it causes listeriosis, disease to which are more susceptible pregnant women and those with depressed immune systems. Because of this, the aim of this study was to evaluate the effect of lactic acid at 2.5% of concentration as decontaminating beef carcasses on the presence of L. monocytogenes on the surface of beef carcasses and to assess the behavior of this bacterium in cooling and under the action of lactic acid. The study was conducted in a profit center of Lima where a total 58 bovine carcasses were sampled randomly divided into two groups. The samples were enclosed by sterile frameworks and corresponded to a total area of 400 cm² per bovine carcasses, taken from 4 zones (hip, flank, brisket and neck) by nondestructive method of swabbing. These samples were enriched in specific culture media and finally subjected to incubation for the presence of L. monocytogenes by use of a diagnostic kit. In the results were observed carcasses who had changes for the presence of bacteria on the surface of carcasses before and after chilling and the action of lactic acid, however when evaluating the results by McNemar statistical test notes that these changes were randomized (p>0.05) to group 1 and group 2, which was subjected to the action of lactic acid, it appears that no statistically significant difference (p <0.05) so the bactericidal action of lactic acid 2.5% is shown against L. monocytogenes on the surface of carcasses. We conclude that treatment with 2.5% lactic acid is useful as a decontaminant but its use would not replace good hygiene practices or profit during storage or refrigerated chambers because the treatment had a bactericidal effect in over 50% but not all of bovine carcasses evaluated. Keywords: carcasses, L. monocytogenes, listeriosis, acid, lactic, decontamination / Tesis

Experiments were performed to investigate the effects of wash treatments, consisting of hot water, 2% lactic, 2% acetic, or 2% levulinic acid, for decontamination of pathogenic bacteria previously inoculated onto meat surfaces, to inhibit growth of pathogenic bacteria inoculated onto previously washed meat surfaces, and on the organoleptic quality of sliced turkey roll and beef trim. Acid washes were no more effective at reducing Escherichia coli O157:H7 on beef plate, Listeria monocytogenes on sliced turkey roll, and Salmonella on pork belly than was water wash. Only lactic acid treatment was more effective than water at reducing Salmonella on chicken skin, but by less than 1 log CFU/cm2. Increasing wash temperatures with 2% levulinic acid did not reduce E. coli O157:H7 on beef plate. Organic acid washes did not protect against growth of L. monocytogenes and E. coli O157:H7. Acetic acid prevented growth of Salmonella, but only on chicken skin. Organic acid spray treatments of sliced turkey roll and beef trim did not affect consumer liking of turkey roll or cooked ground beef patties. Acid treatments had some effect on instrumental color measurements, but these appear to have little practical significance. Overall, washing with 2% organic acid solutions was no more effective at reducing pathogenic bacteria on meat surfaces than washing with water.

No description available.

Acid-stress adaptation in Listeria monocytogenes (Lm) serotype 4b and 1/2a occurred when cells were pre-exposed to pH 5.0 tryptic soy broth supplemented with yeast extract (TSB-YE) at 22°C or 37°C but not at 4°C. Prolonged time, varied sublethal acid pH, substitute of acidulants and addition of sodium chloride during 4°C mild acid pre-exposure still did not induce acid-stress adaptation in Lm. This finding was also validated using an acidic cheese, similar to what has seen for Gram-negative bacteria E. coli and Salmonella. Further investigation revealed that major cold shock protein in Lm CspL was not responsible for repressed acid-stress adaptation at 4°C. A bead beating treatment prior to mild acid pre-exposure at 4°C partially induced acid-stress adaptation after pre-exposure in 4°C to mild acid stress. Our data suggests that cold processing or cold storage temperature can lower the possibility of activating acid-stress adaptation in Lm.

Previous findings showed that Listeria monocytogenes exhibits higher heat tolerance to thermal exposure at 60°C when pre-exposed to sublethal heat stress at 48°C for 30/60 min. The objective of this study was to investigate the morphological changes that occurred in L. monocytogenes serotype 1/2a cells as visualized by transmission electron microscopy (TEM) after exposure to sublethal heat stress at 48°C for 60 min and in combination with lethal concentration of carvacrol for 30 min. The TEM micrographs revealed thickening of cell wall and cell membrane, and clumping of cytoplasm when subjected to sublethal heat stress followed by carvacrol treatment. These studies indicate that L. monocytogenes cells when adapted to heat stress may alter its morphology to protect themselves against carvacrol. Further studies will investigate the effect of increased contact time with carvacrol and with other essential oils on the ultrastructural changes in L. monocytogenes cells adapted to heat stress.

The objective of this study was to investigate the differences in survival of L. monocytogenes EGD (Bug600) (serotype 1/2a) in various disinfectants and essential oils after sublethal heat stress at 48°C for 60 min. The survival of heat stressed L. monocytogenes cells was decreased in lethal acid (HCl or H3PO4 pH 2.5), lethal oxidative stress (H2O2 1000 ppm) and lethal quaternary ammonium compounds (QAC 2.5-3.5 ppm) compared to non-stressed control cells. By contrast, the survival of heat stressed L. monocytogenes cells was higher in lethal alkali (NaOH or KOH pH 12) than the control cells. Also, the survival of heat stressed L. monocytogenes cells was higher in lethal carvacrol (428 ppm) and bay oil (1100 ppm) but was decreased in lethal red thyme oil (300 pm) compared to control. These findings indicate that the heat stressed cells of L. monocytogenes are not easily killed by alkali-based disinfectants and essential oils containing carvacrol and bay oil. Therefore, disinfectants and essential treatments should be carefully considered when heat stressed cells of L. monocytogenes may be present.

According to USDA-FSIS reports, L. monocytogenes and Salmonella are two important foodborne pathogens that are prevalent throughout catfish environment. Channel catfish (Ictalurus punctatus) is the most important aquaculture species in the United States accounting for more than 60% of its aquaculture production. The objectives of this study were to determine the effect of strain, temperature concentration on the growth, survival and biofilm formation of L. monocytogenes and Salmonella using catfish mucus extract on different food-contact surfaces. Growth and survival of L. monocytogenes and Salmonella was greater at the higher concentration of mucus extract at both 10°C and 22°C. In 15 micrograms/ml catfish mucus extract L. monocytogenes and Salmonella counts increased to 4.5 log CFU/ml after 72 h at 10°C and 5-7 log CFU/ml counts after 32-48 h at 22°C. In 375 micrograms/ml catfish mucus extract L. monocytogenes and Salmonella counts increased to 6-7 log CFU/ml counts after 72 h at 10°C and 8-9 log CFU/ml counts after 32-48 h at 22°C. L. monocytogenes and Salmonella were able to grow and survive for more than 63 days with at least 4-6 log CFU/ml at 10°C and 6-8 log CFU/ml in 375 micrograms/ml and 15 micrograms/ml of catfish mucus extract respectively. No differences (P > 0.05) among L. monocytogenes and Salmonella strains were observed to form biofilms in the presence catfish mucus extract on the stainless steel surface. The biofilm formation by L. monocytogenes and Salmonella in catfish mucus extract was less (P < 0.05) on buna-n rubber when compared to stainless steel, polyethylene and polyurethane surfaces. Therefore, the findings in this study show that catfish mucus promotes L. monocytogenes and Salmonella to grow and subsequently form biofilms on different food-contact surfaces also promotes growth and survival of these pathogens for longer period of time in catfish processing industries.