Outil d’évaluation des risques microbiologiques d’origine alimentaire, et d’optimisation des procédés de transformation, la microbiologie prévisionnelle ne considère pas l’influence de l’état physiologique des microorganismes sur leur comportement. L’objectif de ce travail est d’identifier des marqueurs permettant de rendre compte de l’impact de l’adaptation cellulaire face à un stress acide. Dans ce cadre, la résistance acide de Bacillus weihenstephanensis a été étudiée et des ARNm ont été identifiés en tant que biomarqueurs de résistance.(i) La résistance acide a été quantifiée via des méthodes culturales pour différents passés cellulaires. L’adaptation à un stress salin est défavorable à la résistance acide en comparaison à des conditions optimales de croissance ou à une adaptation spécifique au stress acide. Néanmoins,la résistance acide de B. weihenstephanensis est modulée de la même façon par l’acidité de l’environnement, indépendamment des conditions de culture préalablement rencontrées.(ii) Un outil basé sur la technologie Pall GeneDisc®a été développé pour permettre la quantification de l’expression génique par RT-qPCR.(iii) La corrélation des données Omic et de résistance acide a permis la sélection de biomarqueurs permettant de différencier les cellules les plus résistantes des cellules les plus sensibles présentent au sein d’une population bactérienne.(iv) Des corrélations linéaires et non linéaires ont également permis de définir des ‘direct biomarker’ qui correspondent aux gènes dont l’expression peut être linéairement corrélée à la résistance; et des ‘long-acting biomarker’ qui correspondent aux gènes dont l’expression transitoire est corrélée à une résistance acide stable.(v) Une analyse multivariée a été utilisée afin de prendre en compte les interactions entre l’expression des différents gènes impliqués dans la résistance acide et a permis la sélection de 9 gènes comme biomarqueurs de résistance acide. Enfin, des premières données prometteuses ont été obtenues. Il serait ainsi possible d’utiliser l’expression génique d’une population bactérienne à un instant donné pour prévoir sa survie dans un environnement létal. / Predictive microbiology is a tool to assess food microbiological risks and optimise food processes. However predictive microbiology which predicts the bacterial behaviour does not take bacterial physiological state into consideration. The aim ofthis work is to identify molecular biomarkers to assess the impact of bacterial adaptation on the subsequent acid resistance. In this study, the acid resistance of Bacillus weihenstephanensis wasinvestigated and mRNAs were identified as acid resistance biomarkers.(i) The bacterial acid resistance of different mildstress adapted cells was quantified using cultural methods. Mild salt-adapted cells were less resistant than cells grown in optimal conditions;and the latter less resistant than acid-adaptedcells. However, the bacterial resistance of B.weihenstephanensis followed the same patternwhen facing acidic changes of the environment and that, whatever the environmental condition previously encountered.(ii) For RT-qPCR gene expression quantifications a specific rotative PCR device based on the PallGeneDisc® Technology was developed.(iii) Omic data and bacterial acid resistances correlation allows the selection of biomarkers to track the more resistant and the more sensitive cells present within the bacterial population.(iv) Both linear and non linear correlations allowed to define two types of biomarkers: ‘Directbiomarker’ for which expression patterns uponmild stress treatment were linearly correlated to the subsequent acid resistance and ‘long-actingbiomarkers’ which were transiently up-regulatedduring mild stress exposure and correlated to increased acid resistance over time.(v) A multivariate analysis was performed to correlate the acid bacterial resistance and the gene expression of vegetative cells. This mathematical method provides the advantage to take gene expressions and their interactions into account and allowed the selection of 9 genes as acid resistance biomarkers of B. weihenstephanensis. Finally, some promising results were also obtained. There by, it would be feasible to use gene expression at a given time to predict the bacterialsurvival behaviour in lethal acid conditions.

Escherichia coli O157:H7 é um dos patógenos alimentares mais importantes da atualidade, e, recentemente, diversas cepas desse microrganismo foram isoladas no sul do Brasil. O objetivo deste trabalho foi caracterizar duas cepas de E. coli O157:H7, através da avaliação de sua resistência térmica, ácida e comportamento frente a diferentes desinfetantes e procedimentos de higienização de vegetais folhosos. Resultados demonstraram a redução significativa das contagens bacterianas, após 3 minutos a 60ºC, 2 minutos a 65º, 1 minuto a 70, 72º C e 75ºC. Contudo, após adaptação ácida, nenhuma das cepas foi inativada a 60ºC, por até 3 minutos. A 70ºC foi observado aumento significativo da resistência térmica apenas para a CC. Em relação à resistência ácida, ambas as cepas sobreviveram quando expostas ao meio TSBm a diferentes pH entre 2,0 a 9,5 ajustados com ácido clorídrico e ácido propiônico (AP). Apenas, o meio com pH 2,0, ajustado com AP, foi capaz de reduzir significativamente às contagens de ambas as cepas. Na avaliação da resistência aos desinfetantes, álcool etílico (96ºGl e 70%) e quaternário de amônio foram capazes de inativar ambas as cepas, mesmo na presença de matéria orgânica. O ácido orgânico, na concentração recomendada pelo fabricante e o dobro dessa concentração, reduziu cerca de 90% das contagens, após 5 minutos de exposição. O dicloroisocianurato de sódio só apresentou reduções significativas quando foram utilizadas duas vezes a concentração recomendada pelo fabricante. Todos os procedimentos de lavagem e desinfecção de alfaces, artificialmente contaminadas com E. coli O157:H7, reduziram as contagens em 2,97±1,21logUFC/g, apenas o tratamento conduzido a 10ºC, diferiu significativamente dos demais, apresentando maior redução 6,27±0,50logUFC/g. Esses resultados demonstram que as cepas de E. coli O157:H7 foram resistentes a diferentes situações de estresse, sendo portanto importantes do ponto de vista epidemiológico, visto que estão presentes no sul do Brasil e podem se tornar agentes etiológicos de surtos alimentares. / Escherichia coli O157:H7 is a major food pathogens today, and recently, several strains of this organism were isolated in southern Brazil. The objective of this study was to characterize two strains of E. coli O157: H7, by evaluating its thermal and acid resistance and behavior among different disinfectants and cleaning procedures on leafy vegetables. The results showed a significant reduction in bacterial counts after 3 minutes at 60 ° C, 2 min at 65 ° and 1 min at 70, 72 º C and 75 º C. However, after acid adaptation, none of the strains was inactivated at 60 ° C among 3 minutes. At 70 ° C significant increase in heat resistance was observed only for CC. Regarding acid resistance, both strains survived when exposed to TSBm medium at different pH adjusted from 2.0 to 9.5 with hydrochloric acid and propionic acid (PA). Only when the medium was adjusted with AP at pH 2.0 it was able to significantly reduce the counts of both strains. Evaluating the resistance to disinfectants, Ethyl Alcohol Free (96ºGl and 70 %) and quaternary ammonium were able to inactivate both strains, even in the presence of organic matter. For the orgnic acid in the concentration recommended by the manufacturer and at the double concentration there was reduced around 90 % of the counts after 5 minutes of exposure. The sodium dichloroisocyanurate only showed significant reductions when used in the double concentration recommended by the manufacturer. All procedures for cleaning and disinfecting the lettuces artificially contaminated with E. coli O157 : H7 counts decreased by 2.97 ± 1.21 log CFU / g , only the treatment conducted at 10 ° C reduced significantly from the others (6 , 27 ± 0.50 log CFU / g). The results demonstrate that the strains of E. coli O157 : H7 were resistant to different stress situations and therefore are important as an epidemiological point of view, as it is present in southern Brazil and can become etiologic agents of food borne outbreaks.

Escherichia coli O157:H7 é um dos patógenos alimentares mais importantes da atualidade, e, recentemente, diversas cepas desse microrganismo foram isoladas no sul do Brasil. O objetivo deste trabalho foi caracterizar duas cepas de E. coli O157:H7, através da avaliação de sua resistência térmica, ácida e comportamento frente a diferentes desinfetantes e procedimentos de higienização de vegetais folhosos. Resultados demonstraram a redução significativa das contagens bacterianas, após 3 minutos a 60ºC, 2 minutos a 65º, 1 minuto a 70, 72º C e 75ºC. Contudo, após adaptação ácida, nenhuma das cepas foi inativada a 60ºC, por até 3 minutos. A 70ºC foi observado aumento significativo da resistência térmica apenas para a CC. Em relação à resistência ácida, ambas as cepas sobreviveram quando expostas ao meio TSBm a diferentes pH entre 2,0 a 9,5 ajustados com ácido clorídrico e ácido propiônico (AP). Apenas, o meio com pH 2,0, ajustado com AP, foi capaz de reduzir significativamente às contagens de ambas as cepas. Na avaliação da resistência aos desinfetantes, álcool etílico (96ºGl e 70%) e quaternário de amônio foram capazes de inativar ambas as cepas, mesmo na presença de matéria orgânica. O ácido orgânico, na concentração recomendada pelo fabricante e o dobro dessa concentração, reduziu cerca de 90% das contagens, após 5 minutos de exposição. O dicloroisocianurato de sódio só apresentou reduções significativas quando foram utilizadas duas vezes a concentração recomendada pelo fabricante. Todos os procedimentos de lavagem e desinfecção de alfaces, artificialmente contaminadas com E. coli O157:H7, reduziram as contagens em 2,97±1,21logUFC/g, apenas o tratamento conduzido a 10ºC, diferiu significativamente dos demais, apresentando maior redução 6,27±0,50logUFC/g. Esses resultados demonstram que as cepas de E. coli O157:H7 foram resistentes a diferentes situações de estresse, sendo portanto importantes do ponto de vista epidemiológico, visto que estão presentes no sul do Brasil e podem se tornar agentes etiológicos de surtos alimentares. / Escherichia coli O157:H7 is a major food pathogens today, and recently, several strains of this organism were isolated in southern Brazil. The objective of this study was to characterize two strains of E. coli O157: H7, by evaluating its thermal and acid resistance and behavior among different disinfectants and cleaning procedures on leafy vegetables. The results showed a significant reduction in bacterial counts after 3 minutes at 60 ° C, 2 min at 65 ° and 1 min at 70, 72 º C and 75 º C. However, after acid adaptation, none of the strains was inactivated at 60 ° C among 3 minutes. At 70 ° C significant increase in heat resistance was observed only for CC. Regarding acid resistance, both strains survived when exposed to TSBm medium at different pH adjusted from 2.0 to 9.5 with hydrochloric acid and propionic acid (PA). Only when the medium was adjusted with AP at pH 2.0 it was able to significantly reduce the counts of both strains. Evaluating the resistance to disinfectants, Ethyl Alcohol Free (96ºGl and 70 %) and quaternary ammonium were able to inactivate both strains, even in the presence of organic matter. For the orgnic acid in the concentration recommended by the manufacturer and at the double concentration there was reduced around 90 % of the counts after 5 minutes of exposure. The sodium dichloroisocyanurate only showed significant reductions when used in the double concentration recommended by the manufacturer. All procedures for cleaning and disinfecting the lettuces artificially contaminated with E. coli O157 : H7 counts decreased by 2.97 ± 1.21 log CFU / g , only the treatment conducted at 10 ° C reduced significantly from the others (6 , 27 ± 0.50 log CFU / g). The results demonstrate that the strains of E. coli O157 : H7 were resistant to different stress situations and therefore are important as an epidemiological point of view, as it is present in southern Brazil and can become etiologic agents of food borne outbreaks.

Escherichia coli O157:H7 é um dos patógenos alimentares mais importantes da atualidade, e, recentemente, diversas cepas desse microrganismo foram isoladas no sul do Brasil. O objetivo deste trabalho foi caracterizar duas cepas de E. coli O157:H7, através da avaliação de sua resistência térmica, ácida e comportamento frente a diferentes desinfetantes e procedimentos de higienização de vegetais folhosos. Resultados demonstraram a redução significativa das contagens bacterianas, após 3 minutos a 60ºC, 2 minutos a 65º, 1 minuto a 70, 72º C e 75ºC. Contudo, após adaptação ácida, nenhuma das cepas foi inativada a 60ºC, por até 3 minutos. A 70ºC foi observado aumento significativo da resistência térmica apenas para a CC. Em relação à resistência ácida, ambas as cepas sobreviveram quando expostas ao meio TSBm a diferentes pH entre 2,0 a 9,5 ajustados com ácido clorídrico e ácido propiônico (AP). Apenas, o meio com pH 2,0, ajustado com AP, foi capaz de reduzir significativamente às contagens de ambas as cepas. Na avaliação da resistência aos desinfetantes, álcool etílico (96ºGl e 70%) e quaternário de amônio foram capazes de inativar ambas as cepas, mesmo na presença de matéria orgânica. O ácido orgânico, na concentração recomendada pelo fabricante e o dobro dessa concentração, reduziu cerca de 90% das contagens, após 5 minutos de exposição. O dicloroisocianurato de sódio só apresentou reduções significativas quando foram utilizadas duas vezes a concentração recomendada pelo fabricante. Todos os procedimentos de lavagem e desinfecção de alfaces, artificialmente contaminadas com E. coli O157:H7, reduziram as contagens em 2,97±1,21logUFC/g, apenas o tratamento conduzido a 10ºC, diferiu significativamente dos demais, apresentando maior redução 6,27±0,50logUFC/g. Esses resultados demonstram que as cepas de E. coli O157:H7 foram resistentes a diferentes situações de estresse, sendo portanto importantes do ponto de vista epidemiológico, visto que estão presentes no sul do Brasil e podem se tornar agentes etiológicos de surtos alimentares. / Escherichia coli O157:H7 is a major food pathogens today, and recently, several strains of this organism were isolated in southern Brazil. The objective of this study was to characterize two strains of E. coli O157: H7, by evaluating its thermal and acid resistance and behavior among different disinfectants and cleaning procedures on leafy vegetables. The results showed a significant reduction in bacterial counts after 3 minutes at 60 ° C, 2 min at 65 ° and 1 min at 70, 72 º C and 75 º C. However, after acid adaptation, none of the strains was inactivated at 60 ° C among 3 minutes. At 70 ° C significant increase in heat resistance was observed only for CC. Regarding acid resistance, both strains survived when exposed to TSBm medium at different pH adjusted from 2.0 to 9.5 with hydrochloric acid and propionic acid (PA). Only when the medium was adjusted with AP at pH 2.0 it was able to significantly reduce the counts of both strains. Evaluating the resistance to disinfectants, Ethyl Alcohol Free (96ºGl and 70 %) and quaternary ammonium were able to inactivate both strains, even in the presence of organic matter. For the orgnic acid in the concentration recommended by the manufacturer and at the double concentration there was reduced around 90 % of the counts after 5 minutes of exposure. The sodium dichloroisocyanurate only showed significant reductions when used in the double concentration recommended by the manufacturer. All procedures for cleaning and disinfecting the lettuces artificially contaminated with E. coli O157 : H7 counts decreased by 2.97 ± 1.21 log CFU / g , only the treatment conducted at 10 ° C reduced significantly from the others (6 , 27 ± 0.50 log CFU / g). The results demonstrate that the strains of E. coli O157 : H7 were resistant to different stress situations and therefore are important as an epidemiological point of view, as it is present in southern Brazil and can become etiologic agents of food borne outbreaks.

No description available.