VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

FMV001Influence of osmotic and pH stress on the alternariolbiosynthesis in Alternaria alternataE. Graf*, M. Schmidt-Heydt, R. GeisenDepartment of Safety and Quality of Fruit and Vegetables, Max RubnerInstitute, Karlsruhe, GermanyMycotoxin producing Alternaria moulds are ubiquitously present andcommonly found on fruits, vegetables and grains. Some Alternaria specieshave teratogenic, mutagenic or cytotoxic potential due to their production ofmycotoxins such like alternariol, alternariol methyl ether and tenuazonicacid. To date, only limited knowledge is available about the regulation of thesynthesis of Alternaria toxins, especially under food relevant conditions. Infoods fungi are exposed to osmotic stress due to a high concentration ofdifferent solutes, and especially in fruits and vegetables fungi encounter acidenvironments. PacC is a key element in pH gene regulation. At alkaline pHvalues,activation of PacC leads to suppression of acidity-expressed genesand at acid pH-values, alkaline-expressed genes are suppressed by inactivePacC. In the current analysis it could be demonstrated that the alternariolbiosynthesis is consistent during a wide pH range, whereas osmoticcomponents like NaCl show a deep impact on the alternariol biosynthesisresulting in complete inhibition already at low concentrations. Highosmolarity in the environment is usually transmitted to the transcriptionallevel of downstream regulated genes by the HOG signal cascade, which is aMAP kinase transduction pathway. The inhibition of alternariol biosynthesisby changes in the osmolarity of the substrate might be regulated by this highosmolarity cascade. It therefore became essential to analyse the HOG1signal transduction pathway of A. alternata in more detail. Expression of theMAP kinase genes hog1 and pbs2 and phosphorylation of the HOG1 proteinwere analyzed. A clear correlation between HOG1 phosphorylation andalternariol biosynthesis could be established. In addition, knock-down ofhog1 by protoplast transformation lead to an non-toxic phenotype withreduced HOG1 phosphorylation. However, a combined alkaline and osmoticstress situation induced the alternariol biosynthesis of the transformant,which leads to the conclusion that hog1 is not involved into alternariolbiosynthesis under alkaline conditions, but pacC. This assumption could beconfirmed by a pacC-knock-down transformant being incapable ofalternariol biosynthesis exclusively at alkaline conditions.FMV002Differential proteomic expression of enterohaemorrhagicE. coli O157:H7 EDL933 grown in intestinal simulatingmediaS. Polzin*, I. Elsenhans, H. SchmidtDepartment of Food Microbiology, University of Hohenheim, Stuttgart,GermanyEnterohaemorrhagic E. coli (EHEC) are serious causative agents of foodborneinfections and can cause a broad range of intestinal and extraintestinaldiseases. The survival of EHEC in the food chain and in the gut of thehuman host may be linked to stress resistance and is dependent of thenutrients available. In different environment EHEC may adapt theirproteomic appearance, this leads to a shift in metabolism and maybeincreased virulence. However, these mechanisms are largely undescribed.To test the metabolic properties of EHEC O157:H7 EDL933 grown in theintestine, we used the simulating ileal environment medium (SIEM) and thesimulating colon environment medium (SCEM) and compared the growth inthese media with that in the rich medium tryptic soy broth (TSB). By the useof 2D-Gelelectrophoresis we determined differentially expressed cytosolicproteins with the least amount of 2 fold higher expression. After growth indifferent media the proteins were evaluated by Delta2D-Analysing Softwareand identified via MALDI-TOF-Analysis. Beside proteins involved inmetabolic pathways, we found overexpressed flagellin and the autoinducer-2synthase LuxS, which mediates cell-cell communication during quorumsensing, as well as many stressinduced proteins like chaperons and aglutamate decarboxylase enzyme. Further we could find the global regulatorHns, responsible for stress adaption and regulation LEE-proteins. These firstresults points to increased pathogenicity after growth in different mediaexhibit different nutrient supply and interfering substances like bile salt andenzymes.To investigate the carbonflux in E. coli EDL933 we established cultures inSIEM, SCEM and TSB containing ubiquitous 13 C 6-Glucose. In cooperationwith the working group of Dr. Eisenreich we could show differentincorporation rates of labelled carbohydrates respecting the synthesisedaminoacids. Because of the acquired label at very high rates especiallyalanine and serine seems to be important for growth under aerobicconditions in intestinal simulating media. The results of these experimentshave shown that growth of EHEC O157:H7 EDL933 under certainenvironmental conditions favors expression of virulence-associated proteins.Further investigations are necessary to describe those metabolic pathways inmore detail.FMV003Characterisation of the incorporation of Listeriamonocytogenes in a raw milk-biofilmC. Weiler 1 , A. Ifland 1 , S. Sigiel 1 , A. Naumann 2 , M. Noll* 11 Federal Institute for Risk Assessment, Division 74 Hygiene andMicrobiology, Berlin, Germany2 Federal Institute for Materials Research and Testing, Division IV.1Biology in Materials Protection and Environmental Issues, Berlin, GermanyA successive establishment of biofilms derived from the microbialcommunity of raw milk is found on devices of milk production withoutsufficient cleaning. Such microbial communities establish biofilm matricesthat enable incorporation of pathogens like Listeria monocytogenes and as aconsequence a continuous contamination of food processing plants. L.monocytogenes was frequently found in raw milk and non-pasteurized rawmilk products and as part of a biofilm community in milk meters and bulkmilk tanks. Listeria-contaminated products are known to cause listeriosis, asevere infection with high mortality for persons at risk, such as pregnantwomen, elderly or children.The aim of this project was to identify at which temporal stage of biofilmformation members of L. monocytogenes settle best and if there was aninteraction with the microbial community of the raw milk. Quantification ofsettled L. monocytogenes in raw milk-biofilm was carried out byfluorescence in situ hybridization (FISH). Microbial interaction onpopulation level was determined by terminal restriction fragment lengthpolymorphism analysis (T-RFLP) while on polymer / physiological levelFourier-Transform Infrared Spectroscopy (FTIR) was employed. The resultsobtained from the experiments revealed that an early addition of L.monocytogenes to raw milk caused a fast and dense biofilm formation andan enriched attachment of milk compounds. The later L. monocytogenes wasadded to the raw milk the lower were their attached cell numbers.Furthermore L. monocytogenes interacted with the raw milk-biofilmcommunity depending on their temporal addition. Particularly in the earlystage of raw milk-biofilm formation L. monocytogenes was a strongcompetitor.FMV004Survival of Listeria monocytogenes in lubricants appliedin the food industryA. Weiss*, S. Wild, H. SchmidtDepartment of Food Microbiology, University of Hohenheim, Stuttgart,GermanyListeria monocytogenes is a food-borne pathogen that is quite frequentlyassociated with fish products. Due to its tenacity and psychrotrophic growthit may persist on the food-processing equipment and machinery for longperiods. The lubricants applied in these machines have been hypothesized aspossible reservoirs and sources of L. monocytogenes due to their extendedservice life. In this project we surveyed the survival of three L.monocytogenes strains in four H1-lubricants applied in the food, especiallyfish, industry. For the determination of the viable counts the nativelubricants were first suspended in sterile glycerol, and then dilution serieswere carried out in physiological sodium chloride solution. The sampleswere spread-plated on Brain Heart Infusion agar and incubated underaerobic conditions. None of the native lubricants contained Listeria. Thenthe lubricants were contaminated with the L. monocytogenes strains andtheir survival was monitored when the lubricants were stored at 37°C. Theviable counts of the L. monocytogenes strains decreased within 14 days, andthe reduction rates were found to depend on the lubricant as well as on thestrain. Interestingly, the isolate from smoked salmon tolerated thisenvironment better than the reference and the type strain. The viable countsof all strains were reduced by 99.995% within seven days after theinoculation. The water content of the lubricant (0, 1 and 5%) had noinfluence on the results. Thus we conclude that the investigated strainscannot survive in the H1-lubricants.spektrum | Tagungsband 2011