VAAM-Jahrestagung 2012 18.–21. März in Tübingen

111MPP007Comparative genomic analysis of 44 Clostridium difficile strainsH. Kurka* 1 , A. Ehrenreich 1 , M. Rupnik 2 , B. Dupuy 3 , M. Monot 3 ,W. Ludwig 1 , W. Liebl 11 Technical University, Departement of Microbiology, Munich, Germany2 Institute of Public Health, Centre for Microbiology, Maribor, Slovenia3 Institute Pasteur, Microbiology, Paris, FranceClostridium difficilei s the main cause of healthcare associated diarrheaworldwide. The Clostridium difficile associated disease ranges from selflimiteddiarrhea to life-threatening colitis. One approach to classifydifferent Clostridium difficile strains is the determination of the ribotype.The ribotype depends on differences in the length of the 16S-23S rRNAintergenic spacer region. Current publications on comparative analysis ofClostridium difficile strains focused mainly on strains of one ribotype. Incontrast we present here a comparative genome analysis of genomesequences of 44 different Clostridium difficile strains, belonging to 22distinct ribotypes.To investigate the phylogenetic diversity among the 44 Clostridiumdifficile strains we computed 14 different trees based on the nucleotidesequence of 14 different highly conservative marker genes using the ARBsoftware package. For each tree we unexpectedly found that strains of thesame ribotype belong to one node of the trees.Within our dataset we elucidated differences and similarities in genecontent within strains of the same ribotype and within strains of differentribotypes by implementing a bidirectional NCBI BLAST. Using thisapproach we computed conserved and specific genes for the Clostridiumdifficile genomes. We found that the number of conserved genes dependson the ribotype of the strain. In accordance to the tree analysis there is astrong correlation between strains of the same ribotype.Knowing similarities and differences on the gene level, the third aspect ofour analysis covers the detection of Single Nucleotide Polymorphisms(SNP). Using the software MUMmer for the SNP analysis we clarifywhich genomic regions are more susceptible to SNPs than others. Forexample we identified one region that seems to be specific for strains ofribotype 078. Generally we found that the number of SNPs depends on theribotype of the genome.Altogether the tree analysis unexpectedly proved so far that strains of thesame ribotype are more related to each other.MPP008Novel strategies for biofilm disruption from metagenomesH. Henke* 1,2 , I. Krohn-Molt* 1 , A. Pommerening-Röser 1 , W. Streit 1 , H. Rohde 21 Biozentrum Klein Flottbek, Microbiology, Hamburg, Germany2 Universitätsklinikum Hamburg-Eppendorf, Medical Microbiology, Hamburg,GermanyStaphylococcus- and Pseudomonas species` biofilms on medical deviceslead to huge hospital associated problems and are difficult to treat [1,2].We report on metagenomic screening methods and partial characterizationof metagenome clones that either inhibit the development of microbialbiofilms or hydrolyze established microbial biofilms. A total of 30.000fosmid clones of two metagenomic libraries have been analyzed for clonesthat encode proteins interfering with the inhibition of de novo formation ofbiofilms and lysis of already established microbial biofilms. Tests wereperformed using S. epidermidis strain 1457 and P. aeruginosa strainPA028. The screenings have been accomplished via an overlay-assay andin micro titer plates. Altogether 10 fosmid clones were identified thatstrongly inhibited the formation of P. aeruginosa PA028 and S. epidermidis1457 biofilm formation. 14 fosmid clones inhibit only S. epidermidis 1457biofilm formation. Furthermore 3 fosmid clones were identified that disruptalready established S. epidermidis 1457 biofilms. Furthermore all 27 identifiedfosmid clones have been sequenced via Illumina and the ORFs and proteinsinvolved in biofilm phenotypes are currently in characterization.1. Rohde H, Mack D, Christner M, Burdelski C, Franke GC et al. (2006) Pathogenesis of staphylococcaldevice-related infections: from basic science to new diagnostic, therapeutic and prophylactic approaches.Rev Med Microbiol 17: 45-54.2. Rupp ME, Archer GL (1994) Coagulase-negative staphylococci: pathogens associated with medicalprogress. Clin Infect Dis 19: 231-243.MPP009Co-regulation of multidrug resistance and pathogenicity inErwinia amylovoraD. Pletzer*, H. WeingartJacobs University Bremen, School of Engineering and Science, Bremen,GermanyErwinia amylovora, a plant pathogenic member of the Enterobacteriaceae,causes fire blight on rosaceous plants, especially pear and apple. Fireblight is one of the most devastating plant diseases caused by bacteria inGermany. Especially apple orchards in South Germany are severelyaffected by this existence-threatening disease, due to the warmer weatherconditions favoring disease development. The commercial implications ofthis plant disease are aggravated by the limited effectiveness of currentcontrol measures. A major pathogenicity factor of E. amylovora ismultidrug efflux mediated by the RND-type pump AcrAB-TolC. It waspreviously shown that this efflux system confers resistance to a broadrange of structurally unrelated compounds including antibiotics, dyes andplant-derived antimicrobial toxins. Moreover, acrB- and tolC-deficientmutants showed a dramatically reduced virulence on apple rootstocks. Theaim of this project is to explore the cause of the attenuated pathogenicity inmutants of E. amylovora lacking a component of the AcrAB-TolC system.In Salmonella enterica, a human pathogenic enterobacterium, it was shownthat a transcriptional activator was responsible for downregulation ofnumerous genes encoding proteins involved in pathogenicity in an acrBdeficientmutant. We will determine whether such a global regulator,responsible for the co-regulation of pathogenicity and multidrug resistance,exists in E. amylovora. Beside AcrAB-TolC, three additional RND-typepumps are present in the annotated genome sequences of E. amylovora. Todetermine the role of these multidrug transporters in antibiotic resistanceand virulence of E. amylovora, transporter-deficient mutants will begenerated and characterized.MPP010Sublethal concentration of benzalkonium chloride increases theintracellular proliferation of Listeria monocytogenes in vitroL. Pricope* 1,2 , A. Nicolau 1 , M. Wagner 2 , K. Rychli 21 Dunarea de jos University, Galati, Romania2 Institute for Milk Hygiene, University of Veterinary Medicine, Vienna, AustriaQuestion: Listeria monocytogenes(L.monocytogenes) is a foodbornepathogen able to persist in the food processing environment for months oreven years. Some L.monocytogenes strains are more resistant than othersto certain sanitizers, like benzalkonium chloride (BAC), and thereforerepresent a continuous source of recontamination of food products. Arecent study indicates that BAC affects the expression of stress proteinsand also of proteins related to virulence in L. monocytogenes (Kastbjerg etal, 2010).The aim of our study was to assess the effect of sublethal concentration ofBAC on the virulence potential of L.monocytogenes.Methods: Three L.monocytogenes strains isolated from cheese smearwater - Austria (Lm1), cheese - Ireland (Lm2), and smoked salmon -Denmark (Lm3) and the clinical strain EGDe, all serovar 1/2a, wereincubated with or without 1.25mg/l BAC for 30 minutes. Invasion andintracellular proliferation after 4 hours were determined in a cell cultureassay using Caco-2, a human colonic carcinoma, HepG2, a humanhepatocellular liver carcinoma and THP-1, a human acute monocyticleukemia cell line.Results: The four L.monocytogenes strains vary significantly in invasionand proliferation efficiency, in respect to all three human cell types. EGDeshowed the highest ability to invade all cell types, followed by Lm 3,whereas for Lm1 and Lm 2 a significant lower invasion rate was detected.Incubation with BAC significantly reduced the invasion rate only of EGDeand Lm3, while the invasion efficiency of Lm1 and Lm 2 was only slightlybut not significantly decreased by incubation with BAC.Surprisingly, 30 minutes exposure to 1.25mg/l BAC increasedsignificantly the intracellular proliferation for all four strains in all threedifferent human cell types.Conclusions: These results suggest that through the exposure to ‘stress’caused by sublethal concentrations of disinfectants L.monocytogenes mighteasier adapt to the intracellular environment of the human cells whichleads to a higher intracellular proliferation.Kastgjerg VG, Halberg Larsen M, Gram L, Ingmer H, (2010), Influence of sublethal concentrationof common disinfectants on expression of virulence genes in Listeria monocytogenes, Appl. andEnvironmental Microbiology 76(1): 303-309MPP011Occurence of culturableVibrio choleraefrom LakeVictoriaand two rift valley lakes Albert and George, UgandaM. Kaddumukasa* 1,2 , F. Muyodi 31 Makerere University, Biological Sciences, Kampala, Uganda2 University, Biology, Kampala, Uganda3 University, Biological sciences, Kampala, UgandaIn Uganda the quality and quantity of clean water are already threatenedby poor sanitation, pollution, increasing population pressure anddeforestation. Links between climate change impacts, clean water andsanitation and human health are significant in Uganda. An investigationinto the occurrence ofVibrio choleraeand correlation with environmentalfactors was conducted from September 2009 to August 2010 in three lakes.Water samples were collectedmonthly from three shore sampling sites inLakes Victoria (Gabba), Albert (Butiaba), George (Kayanzi) sites. Variousenvironmental parameters were monitored over this period. Enrichmenttechniques and standard tests were used to detect the presence ofV.cholerae.Seventy five percent (n= 90) of the samples were positive forV.cholerae. Environmental parameters were found to vary with theabundance ofV. choleraeover the seasons. V. choleraewas morefrequentlydetected during the dry than in the wet season. Results revealBIOspektrum | Tagungsband 2012