TYPING OF LEGIONELLA PNEUMOPHILA SEROGROUP 1BY SBT, MLVA AND SPOLIGOTYPING

Introduction: The current typing methodof Legionella pneumophila recommended bythe European EWGLI Consortium is a multilocus sequence typing (MLST)-like protocolcalled sequence-based typing (SBT).This approachis highly portable and widely used toperform epidemiological surveys and outbreakinvestigation. Recent studies have demonstratedthe values of using the multiple-locusvariable-number tandem repeat (VNTR)analysis (MLVA) and the diversity of clusteredregularly interspaced short palindromic repeat(CRISPR)-based typing technique (Spoligotyping)as genotyping markers.In this study we used these three methods fortyping the L. pneumpophila isolates and for investigatingthe origin of two cases of legionellosisin Palermo, Italy.Materials and Methods: A total of 40 strainsof L. pneumophila was investigated. The strainsanalyzed were obtained from 2 clinical samplesand 38 environmental samples (hotels,hospitals and private home); all isolates belongedto serogroup 1.SBT alleles were coded following the EWGLIconvention and minimum spanning tree wasmade using BioNumerics 6.5. For VNTRs typing12 VNTR loci followed by a number wereused and MLVA clustering was performed byHamming’s distance and UPGMA. In additionthe Spoligotyping was performed by Luminexdevices.Results: SBT analysis discriminated ten differentgenotypes (STs). In particular, only oneclinical isolate showed the same ST of environmentalstrains obtained from the structure inwhich the subject was hospitalized.The MLVA and Spoligotyping did not confirmedthe source of contamination.Moreover through the MLVA analysis four differentclonal complex (VACCs) were identified.Discussion and Conclusion: Phylogeneticanalysis in L. pneumophila is a difficult taskconsidering the enormous genetic differencesobserved in the accessory genome of this species.However, by using VNTR polymorphismand Spoligotyping is possible to group isolateswithin complex which are highly congruentwith SBT clustering results and to providefurther insight into relationships among thesegroups.In this study we demonstrate that typing by thethree methods provides valuable informationfor epidemiological studies and for identificationof clonal complex in L. pneumophila.48 Oral CommunicationsCO 07MECHANISM OFSITE-SPECIFICINTEGRATION OF ICE Tn5253IN THE CHROMOSOMEOF STREPTOCOCCUSPNEUMONIAEFrancesco Santoro1, Alessandra Romeo1,Marco Oggioni2, Gianni Pozzi1,Francesco Iannelli11LAMMB, Dip. di Biotecnologie Mediche,Università di Siena - Italy; 2Department of Genetics,University of Leicester, Leicester - United KingdomIntroduction: Integrative Conjugative Elements(ICEs) are mobile genetic elements thatcan integrate into bacterial genome, excisefrom it to form a closed circular intermediatecapable of intracellular transposition in a newgenomic location or intercellular transpositionin a new genome host upon conjugative transfer.ICE Tn5253 of Streptococcus pneumoniae isa composite element which carries the tet(M)bearing Tn5251 and the resistance genes andthe cat bearing Ωcat(pC194), conferring resistanceto tetracycline and chloramphenicol, respectively.Tn5253 is found integrated at a 83-bp specific target site (attB) into the R6 pneumococcalchromosome.Materials and Methods: Bacterial strainsused for conjugation are standard laboratorystrains whose genome sequence is available inGenBank. For plate conjugation experiments,donor and recipient cells were grown separatelyand mixed at 1:10 ratio, plated and incubatedfor 4 h at 37°C. Scoring of transconjugants wasobtained in presence of appropriate antibioticswith a multilayer plating procedure.