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

Abstract

Introduction: The current typing method of Legionella pneumophila recommended by the European EWGLI Consortium is a multi locus sequence typing (MLST)-like protocol called sequence-based typing (SBT).This approach is highly portable and widely used to perform epidemiological surveys and outbreak investigation. Recent studies have demonstrated the values of using the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and the diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (Spoligotyping) as genotyping markers. In this study we used these three methods for typing the L. pneumpophila isolates and for investigating the origin of two cases of legionellosis in Palermo, Italy. Materials and Methods: A total of 40 strains of L. pneumophila was investigated. The strains analyzed were obtained from 2 clinical samples and 38 environmental samples (hotels, hospitals and private home); all isolates belonged to serogroup 1. SBT alleles were coded following the EWGLI convention and minimum spanning tree was made using BioNumerics 6.5. For VNTRs typing 12 VNTR loci followed by a number were used and MLVA clustering was performed by Hamming’s distance and UPGMA. In addition the Spoligotyping was performed by Luminex devices. Results: SBT analysis discriminated ten different genotypes (STs). In particular, only one clinical isolate showed the same ST of environmental strains obtained from the structure in which the subject was hospitalized. The MLVA and Spoligotyping did not confirmed the source of contamination. Moreover through the MLVA analysis four different clonal complex (VACCs) were identified. Discussion and Conclusion: Phylogenetic analysis in L. pneumophila is a difficult task considering the enormous genetic differences observed in the accessory genome of this species. However, by using VNTR polymorphism and Spoligotyping is possible to group isolates within complex which are highly congruent with SBT clustering results and to provide further insight into relationships among these groups. In this study we demonstrate that typing by the three methods provides valuable information for epidemiological studies and for identification of clonal complex in L. pneumophila. 48 Oral Communications CO 07 MECHANISM OF SITE-SPECIFIC INTEGRATION OF ICE Tn5253 IN THE CHROMOSOME OF STREPTOCOCCUS PNEUMONIAE Francesco Santoro1, Alessandra Romeo1, Marco Oggioni2, Gianni Pozzi1, Francesco Iannelli1 1LAMMB, Dip. di Biotecnologie Mediche, Università di Siena - Italy; 2Department of Genetics, University of Leicester, Leicester - United Kingdom Introduction: Integrative Conjugative Elements (ICEs) are mobile genetic elements that can integrate into bacterial genome, excise from it to form a closed circular intermediate capable of intracellular transposition in a new genomic location or intercellular transposition in a new genome host upon conjugative transfer. ICE Tn5253 of Streptococcus pneumoniae is a composite element which carries the tet(M) bearing Tn5251 and the resistance genes and the cat bearing Ωcat(pC194), conferring resistance to tetracycline and chloramphenicol, respectively. Tn5253 is found integrated at a 83- bp specific target site (attB) into the R6 pneumococcal chromosome. Materials and Methods: Bacterial strains used for conjugation are standard laboratory strains whose genome sequence is available in GenBank. For plate conjugation experiments, donor and recipient cells were grown separately and mixed at 1:10 ratio, plated and incubated for 4 h at 37°C. Scoring of transconjugants was obtained in presence of appropriate antibiotics with a multilayer plating procedure. PCR and direct PCR sequencing were carried out following standard protocols. DNA sequence alignments were performed using Clustalw2 and Lalign. Results: Tn5253