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A carbapenem resistant sequence type (ST) 512 KPC 3 producing Klebsiella pneumoniae strain showing a novel variant plasmid content was isolated in Palermo Italy in 2014. ST512 is a worldwide successful clone associated with the spread of blaKPC genes located on the IncFIIk pKpQIL plasmid. In our ST512 strain the blaKPC 3 was exceptionally located on an IncX3 plasmid whose complete sequence was determined. Two copies of the Tn44011a:blaKPC 3 transposon due to a intramolecular transposition events were detected in the plasmid.

L’epidemiologia della Tubercolosi (TB) in Sicilia è in rapida evoluzione con un aumento dei casi nella popolazione di nazionalità non italiana. La tipizzazione molecolare dei ceppi di M. tuberculosis complex (MTBC) costituisce un insostituibile supporto alla conoscenza dei ceppi circolanti e delle correlazioni tra i casi di TB. MATERIALI E METODI: Sono stati presi in considerazione i flussi di dati correnti, in particolare i dati di notifica delle malattie infettive del Sistema Informativo delle Malattie Infettive (SIMI) regionale incrociando questi con i dati provenienti dalle Schede di Dimissione Ospedaliera (SDO) attraverso il metodo del capture - recapture. Grazie alle attività del Laboratorio di Riferimento Regionale per la Sorveglianza e il Controllo della Tubercolosi è stata creata una ceppoteca regionale per lo studio dell’antibiotico-resistenza e per la tipizzazione mediante metodiche MIRU/VNTR a 24 loci e Spoligotyping di tutti i ceppi di Mycobacterium tuberculosis complex ricevuti dalle strutture ospedaliere regionali nel biennio 2014 - 2015, per avere contezza della circolazione dei ceppi di MTBC e per fornire un supporto all’epidemiologia clinica basata sul contact-tracing in modo da avere a disposizione dati epidemiologici più approfonditi, oggettivi e confrontabili. RISULTATI: Attraverso il linkage tra notifica e primo ricovero riportato sulle SDO di ciascun paziente si è riusciti a valutare per il 2016 la percentuale di notifica a livello regionale dei casi ricoverati per TBC, attestandosi intorno al 65%. Sono stati collezionati e sottoposti a tipizzazione molecolare 307 ceppi di MTBC da 307 pazienti, mostrando una grande eterogeneità dei principali lineage di MTBC rappresentati e identificando 28 clusters. 8 ceppi erano multi-resistenti e provenivano da pazienti di 5 diverse nazionalità. Sono stati inoltre rivalutati alcuni focolai epidemici attraverso i dati molecolari dei ceppi isolati. CONCLUSIONI: Una sorveglianza continua e sistematica della TB in Sicilia con l’ausilio di metodologie molecolari è condizione essenziale ai fini di una valutazione accurata del mutevole scenario epidemiologico e dell’efficacia delle misure di prevenzione e controllo.

L’incidenza delle infezioni micotiche ha subito negli ultimi decenni un incremento rilevante, sia in ambito ospedaliero che comunitario. In ambiente ospedaliero il problema sta assumendo dimensioni preoccupanti poiché, sebbene Candida spp. si debba considerare il principale responsabile di infezioni correlate ai miceti, emergono altre specie il cui ruolo deve essere accertato. In particolare Dipodascus capitatus, forma teleomorfica di Geotrichum capitatum, microrganismo che può risiedere nel suolo, sulla pelle e nel tratto respiratorio e gastroenterico, nel passato considerato un contaminate o un innocuo colonizzatore, da qualche anno è stato associato ad infezioni soprattutto nei pazienti immunocompromessi. Sebbene le infezioni da tale microrganismo siano spesso favorite dall’uso di antibiotici e di cateteri, le forme invasive sembrano verificarsi in pazienti neutropenici e nei soggetti sottoposti a chemioterapia intensiva per leucemia acuta. Dai reparti ad alto rischio sono stati anche rilevati episodi epidemici D. capitatus-correlati. Il suo riscontro non deve quindi essere sottovalutato ed è necessario poter disporre di test semplici che ne permettano una rapida identificazione. Tra i metodi fenotipici quello che si basa sulla valutazione della crescita in terreno cromogeno è considerato ottimale, soprattutto se si utilizza il CCA (Chromogenic Candida Agar, Oxoid); ed è stato già descritto quale metodo molecolare il sequenziamento. Anche dai reparti dell’A.O.U.P. “P. Giaccone” di Palermo, nel corso dell’ultimo anno, sono stati isolati stipiti di D. capitatus. I ceppi, provenienti da tre pazienti, da campioni diversi e da differenti reparti, sono stati identificati fenotipicamente, utilizzando il CHROMagar Candida (BD), e la loro identificazione è stata confermata tramite sequenziamento. Inoltre, al fine di valutare l’eventuale diffusione epidemica di questi isolati, sono stati utilizzati la PCR fingerprinting che prevede l’utilizzo della sequenza core del fago M13 e la RAPD (Random Amplification of Polymorphic DNA) utilizzando il primer Ope-4 e la combinazione dei primer W80A-Ap12h.

Objectives. We performed a one-year prospective surveillance study on MRSA colonization within the five NICUs of the metropolitan area of Palermo, Italy. The purpose of the study was to assess epidemiology of MRSA in NICU from a network perspective. Methods. Transfer of patients between NICUs during 2014 was traced based on the annual hospital discharge records. In the period February 2014 – January 2015, in the NICU B, at the University teaching hospital, nasal swabs from all infants were collected weekly, whereas in the other four NICUs (A, C, D, E) at four week-intervals of time. MRSA isolates were submitted to antibiotic susceptibility testing, SCCmec typing, PCR to detect lukS-PV and lukF-PV (lukS/F-PV) genes and the gene encoding the toxic shock syndrome toxin (TSST-1), multilocus variable number tandem repeat fingerprinting (MLVF) and multilocus sequence typing (MLST). Results. In the period under study, 587 nasal swabs were obtained from NICU B, whereas 218, 180, 157 and 95 from NICUs A, C, D and E, respectively. Two groups of NICUs at high prevalence and low prevalence of MRSA colonization were recognized. Overall, 113 isolates of MRSA were identified from 102 infants. Six MLVF types (A-F) were detected, with type C being subdivided into five subtypes. Five sequence types (STs) were found with ST22-IVa being the most frequent type in all NICUs. All the MRSA molecular subtypes, except for ST1-IVa, were identified in NICU B. Conclusions. Our findings support the need to approach surveillance and infection control in NICU in a network perspective, prioritizing referral healthcare facilities.

Colonization and infection by multidrug-resistant gram-negative bacilli (MDR GNB) in neonatal intensive care units (NICUs) are increasingly reported. We conducted a 5-year prospective cohort surveillance study in a tertiary NICU of the hospital “Paolo Giaccone,” Palermo, Italy. Our objectives were to describe incidence and trends of MDR GNB colonization and the characteristics of the most prevalent organisms and to identify the risk factors for colonization. Demographic, clinical, and microbiological data were prospectively collected. Active surveillance cultures (ASCs) were obtained weekly. Clusters of colonization by extended spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae were analyzed by conventional and molecular epidemiological tools. During the study period, 1152 infants were enrolled in the study. Prevalences of colonization by MDR GNB, ESBL-producing GNB and multiple species/genera averaged, respectively, 28.8%, 11.7%, and 3.7%. Prevalence and incidence density of colonization by MDR GNB and ESBL-producing GNB showed an upward trend through the surveillance period. Rates of ESBL-producing E coli and K pneumoniae colonization showed wide fluctuations peaking over the last 2 years. The only independent variables associated with colonization by MDR GNB and ESBL-producing organisms and multiple colonization were, respectively, the days of NICU stay (odds ratio [OR] 1.041), the days of exposure to ampicillin–sulbactam (OR 1.040), and the days of formula feeding (OR 1.031). Most clusters of E coli and K pneumoniae colonization were associated with different lineages. Ten out of 12 clusters had an outborn infant as their index case. Our study confirms that MDR GNB are an increasing challenge to NICUs. The universal once-a-week approach allowed us to understand the epidemiology of MDR GNB, to timely detect new clones and institute contact precautions, and to assess risk factors. Collection of these data can be an important tool to optimize antimicrobials use and control the emergence and dissemination of resistances in NICU.

Objectives Surveillance cultures are of great importance to detect the emergence of multidrug resistant (MDR) bacteria in high-risk settings and prevent the occurrence of colonization and clinical infection cases. We report the isolation of OXA-48-harbouring Klebsiella pneumoniae sequence type (ST) 530 in a Neonatal Intensive Care Unit (NICU) in Palermo, Italy. Methods Since June 2009, a routine surveillance protocol is in place in the NICU of the University Hospital of Palermo, including nasal and rectal swabs obtained on a weekly basis to monitor the prevalence of colonization by methicillin- resistant Staphylococcus aureus, MDR Gram-negatives and glycopeptide-resistant enterococci. Enteric colonization by antibiotic-resistant Gram negatives is assessed by culturing overnight broth-enriched rectal swabs onto MacConkey agar plates with disks of meropenem (10 mg) and ceftazidime (30 mg). Colonies growing within antibiotic inhibition zones are subcultured, biochemically identified and submitted to antimicrobial susceptibility testing by disk diffusion method and E-test and to ESBL detection according with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Beta-lactamase genes are identified by polymerase chain reaction (PCR) amplification and sequencing. Multilocus sequence typing (MLST) is performed and STs are determined using the K. pneumoniae MLST database (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). Results In September 2013 an isolate of OXA-48 carbapenemase producing K. pneumoniae was identified from a newborn admitted from another hospital. Antimicrobial susceptibility testing showed susceptibility to 3rd and 4th generation cephalosporins and to aminoglycosides, intermediate suceptibility to imipenem and meropenem and resistance to ertapenem. PCR amplification tested positive for blaOXA-48 gene, and negative for other beta-lactamase genes. The isolate was identified as K. pneumoniae ST530 by MLST. The patient stayed in the NICU for five weeks, and OXA-48 K. pneumoniae colonization was confirmed at every weekly screening. During the last two weeks a second newborn tested positive for K. pneumoniae with a similar antimicrobial resistance pattern, which was confirmed to belong to ST530. Infection control prevention measures were strenghtened. The two colonized patients were discharged in mid-October and no further cases were detected. Conclusions Carbapenem-resistant Enterobacteriaceae are an emerging threat in Europe, particularly in the Mediterranean basin. The frequent horizontal transmission of plasmid-related resistance genes among different strains and species is allowing the epidemic diffusion of such MDR organisms in Europe in the last years. The remarkable potential for causing invasive infections and the limited therapeutic options associated with high mortality rates are of great concern. The implementation of surveillance programmes and infection control measures particularly in high-risk settings should be considered an essential strategy for public health interventions.

Background: Extraintestinal pathogenic Escherichia coli (ExPEC) bacteria have the ability to cause diverse and serious diseases, such as urinary tract infections (UTIs) and bacteremia; incidence of bacteremia is increasing globally. The emergence of multidrug resistance in E. coli is also becoming a global concern, with particular emphasis on E. coli sequence type (ST) 131, which is being increasingly reported in UTIs. Drug resistance is mediated by extended- spectrum β-lactamases (ESBLs), mainly of the CTX-M family, particularly CTX-M-15, and less frequently of the SHV and OXA families. Few studies are available regarding the characterization of E. coli strains causing bacteremia. Methods: We characterized 52 selected fluoroquinolones and cephalosporins-resistant E. coli isolates from bacteremia patients treated at Policlinico University Hospital (Palermo, Italy) over a 6-years period, with the aim of developing an epidemiologic profile of the population of ExPEC that causes bacteremia. Multiplex PCR to determine the phylogenetic group, multiplex ERIC PCR to analyze the epidemiological correlations, multiplex PCR detection of ESBL genes, multilocus sequence typing using the Achtman scheme (http://mlst.ucc.ie/ mlst/dbs/Ecoli), and virulence-associated gene (VAG) carriage screening by multiplex PCR were performed on isolates. Results: The 86.5% of our strains belonged to phylogenetic group B2, while the phylogenetic group D appeared to be under-represented (13.5%) and no strain belonged to group A nor B1. Clustering analysis by ERIC-PCR showed a high heterogeneity of the strains that did not allow a significant grouping and PCR screening for ESBL carriage showed significantly higher carriage for CTX-M 15. Multilocus sequence types were determined in all our bacteremia E. coli isolates: ST131 dominated and was significantly higher in prevalence (75%) followed by ST167, 58, 38, 744, 405, 453, 707 and 1447. VAGs screening of all isolates showed that the most represented were kpsM II and iutA coding for the capsule and an aerobactin (siderophore) receptor, respectively. Conclusions: Overall, our results show that ST131 is the predominant E. coli lineage among the 52 extraintestinal pathogenic E. coli (ExPEC) isolates from bacteremia patients included in this study. Our group B2 ExPEC ST131 isolates produce extended-spectrum b-lactamases, especially CTX-M-15, and almost all are resistant to fluoroquinolones. 42ND

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 was transferred by conjugation to pneumococcal strains with different genetic backgrounds and to bacterial species such as Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes, and Enterococcus faecalis, with conjugation frequencies ranging from