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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.

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

In recent years, anti-microbial peptides have emerged as a critical component of host innate defense. These peptides are gene-encoded natural antibiotics expressed by immune and non-immune cell types including epithelia. Human beta defensin are disulphide linked, low molecular weight cationic peptides that are known to be a major components of innate immune defense mechanisms at mucosal surfaces. Their expression level differs between those that are constitutively expressed and those that are induced upon challenge with inflammatory or pathogen-derived stimuli. To date six members of the human-β-defensins (hBD1-6) family have been identified. Helicobacter pylori (H. pylori), a pathogenic but noninvasive Gram-negative bacterium, appears to be unique in its adaptation to long-term survival in the acidic environment of human stomach. hBD3 as well hBD2 is known to be induced in gastric epithelial cells in infection by the H. pylori and may be involved in the pathogenesis of H. pylori-associated gastritis, possibly through its function as immune and inflammatory mediator. In our study we reported the analysis of HBD-2 and 3 in a sample sera of 45 patients by ELISA, the patients were divided as follows: 12 with an active infection, 11 uninfected, 11 with chronic infection and 11 that have eradicated the infection.

Cephalosporin-resistant Escherichia coli has been increasingly reported worldwide. In this study, 32 cephalosporin resistant E. coli isolates identified from cancer patients in Cairo, Egypt in 2009-2010 were analyzed. Twenty-three were of phylogenetic group D, seven A and one each B1 and B2. By rep-PCR 15 phylogroup D isolates were grouped in four clusters, one with sequence type (ST) 405 and three ST68. Seventeen isolates showed single patterns. blaCTX-M-15 and aac(6')-Ib-cr were the most common resistance determinants. blaOXA-48 and blaVIM were also detected. Multidrug resistant E. coli seriously affects healthcare, especially in immunocompromised hosts, such as cancer patients.

CagA is a major virulence factor of Helicobacter pylori that, once injected into the epithelial cells and phosphorylated on specific bacterial tyrosine residues within repeating EPIYA-A,-B,-C, and -D motifs, localizes to the plasma membrane and interacts with a number of intracellular effectors suggested to play an important role in Helicobacter pylori pathogenesis. EPIYA-D (in East Asian CagA) and EPIYA-C motifs (in Western CagA) are the main sites of CagA phosphorylation and the presence both of EPIYA-D or an increasing number of EPIYA-C motifs, rather than the general CagA positivity, has been associated with more severe gastroduodenal disease. With the aim to analyze EPIYA motifs in 24 cagA+ H.pylori isolates and in a number of recently collected, frozen, or, in particular, deparaffinized biopsies and clinical sample, all obtained from 62 patients with different H.pylori pathology, we comparatively evaluated EPIYA profiles by polymerase chain reaction amplification using single sets of primers flanking the variable EPIYA coding region, or a single forward primer and multiple reverse primers specific for the individual EPIYA motifs. Only the primers originally employed by Rudi et al. (1988)to amplify the variable 3'' region of the cagA gene allowed identification of EPIYA motifs in all biopsies and clinical samples. Multiple infections and EPIYA profiles with more than one EPIYA-C motif, in some cases confirmed by DNA sequencing, were observed in 12 and 22 patients, respectively. As expected, the increasing numbers of EPIYA-C motifs were associated with more severe gastric pathologies.

Background: In Italy, Klebsiella pneumoniae carbapenemase producing K. pneumoniae (KPC-Kp) strains are highly endemic and KPC producing CC258 is reported as the widely predominating clone. In Palermo, Italy, previous reports have confirmed this pattern. However, recent preliminary findings suggest that an epidemiological change is likely ongoing towards a polyclonal KPC-Kp spread. Here we present the results of molecular typing of 94 carbapenem non susceptible K. pneumoniae isolates detected during 2014 in the three different hospitals in Palermo, Italy. Methods and Results: Ninety-four consecutive, non replicate carbapenem non susceptible isolates were identified in the three largest acute general hospitals in Palermo, Italy, in the six-month period March-August 2014. They were characterized by PCR for β-lactam, aminoglycoside and plasmid mediated fluoroquinolone resistance genetic determinants. The mgrB gene of the colistin resistant isolates was amplified and sequenced. Clonality was assessed by pulsed field gel electrophoresis and multilocus sequence typing. Eight non-CC258 sequence types (STs) were identified accounting for 60% of isolates. In particular, ST307 and ST273 accounted for 29% and 18% of isolates. CC258 isolates were more frequently susceptible to gentamicin and non-CC258 isolates to amikacin. Colistin non susceptibility was found in 42% of isolates. Modifications of mgrB were found in 32 isolates. Conclusions: Concurrent clonal expansion of some STs and lateral transmission of genetic resistance determinants are likely producing a thorough change of the KPC-Kp epidemiology in Palermo, Italy. In our setting mgrB inactivation proved to substantially contribute to colistin resistance. Our findings suggest the need to continuously monitor the KPC-Kp epidemiology and to assess by a nationwide survey the possible shifting towards a polyclonal epidemic.

Acalculous cholecystitis is a syndrome of gallbladder inflammation without gallstones, recognized within the setting of critically ill patients. Acalculous cholecystitis associated with infectious agents is reported in the literature to be rare. Herein we describe a case of acalculous cholecystitis in a patient with malaria caused by Plasmodium falciparum and apparent cytomegalovirus infection, and discuss the possible role of CMV in the pathogenesis of acalculous cholecystitis in patients with malaria.

Mycetoma (or "madura foot") is characterized by deformation, cutaneous lesions, infection of tissues extending from the cutaneous layer to the underlying fascia, and an indolent course. A number of fungal or bacterial agents that are introduced through traumatic inoculation can be responsible for the disease, but Actinomadura madurae is among the most common agents of mycetoma occurring worldwide. We report a case of madura foot caused by A. madurae in an immunocompetent young Somali man who was admitted with a diagnosis of skin and soft tissue infection of the left foot with osteomyelitis. The present report emphasizes the importance of the knowledge of this infection, which is sporadic but problematic to treat and, above all, difficult to diagnose. Moreover, a multidisciplinary approach with involvement of an infectious diseases specialist with experience in tropical diseases and a microbiology unit performing rapid molecular diagnostic tests is required for early diagnosis and an optimal antibiotic therapy.

Helicobacter pylori