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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

Background: Methicillin resistant Staphylococcus aureus (MRSA) is a major etiological agent of infection in neonatal intensive care units (NICUs). Routes of entry of this organism can be different and the transmission pathway complex. Colonized neonates are the main endogenous reservoir. Methods and Results: We conducted a prospective three-year study on MRSA colonization recruiting 722 neonates admitted between 2009 and 2012. Nasal swabs were cultured weekly and MRSA isolates were submitted to molecular typing. The annual incidence density of acquisition of MRSA ranged from a maximum of 20.2 cases for 1000 patient-days during the first year to a minimum of 8.8 cases in the second one to raise again up to 13.1 cases during the third year. The mean weekly colonization pressure fluctuated from 19.1% in the first year to 13.4% in the second year and 16.8% in the third year. It significantly correlated with the number of MRSA acquisitions in the following week. Overall, 187 (25.9%) subjects tested positive for MRSA. A non multiresistant, tst positive, ST22-MRSA-IVa spa t223 strain proved to be endemic in the NICU, being identified in 166 (88.8%) out of 187 colonized neonates. Sporadic or epidemic occurrence of other strains was detected. Conclusions: An MRSA strain belonging to the tst1 positive, UK-EMRSA-15/ ‘‘Middle Eastern Variant’’ appeared to be endemic in the NICU under investigation. During the three-year period, substantial changes occurred in case-mix of patients moving towards a higher susceptibility to MRSA colonization. The infection control procedures were able to decrease the colonization rate from more than 40% to approximately 10%, except for an outbreak due to a CA-MRSA strain, ST1-MRSAIVa, and a transient increase in the colonization prevalence rate coincident with a period of substantial overcrowding of the ward. Active surveillance and molecular typing contributed to obtain a reliable picture of the MRSA dissemination in NICU.

Introduction: Infection with Helicobacter pylori (H. pylori) evokes inflammatory and immune responses of the host, which most likely determine the clinical outcome of H. pylori infection. In the gastric epithelium, expression of human alpha-defensins, beta-defensins (hBD)-1, -2 and -3 has been detected in vivo. In particularly hBD3 as well hBD2 is known to be induced in gastric epithelial cells 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. However, during prolonged infection, hBD3 was subsequently downregulated by the H. pylori virulence determinant CagA. Materials and Methods: Patients. In this study, serum and biopsies of 33 healthy individuals stratified according to the H. pylori infection (17 positive and 11 negative) was analyzed. Elisa Assay and Real Time PCR. Sera of patients were assayed by enzyme-linked immunosorbent (ELISA) assay for detection of both hBD- 2 (Phoenix Pharmaceuticals, Inc.) and hBD-3 (Alpha Diagnostic International) levels, according to the manufacture’s protocol. Protein concentration was expressed as pg/ml of serum volume. Real time PCR was carried out with the LC Fast Start DNA Master SYBR Green kit (LightCycler 2.0 Instrument, Roche; Milano, Italy) and PCR products were examined on 1.4% agarose gel. 192 Posters P 136 IMPACT OF HBV GENOTYPES A AND D GENETIC VARIABILITY ON INFECTION EVOLUTION Noemi Urone, Donatella Ferraro Sezione di Microbiologia “A. Chiarini”, Dipartimento di Scienze per la Promozione della Salute e Materno Infantile “G. D’Alessandro”, Università degli Studi di Palermo - Italy Introduction: HBV is characterized by a high genetic variability and several studies have demonstrated the correlation between viral variants and severe forms of acute and chronic liver disease. Few data are available about the impact of genetic variability on the evolution of acute infection. Recently, 5 SNPs (C504, C801, G1171, T1785 and A1786) into HBV genome were reported as potential novel markers linked to a lower rate of chronicity. The aim of this study was to analyze the genetic variability of HBV genotypes A and D isolates from cases of self-limiting acute (AHB) and chronic hepatitis (CHB) in order to identify HBV variants associated with chronicity or resolution of infection. Patients and Methods: The preS-S, preC-C and the overlapped P and X genes, were sequenced from sera of 33 AHB and 46 CHB Sicilian patients and analyzed with 92 HBV-A and D sequences from Gene Bank in order to identify HBV geno/subtype by phylogenetic analysis and to study the genetic variability. Results: Eighteen strains of HBV-D and 15 of HBV-A from AHB, 42 of HBV-D and 4 of HBVA2 from CHB patients were obtained. Two in frame deletions, between preS1 and preS2 regions (Δ173-321, Δ1-29) and in the S gene (Δ489-516) were detected in an AHB-D3 strain while 2 CHB-D1 strains showed the deletions Δ37-52 and Δ36-57 into preS2 region. The mutation M01I at preS2 start codon was detected in 2 AHB-D1 and a CHB-D1strains. Mutations of immune-escape were not found but >30% of strains showed many amino acid polymorphisms in “a determinant” domain. Secondary drug resistance mutations on RT-domains were identifiedGenotyping of H.pylori. The DNA of each biopsy, estracted using a High Pure Template Preparation Kit (Roche), was used to amplify following genes: ureA, cagA. Results: Protein levels of both studied defensins, hBD-2 and hBD-3, are lower or unchanged in serum of H. pylori- positive compared to those H. pylori- negative subjects. However, mRNA levels of hBD-2 and hBD-3 are up-regulated in most gastric biopsies of H. pylori-positive subjects, except in patients belonging to the genotype cagA+ vacA s1m1 EPIYA ABC. Conclusion: Although it has been demonstrated that H.pylori infection was associated with significant upregulation of hBD-2 and hBD-3 transcript levels in gastric mucosa, our data suggest that no similar association was found on protein level in serum of patients. In addition to, the upregulation of hBD-2 and hBD-3 transcript levels is absent in gastric biopsies of some H. pylori- positive subjects. These results suggest that, most likely, H. pylori strains belonging to a particular genotype not only inhibit pathogen-derived hBD-2 and hBD- 3 induction, but also block their constitutive expression to escape from the strong antimicrobial effects.

A survey was performed in May 2013 to assess methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization in healthy children attending 26 municipal daycare centres in Palermo, Italy. Of the 500 children, ten (2 %) tested positive. Eight MRSA isolates were tst1-positive ST22-MRSA-IVa, spa t223; the other two isolates were identified as ST1-IVa and ST398-V, respectively. tst1-positive ST22-MRSA, spa t223 has been previously identified only in the Middle Eastern area.

In developed countries about 10% of the hospitalizations are complicated by a healthcare-associated infection [1]. Up to 75% of these infections are due to multidrug-resistant organisms (MDROs) [1]. Antimicrobial resistant bacterial infections are associated to higher morbidity, mortality and healthcare costs than those caused by susceptible organisms [1]. The findings of the point prevalence survey in European acute care hospitals published in 2013 by the European Centre for Disease Control and Prevention (ECDC) show large variations between countries and between different regions of the same country, with Italy being allocated within the high-endemic areas for both MRSA and MDROs [2]. Despite antimicrobial resistance affects most bacterial species, MDR Gram negatives represent the most serious threat. In a few years Enterobacteriaceae, mainly Escherichia coli and Klebsiella pneumoniae, have evolved from extended spectrum β-lactamase (ESBL) producing to carbapanem-resistant organisms [3]. Simultaneously, Acinetobacter baumannii has quickly become extremely or pan-drug resistant [4]. Carbapenem resistant Gram negatives heavily impact on clinical outcomes with mortality rates significantly higher than the susceptible strains of the same species [1]. Of further concern, very few antimicrobial agents are available for an effective treatment of these infections and new agents active against these organisms are not currently in development. Many intertwining factors are driving these epidemiological changes, involving patients, healthcare delivery systems, infection control practices and, most important, misuse and inappropriate use of antibiotics in all healthcare facilities, in community and in animal husbandry. In particular, the transition of the healthcare delivery systems from a hospital-centered model to a healthcare facility network has gradually blurred the borders between hospital and community and the patients’ travel within this network has critically contributed to disseminate MDROs [5]. As a consequence, antimicrobial resistance is now as common, if not more so, in post-acute clinical facilities, such as long term care settings and nursing homes [5]. The “revolving door” is the very efficacious image used as the paradygm of the spreading routes of organisms with hospital and community reservoirs, as E. coli or MRSA. The revolving door, indeed, enlightens how the colonized patients entering back and forth several healthcare settings drive the amplification of the antibiotic resistance [6]. Stringent infection control and prevention practices and wise use of antibiotics are unanimously agreed as the key actions to fight MDROs. Of course, we need new antibiotics, but first we have to learn how to protect them from a precipitous erosion of their effectiveness.

To the Editor,Human infections caused by methicillin-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) have been emerging in recent y in Europe [1–3]. Most studies have shown a stron...

Here we describe carbapenem resistance determinants in two Klebsiella pneumoniae isolates recovered from two hospitalised patients in the same intensive care unit of a cancer hospital in Cairo, Egypt. PCR and sequencing were used to detect and characterise β-lactamase genes. Clonal relationships between the isolates were analysed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The first K. pneumoniae isolate carried the blaNDM-1 gene and the second isolate carried the blaOXA-163 gene. Both isolates co-expressed the extended-spectrum β-lactamase CTX-M-15. The two isolates belonged to different sequence types (STs), ST11 and ST16, respectively. No history of travel was established for the two patients. The first identification of NDM-1-producing K. pneumoniae in Egypt adds further evidence to the spread of NDM-1-producing Gram-negative micro-organisms in North Africa. The additional detection of blaOXA-163 in a K. pneumoniae isolate confirms its endemic presence in a critical healthcare setting of this geographic area.

Staphylococcus pseudintermedius is a coagulase-positive specie similar to Staphylococcus intermedius , frequently associated with pyoderma, otitis and urinary tract infections of dogs and cats (van Duijkeren and others 2011). No information about bovine mastitis caused by S pseudintermedius is available in the literature. Antimicrobial resistance among S pseudintermedius strains is increasing: in the past, susceptibility to most antibiotics was common (Bond and Loeffler 2012), but in the last few years methicillin-resistant S pseudintermedius (MRSP) strains have emerged as a significant animal health problem in veterinary medicine (Schwarz and others 2008, van Duijkeren and others 2008, Weese and van Duijkeren 2010). The methicillin-resistance of MRSP is mediated by the mecA gene, such as in Staphylococcus aureus (MRSA). The gene is located on a chromosomal mobile element (staphylococcal chromosomal cassette, SCC mec ), which can be horizontally transferred (Weese and others 2010), raising concern about spreading of resistance among species and hosts (Bond and Loeffler, 2012). Therefore, the aim of this study was to report the occurrence of bovine mastitis by MRSP in a dairy herd and to investigate the characteristics of the isolates collected during a control programme for contagious pathogens. The herd is a free-stall located in Lombardy region, Italy, and consists of 250 lactating cows. Due to high bulk milk somatic cell counts (SCC), the farmer decided to participate in the voluntary control programme for …

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.

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.

Introduzione. Negli ultimi anni si è assistito ad un generale incremento e ad una sempre più estesa diffusione, sia in ambito ospedaliero che comunitario, di microrganismi multiresistenti. In assenza ed in attesa di trattamenti farmacologici efficaci maggiore attenzione deve essere impiegata alla realizzazione di strategie di sorveglianza mirate. Il primo isolamento di un ceppo di Staphylococcus aureus meticillinoresistente (MRSA) risale al 1961 e fu seguito da una repentina diffusione negli ospedali di tutto il mondo. Ad oggi MRSA è riconosciuto come uno dei principali agenti responsabili delle infezioni nosocomiali e le percentuali di isolamento variano da Paese in Paese in maniera notevole. In Europa la prevalenza di MRSA aumenta essenzialmente da Nord a Sud raggiungendo valori tra il 40% e il 50% in Italia. Intorno al 1990 si registrò il primo isolamento di MRSA in comunità e da allora la diffusione e gli isolamenti in comunità sono sempre più comuni. Nell'arco di pochi anni i ceppi MRSA hanno dimostrato una notevole capacità evolutiva e adattativa che desta molte preoccupazioni. Oggi è di uso comune la distinzione tra HA-MRSA e CA-MRSA utilizzata per tentare un approccio più rapido e mirato al problema ma che nelle nostre realtà non è sempre così netta. Metodi. Lo studio che abbiamo svolto presso il Dipartimento di Scienze per la Promozione delle Salute e Materno-Infantile 'G. D'Alessandro' ha come obiettivo la valutazione dell'epidemiologia di MRSA sia in ospedale che in comunità confrontando la circolazione di MRSA all'interno dell'UTIN (Unità di Terapia Intensiva Neonatale) del Policlinico 'Paolo Giaccone' e quella negli asili nido comunali di Palermo. Abbiamo raccolto in totale circa 1000 tamponi nasali dai bambini degli asili e dai neonati ricoverati in UTIN. Tutti sono stati analizzati per la ricerca di MRSA. Risultati. La frequenza di isolamento di MRSA è circa del 10% tra i tamponi provenienti dall'UTIN contro il 2% dei tamponi prelevati negli asili. La frequenza di isolamento di ceppi MSSA è più elevata ma molto simile in entrambi i contesti e pari al 12% e il 15%. L'attività svolta in UTIN rientra in un programma di sorveglianza iniziato nel giugno 2009 e ad oggi ha dato risultati molto positivi riducendo la frequenza delle colonizzazioni da MRSA dal 27% (2009) al 10% (2013). Conclusioni. Le strutture assistenziali rappresentano un contesto particolarmente a rischio per la circolazione di microrganismi multiresistenti e come tali devono essere tenuti sempre sotto stretta osservazione. La sorveglianza attiva delle colonizzazioni consente di monitorare in maniera semplice, poco costosa e tempestiva i principali agenti responsabili di infezioni correlate all'assistenza. Il confronto con la situazione in comunità ha consentito di conoscere con maggiore accuratezza l'epidemiologia di MRSA. La sorveglianza è necessaria per realizzare interventi di prevenzione e controllo mirati ed efficaci.

We describe polyclonal spread of colistin-resistant Klebsiella pneumoniae in an acute general hospital in Italy. Between June and December 2011, 58 colistin-resistant K. pneumoniae isolates were recovered from 28 patients admitted to different wards, but mainly in the intensive care units. All isolates were tested for drug susceptibility and the presence of beta-lactamase (bla) genes. Clonality was investigated by repetitive extragenic palindromic (rep)-PCR and multilocus sequence typing (MLST). Fifty-two isolates had minimum inhibitory concentrations (MICs) for colistin of 6-128 mg/L, carried blaKPC3 and were attributed to sequence type ST258. The remaining six isolates were susceptible to carbapenems, exhibited MICs for colistin of 3-32 mg/L, and belonged to two different types, ST15 and ST273. Rep-PCR included all isolates in three clusters, one containing all ST258 KPC-3-producing isolates and two containing ST15 and ST273 isolates. Cross-transmission containment measures and intensification of staff and environmental hygiene could not stop the outbreak. Selective pressure and horizontal transmission probably contributed to emergence and spread of three different strains of colistin-resistant K. pneumoniae in the hospital. Strict implementation of the above measures and a wider awareness of the antimicrobial resistance threat are crucial to preserve the last therapeutic options of the multidrug-resistant Gram-negative infections. .

Background Multidrug-resistant Acinetobacter baumannii, initially considered as having a poor clinical relevance, is frequently isolated from infection cases in intensive care units. We describe the epidemiology of carbapenem resistant A. baumannii (CRAB) in a general ICU in Palermo, Italy, from October 2010 to March 2011. Findings 58 of 61 isolates exhibited MICs for meropenem or imipenem ≥16 mg/L. Forty-nine carried blaOXA-23 and two blaOXA-58 genes. Five subtype clusters were detected by rep-PCR. Clusters D and E included 10 isolates that tested negative for the carbapenem resistance genes. MLST attributed all isolates, but two, with sequence type (ST)2, whereas the two remaining isolates with ST78. The respiratory tract was the most common site of infection (26 out of 36 cases. 72.2%). A high infection related mortality rate was observed (18 out of 35 patients, 51.4%). Nineteen patients tested positive for other multidrug resistant organisms in addition to CRAB. In eight cases isolates belonging to distinct subtype clusters and/or with distinct carbapenemase profiles were identified. Conclusions Carbapenem resistance was prominently driven by the dissemination of CRAB isolates belonging to ST2, carrying the carbapenemase gene blaOXA-23. The colonization/infection of some patients by multiple strains is suggestive of an endemic circulation of CRAB.

Klebsiella pneumoniae is one of the most common multidrug-resistant (MDR) Gram-negative organisms worldwide, responsible for high morbidity and mortality both in hospitals and alternative healthcare settings. Recently, increasing use of carbapenems has promoted the emergence and dissemination of carbapenem-non-susceptible MDR K. pneumoniae strains. All of the CNSKP strains isolated between January 2009 and December 2011 in the General Hospital of Prato (Prato, Italy) were studied. Our findings indicate that, unlike previously reported by other authors from different regions of Italy, K. pneumoniae producing KPC-2 and belonging to ST101 have been established in the area of Prato, Tuscany, Italy. This study confirms the previously reported involvement of multiple clones of K. pneumoniae in the spread of carbapenem resistance in Italy.

Introduction Rapidly growing mycobacteria are opportunistic pathogens in patients with underlying risk factors. Mycobacterium abscessus subsp. bolletii is a newly recognized member of rapidly growing mycobacteria, isolated from respiratory tract and cutaneous infections. Case presentation We describe a case of chronic disseminated infection caused by M. abscessus subsp. bolletii in a 38-year-old Sri Lankan man with idiopathic CD4+ T lymphocytopenia. Idiopathic CD4+ T lymphocytopenia is a rare cause of immunodysfunction that, similar to human immunodeficiency virus infection, causes a depletion of CD4+ T lymphocytes. M. abscessus subsp. bolletii infection was diagnosed by culture isolation from two sputum samples. Conclusions To the best of our knowledge this is the first report of M. abscessus subsp. bolletii disseminated infection in a patient affected by idiopathic CD4+ T lymphocytopenia. In contrast to previous reports, the isolate of M. abscessus subsp. bolletii presented intermediate resistance to clarithromycin and was susceptible to cefoxitin and imipenem.

INFECTIONS CAUSED BY KLEBSIELLA PNEUMONIAE SEQUENCE TYPE 258 PRODUCING K. PNEUMONIAE CARBAPENEMASE 3 (KPC-Kp)HAVE WIDELY EMERGED AND BOTH INDIVIDUAL CASES AND OUTBREAKS OF COLONIZATION OR INFECTION HAVE BEEN REPORTED IN PALERMO, ITALY.OBIETTIVO: THIS IS A RETROSPECTIVE CASE SERIES THAT DESCRIBES THE CLINICAL AND MICROBIOLOGIC OUTCOMES OF 16 PATIENTS WHO RECEIVED A COMBINATION OF HIGH-DOSE TIGECYCLINE AND COLISTIN FOR TREATMENT OF VAP (4 CASES) AND SEVERE BACTERAEMIA (12 CASES) DURING THE YEARS 2009-2011. 11 OUT OF THE 16 CASES WERE POST SURGICAL PATIENTS WITH INTRABDOMINAL ABSCESSES DUE TO KPC-Kp. IN 5 CASES COMBINED KPC-Kp AND CARBAPENEM-RESISTANT ACINETOBACTER BAUMANNII INFECTION WAS ALSO OBSERVED.RISULTATI: CLINICAL EFFICACY WAS ASSESSED IN ALL 16 PATIENTS, WITH 14 OF THEM HAVING A POSITIVE OUTCOME, DEFINED AS EITHER CLINICAL IMPROVEMENT OR CLINICAL CURE. a CLINICAL SUCCESS APPEARED TO BE MUCH MORE FREQUENT THA THAT PREVIOUSLY REPORTED WITH COLISTIN MONOTHERAPY.CONCLUSIONE: STUDIES EVALUATING COMBINATION THERAPY AND WELL-CONTROLLED CLINICAL TRIALS ARE NEEDED TO DEFINED THE OPTIMAL TREATMENT OF INFECTIOUS CAUSED BY KPC-KpAND, MORE GENERALLLY, CARBAPENEM-RESISTANT BACTERIA.

Gram-negative pathogens producing carbapenemases represent an alarming clinical threat with serious effects on patient outcomes ([3][1], [7][2]). In 2001, Yigit et al. ([11][3]) reported a novel β-lactamase termed “ K lebsiella pneumoniae carbapenemase” (KPC-1) in North Carolina. KPC-producing

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of infections outside of health care settings. We carried out a survey to determine the prevalence and characteristics of MRSA isolates (CA-MRSA, HA-MRSA) identified among in- and outpatients by the clinical microbiology laboratories of four general hospitals in Palermo, Italy during the period February-January 2010. Methods: Participating laboratories performed isolation, confirmed methicillin-resistance by their routine method and weekly sent their strains to the coordinating laboratory at the Department of Sciences for Health Promotion “G. D’Alessandro”, University of Palermo, Italy. The isolates were test for antimicrobial susceptibility by E-test and using the disk diffusion test. Presence of the mecA gene was investigated by PCR using primers and standard conditions. Multiplex PCR was performed to determine SCCmec types I to V.9 Strains assigned to SCCmec type IVa were submitted to polymerase chain reaction (PCR) for detection of the Panton-Valentine leukocidin toxin genes lukS-PV and lukF-PV. Multiple-locus variable-number tandem-repeat analysis (MLVA) was performed. Pulsed field gel electrophoresis (PFGE) was performed as previous reported. Multilocus sequence typing (MLST) was performed on the MRSA strains following the recommended procedure at the S. aureus MLST. Results: we collected 227 isolates from 185 patients. The distribution of MRSA from different wards was: intensive-care 25%, surgery 20%, internal medicine 33%, other 15,5% unknown 6,5%. 69% of MRSA strains were resistant to ciproflox and/or levofloxacin, 40% to macrolide and 38% to gentamycin. SCCmec type IVa has been found in 42 isolates from hospitalized patients. PFGE analysis showed 81 MLVA different banding patterns. Strains of MRSA ST398 were found. Conclusions: Detect circulation of MRSA clinically relevant strains through surveillance, timely hygienic interventions and cooperation by health care personnel are crucial to minimize or control health care-associated infections. The changing epidemiology of MRSA indicates that collaborative surveillance plans integrating human and animal information should be increased.

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.

In a bacterium like Helicobacter pylori, which is characterized by a recombinant population structure, the associated presence of genes encoding virulence factors might be considered an expression of a selective advantage conferred to strains with certain genotypes and, therefore, a potentially useful tool for predicting the clinical outcome of infections. However, differences in the geographical and ethnic prevalence of the H. pylori virulence-associated genotypes can affect their clinical predictive value and need to be considered in advance. In this study we carried out such an evaluation in a group of patients living in Sicily, the largest and most populous island in the Mediterranean Sea. cagA, vacA, babA2, hopQ, oipA, sabA, and hopZ were the H. pylori virulence-associated genes assayed; their presence, expression status or allelic homologs were detected in H. pylori DNA samples and/or isolated strains, obtained by gastric biopsy from 90 Sicilian patients with chronic gastritis, inactive (n = 37), active (n = 26), or active with peptic ulcer (n = 27). Genotypes cagA (+), vacAs1, vacAm1, babA2 (+), and hopQ I, I/II were identified in 51.8, 80.4, 35.2, 47.3, and 67.7% of the different samples respectively. Only these genotypes were associated with each other and with the active form of chronic gastritis, irrespective of the presence of a peptic ulcer. In our isolates their prevalence was more similar to values observed in the north of Italy and France than to those observed in Spain or other Mediterranean countries that are closer and climatically more similar to western Sicily.

The association of Helicobacter pylori (Hp) infection with gastric cancer is well known and might be considered a paradigmatic example of the role that interaction among environmental factors and individual background might play in inducing age-associated disease. To evaluate the role of interaction of Hp infection with genetic background, gastric cancer and chronic gastritis patients as well as random selected controls were typed for five inflammation-related polymorphisms of IL-1 and IL-10 cytokine genes. No association among IL-10 or IL-1 variants with an increased risk of gastric cancer was found, whereas an Hp-independent association of IL-1beta -511T positive genotypes to an increased risk of chronic gastritis was found (Hp-/511T+ OR 1.89, 95% CI: 1.01-3.54; Hp+/-511T+ OR 1.83, 95% CI: 1.05-3.19). Stratification of gastric cancer group according to Hp infection does not allow finding a statistically significant association of Hp+ to the higher histological grading (G3) of gastric cancer (OR 1.54, 95% CI: 0.46-5.11). Our findings seem to confirm that cytokine genetic variants might contribute to determining the background for inflammaging in which H. pylori infection might facilitate cancer development.

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.