pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401b Inserted into an Xer Site-Specific Recombination Locus

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported

Deferral of assessment of pulmonary embolism

We evaluated a simplified algorithm for safely postponing diagnostic imaging for pulmonary embolism (PE). At the index visit, patients were identified as being at high or low risk of PE; the former received full dosage low molecular weight heparin while the latter were left untreated until performance of diagnostic imaging (max 72 hours). During this period, no thromboembolic events occurred in low-risk patients (0/211, 0.% [upper 95% CI 0.9%]); only one event occurred in those at high-risk (1/125, 0.8% [upper 95% CI, 1.2]). Our study demonstrates that diagnostic imaging for PE can be safely deferred for up to 3 days

Aspergillus fumigatus biofilm formation on different bone substitutes used in maxillary sinus augmentation: an in vitro analysis

BACKGROUND: Fungus ball (FB) typically affects healthy adults, and Aspergillus fumigatus is the most frequent etiologic agent: iatrogenic factors represent an important issue in FB pathogenesis. Moreover, a recent study suggested a significant association between the use of anorganic bovine bone as sinus grafting material and subsequent development of FB. The aim of the present investigation is to evaluate in vitro eventual differences in the ability of Aspergillus fumigatus to colonize different bone grafting materials and grow on them as biofilm. FINDINGS: Five different bone substitutes (demineralized bone matrix, anorganic bovine bone, f-tricalcium phosphate, synthetic nano-hydroxyapatite, and synthetic hydroxyapatite), commonly used in sinus floor augmentation procedures, were inoculated with conidia suspensions of A. fumigatus and incubated at 37\u2009\ub0C for 4 and 8\u2009h, in standardized conditions. Biofilm bound to the different materials underwent quantitative and qualitative analysis by confocal and scanning electron microscopy. A. fumigatus proved to be able to adhere and form biofilm on all the tested bone substitutes. The surface plot representation of the samples displayed some differences in the density of the superficial layer, due to the physical characteristics of the biomaterials. Nevertheless, Kruskal-Wallis test showed no significant differences in biomass amount among the five bone substitutes (p\u2009=\u20090.236 and p\u2009=\u20090.55 after 4 and 8\u2009h adhesion, respectively). CONCLUSIONS: All the bone substitutes normally used in sinus floor augmentation represent a favorable substrate for fungal growth, due to their physical and chemical characteristics. During sinus floor elevation procedures, Schneiderian membrane integrity should be maintained in order to avoid the exposure of the grafting material at the respiratory environment, with potential risks of fungal colonization

Interplay of OpdP Porin and Chromosomal Carbapenemases in the Determination of Carbapenem Resistance/Susceptibility in Pseudomonas aeruginosa

8noCarbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.openopenAtrissi, Jad; Milan, Annalisa; Bressan, Raffaela; Lucafò, Marianna; Petix, Vincenzo; Busetti, Marina; Dolzani, Lucilla; Lagatolla, CristinaAtrissi, Jad; Milan, Annalisa; Bressan, Raffaela; Lucafò, Marianna; Petix, Vincenzo; Busetti, Marina; Dolzani, Lucilla; Lagatolla, Cristin

Fitness cost associated with the chromosomal integron In70.2 in Pseudomonas aeruginosa clinical isolates

An epidemiologic survey performed at the Trieste University Hospital (northeastern Italy) between 1999 and 2002 revealed a remarkable spread of an MDR Pseudomonas aeruginosa strain, named TS-832035, which carried the chromosomal integron In70.2 containing four gene cassettes (blaVIM-1, aacA4, aphA15 and aadA1) in its variable region and conferring resistance to ß-lactams, including carbapenems, and to several aminoglycosides. Moreover, some other P. aeruginosa isolates, strictly related to TS-832035 but lacking in the integron In70.2, were detected, but they remained a minor component within the cluster during the three years of surveillance.They showed an MDR phenotype like TS- 832035, differing only for the susceptibility level to carbapenems. The genomic relatedness between TS-832035 and TS-103 was investigated by random amplification of polymorphic DNA (RAPD) typing, pulsed-field gel electrophoresis (PFGE) analysis of SpeI-digested genomic DNA, and multilocus sequence typing (MLST). The cost of the integron In70.2 on the fitness of TS-832035 was determined by performing growth kinetics and direct competition assays against the clonal isolate TS-103 in three media differing for nutrient availability: a rich medium (Luria Bertani (LB) Broth) and a minimal medium (28 g/l K2HPO4, 12 g/l KH2PO4, 0.4 g/l MgSO4, 7H2O, 4 g/l (NH4)2SO4) added with a rich carbon source (0.4% w/v glucose) or with a poorer carbon source (0.4% w/v sodium acetate). Growth kinetic data were obtained by measuring optical density at 600 nm (OD600). For competition assays, the number of CFU/ml of each isolate was estimated by colony-hybridization. We proved the clonality of the two isolates by molecular investigations.The results of the growth kinetics showed the existence of a significant in vitro fitness cost associated with the integron In70.2, more evident in a poorer medium.The sensitivity of the two isolates to the antimicrobial agents tested was the same, except for the different levels of resistance to carbapenems (MIC 16 μg/ml versus 64-128 μg/ml). Although we can not exclude that other factors may have favoured the in vivo spread of TS-832035, our results suggest that the increased level of resistance to carbapenems has conferred on this isolate a selective advantage able to compensate metabolic cost associated to the integron

Isolamento di Klebsiella pneumoniae resistente ai carbapenemi e Klebsiella pneumoniae sensibile da un campione urinario.

Introduzione. La diffusione di enterobatteri resistenti ai carbapenemi (CRE) \ue8 in preoccupante aumento in ospedali e lungodegenze, dove la selezione operata dalle terapie crea condizioni ideali per la loro moltiplicazione e trasmissione. Nel 2009 \ue8 comparso all\u2019Ospedale di Cattinara di Trieste un cluster di K.pneumoniae con resistenza ai carbapenemi dovuta a modifica della permeabilit\ue0 di membrana. Mancavano finora all\u2019appello ceppi produttori di KPC-carbapenemasi gi\ue0 diffusi in tutta Italia. Scopo dello studio \ue8 caratterizzare il meccanismo di resistenza in un ceppo di K.pneumoniae isolato di recente e valutare strategie per facilitare l\u2019isolamento dei ceppi resistenti in campioni con flora polimicrobica. Metodi. In luglio 2013 \ue8 stato isolato da un campione di urine un ceppo di K.pneumoniae CRE associato a K.pneumoniae sensibile. L\u2019isolato resistente \ue8 stato confrontato con i ceppi CRE del 2009 conservati a -40\ub0C. Identificazione e antibiogramma sono stati eseguiti utilizzando il sistema Vitek2; per conferma della produzione di carbapenemasi sono stati usati test di sinergia: meropenem/meropenem+EDTA; test KPC/MBL/OXA-48 (ROSCO diagnostica); test di Hodge modificato. I ceppi sono stati testati con PCR specifica per il gene blaKPC. La tipizzazione dei ceppi \ue8 stata effettuata mediante macrorestrizione con XbaI. Risultati. L\u2019isolamento del ceppo CRE dal ceppo sensibile \ue8 stato possibile dopo passaggi in piastra con utilizzo di dischetti di meropenem. L\u2019antibiogramma \ue8 sovrapponibile a quello dei ceppi CRE del 2009. I test fenotipici rilevano produzione di \u3b2-lattamasi tipo KPC, confermata da PCR. I profili PFGE del ceppo CRE e di quello sensibile differiscono per tre bande. Il lignaggio dei due isolati verr\ue0 ulteriormente indagato mediante MLST. Conclusioni. Campioni con flora polimicrobica rendono difficile l\u2019isolamento di eventuali microrganismi MDR; una semplice strategia che utilizzi dischetti di meropenem in terreni selettivi per Gram negativi, risolve la differenziazione fra batteri CRE e ceppi fenotipicamente non distinguibili ma sensibili. I test di conferma con dischetti sono di facile uso e in grado di identificare la produzione di carbapenemasi tipo MBL o KPC, ma richiedono tempi di risposta maggiori rispetto al test molecolare

Relationship between antibiotic susceptibility and biofilm production in a clinical strain of Burkholderia cepacia complex

Infections caused by Burkholderia cepacia complex in patients with cystic fibrosis (CF) are often related to increased mortality. One of the mechanisms that makes the bacteria more resistant to the action of antibiotics is the ability to produce biofilm. It has been compared the antibiotic resistance of two clinical isolates of a biofilm-producing strain called BTS-2, responsible for chronic pulmonary infection in a CF patient. Bacteria were incubated in both Yeast Extract Mannitol Medium (YEM) and Mueller Hinton Broth (MHB) at 30°C for 24 hours. Biofilm was quantified by spectrophotometer readings (OD570) of the extracted crystal violet. By microdilution broth, we evaluated the minimum concentration of antibiotic inhibiting the growth of the planktonic form (MIC); by the technology “Calgary Biofilm Device” and by Resazurin, we evaluated the susceptibility of sessile forms (BIC). Data demonstrate that the microorganism in the course of time changed its sensitivity to some of the antibiotics tested. The comparison between sessile and planktonic forms of BTS2 pre-incubated in MHB showed that sessile forms increased their resistance to antibiotics in a few cases only. BICs of BTS2 pre-incubated in YEM, where the strain produced an amount of biofilm significantly lower than in MHB, showed a higher susceptibility to 4 of the 10 antibiotics tested. Our data showed that the susceptibility of sessile forms of a strain is not always lower than the susceptibility of its planktonic forms and that culture medium can affect significantly the biofilm production

Standardization and Interlaboratory Reproducibility Assessment of Pulsed-Field Gel Electrophoresis-Generated Fingerprints of Acinetobacter baumannii

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains

Developing Policies and Actions in Response to Missed Nursing Care: A Consensus Process

16siAIM: To support the development of appropriate policies and actions in the field of Missed Nursing Care (MNC). BACKGROUND: There has been an ever-growing international debate on MNC, interventions that nurses have identified as necessary for their patients, but which for various reasons they are unable to provide or are forced to delay. Despite MNC's relevance, its translation into policies and actions has not been documented to date. METHOD: A Consensus Development Method was employed involving (1) a Nominal Group composed of experts in the field, policymakers, and the President of the Regional Nursing Professional Boards, and (2) 218 nurses appointed primarily at the managerial levels. RESULTS: A total of eight Consensus Statements were approved and organised in a series of sub-statements designed to: (1) Render the concept of MNC culturally acceptable in the Italian context, with the agreement that Compromised Nursing Care (CNC) is the best term to be used in this field, as a synonym for MNC; (2) Measure CNC as a strategy to increase patient safety; (3) Select an appropriate CNC measurement tool; (4) Optimise CNC measurement; (5) Conduct effective CNC data analysis; (6) Design and implement interventions to prevent and/or minimise CNC; (7) Assess and disseminate findings on interventions' effectiveness, and (8) provide final remarks on the way to move forward. CONCLUSIONS: We developed a process to introduce the phenomenon of MNC in the Italian culture and agreed firstly on the term Compromised Nursing Care, which better reflects MNC's meaning according to the context, and facilitates an open discussion on the phenomenon both within and outside the profession. The following Consensus Statements emerged represent a systematic approach, starting from the measurement and finishing with the re-measurement of the occurrence of MNC after having implemented concrete actions. IMPLICATIONS FOR NURSING MANAGEMENT: The approved Consensus Statements can guide decision-makers to develop concrete policies and actions that promote the improvement of quality of care and patients' safety by minimising and/or preventing MNC's occurrence.partially_openembargoed_20200726Palese, Alvisa; Bassi, Erika; Tommasini, Cristina; Vesca, Roberta; Di Falco, Achille; De Lucia, Paola; Mulloni, Giovanna; Paoletti, Flavio; Rissolo, Raffaela; Sist, Luisa; Sanson, Gianfranco; Guardini, Ilario; Bressan, Valentina; Mesaglio, Maura; Papastavrou, Evridiki; Blackman, IanPalese, Alvisa; Bassi, Erika; Tommasini, Cristina; Vesca, Roberta; Di Falco, Achille; De Lucia, Paola; Mulloni, Giovanna; Paoletti, Flavio; Rissolo, Raffaela; Sist, Luisa; Sanson, Gianfranco; Guardini, Ilario; Bressan, Valentina; Mesaglio, Maura; Papastavrou, Evridiki; Blackman, Ia

Epidemic Dissemination of a Carbapenem-Resistant Acinetobacter baumannii Clone Carrying armA Two Years After Its First Isolation in an Italian Hospital

This study describes the dissemination of a carbapenem-resistant Acinetobacter baumannii (CRAB) strain in a university hospital in Northeast Italy. Characterization of the outbreak strain was combined with a retrospective analysis of all CRAB isolates collected in the same hospital during the 5 years preceding the outbreak, with the aim of elucidating the origin of the epidemic spread. The outbreak strain was shown to belong to the International Clone II and carry the blaOXA-23 gene, flanked by two ISAba1 sequences in opposite orientation (Tn2006 arrangement). The epidemic clone harbored also the blaOXA-66 allele of the carbapenemase intrinsic to A. baumannii, the determinant of ArmA 16S rRNA methylase and a class 1 integron, with the aacA4, catB8, and aadA1 cassette array. Genotype analysis, performed by macrorestriction analysis and VRBA, revealed that isolates related to outbreak strain had been sporadically collected from inpatients in the 2 years preceding outbreak start. Carriage of blaOXA-66, armA, and the integron further supported relatedness of these isolates to the outbreak clone. Outbreak initially involved three medical wards, typically hosting elderly patients with a history of prolonged hospitalization. The study highlights the need to adopt strict infection control measures also when CRAB isolation appears to be a sporadic event