Genetic analysis of exopolysaccharide acetilation product by Burkholderia cepacia

Bacteria belonging to the Burkholderia cepacia complex are mainly isolated from the sputum of cystic fibrosis patients and frequently show a mucoid phenotype.Most of them produce an exopolysaccharide called cepacian, whose repeating unit consists of a branched heptasaccharide carrying from one to three acetyl esters. Two genetic loci, bceI and bceII, consisting of 11 and 9 genes respectively, are involved in cepacian biosynthesis.Three genes located in the bceII locus, named bceOSU, code for different acyltransferases. As the presence of acetyl groups influences the viscosity of cepacian, we compared three strains (two clinical isolates named BTS2 and BTS7, and the reference strain Burkholderia sp. 383) exhibiting differences both in the acetylation pattern and at the genomic level, for the presence of insertion sequences adjacent to bceU

Colonization of the tip of a thoracic catheter by Enterococcus faecalis resistant to vancomycin and linezolid

We report the isolation ofEnterococcus faecalisresistant to vancomycin and linezolid from the tip of a thoracic drainage catheter in an elderly patient. He was treated with vancomycin for a pleural empyema due to a meticillin-resistantStaphylococcus aureusbut never received linezolid. A surveillance rectal swab yielded both linezolid-susceptible and -resistant strains, and the two isolates were not genotypically related. Careful monitoring for linezolid-resistance is critical to avoid potential therapy failure and transmission of resistantE. faecalis

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

L’ambito biologico. Il PAS A057 - Scienza degli alimenti

L’organizzazione del Piano dell’Offerta Formativa per il PAS A057 è stata influenzata sia dalla varietà di titoli di accesso che dalla diversità dei programmi oggetto d’insegnamento relativi a questa classe. Nei 18 crediti previsti per la Didattica disciplinare sono stati inclusi corsi di Chimica, Tecnologia, Qualità e Sicurezza, Microbiologia e Tracciabilità genetica degli Alimenti. Inoltre, sono stati inseriti un corso di Didattica delle Scienze e alcuni Laboratori, basati su argomenti disciplinari, con esempi di progettazione di percorsi didattici e simulazioni di lezioni. I laboratori, che ponevano anche l’accento su Bisogni Educativi Speciali e Tecnologie dell’Informazione e della Comunicazione, erano affidati a docenti di scuola secondaria di secondo grado, in grado di trasmettere esperienza d’insegnamento e conoscenza del mondo della scuola.The study plan of this course has been developed with the aim to differentiate features of the A057 candidates, i.e., the diversity of their backgrounds and the variety of subjects that they can teach with their teaching qualifications. The course workload consisted of 18 university credits, which included topics on Food Chemistry, Food Technology, Food Quality and Safety, Food Microbiology and Food Genetics. There was also an introduction to Science Teaching and some workshops were organized. Secondary school teachers led the workshops in order to have the possibility to transfer their professional experience and their knowledge of the school environment. During these workshops, students became familiar with lesson planning and performed lesson simulations on Food Chemistry and Food Technology, with specific regards to Special Educational Needs and to Information and Communication Technology

Semplici esperienze pratiche per introdurre lo studio della Microbiologia

Due semplici esperienze di laboratorio consentono agli studenti di scoprire il microscopico mondo invisibile che li circonda. La prima esperienza consiste nel coltivare i batteri normalmente presenti sulle superfici corporee e su piccoli oggetti, mentre la seconda punta a valutare il numero di batteri contenuti in una porzione di bevanda probiotica. Le esperienze sono utili per introdurre lo studio della microbiologia e per facilitare la comprensione di temi importanti, quali la moltiplicazione mediante fissione binaria, l’uso dei terreni di coltura, la quantificazione dei batteri di un campione. La scelta di coltivare microrganismi da elementi che sono parte dell’esperienza quotidiana stimola l’interesse degli studenti ed elimina la necessità di conservare ceppi batterici in laboratorio.Two simple laboratory experiences allow students to discover the unseen microbial world in everyday life. The aim of the first experience is to culture bacteria from body surfaces and small objects, while the second experience consists of estemating the number of bacteria within a probiotic drink serving. The two experiences may be used to introduce the study of microbiology and to support the understanding of important issues, such as bacterial growth (binary fission), culture media and the measurement of bacterial populations. Culturing bacteria from everyday items stimulates students’ interest and eliminates the need to store bacterial strains in the teaching laboratory

Pseudomonas aeruginosa e altri bacilli gram-negativi non fermentanti

Questo capitolo tratta un insieme tassonomicamente eterogeneo di microrganismi aerobi, accomunati dal fatto di utilizzare i carboidrati attraverso la via ossidativa. Nel loro insieme, essi sono indicati come bacilli gram-negativi non fermentanti (BGNNF), una definizione che ha valenza operativa, piuttosto che tassonomica. Pseudomonas aeruginosa è il rappresentante più rilevante del gruppo, al quale appartengono inoltre batteri dei generi Acinetobacter, Burkholderia, Stenotrophomonas, Alcaligenes, Flavobacterium e altri

Biofilms Developed on\ua0Dental Implant Titanium Surfaces with Different Roughness: Comparison Between In Vitro and In Vivo Studies

Microbial biofilms developed on dental implants play a major role in perimplantitis\u2019 pathogenesis. Many studies have indicated that surface roughness is the main feature favoring biofilm development in vitro, but its actual influence in vivo has still to be confirmed. In this study, the amount of biofilm formed on differently treated titanium surfaces, showing distinct roughness, has been examined both in vivo and in vitro by Confocal Laser Scanning Microscopy. In vitro studies availed of biofilm developed by Pseudomonas aeruginosa or by salivary bacteria from volunteer donors. In vivo biofilm production was obtained by exposing titanium discs to the oral cavity of healthy volunteers. In vitro experiments showed that P. aeruginosa and, to a lesser extent, salivary bacteria produce more biomass and develop thicker biofilms on laser-treated and sandblasted titanium surfaces with respect to machined ones. In vivo experiments confirmed that bacterial colonization starts on sites of surface unevenness, but failed to disclose biomass differences among biofilms formed on surfaces with different roughness. Our study revealed that biofilm developed in vitro is more easily influenced by surface features than biofilm formed by complex communities in the mouth, where the cooperation of a variety of bacterial species and the presence of a wide range of nutrients and conditions allow bacteria to optimize substrate colonization. Therefore, quantitative differences observed in vitro among surfaces with different characteristics may not be predictive of different colonization rates in vivo

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

Characterization of integrons in Burkholderia cepacia clinical isolates

Burkholderia cepacia is an opportunistic pathogen able to colonize the airways of Cystic Fibrosis (CF) patients, frequently developing chronic infections. In 20% of cases these infections cause severe and poorly controlled pathological situations because of the intrinsic antibiotic resistance expressed by the microorganism. CF patients are often subjected to antibiotic therapy: this facilitates the acquisition of antibiotic resistance determinants by the infecting bacteria. Integrons are mobile genetic elements that are widespread in bacterial populations and favor the acquisition of gene cassettes coding for these determinants.The presence of class 1 integrons was investigated by PCR with primers specific for the 5’ and 3’ ends in Burkholderia isolates recovered from patients in treatment at the CF center of Friuli Venezia Giulia. The same integron, carrying an uncommon allelic form (Ib) of the aacA4 gene in its cassette array and conferring resistance to some aminoglycosides, was found in two independent isolates (different RAPD profiles) infecting two different patients. In both isolates the integron was carried by plasmids and was still present 3 and 6 years later the first finding. Despite the exchange of integrons between bacterial pathogens is fully described, these items were not frequently found in Burkholderia isolates. Although the clinical relevance of the integron we identified is low (a single gene cassette encoding a widespread resistance),we feel concerned that these genetic elements begin to circulate in this bacterial species, as this could make more and more troublesome the treatment of infections notoriously difficult to eradicate

The biofilm produced by Burkholderia cepacia complex: molecular aspects and relationship with exopolysaccharides

Introduction. In cystic fibrosis patients, Burkholderia cepacia complex (Bcc) can cause serious pulmonary chronic infections thanks in part to the ability to form biofilm, matrix rich in exopolysaccharides. In Bcc grown in the planktonic state, the main exopolysaccharide is cepacian while in biofilm its presence is controversial. Methods and Results. Two clinical isolates, named BTS7 and BTS2, were studied. BTS7 produces abundant cepacian but not much biofilm (quantified by colorimetric method).At least two of the genes involved in cepacian biosynthesis are not necessary for biofilm production as two BTS7 derivatives, bceB and bceQ knocked out by transposon mutagenesis, produce biofilm levels comparable to the wild-type. BTS2 sinthesyzes cepacian only if cultured on a specific medium. It has been colonizing a patient for almost ten years, showing a significant reduction of biofilm production during this period. This reduction did not appear together with the lack of factors required for the initial adhesion to the surface, or to differences in some of the Bcc genes involved in biofilm formation. Moreover, sequencing of its bce locus revealed a bceX gene, absent in BTS7, coding for a trascriptional regulator. Its product may negatively regulate the production of cepacian but not the one of other polysaccharides, promoting the formation of biofilm. Conclusions. Cepacian seems to be marginal in the production of biofilm.The reduced ability to produce biofilm of BTS2 suggests possible gene mutations occurred over time. Using custom arrays we will compare the gene expression of the BTS2 isolates, to identify the genes responsible for the observed phenotypic changes