Phenotypic and genotypic characterisation of Burkholderia cenocepacia J2315 mutants affected in homoserine lactone and diffusible signal factor-based quorum sensing systems suggests interplay between both types of systems

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid ("Burkholderia diffusible signal factor'', BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (Delta cepI Delta BCAM0581, Delta cciI Delta BCAM0581 and Delta cepI Delta cciI) mutants and a triple (Delta cepI Delta cciI Delta BCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The Delta BCAM0581 mutant and the Delta cepI Delta BCAM0581 and Delta cciI Delta BCAM0581 double mutants produced significantly less AHL compared to the WT strain and the Delta cepI and Delta cciI single mutant, respectively. The expression of cepI and cciI in Delta BCAM0581, was approximately 3-fold and 7-fold (p < 0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the Delta BCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system

Management of Patients Diagnosed with Endometrial Cancer: Comparison of Guidelines

: Endometrial cancer is the most common gynecological malignancy in Europe and its management involves a variety of health professionals. In recent years, big discoveries were made concerning the management of patients diagnosed with endometrial cancer, particularly in the field of molecular biology and minimally invasive surgery. This requires the continuous updating of guidelines and protocols over the years. In this paper, we aim to summarize and compare common points and disparities among protocols for management of patients diagnosed with endometrial cancer by leading international gynecological oncological societies. We therefore systematically report the parallel among the guidelines based on the various steps patients with endometrial cancer usually undergo. The comparison between American and European protocols revealed some relevant disparities, in particular regarding surgical staging, molecular biology application as a prognostic tool and follow up regimens. This could possibly cause differences in interpreting and applying protocols in clinical practice in small centers, leading to a lack of adherence to guidelines or even prompting a confusing mix of them

Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in Gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4–D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis

Primers used in this work.

a<p>Restriction endonuclease sites incorporated in the oligonucleotide sequences are underlined.</p

Protease production in various <i>B. cenocepacia</i> strains in the abscence (CTRL) and presence of AHL (5 µM), BDSF (5 µM) or both AHL/BDSF (5 µM).

<p>*: significantly different protease production compared to no signal (p<0.05).</p

Percent survival of <i>C. elegans</i> (average ± standard deviation) infected with various <i>B. cenocepacia</i> strains in the absence (CTRL) or presence of signalling molecules (AHL, BDSF or both; 5 µM).

<p>The results are expressed as the percent survival after 24 h (black bars) or 48 h (grey bars) of infection and treatment. *: significantly different survival compared to uninfected control (p<0.0001); **: significantly different survival compared to infection with WT (p<0.0001).</p

MIC (µg/ml) of various antibiotics towards <i>B. cenocepacia</i> J2315 WT and mutant strains.

<p>CAZ, ceftazidime; FOX, cefoxitin; CIP, ciprofloxacin; MEM, meropenem; TOB, tobramycin.</p

Production of AHL molecules by various <i>B. cenocepacia</i> strains A) in the absence and B) presence of BDSF (5 µM).

<p>*: significantly different from no AHL production (p<0.001); **: significantly different from control receiving no BDSF (p<0.001).</p

Evaluation of fluoroquinolone resistance mechanisms in Pseudomonas aeruginosa MDR clinical isolates.

Efflux transporters have a considerable role in the multidrug resistance (MDR) of Pseudomonas aeruginosa, an important nosocomial pathogen. In this study, 45 P. aeruginosa clinical strains, with an MDR phenotype, have been isolated in a hospital of Northern Italy and characterized to identify the mechanisms responsible for their fluoroquinolone (FQ) resistance. These isolates were analyzed for clonal similarity, mutations in genes encoding the FQ targets, overexpression of specific Resistance Nodulation-cell Division efflux pumps, and search for mutations in their regulatory genes. The achieved results suggested that the mutations in genes encoding ciprofloxacin targets represented the main mechanism of FQ resistance of these strains; 97.8% of these isolates showed mutations in gyrA, 28.9% in gyrB, 88.9% in parC, and 6.7% in parE. Another mechanism of resistance was overexpression of the efflux pumps in some representative strains. In particular, overexpression of MexXY-OprM drug transporter was found in five isolates, whereas overexpression of MexCD-OprJ was detected in two isolates; surprisingly, in one of these last two isolates, also overexpression of MexAB-OprM pump was identified

Production of BDSF by the Δ<i>cepI</i>Δ<i>cciI</i> double mutant.

<p>Biofilm formation of the ΔBCAM0581 without supplementation, supplemented with supernatant of the Δ<i>cepI</i>Δ<i>cciI</i> mutant or ΔBCAM0581 mutant or supplemented with BDSF (5 µM). The relative amount of metabolically active cells was quantified with CellTiter Blue (black bars), while the relative amount of total biomass was quantified with crystal violet (grey bars).</p