Burkholderia anthina sp. nov. and Burkholderia pyrrocinia,two additional Burkholderia cepacia complex bacteria,may confound results of new molecular diagnostic tools

Nineteen Burkholderia cepacia-like isolates of human and environmental origin could not be assigned to one of the seven currently established genomovars using recently developed molecular diagnostic tools for B. cepacia complex bacteria. Various genotypic and phenotypic characteristics were examined. The results of this polyphasic study allowed classification of the 19 isolates as an eighth B. cepacia complex genomovar (Burkholderia anthina sp. nov.) and to design tools for its identification in the diagnostic laboratory. In addition, new and published data for Burkholderia pyrrocinia indicated that this soil bacterium is also a member of the B. cepacia complex. This highlights another potential source for diagnostic problems with B. cepacia-like bacteria.     

Opinion: Re-evaluating prokaryotic species

There is no widely accepted concept of species for prokaryotes, and assignment of isolates to species is based on measures of phenotypic or genome similarity. The current methods for defining prokaryotic species are inadequate and incapable of keeping pace with the levels of diversity that are being uncovered in nature. Prokaryotic taxonomy is being influenced by advances in microbial population genetics, ecology and genomics, and by the ease with which sequence data can be obtained. Here, we review the classical approaches to prokaryotic species definition and discuss the current and future impact of multilocus nucleotide-sequence-based approaches to prokaryotic systematics. We also consider the potential, and difficulties, of assigning species status to biologically or ecologically meaningful sequence clusters.     

Identification of genomic groups in the genus Stenotrophomonas using gyrB RFLP analysis

Stenotrophomonas maltophilia isolates have been recovered from various clinical samples, including the respiratory tract of cystic fibrosis (CF) patients, but this organism is also widespread in nature. Previously it has been shown that there is a considerable genomic diversity within S. maltophilia. The aims of our study were to determine the taxonomic resolution of restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction-amplified gyrB gene for the genus Stenotrophomonas. Subsequently, we wanted to use this technique to screen a set of S. maltophilia isolates (with emphasis on a specific subset, isolates recovered from CF patients), to assess the genomic diversity within this group. In this study we investigated 191 Stenotrophomonas sp. isolates (including 40 isolates recovered from CF patients) by means of gyrB RFLP. The taxonomic resolution of gyrB RFLP, and hence its potential as an identification tool, was confirmed by comparison with results from published and novel DNA-DNA hybridisation experiments. Our data also indicate that the majority of CF isolates grouped in two clusters. This may indicate that isolates from specific genomic groups have an increased potential for colonisation of the respiratory tract of CF patients.     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.     

Efficacy of silver-releasing rubber for the prevention of Pseudomonas aeruginosa biofilm formation in water

The aim of the present study was to evaluate the efficacy of Elastoguard silver-releasing rubber in preventing Pseudomonas aeruginosa biofilm formation in water. Biofilm formation by P. aeruginosa under various conditions in an in vitro model system was compared for silver-releasing and conventional rubber. Under most conditions tested, the numbers of sessile cells attached to silver-releasing rubber were considerably lower with reference to conventional rubber, although the effect diminished with increasing volumes. The release of silver also resulted in a decrease in planktonic cells. By exposing both materials simultaneously to conditions for biofilm growth, it became obvious that the antibiofilm effect was due to a reduction in the number of planktonic cells, rather than to contact-dependent killing of sessile cells. The data demonstrate that the use of silver-releasing rubber reduces P. aeruginosa biofilm in water and reduces the number of planktonic cells present in the surrounding solution.     

Prevention of Candida albicans Biofilm Formation by Covalently Bound Dimethylaminoethylmethacrylate and Polyethylenimine

Candida albicans biofilms are a major cause of voice prosthesis deterioration in laryngectomized patients. The aim of this study was to produce a surface capable of inhibiting C. albicans biofilm formation. Dimethylaminoethylmethacrylate (DMAEMA) and polyethylenimine (PEI) moieties were covalently bound to the surface of polydimethylsiloxane (PDMS) or polymethylmethacrylate (PMMA) and subsequently quaternized. Physicochemical characterization of the grafted surfaces was carried out and their effect on C. albicans cell numbers was assessed using a modified Robbins device to grow the biofilms. Covalently bound quaternized polyDMAEMA (polyDMAEMAq) and PEI (PEIq) inhibited biofilm growth, with reductions up to 92%. Our approach may show promise for future application in medical devices such as catheters and prostheses.     

Antiseptic cyclodextrin-functionalized hydrogels and gauzes for loading and delivery of benzalkonium chloride

Prevention and management of wound infections receive a lot of attention, since the presence of micro-organisms interferes with the wound-healing process. The aim of this work was to use cyclodextrins (CDs) to endow hydrogels and gauzes with the ability to take up antiseptics and sustain their delivery for several hours. Benzalkonium chloride (BzCl) can form inclusion complexes with cross-linked CDs that regulate the release through an affinity-driven mechanism. Grafting of CDs to cotton gauzes using citric acid as the linker, at 190?°C and for 15?min, led to grafting yields of about 148%, much larger than those obtained at 180?°C or with shorter reaction times. Microbiological tests revealed that the BzCl-loaded networks can inhibit the growth of Staphylococcus epidermidis and Escherichia coli both on agar plates and in liquid medium. Furthermore, the antiseptic-loaded gauzes were able to inhibit biofilm formation by Staphylococcus aureus RN1HG pMV158GFP when applied in early stages of biofilm formation and could reduce the number of living cells in preformed biofilms grown in a chronic wound biofilm model. These findings highlight the role of CDs as main components of hydrogels and gauzes for the efficient delivery of antiseptics.     

Real-time PCR expression profiling of genes encoding potential virulence factors in <Emphasis Type="Italic">Candida albicans</Emphasis> biofilms: identification of model-dependent and -independent gene expression

Background  

Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.