Anti-biofouling and antibacterial surfaces via a multicomponent coating deposited from an up-scalable atmospheric-pressure plasma-assisted CVD process.

Prevention of bacterial adhesion and biofilm formation on the surfaces of materials is a topic of major medical and societal importance. In this study, an up-scalable atmospheric-pressure plasma assisted deposition method is introduced to produce a multicomponent coating towards the elaboration of antibacterial and anti-biofilm surfaces. Interestingly, from a single catechol-based monomer, high deposition rates of highly chemically reactive functional thin films bearing catechol as well as quinone groups are achieved. The catechol-bearing thin film allows the in situ silver nanoparticle formation, assessed by scanning electron microscopy and EDX, whilst the enriched-quinone thin film is exploited for immobilizing dispersine B, an enzyme. In vitro functional assays demonstrated the dual antibacterial and anti-biofouling resistance properties of the coatings due to the antibacterial effect of silver and the fouling resistance of grafted dispersine B, respectively. Surfaces coated only with silver provide an antibacterial effect but fail to inhibit bacterial attachment, highlighting the usefulness of such dual-action surfaces. The approach presented here provides a simple and effective chemical pathway to construct powerful antibacterial surfaces for various industrial applications.

Significance of a Noble Metal Nanolayer on the UV and Visible Light Photocatalytic Activity of Anatase TiO2 Thin Films Grown from a Scalable PECVD/PVD Approach.

UV and visible light photocatalytic composite Pt and Au-TiO2 coatings have been deposited on silicon and glass substrates at low temperature using a hybrid ECWR-PECVD/MS-PVD process. Methylene blue, stearic acid, and sulfamethoxazole were used as dye, organic, and antibiotic model pollutants, respectively, to demonstrate the efficiency of these nanocomposite coatings for water decontamination or self-cleaning surfaces applications. Raman investigations revealed the formation of anatase polymorph of TiO2 in all synthesized coatings with a shifting of the main vibrational mode peak to higher wavenumber in the case of Au-TiO2 coating, indicating an increase number of crystalline defects within this coating. Because of the difference of the chemical potentials of each of the investigated noble metals, the sputtered metal layers exhibit different morphology. Pt sputtered atoms, with high surface adhesion, promote formation of a smooth 2D layer. On the other hand, Au sputtered atoms with higher cohesive forces promote the formation of 5-10 nm nanoparticles. As a result, the surface plasmon resonance phenomenon was observed in the Au-TiO2 coatings. UV photoactivity of the nanocomposite coatings was enhanced 1.5-3 times and 1.3 times for methylene blue and stearic acid, respectively, thanks to the enhancement of electron trapping in the noble metal layer. This electron trapping phenomenon is higher in the Pt-TiO2 coating because of its larger work function. On the other hand, the enhancement of the visible photoactivity was more pronounced (3 and 7 times for methylene blue and stearic acid, respectively) in the case of Au-TiO2 thanks to the surface plasmon resonance. Finally, these nanocomposite TiO2 coatings exhibited also a good ability for the degradation of antibiotics usually found in wastewater such as sulfamethoxazole. However, a complementary test have showed an increase of the toxicity of the liquid medium after photocatalysis, which could be due the presence of sulfamethoxazole's transformation byproducts.

Demonstrating plasmid-based horizontal gene transfer in complex environmental matrices: a practical approach for a critical review.

Plasmid-based dissemination of antibiotic resistance genes in environmental microbial communities is a matter of concern for public health, but it remains difficult to study for methodological reasons. In this study, we used the broad host range plasmid pB10 to compare and to point out the main drawbacks of the three different approaches currently used to evaluate plasmid transfer in natural communities. Culture-based selection of transconjugants appeared to be compromised by high prevalence of antibiotic resistances among natural communities, unless high loads of initial pB10-donor inocula were used. Fluorescence-based detection of transconjugants reached a dead-end consequently to the narrow host range of bacteria expressing fluorescent proteins from a genetically modified pB10 plasmid, in addition to the relatively high background level of fluorescence exhibited by some environmental matrices. The molecular-based approach was the only one to provide a mean to detect rare plasmid transfer events following a low but realistic initial pB10-donor inoculation. Whatever the method, culture-based or molecular-based, the detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events.

Development of a quantitative immunocapture real-time PCR assay for detecting structurally intact adenoviral particles in water.

Development of rapid, sensitive and specific methods for detection of infectious enteric viruses in water is challenging but crucial for gaining reliable information for risk assessment. An immunocapture real-time PCR (IC-qPCR) was designed to detect jointly the two major viral particle components, i.e. the capsid protein and the viral genome. Targeting both constituents helps circumventing the technical limits of cell culture approaches and the inability of PCR based methods to predict the infectious status. Two waterborne pathogenic virus models, human adenovirus types 2 and 41, were chosen for this study. IC-qPCR showed a detection limit of 10MPNCU/reaction with a dynamic range from 10(2) to 10(6)MPNCU/reaction. Sensitivity was thus 100-fold higher compared to ELISA-based capture employing the same anti-hexon antibodies. After optimisation, application on environmental water samples was validated, and specificity towards the targeted virus types was obtained through the qPCR step. Heat-treated pure samples as well as surface water samples brought evidence that this method achieves detection of encapsidated viral genomes while excluding free viral genome amplification. As a consequence, adenovirus concentrations estimated by IC-qPCR were below those calculated by direct qPCR. The results demonstrate that the IC-qPCR method is a sensitive and rapid tool for detecting, in a single-tube assay, structurally intact and thus potentially infectious viral particles in environmental samples.

Persistence and dissemination of the multiple-antibiotic-resistance plasmid pB10 in the microbial communities of wastewater sludge microcosms.

Plasmid-mediated dissemination of antibiotic resistance genes is widely recognized to take place in many environmental compartments but remains difficult to study in a global perspective because of the complexity of the environmental matrices considered and the lack of exhaustive tools. In this report, we used a molecular approach based on quantitative PCR to monitor the fate of the antibiotic resistance plasmid pB10 and its donor host in microbial communities collected from various wastewater treatment plant (WWTP) sludges and maintained in microcosms under different conditions. In aerated activated sludge microcosms, pB10 did not persist because of an apparent loss of the donor bacteria. The persistence of the donor bacteria noticeably increased in non-aerated activated sludge microcosms or after amending antibiotics (sulfamethoxazole or amoxicillin) at sub-inhibitory concentrations, but the persistence of the donor bacteria did not stimulate the dissemination of pB10. The dissemination of the plasmid appeared as an increasing plasmid to donor ratio in microcosm setups with microbial communities collected in anaerobic digesters or the spatially organized communities from fixed biofilm reactors. As a whole, the data collected suggest that some WWTP processes, more than others, may sustain microbial communities that efficiently support the dissemination of the multiple-antibiotic-resistance plasmid pB10.

Improving the recovery of qPCR-grade DNA from sludge and sediment.

DNA extraction is often considered as the limiting step of most molecular approaches in ecology and environmental microbiology. Ten existing DNA extraction protocols were compared for recovery of DNA from sludge and a modified version of the protocol described by Porteous et al. (Mol Ecol 6:787-791, 1997) was determined to be the best method for recovery of DNA suitable for PCR. In this respect, it appeared that the commonly used guanidine isothiocyanate could impair the quality of the extracted DNA unless its concentration is lowered. Second, conditioning the samples as liquors as opposed to pellets critically impacts the outcome of the extraction. The suitability of the modified Porteous protocol for quantitative PCR applications is demonstrated in a series of experiments showing the absence of interfering coextracted inhibitors and the linear correspondence between the concentrations of input target DNA and PCR product. Interestingly, it is also shown that the nature of the environmental matrices affects the recovery yield of both circular plasmids and chromosomal DNA, resulting in an apparent fluctuation of the plasmid copy number per cell. This means that quantitative data obtained by PCR remain comparable as long as they apply to an identical target sequence extracted from a similar environment and amplified under the same conditions.

Monitoring the dissemination of the broad-host-range plasmid pB10 in sediment microcosms by quantitative PCR.

Studying the transfer of specific mobile genetic elements in complex environmental matrices remains difficult because suitable molecular tools are not yet available to back up classical culture-dependent approaches. In this report, we show that quantitative PCR could be used to monitor the dissemination of the broad-host-range plasmid pB10 in sediment microcosms. This approach lies in the differential measurement of the host and plasmid DNAs used to inoculate the microcosms, using a particular design of quantitative PCR primers/probes where we took advantage of the mosaic aspect of the bacterial genomes to achieve a highly specific quantitative PCR detection system.