Development of a rapid method to detect and to monitor bacteriophage amplification in contact with viable but non-culturable bacteria.

We propose in this study to develop a rapid, reliable, and non-culture method to detect and estimate bacteriophage (phage) titre as an alternative to the routine use of the double agar overlay assay (DLA). The present method is based on the analysis of nanoparticle (NPs) dispersion/aggregation dynamic in interaction with the phage. Titanium dioxide nanoparticles (TiO2-NPs) were used as nanosensors to detect and monitor virions' titres in aqueous samples. Dispersion stability of TiO2-NPs in aqueous suspension was investigated using a UV-Visible spectrophotometer. The comparison of NP spectral profiles with and without phage elucidated the impact of phage's titre on NP dispersion/aggregation behaviour in an aqueous solution. Indeed, the increase of nanoparticle dispersion stability is correlated with the increase of phage titre. Thus, based on this result, the phage was considered as a bio-dispersant agent. The determination of area under spectral profiles limiting the UV region [200-400 nm] was allowed to quantify, and compare the NPs bio-dispersion rate, in relation with added phage at different titres. In this study, this method was applied to monitor the phage amplification cycle for the detection of bacteria in viable but non-culturable (VBNC) state after water treatment by photocatalysis. The analysis of NP bio-dispersion rate shows an increase of TiO2-NP dispersion stability correlated with an increase of free phage titration, mainly after the entry of target bacteria in VBNC state underestimated using a conventional method. Thus, this method could allow the establishment of new recommendations of wastewater treatment and assessment.

Use of bacteriophage to inactivate pathogenic bacteria from wastewater.

The aim of this study was to enhance the rhizobacterium potential in horizontal subsurface flow constructed wetland (CW) system planted by Phragmites australis using specific and lytic phages. The bioinoculation of specific bacteriophage for target bacteria; Salmonella typhi, and the monitoring of bacterial inactivation under different conditions showed the effectiveness of this methodology to enhance bacteria reduction and consequentially ameliorate purification performance of this studied biological treatment system. The injection of the phage at a concentration equal to 103 UFP/mL within the rhizosphere of the inoculated filter (F) was allowed 1 U-Log10 of improvement of bacterial inactivation compared to the control filter (T) nearly 1 logarithmic unit thus, a 90% improvement of bacteria reduction. When we increased the phage titer (105 UFP/mL), the bacterial reduction equal to 2.75 U-Log10 (N/N0) was registered that corresponds to a decrease of nearly 99.9%. According to the first-order model, the inactivation coefficient is equal to 2.29 min-1 (0.88 min-1 for the first experiment) and the bacterial reduction rate is 5 times higher than that determined for the control filter. This results show the positive impact of the phage in the bacterial inactivation and the improvement of water treatment of the biofilter C.

Effect of photocatalysis (TiO2/UVA) on the inactivation and inhibition of Pseudomonas aeruginosa virulence factors expression.

Water disinfection using visible light-active photocatalyst has recently attracted more attention due to its potential to inactivate microbes. In this study, we have investigated the efficiency of photocatalysis (TiO2/UVA) on the inactivation of Pseudomonas aeruginosa and the attenuation of its virulence factors. For this aim, the photocatalytic effects of TiO2/UVA on the cultivability and viability of P. aeruginosa were investigated. Furthermore, during the photocatalysis, the morphology of the bacterial cells was examined by atomic force microscopy (AFM) while the virulence factors were assessed by protease and lipase activities in addition to the mobility and communication of cells. The results revealed that during the photocatalysis the bacterial cells lost their cultivability and viability on agar under the action of the reactive oxygen species generated by the photocatalytic reaction. In addition, AFM observations have shown a damage of the bacterial membrane and a total disruption of the bacterial cells. Moreover, the major virulence factors such as biofilm, lipase and protease expression have been markedly inhibited by TiO2/UVA treatment. In addition, the bacteria lost their ability of communication 'quorum sensing' and mobility with twitching and swarming types after 60 min of photocatalytic treatment. Accordingly, TiO2/UVA is an effective method to reduce P. aeruginosa virulence and to prevent biofilm formation.

Monitoring of methylene blue monomers and dimers to control the bacterialogical water quality including application to photocatalysis.

In this study, we propose the development of a rapid and reliable method to control and to monitor microbial water quality. The methylene blue (MB) decolorization assay was based on the analysis of spectral profiles of dye in interaction with a different bacterial concentration. The determination of dye decolorization rate (DDR) shows a correlation between the MB reduction rate and the bacterial density. Moreover, the kinetic of the monomer and dimer equilibrium of MB in water mainly, the monitoring of bounded MB species in relationship with a knowed concentration of target bacteria, was allowed to establish a relationship between MB decolorization rate and bacterial density. Furthermore, this method was applied to evaluate the water quality after photocatalysis. Based on this method, the photocatalytic effects on bacterial density was highlighted by the decrease in DDR after photocatalytic treatment with fractioned times (0 to 5 h); this increase was followed by a decrease of bounded MB species and, an increase in free MB forms miming the reduction of bacterial density due to the biocide effects of photocatalysis process. However, the analysis of spectra profiles shows a weak but a continuous decrease in bounded MB dimer and monomer forms in the treated water samples exempt of culturable bacteria. Moreover, the MB spectra profiles were tended toward a negative control spectrum without superposition. Thus, the possibility of the presence of viable but non-culturable bacteria was expected; therefore, to optimize this tertiary water treatment process, an extending on proceeding time was recommended to avoid the bacterial resuscitation after photocatalysis.

The application of phage reactivation capacity to sens bacterial viability and activity after photocatalytic treatment.

We purpose in this study to develop a reliable and low-cost method for the detection of Viable but nonculturable (VBNC) bacteria. Indeed, after water disinfection, injured-VBNC bacteria can be underestimated using conventional assessment methods, causing false-negative results and, posing a significant and potential health risk. The VBNC bacterial survival strategy can hide the real microbial quality of treated water. To overcome this bacterial assessment limitation, we were used a specific and lytic phage to monitor the presence of active bacteria; Pseudomonas aeruginosa after photocatalytic treatment. Within 2 h of phage-target bacteria contact, the reduction of phage amplification rate (At) can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of new infectious phages release despite their lose of cultivability in the usual media. The determination of phage reactivation coefficient (Rt) after 2 and 8 h of phage-target cell contact time reveals the ability of phages to reactive their infectivity and their amplification in positive correlation with their host cells viability and activity. The increase in phage reactivation coefficient (Rt) after an extension of the latent period was directly related to the positive interaction between infectious phages and potential active bacteria. The use of this method can improve the water disinfection process and avoid public health-hazardous especially related to the resuscitation of active-nonculturable bacteria mainly for pathogenic bacteria.

Use of the catalytic complex TiO2/red cabbage anthocyanins to reduce the biofilm formation by planktonic bacteria.

The bacterial cells dwelling within the biofilm usually develop resistance against common disinfectants. In this current study, to improve the effectiveness of photocatalytic treatment, a natural sensitizer in combination with unsupported titanium dioxide nanoparticles (TiO2-NPs) was used to optimize the absorbance of NPs in the visible region and, to enhance the catalytic activity of the semiconductor. Different kinetic parameters were determined according to the first-order and the biphasic models to evaluate the ability of tested bacteria to form biofilm under different photocatalytic treatment conditions. As a result, the addition of red cabbage anthocyanins (RCA) as photosensitizer allows the enhancement of biocide activity of TiO2-NPs and the reduction of biofilm formation by tested bacteria.

Detection of active pathogenic bacteria under stress conditions using lytic and specific phage.

In this study, we have monitored the potential activity of a foodborne and waterborne pathogenic bacterium, Salmonella typhi, under starvation conditions. The interaction between lytic phage and starved-VBNC pathogenic bacteria was studied to establish reliable methods for the detection of active cells before resuscitation. The analysis of phage kinetic parameters has demonstrated the flexibility of lytic with the quantity and mainly the quality of host cells. After 2 h of phage-starved-VBNC bacteria interaction, the reduction of phage amplification rate can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of burst and release of infectious phages by active bacteria. After an extension of the latent period, the boost of the phage amplification rate was directly related to the positive interaction between potential intracellular 'engaged' phages and potential active bacteria. Furthermore, the modeling of the Salmonella-specific phage growth cycle in relationship with starved host cells can highlight the impact of the viability and the activity state of the host cells on the phage's growth cycle.

Enhancement of rhizocompetence in pathogenic bacteria removal of a constructed wetland system.

The main goal of the present study was to enhance the rhizobacterium potential in a horizontal subsurface flow constructed wetland system planted with Phragmites australis, through environmentally friendly biological approaches. The bioinoculation of antagonist bacteria has been used to promote higher rhizosphere competence and improve pathogenic bacteria removal from wastewater. The experiment was performed both with single and sequential bioinoculation. The results showed that strain PFH1 played an active role in pathogenic bacteria removal, remarkably improving inactivation kinetics of the pathogenic tested bacterium Salmonella typhi in the plant rhizosphere. The single bioinoculation of selected bacteria into the rhizosphere of P. australis improved the kinetics of S. typhi inactivation by approximately 1 U-Log10 (N/N0) (N is the number of viable cultured bacteria at time t, N0 is the number of viable and cultivable bacteria at time t0) compared to the control. By a series of multi-bioinoculations, the enhancement of pathogenic bacteria reduction compared to the inhibition rate in the pilot-scale control was of 2 U-Log10(N/N0). These findings suggested that this strain represents a promising candidate to enhance water purification in constructed wetlands.

ZnO Nanorods with High Photocatalytic and Antibacterial Activity under Solar Light Irradiation.

ZnO nanorods (NRs) with an average length and diameter of 186 and 20 nm, respectively, were prepared through a mild solvothermal route and used as photocatalysts either as dispersed powder or immobilized on glass slides. The ZnO NRs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Dispersed ZnO NRs and, to a lesser extent, immobilized ZnO NRs were demonstrated to exhibit high photocatalytic activity under simulated sunlight of low intensity (5.5 mW/cm²) both for the degradation of the Orange II dye and for Escherichia coli bacterial decontamination (2.5-fold survival decrease after 180 min irradiation for immobilized NRs). SEM, atomic force microscopy (AFM), fluorescence spectroscopy, and epifluorescence microscopy demonstrate that cell surface damages are responsible of bacterial inactivation. The immobilized ZnO NRs could be reused up to five times for bacterial decontamination at comparable efficiency and therefore have great potential for real environmental applications.

Cannabinoid receptor 1 is a major mediator of renal fibrosis.

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-ß1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

Detection of viable but non cultivable Escherichia coli after UV irradiation using a lytic Qbeta phage.

In order to qualify the germicidal efficacy of ultraviolet (UV) disinfection system, we generally determine the reduction of viable bacteria after UV-C irradiation. However, the simple count of viable and cultivable bacteria in usual media cannot reflect whether or not the UV dose applied to disinfect water is sufficient to inactivate bacteria. Indeed, there is a bacterial mix in the UV-treated water: dead bacteria, viable and cultivable bacteria and viable but noncultivable bacteria (VBNC). The third type of bacteria can constitute a potential risk for public health. In fact, VBNC bacteria can be active and cause diseases. Consequently, the combination of a conventional method used to measure colony-forming ability after UV disinfection and the determination of adsorption constants of a lytic Qbeta phage in relation to irradiated host cells by an increased UV dose (Escherichia coli ATCC 13965) allows the detection of active bacteria, which lose their cultivability in usual growth media, but keep the phage susceptibility.