Efficient isolation of Campylobacter bacteriophages from chicken skin, analysis of several isolation protocols.

The application of Campylobacter specific bacteriophages appears as a promising food safety tool for the biocontrol of this pathogen in the poultry meat production chain. However, their isolation is a complicated challenge since their occurrence appears to be low. This work assessed the efficiency of seven protocols for recovering Campylobacter phages from chicken skin samples inoculated at phage loads from 5.0 × 101 to 5.0 × 106 PFU/g. The enrichment of chicken skin in selective Bolton broth containing target isolates was the most efficient procedure, showing a low detection limit of 5.0 × 101 PFU/g and high recovery rates of up to 560%. This method's effectiveness increased as phage concentration decreased, showing its suitability for phage isolation. When this method was applied to isolate new Campylobacter phages from retail chicken skin, a total of 280 phages were recovered achieving an isolation success rate of 257%. From the 109 samples 68 resulted phage positive (62%). Chicken skin could be, therefore, considered a rich source in Campylobacter phages. This method is a simple, reproducible and efficient approach for the successful isolation of both group II and III Campylobacter specific bacteriophages, which could be helpful for the enhancement of food safety by reducing this pathogen contamination in broiler meat.

Static and Continuous flow-through pulsed light technology for pesticide abatement in water.

Perspectives concerning pulsed light (PL) technology as a novel water decontamination treatment are overviewed in this work. Degradation of atrazine, malathion, chlorpyriphos-methyl and bromopropylate in aqueous solutions at different concentrations was performed employing static and continuous flow-through PL units. Results for both PL systems were compared in terms of efficacy of pesticides degradation and derived photoproducts formed. The pesticides degradation increased with the applied total fluence for both PL systems. In general, PL induced rapid degradation of all studied compounds although some differences were observed in the rate of degradation among them. The most intense treatment in the static unit (11J/cm2) yielded degradations between 60 and 85% whereas degradations between 74 and 93% were observed after exposure to slightly lower fluences (10J/cm2) in the dynamic unit. For both PL systems bromopropylate was the most affected compound. Photodegradation products arose from chemical reactions involving structural changes like dehalogenation, desulfuration, dealkylaytion and oxidation of the alkyl chains. Although further research is needed before an efficient application in full-scale systems, PL appears as a promising technology in order to reduce the presence of pesticides residues in process or waste waters, allowing thereby recirculation of these waters with the subsequent economic and environmental advantages.

Survival and growth of Listeria innocua treated by pulsed light technology: impact of post-treatment temperature and illumination conditions.

Inactivation of Listeria innocua by pulsed light (PL) was evaluated at different post-treatment temperature and illumination conditions. The impact of post-PL-treatment temperature on L. innocua culturability was evaluated for cells cultured at 37 °C (optimal growth temperature) and 4 °C (classical refrigerated food temperature). For both culture conditions, significant higher reductions (up to 3 log) were observed after post-PL-treatment temperature of 4 °C than of 37 °C. Contrarily, L. innocua culturability after PL treatment increased up to 2.2 log in presence of daylight illumination in comparison to dark storage. This photorepair mechanism was quickly activated reaching the maximum photoreactivation level after only 30 min of illumination. Moreover, photorepair capacity was rapidly reduced by increasing the time in darkness from PL treatment to samples illumination, being completely lost after time in darkness equal or greater than 5 h. According to these findings, the combination of PL with post-treatment temperature of 4 °C has a synergistic effect on the inactivation of L. innocua, whereas post-treatment daylight illumination has an antagonic effect on PL antimicrobial efficacy. Post-PL-treatment temperature and illumination conditions could be thereby considered important environmental factors to activate, inhibit or control the repair and/or growth of L. innocua survivors after PL treatment.

Sensitivity to pulsed light technology of several spoilage and pathogenic bacteria isolated from fish products.

The effect of pulsed light (PL) on the inactivation of six fish spoilage bacteria (Photobacterium phosphoreum, Serratia liquefaciens, Shewanella putrefaciens, Brochothrix thermosphacta, Pseudomonas group I, and Pseudomonas groups III and IV), six Listeria monocytogenes isolates from fish products, and one strain of Listeria innocua was evaluated. For all tested strains, grown at 4°C (temperature to process, store, and distribute convenient, lightly preserved fish products), the maximum detectable inactivation (6 to 6.5 log) was observed after treatments lower than 0.3 J/cm(2). The most sensitive bacteria were P. phosphoreum and S. liquefaciens, while both L. innocua and L. monocytogenes were the most resistant species to PL. Bacterial inactivation kinetics depended on their susceptibility to PL. Typically, sigmoidal curves were observed, with an initial shoulder followed by an exponential loss of cell culturability down to the maximum detectable inactivation. However, no initial shoulder was pointed out for the most sensitive species (P. phosphoreum and S. liquefaciens). Since the PL resistance of all tested Listeria strains (L. innocua and L. monocytogenes isolates) was similar, we propose the use of L. innocua as a surrogate for L. monocytogenes in PL technology validation.

Fast atrazine photodegradation in water by pulsed light technology.

Pulsed light technology consists of a successive repetition of short duration (325µs) and high power flashes emitted by xenon lamps. These flashlamps radiate a broadband emission light (approx. 200-1000 nm) with a considerable amount of light in the short-wave UV spectrum. In the present work, this technology was tested as a new tool for the degradation of the herbicide atrazine in water. To evaluate the presence and evolution with time of this herbicide, as well as the formation of derivatives, liquid chromatography-mass spectrometry (electrospray ionization) ion trap operating in positive mode was used. The degradation process followed first-order kinetics. Fluences about 1.8-2.3 J/cm(2) induced 50% reduction of atrazine concentration independently of its initial concentration in the range 1-1000 µg/L. Remaining concentrations of atrazine, below the current legal limit for pesticides, were achieved in a short period of time. While atrazine was degraded, no chlorinated photoproducts were formed and ten dehalogenated derivatives were detected. The molecular structures for some of these derivatives could be suggested, being hydroxyatrazine the main photoproduct identified. The different formation profiles of photoproducts suggested that the degradation pathway may include several successive and competitive steps, with subsequent degradation processes taking part from the already formed degradation products. According to the degradation efficiency, the short treatment time and the lack of chloroderivatives, this new technology could be considered as an alternative for water treatment.

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR.

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.