P. fluorescens biofilm control using bacteriophage ΦS1

Pseudomonas fluorescens biofilms contribute to the spoilage of dairy industry products due to the proteolytic activity of some Pseudomonas fluorescens strains. The eradication of these biofilms is difficult using the traditional chemical biocides due to the low removal action of these agents. Additionally chemical control leaves inactivated cells attached to the surface that tends to provide an ideal environment for further bacterial adhesion and growth. Bacteriophages can be seen as good alternative biofilm control agents due to their high specificity, efficacy against biocide resistant bacteria and because they are innocuous to the environment. The use of the bacteriophage phi-S1 as control agent of P. fluorescens biofilms at early stages of formation and in the mature stage was investigated in this work. Phage infection of attached cells was studied using a parallel plate flow cell mounted on an inverted microscope and with an automatic image analysis system that enables in situ and real time enumeration of cells. After phage infection the recolonization of the surfaces was also assessed with this system. The results obtained showed that phage infection caused a removal of about 93.5 ± 1.51% of the adhered cells. Additionally P. fluorescens recolonization of the surfaces was no longer possible due to the adsorption of phages (assessed by EDS) that remained on the glass surfaces after being washed during one hour with buffer. Five days old biofilms and planktonic cells in different growth phases were infected with phage (with initial MOI of 0.5). The infection was monitored by measuring the amount of cell lysis, PFU release and decrease in biomass. The results obtained revealed that although the rate of cell lysis and PFU release was lower in biofilms than those in exponential planktonic cultures (p<0.05, ANOVA), the decrease in the biomass after 200 minutes was approximately the same of infection and about 85.27 ± 1.41%. This work demonstrated that phages are important tools to be considered in industrial control of biofilms because they are able to cause significant biomass reduction of early stages and mature biofilms and prevent cell recolonization of the surfaces

Prevention of P. fluorescens adhesion to surfaces using bacteriophage ΦS1

The presence of biofilms in industrial settings is problematic as bacteria are responsible for several losses including product spoilage. Biofilms are difficult to eradicate and their removal and destruction has been for long attempted using chemical biocides. These agents usually yield very low biofilm removal amounts, have negative environmental impacts and the emergence of biocide-resistant bacteria represents one of the major drawback on their use. Therefore there is an increase interest in the utilization of bacteriophages (phages). Phages are specific for a host or a range of hosts, active against biocide-resistant bacteria and considered innocuous to the environment. This work focuses on the use of bacteriophage ΦS1 to prevent P. fluorescens biofilm formation. Glass and stainless steel surfaces were coated with a suspension of 10^9 PFUmlˉ¹ prior to bacterial attachment. The results obtained showed, that P. fluorescens were no longer able to adhere to these surfaces. Moreover, after several washings of the surface the remaining attached phages (10³ PFUmlˉ¹) were still able to prevent biofilm formation on the surfaces

Bacteriophage Ф S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms

This communication focuses on the efficacy of a specific lytic phage, phage Ф S1, as a control agent of Pseudomonas fluorescens biofilms. The effect of phage infection temperature and the host growth temperature were evaluated. The results obtained showed that the phage infection process was temperature dependent and that the optimum temperature of infection of planktonic cells and biofilms was 26ºC. At this temperature, bacteriophage Ф S1, at a multiplicity of infection (MOI) of 0.5 infected both planktonic cells and biofilms causing a biomass reduction of about 85% in both cases.Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI) - POCTI/BIO/35683/99

Pseudomonas fluorescens infection by bacteriophage ФS1 : the influence of temperature, host growth phase and media

The influence of host growth temperature, phase and media, together with the effect of infection temperature on bacteriophage ФS1 infection of Pseudomonas fluorescens were examined. The rates of cell lysis and phage release were determined and showed that the efficacy of phage infection was optimal with host cells grown and infected at 26 ºC. The host physiological state also affected these rates. Infection was dependent on the presence of cell wall proteins with molecular weights of 17.5 ± 1 and 99 ± 5 kDa.Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI) - POCTI/BIO/35683/99

Counter-current chromatography for the separation of terpenoids: A comprehensive review with respect to the solvent systems employed

Copyright @ 2014 The Authors.This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.Natural products extracts are commonly highly complex mixtures of active compounds and consequently their purification becomes a particularly challenging task. The development of a purification protocol to extract a single active component from the many hundreds that are often present in the mixture is something that can take months or even years to achieve, thus it is important for the natural product chemist to have, at their disposal, a broad range of diverse purification techniques. Counter-current chromatography (CCC) is one such separation technique utilising two immiscible phases, one as the stationary phase (retained in a spinning coil by centrifugal forces) and the second as the mobile phase. The method benefits from a number of advantages when compared with the more traditional liquid-solid separation methods, such as no irreversible adsorption, total recovery of the injected sample, minimal tailing of peaks, low risk of sample denaturation, the ability to accept particulates, and a low solvent consumption. The selection of an appropriate two-phase solvent system is critical to the running of CCC since this is both the mobile and the stationary phase of the system. However, this is also by far the most time consuming aspect of the technique and the one that most inhibits its general take-up. In recent years, numerous natural product purifications have been published using CCC from almost every country across the globe. Many of these papers are devoted to terpenoids-one of the most diverse groups. Naturally occurring terpenoids provide opportunities to discover new drugs but many of them are available at very low levels in nature and a huge number of them still remain unexplored. The collective knowledge on performing successful CCC separations of terpenoids has been gathered and reviewed by the authors, in order to create a comprehensive document that will be of great assistance in performing future purifications. © 2014 The Author(s)

Characterisation of the NRF2 transcriptional network and its response to chemical insult in primary human hepatocytes: implications for prediction of drug-induced liver injury

The transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical toxicities such as drug-induced liver injury (DILI). However, very little is known about the makeup of the NRF2 transcriptional network and its response to chemical perturbation in primary human hepatocytes (PHH), which are often used as a translational model for investigating DILI. Here, microarray analysis identified 108 transcripts (including several putative novel NRF2-regulated genes) that were both downregulated by siRNA targeting NRF2 and upregulated by siRNA targeting KEAP1 in PHH. Applying weighted gene co-expression network analysis (WGCNA) to transcriptomic data from the Open TG-GATES toxicogenomics repository (representing PHH exposed to 158 compounds) revealed four co-expressed gene sets or ‘modules’ enriched for these and other NRF2-associated genes. By classifying the 158 TG-GATES compounds based on published evidence, and employing the four modules as network perturbation metrics, we found that the activation of NRF2 is a very good indicator of the intrinsic biochemical reactivity of a compound (i.e. its propensity to cause direct chemical stress), with relatively high sensitivity, specificity, accuracy and positive/negative predictive values. We also found that NRF2 activation has lower sensitivity for the prediction of clinical DILI risk, although relatively high specificity and positive predictive values indicate that false positive detection rates are likely to be low in this setting. Underpinned by our comprehensive analysis, activation of the NRF2 network is one of several mechanism-based components that can be incorporated into holistic systems toxicology models to improve mechanistic understanding and preclinical prediction of DILI in man

The vaccination of 35,000 dogs in 20 working days using combined static point and door-to-door methods in Blantyre, Malawi

An estimated 60,000 people die of rabies annually. The vast majority of cases of human rabies develop following a bite from an infected dog. Rabies can be controlled in both human and canine populations through widespread vaccination of dogs. Rabies is particularly problematic in Malawi, costing the country an estimated 13 million USD and 484 human deaths annually, with an increasing paediatric incidence in Blantyre City. Consequently, the aim of this study was to vaccinate a minimum of 75% of all the dogs within Blantyre city during a one month period. Blantyre's 25 administrative wards were divided into 204 working zones. For initial planning, a mean human:dog ratio from the literature enabled estimation of dog population size and dog surveys were then performed in 29 working zones in order to assess dog distribution by land type. Vaccination was conducted at static point stations at weekends, at a total of 44 sites, with each operating for an average of 1.3 days. On Monday to Wednesday, door-to-door vaccination sessions were undertaken in the areas surrounding the preceding static point stations. 23,442 dogs were vaccinated at static point stations and 11,774 dogs were vaccinated during door-to-door vaccinations. At the end of the 20 day vaccination programme, an assessment of vaccination coverage through door-to-door surveys found that of 10,919 dogs observed, 8,661 were vaccinated resulting in a vaccination coverage of 79.3% (95%CI 78.6-80.1%). The estimated human:dog ratio for Blantyre city was 18.1:1. Mobile technology facilitated the collection of data as well as efficient direction and coordination of vaccination teams in near real time. This study demonstrates the feasibility of vaccinating large numbers of dogs at a high vaccination coverage, over a short time period in a large African city