Viability, Stability and Biocontrol Activity in Planta of Specific Ralstonia solanacearum Bacteriophages after Their Conservation Prior to Commercialization and Use.

Ralstonia solanacearum is a pathogen that causes bacterial wilt producing severe damage in staple solanaceous crops. Traditional control has low efficacy and/or environmental impact. Recently, the bases of a new biotechnological method by lytic bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with specific activity against R. solanacearum were established. However, some aspects remain unknown, such as the survival and maintenance of the lytic activity after submission to a preservation method as the lyophilization. To this end, viability and stability of lyophilized vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 and their capacity for bacterial wilt biocontrol have been determined against one pathogenic Spanish reference strain of R. solanacearum in susceptible tomato plants in different conditions and making use of various cryoprotectants. The assays carried out have shown satisfactory results with respect to the viability and stability of the bacteriophages after the lyophilization process, maintaining high titers throughout the experimental period, and with respect to the capacity of the bacteriophages for the biological control of bacterial wilt, controlling this disease in more than 50% of the plants. The results offer good prospects for the use of lyophilization as a conservation method for the lytic bacteriophages of R. solanacearum in view of their commercialization as biocontrol agents.

Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types.

To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).

Sewage-specific enterococcal bacteriophages and multiple water quality parameters for coastal water quality assessment.

Coastal water quality is deteriorating worldwide. Water quality monitoring is therefore essential for public health risk evaluation and the management of water bodies. This study investigated the feasibility of using bacteriophages of Enterococcus faecalis as sewage-specific faecal indicators, together with physicochemical (dissolved oxygen, pH, temperature and total suspended solids) and biological parameters, to assess coastal water quality using multivariate analysis incorporating non-detects. The principal component and cluster analyses demonstrated that coastal water quality was mostly influenced by biological parameters, including Escherichia coli and total coliforms, which were found in all 31 sampling sites, and enterococci, which was found in all but two sampling sites. The enterococcal bacteriophages AIM06 and SR14 were detected in 17 and 18 samples at concentrations up to 1,815 and 2,790 PFU/100 mL, respectively. Both bacteriophages co-presented in approximately 80% of phage-positive samples, and the concentrations at each site were not significantly different. Overall, either bacteriophage could be used to differentiate high- and low-level coastal water pollution, as grouped by cluster analysis. This study is the first to investigate the suitability of sewage-specific bacteriophages of E. faecalis for monitoring coastal water quality and emphasises the importance of a multivariate analysis with non-detects to facilitate coastal water quality monitoring and management.

Bacteriophage production processes.

High quantities of bacteriophages are currently used in the food industry and agriculture. However, growing antibiotic resistance of bacteria has recently awakened the interest to use bacteriophages for the treatment of bacterial infections in humans indicating that even higher quantities will be required in the future. High demand combined with a wide range of applications requires also efficient bacteriophage production processes operating at low production costs and with high productivity. To achieve this goal, different approaches were introduced and extensive studies of various parameters affecting bacteriophage formation were investigated. In this mini-review, we provide a short overview about different operation modes of bacteriophage production such as batch, semi-continuous and especially continuous with the pros and cons of each. We present factors affecting bacterial physiological state, its effect on phage formation and provide a description of methods for determination of bacteriophage growth parameters, through which bacteriophage formation is obtained. Understanding of described phenomena and inclusion of potential occurrence of mutations and selection in continuous systems enables evaluation of continuous process productivity and its optimization.

Bacteriophage Clinical Use as Antibacterial "Drugs": Utility and Precedent.

For phage therapy-the treatment of bacterial infections using bacterial viruses-a key issue is the conflict between apparent ease of clinical application, on the one hand, and on the other hand, numerous difficulties that can be associated with undertaking preclinical development. These conflicts between achieving efficacy in the real world versus rigorously understanding that efficacy should not be surprising because equivalent conflicts have been observed in applied biology for millennia: exploiting the inherent, holistic tendencies of useful systems, e.g., of dairy cows, inevitably is easier than modeling those systems or maintaining effectiveness while reducing such systems to isolated parts. Trial and error alone, in other words, can be a powerful means toward technological development. Undertaking trial and error-based programs, especially in the clinic, nonetheless is highly dependent on those technologies possessing both inherent safety and intrinsic tendencies toward effectiveness, but in this modern era we tend to forget that ideally there would exist antibacterials which could be thus developed, that is, with tendencies toward both safety and effectiveness, and which are even relatively inexpensive. Consequently, we tend to demand rigor as well as expense of development even to the point of potentially squandering such utility, were it to exist. In this review I lay out evidence that in phage therapy such potential, in fact, does exist. Advancement of phage therapy unquestionably requires effective regulation as well as rigorous demonstration of efficacy, but after nearly 100 years of clinical practice, perhaps not as much emphasis on strictly laboratory-based proof of principle.

Numbers of coliforms, Escherichia coli, F-RNA phage, rotavirus, bovine enteric calicivirus and presence of non-O157 STEC on commercial vacuum packaged beef.

The numbers of coliforms, Escherichia coli, F-RNA coliphages, bovine enteric calicivirus (BEC) and rotavirus (RV) and presence of non-O157 shiga toxigenic E. coli (STEC) were determined on commercial vacuum packaged beef subprimals at the retail level from swabs obtained from the entire surfaces of 150 cuts that originated from federally and provincially registered plants. The prevalence and log mean numbers of E. coli were higher in provincially registered plants than in federally registered plants; 64% vs 20%, respectively, and -0.3 vs -1.22 log cfu/100 cm(2), respectively. In contrast, the prevalence and mean log numbers of F-RNA coliphages were lower for the provincially registered plants than for the federally registered plants; 31% vs 68% and -0.86 vs -0.13 log cfu/100 cm(2), respectively. One E. coli sample tested positive for stx2 and eae. F-RNA coliphages associated with human origin (GII/GIII) were detected in 12% and 30% of samples that originated from provincially and federally registered plants, respectively. RV RNA was detected in 4% of samples while BEC RNA was not detected. Although the infectivity of RV is unknown, the presence of viable F-RNA coliphages suggests that consumers could potentially be at risk when consuming undercooked meat that is contaminated with RV.

Application of microbiological quantitative methods for evaluation of changes in the amount of bacteria in patients with wounds and purulent fistulas subjected to phage therapy and for assessment of phage preparation effectiveness (in vitro studies).

PURPOSE: Quantitative microbiological studies may provide important information required for successful phage therapy (PT), however methods for PT monitoring of purulent wounds and fistulas has never been reported before. Therefore our goal was to determine and apply microbiological quantitative methods (MQMs) for monitoring experimental PT. METHODS: Samples from agar plates with growing bacteria were collected using dry and wet sterile compresses, or swabs. After shaking the sample in saline the amount of bacteria in suspension was determined. The method was standardized. The MQM using compress was applied for comparison of in vitro activity of phage preparations with other agents for wound rinsing. The usefulness of this swabbing method was tested in the Phage Therapy Unit for monitoring of experimental PT of patients with chronic wounds or purulent fistulas. RESULTS: Minimum, maximum and standard deviation values used for standardization of the studied method showed that data repeatability was good; thus the method was used for quantitation of bacteria taken both from plates in vitro and patients samples. Effectiveness of phage preparations was compared to gentamicin in vitro. Phages were as effective as antibiotics in reducing the amount of bacteria on agar plates, and this effect was not only due to simple mechanical removal of bacteria, but dependent on their antibacterial activity. We have also observed that the results of bacteria quantitation may correlate with the local status of a wound/fistula in a particular stage of PT. CONCLUSION: The standardized swabbing method of bacteria quantitation can be used for PT monitoring. Presented MQMs are simple and may help to monitor the therapy process and to decide on its duration, frequency and a kind of the phage applied. They can also be applied in other antibacterial treatment strategies.

Physical model of the immune response of bacteria against bacteriophage through the adaptive CRISPR-Cas immune system.

Bacteria and archaea have evolved an adaptive, heritable immune system that recognizes and protects against viruses or plasmids. This system, known as the CRISPR-Cas system, allows the host to recognize and incorporate short foreign DNA or RNA sequences, called 'spacers' into its CRISPR system. Spacers in the CRISPR system provide a record of the history of bacteria and phage coevolution. We use a physical model to study the dynamics of this coevolution as it evolves stochastically over time. We focus on the impact of mutation and recombination on bacteria and phage evolution and evasion. We discuss the effect of different spacer deletion mechanisms on the coevolutionary dynamics. We make predictions about bacteria and phage population growth, spacer diversity within the CRISPR locus, and spacer protection against the phage population.

Assessment of Escherichia coli O157:H7-specific bacteriophages e11/2 and e4/1c in model broth and hide environments.

The efficacy of bacteriophages e11/2 and e4/1c as potential biocontrol agents for Escherichia coli O157:H7 in food applications was assessed under conditions relevant to the food chain environment. The stability of each phage was determined following exposure to varying environmental conditions (pH, temperature, water activity, and sodium chloride) and the ability of each phage to infect and reduce E. coli O157:H7 numbers under selected conditions was also examined. Both e11/2 and e4/1c significantly (p<0.05) reduced numbers of E. coli O157:H7 when exposed to pH values ranging from pH>4 to pH 9, temperatures from 4 °C to 37 °C, water activity values of 0.87 or 0.91 to 1.00 and NaCl concentrations of 1% to 2.5%. Subsequently, a cocktail of both phages was used (e11/2 and e4/1c) to assess reduction of E. coli O157:H7 on cattle hide pieces. This involved inoculating pieces of hide (20×20 cm) with E. coli O157:H7 (approximately 106cfu/cm²) which were subsequently treated with either a suspension of a phage cocktail, consisting of e11/2 and e4/1c (multiplicity of infection of 1000 and 10,000, respectively) or water or not treated. Two different investigations were carried out; immediately or 1h after treatment application was performed in different experiments. Swab samples taken immediately after phage treatment showed no significant (p>0.05) reduction of E. coli O157:H7 numbers compared to the water treated or untreated samples. However, an extended exposure time of 1h following phage application revealed a significant reduction (p<0.05) (1.5 log10 cfu/cm² reduction) in E. coli O157:H7 numbers compared to the numbers recovered on samples treated with water only. These findings demonstrate the potential use of e11/2 and e4/1c phages as a biocontrol agent for E. coli O157:H7 within various stages of the food chain, including on cattle hide.

A colorimetric microtiter plate method for assessment of phage effect on Pseudomonas aeruginosa biofilm.

Bacteriophages have a potential in biofilm control. The aim of the study was to develop a method for selection of the most effective Pseudomonas aeruginosa phages for inhibition of biofilm formation and its eradication. The microtiter plate method is based on crystal violet staining and measuring of optical density.