Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r=0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.This work was supported by the Portuguese Science Foundation [project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124- FEDER-027462)], [project IF/01413/2013]; and DNA mimics [ref. PIC/IC/82815/ 2007] from Fundação para a Ciência e Tecnologia (FCT) and Ministério da Ciência, Tecnologia e Ensino Superior (MCTES)

Bacteriophages to control Shiga toxin-producing E. coli safety and regulatory challenges

Shiga toxin-producing Escherichia coli (STEC) are usually found on food products due to contamination from the fecal origin, as their main environmental reservoir is considered to be the gut of ruminants. While this pathogen is far from the incidence of other well-known foodborne bacteria, the severity of STEC infections in humans has triggered global concerns as far as its incidence and control are concerned. Major control strategies for foodborne pathogens in food-related settings usually involve traditional sterilization/disinfection techniques. However, there is an increasing need for the development of further strategies to enhance the antimicrobial outcome, either on food-contact surfaces or directly in food matrices. Phages are considered to be a good alternative to control foodborne pathogens, with some phage-based products already cleared by the Food and Drug Administration (FDA) to be used in the food industry. In European countries, phage-based food decontaminants have already been used. Nevertheless, its broad use in the European Union is not yet possible due to the lack of specific guidelines for the approval of these products. Furthermore, some safety concerns remain to be addressed so that the regulatory requirements can be met. In this review, we present an overview of the main virulence factors of STEC and introduce phages as promising biocontrol agents for STEC control. We further present the regulatory constraints on the approval of phages for food applications and discuss safety concerns that are still impairing their use.The authors thank the Portuguese Foundation for Scienceand Technology (FCT) through the strategic funding of UID/BIO/04469/2019 unit, and the project PhageSTEC [PTDC/CVT-CVT/29628/2017], under the scope of COMPETE 2020 [POCI-01-0145-FEDER-029628]. The author GP acknowledges theFCT grant [SFRH/BD/117365/2016].info:eu-repo/semantics/publishedVersio

Phage engineering: how advances in molecular biology and synthetic biology are being utilized to enhance the therapeutic potential of bacteriophages

Background The therapeutic potential of bacteriophages has been debated since their first isolation and characterisation in the early 20th century. However, a lack of consistency in application and observed efficacy during their early use meant that upon the discovery of antibiotic compounds research in the field of phage therapy quickly slowed. The rise of antibiotic resistance in bacteria and improvements in our abilities to modify and manipulate DNA, especially in the context of small viral genomes, has led to a recent resurgence of interest in utilising phage as antimicrobial therapeutics. Results In this article a number of results from the literature that have aimed to address key issues regarding the utility and efficacy of phage as antimicrobial therapeutics utilising molecular biology and synthetic biology approaches will be introduced and discussed, giving a general view of the recent progress in the field. Conclusions Advances in molecular biology and synthetic biology have enabled rapid progress in the field of phage engineering, with this article highlighting a number of promising strategies developed to optimise phages for the treatment of bacterial disease. Whilst many of the same issues that have historically limited the use of phages as therapeutics still exist, these modifications, or combinations thereof, may form a basis upon which future advances can be built. A focus on rigorous in vivo testing and investment in clinical trials for promising candidate phages may be required for the field to truly mature, but there is renewed hope that the potential benefits of phage therapy may finally be realised