Soon after their discovery in the early 20th century, bacteriophages were recognized to have great potential as antimicrobial agents, a potential that has yet to be fully realized. The nascent field ...of phage therapy was adversely affected by inadequately controlled trials and the discovery of antibiotics. Although the study of phages as anti-infective agents slowed, phages played an important role in the development of molecular biology. In recent years, the increase in multidrug-resistant bacteria has renewed interest in the use of phages as antimicrobial agents. With the wide array of possibilities offered by genetic engineering, these bacterial viruses are being modified to precisely control and detect bacteria and to serve as new sources of antibacterials. In applications that go beyond their antimicrobial activity, phages are also being developed as vehicles for drug delivery and vaccines, as well as for the assembly of new materials. This review highlights advances in techniques used to engineer phages for all of these purposes and discusses existing challenges and opportunities for future work.
The recent nomination by the World Health Organization of Acinetobacter baumannii as the number one priority pathogen for the development of new antibiotics is a direct consequence of its fast ...evolution of pathogenicity, and in particular of multidrug resistance. While the development of new antibiotics is critical, understanding the mechanisms behind the crescent bacterial antibiotic resistance is equally relevant. Often, resistance and other bacterial virulence elements are contained on highly mobile pieces of DNA that can easily spread to other bacteria. Prophages are one of the mediators of this form of gene transfer, and have been frequently found in bacterial genomes, often offering advantageous features to the host. Here we assess the contribution of prophages for the evolution of A. baumannii pathogenicity. We found prophages to be notably diverse and widely disseminated in A. baumannii genomes. Also remarkably, A. baumannii prophages encode for multiple putative virulence factors that may be implicated in the bacterium's capacity to colonize host niches, evade the host immune system, subsist in unfavorable environments, and tolerate antibiotics. Overall our results point towards a significant contribution of prophages for the dissemination and evolution of pathogenicity in A. baumannii, and highlight their clinical relevance.
Peptidoglycan degrading enzymes are of increasing interest as antibacterial agents, especially against multi-drug resistant pathogens. Herein we present a review about the biological features of ...virion-associated lysins and endolysins, phage-derived enzymes that have naturally evolved to compromise the bacterial peptidoglycan from without and from within, respectively. These natural features may determine the adaptability of the enzymes to kill bacteria in different environments. Endolysins are by far the most studied group of peptidoglycan-degrading enzymes, with several studies showing that they can exhibit potent antibacterial activity under specific conditions. However, the lytic activity of most endolysins seems to be significantly reduced when tested against actively growing bacteria, something that may be related to fact that these enzymes are naturally designed to degrade the peptidoglycan from within dead cells. This may negatively impact the efficacy of the endolysin in treating some infections in vivo. Here, we present a critical view of the methods commonly used to evaluate in vitro and in vivo the antibacterial performance of PG-degrading enzymes, focusing on the major hurdles concerning in vitro-to-in vivo translation.
Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and nowadays, a common infection can be ...fatal. To address this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since Pseudomonas aeruginosa is one of the most common causes of health care-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review further addresses the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.
In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically ...engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.
Due to the rise of multidrug-resistant infections in humans, phage therapy is gaining renewed attention in Western medicine. Despite the increasing number of publications focussed on the isolation, ...characterization and in vitro performance of different phages, there is still a lack of concise pre-clinical information to guide the application of phage therapy in clinical practice. Nevertheless, over the last decade, efforts have been made to conduct more detailed studies of the in vivo efficacy of phages. Here, we review the most relevant in vivo studies performed in the last decade covering phage efficacy in both preclinical and clinical trials. We compare different routes of administration, dosage effect and different animal models of distinct types of infections. Moreover, insights into case studies and results from clinical trials are presented. Challenges and limitations of phage use as evidenced by the current state of research are also discussed in order to improve both the trustworthiness and success of the implementation of phage therapy.
An innovative delivery system based on bacteriophages-loaded alginate-nanohydroxyapatite hydrogel was developed as a multifunctional approach for local tissue regeneration and infection prevention ...and control. Bacteriophages were efficiently encapsulated, without jeopardizing phage viability and functionality, nor affecting hydrogel morphology and chemical composition. Bacteriophage delivery occurred by swelling-disintegration-degradation process of the alginate structure and was influenced by environmental pH. Good tissue response was observed following the implantation of bacteriophages-loaded hydrogels, sustaining their biosafety profile. Bacteriophages-loaded hydrogels did not affect osteoblastic cells' proliferation and morphology. A strong osteogenic and mineralization response was promoted through the implantation of hydrogels system with nanohydroxyapatite. Lastly, bacteriophages-loaded hydrogel showed excellent antimicrobial activity inhibiting the attachment and colonization of multidrug-resistant E. faecalis surrounding and within femoral tissues. This new local delivery approach could be a promising approach to prevent and control bacterial contamination during implantation and bone integration.
An innovative delivery system based on bacteriophages-loaded alginate-nanohydroxyapatite hydrogel was developed as a multifunctional approach to prevent and control bacterial contamination, given the local delivery of phages and their ability to replicate at the site of infection, during the implantation and bone integration, promoted by nanoHA particles. Display omitted
Escherichia coli and Salmonella Enteritidis are foodborne pathogens forming challenging biofilms that contribute to their virulence, antimicrobial resistance, and survival on surfaces. Interspecies ...interactions occur between species in mixed biofilms promoting different outcomes to each species. Here we describe the interactions between E. coli and S. Enteritidis strains, and their control using specific phages. Single-species biofilms presented more cells compared to dual-species biofilms. The spatial organization of strains, observed by confocal microscopy, revealed similar arrangements in both single- and dual-species biofilms. The EPS matrix composition, assessed by Fourier-transform infrared spectroscopy, disclosed that the spectra extracted from the different dual-species biofilms can either be a combination of both species EPS matrix components or that the EPS matrix of one species predominates. Phages damaged more the single-species biofilms than the mixed biofilms, showing also that the killing efficacy was greatly dependent on the phage growth characteristics, bacterial growth parameters, and bacterial spatial distribution in biofilms. This combination of methodologies provides new knowledge of species-species and phage-host interactions in biofilms of these two major foodborne pathogens.
A total of 179 Shiga toxin-producing Escherichia coli (STEC) complete genomes were analyzed in terms of serotypes, prophage coding regions, and stx gene variants and their distribution. We further ...examined the genetic diversity of Stx-converting phage genomes (Stx phages), focusing on the lysis-lysogeny decision and lytic cassettes.
We show that most STEC isolates belong to non-O157 serotypes (73 %), regardless the sources and geographical regions. While the majority of STEC genomes contain a single stx gene (61 %), strains containing two (35 %), three (3 %) and four (1 %) stx genes were also found, being stx2 the most prevalent gene variant. Their location is exclusively found in intact prophage regions, indicating that they are phage-borne. We further demonstrate that Stx phages can be grouped into four clusters (A, B, C and D), three subclusters (A1, A2 and A3) and one singleton, based on their shared gene content. This cluster distribution is in good agreement with their predicted virion morphologies. Stx phage genomes are highly diverse with a vast number of 1,838 gene phamilies (phams) of related sequences (of which 677 are orphams i.e. unique genes) and, although having high mosaicism, they are generally organized into three major transcripts. While the mechanisms that guide lysis-lysogeny decision are complex, there is a strong selective pressure to maintain the stx genes location close to the lytic cassette composed of predicted SAR-endolysin and pin-holin lytic proteins. The evolution of STEC Stx phages seems to be strongly related to acquiring genetic material, probably from horizontal gene transfer events.
This work provides novel insights on the genetic structure of Stx phages, showing a high genetic diversity throughout the genomes, where the various lysis-lysogeny regulatory systems are in contrast with an uncommon, but conserved, lytic system always adjacent to stx genes.
Abstract
The emergence of the biopharmaceutical industry represented a major revolution for modern medicine, through the development of recombinant therapeutic proteins that brought new hope for many ...patients with previously untreatable diseases. There is a ever-growing demand for these therapeutics that forces a constant technological evolution to increase product yields while simultaneously reducing costs. However, the process changes made for this purpose may also affect the quality of the product, a factor that was initially overlooked but which is now a major focus of concern. Of the many properties determining product quality, glycosylation is regarded as one of the most important, influencing, for example, the biological activity, serum half-life and immunogenicity of the protein. Consequently, monitoring and control of glycosylation is now critical in biopharmaceutical manufacturing and a requirement of regulatory agencies. A rapid evolution is being observed in this context, concerning the influence of glycosylation in the efficacy of different therapeutic proteins, the impact on glycosylation of a diversity of parameters/processes involved in therapeutic protein production, the analytical methodologies employed for glycosylation monitoring and control, as well as strategies that are being explored to use this property to improve therapeutic protein efficacy (glycoengineering). This work reviews the main findings on these subjects, providing an up-to-date source of information to support further studies.
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