Viruses of bacteria, known as bacteriophages or phages, were discovered nearly 100 years ago. Their potential as antibacterial agents was appreciated almost immediately, with the first ‘phage therapy’ trials predating Fleming’s discovery of penicillin by approximately a decade. In this review, we consider phage therapy that can be used for treating bacterial infections in humans, domestic animals and even biocontrol in foods. Following an overview of the topic, we explore the common practice – both experimental and, in certain regions of the world, clinical – of mixing therapeutic phages into cocktails consisting of multiple virus types. We conclude with a discussion of the commercial and medical context of phage cocktails as therapeutic agents. In comparing off-the-shelf versus custom approaches, we consider the merits of a middle ground, which we deem ‘modifiable’. Finally, we explore a regulatory framework for such an approach based on an influenza vaccine model.

Phage cocktails and the future of phage therapy

Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology.

/pdf/bacteriophage-endolysins-as-novel-antimicrobials-3jmtpjl7y2.pdf

Bacteriophage endolysins as novel antimicrobials

Bacteriophage (phage) therapy, i.e., the use of viruses that infect bacteria as antimicrobial agents, is a promising alternative to conventional antibiotics. Indeed, resistance to antibiotics has become a major public health problem after decades of extensive usage. However, one of the main questions regarding phage therapy is the possible rapid emergence of phage-resistant bacterial variants, which could impede favourable treatment outcomes. Experimental data has shown that phage-resistant variants occurred in up to 80% of studies targeting the intestinal milieu and 50% of studies using sepsis models. Phage-resistant variants have also been observed in human studies, as described in three out of four clinical trials that recorded the emergence of phage resistance. On the other hand, recent animal studies suggest that bacterial mutations that confer phage-resistance may result in fitness costs in the resistant bacterium, which, in turn, could benefit the host. Thus, phage resistance should not be underestimated and efforts should be made to develop methodologies for monitoring and preventing it. Moreover, understanding and taking advantage of the resistance-induced fitness costs in bacterial pathogens is a potentially promising avenue.

https://serval.unil.ch/resource/serval:BIB_6572B8A55D64.P001/REF.pdf

Resistance Development to Bacteriophages Occurring during Bacteriophage Therapy

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.

/pdf/genetically-engineered-phages-a-review-of-advances-over-the-4adrx3lanr.pdf

Genetically Engineered Phages: a Review of Advances over the Last Decade

Bacteriophages can influence the abundance, diversity, and evolution of bacterial communities. Several bacteriophages have been reported to add virulence factors to their host and to increase bacterial virulence. However, lytic bacteriophages can also exert a selective pressure allowing the proliferation of strains with reduced virulence. This reduction can be explained because bacteriophages use structures present on the bacterial surface as receptors, which can be virulence factors in different bacterial species. Therefore, strains with modifications in these receptors will be resistant to bacteriophage infection and may also exhibit reduced virulence. This mini-review summarizes the reports on bacteriophage-resistant strains with reductions in virulence, and it discusses the potential consequences in phage therapy and in the use of bacteriophages to select attenuated strains for vaccines.

/pdf/virulence-reduction-in-bacteriophage-resistant-bacteria-3ht8zd42ab.pdf

Virulence reduction in bacteriophage resistant bacteria

Background 
Bacteriophage could be an alternative to conventional antibiotic therapy against multidrug-resistant bacteria. However, the emergence of resistant variants after phage treatment limited its therapeutic application.
Methodology/Principal Findings 
In this study, an approach, named “Step-by-Step” (SBS), has been established. This method takes advantage of the occurrence of phage-resistant bacteria variants and ensures that phages lytic for wild-type strain and its phage-resistant variants are selected. A phage cocktail lytic for Klebsiella pneumoniae was established by the SBS method. This phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3) which have different but overlapping host strains. Several phage-resistant variants of Klebsiella pneumoniae were isolated after different phages treatments. The virulence of these variants was much weaker [minimal lethal doses (MLD)>1.3×109 cfu/mouse] than that of wild-type K7 countpart (MLD = 2.5×103 cfu/mouse). Compared with any single phage, the phage cocktail significantly reduced the mutation frequency of Klebsiella pneumoniae and effectively rescued Klebsiella pneumoniae bacteremia in a murine K7 strain challenge model. The minimal protective dose (MPD) of the phage cocktail which was sufficient to protect bacteremic mice from lethal K7 infection was only 3.0×104 pfu, significantly smaller (p<0.01) than that of single monophage. Moreover, a delayed administration of this phage cocktail was still effective in protection against K7 challenge.
Conclusions/Significance 
Our data showed that the phage cocktail was more effective in reducing bacterial mutation frequency and in the rescue of murine bacteremia than monophage suggesting that phage cocktail established by SBS method has great therapeutic potential for multidrug-resistant bacteria infection.

/pdf/a-method-for-generation-phage-cocktail-with-great-2znovb8t11.pdf

A method for generation phage cocktail with great therapeutic potential.

GH15 is a polyvalent phage that shows activity against a wide range of Staphylococcus aureus strains. In this work, the complete genome sequence of GH15 was determined. With a genome size of 139,806 bp (double-stranded DNA), GH15 is the largest staphylococcal phage sequenced to date. The complete genome encodes 214 open reading frames (ORFs) and 4 tRNAs. The closest relatives are the class III staphylococcal myobacteriophages, including K, A5W, ISP, Sb-1, and G1. Interestingly, although corresponding gene sequences demonstrate very high similarity, all the introns and inteins present in the phages listed above are absent in GH15. As such, GH15 can be considered phylogenetically unique among the staphylococcal myobacteriophages, indicating the diversity of this family.

Complete genome sequence of Staphylococcus aureus bacteriophage GH15.

Phage GH15 is a polyvalent phage that shows activity against a wide range of Staphylcoccus aureus strains. This study analysed the genome of GH15. The genome size of GH15 (139 806 bp) was found to be larger than that of the known staphylococcal phages, and the G+C content (30.23 mol%) of GH15 was lower than that of any other staphylococcal myovirus phages. By mass spectrometry, ten structural proteins were identified. Analysis revealed that GH15 was closely related to phages G1, ISP, A5W, Sb-1 and K, and was moderately related to Twort. In light of the variability in identity, coverage, G+C content and genome size, coupled with the large number of mosaicisms, there certainly were close evolutionary relationships from K to Sb-1, A5W, ISP, G1 and finally GH15. Interestingly, all the introns and inteins present in the above phages were absent in GH15 and there appeared to be intron loss in GH15 compared with the intron gain seen in other phages. A comparison of the intron- and intein-related genes demonstrated a clear distinction in the location of the insertion site between intron-containing and intron-free alleles, and this might lead to the establishment of a consensus sequence associated with the presence of an intron or intein. The comparative analysis of the GH15 genome sequence with other phages not only provides compelling evidence for the diversity of staphylococcal myovirus phages but also offers new clues to intron shift in phages.

Genomic characterization of lytic Staphylococcus aureus phage GH15: providing new clues to intron shift in phages.

LysGH15, a phage endolysin, exhibits a particularly broad lytic spectrum against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). Sequence analysis reveals that this endolysin contains a C-terminal cell wall binding domain (SH3b), which causes the endolysin to bind to host strains. In this study, the substrate binding affinity of the SH3b domain (LysGH15B) was evaluated. A fusion protein of LysGH15B and green fluorescent protein (LysGH15B-GFP) were cloned and expressed in Escherichia coli. Laser scanning confocal microscopy was used to detect the fluorescence of the treated cells irradiated at different excitation wavelengths and to determine the binding activity of LysGH15B-GFP and GFP. We found that LysGH15B-GFP not only generated green fluorescence, but, more importantly, also displayed specific affinity to staphylococcal isolates, especially MRSA. In contrast, the single GFP did not display any binding activity. The high affinity was attributed to the portion of LysGH15B and the binding activity of the fusion protein was specific to staphylococci. This study provides an insight into the SH3b domain of LysGH15. The specific binding activity may cause LysGH15B to serve as an anchoring device, and offer an alternative approach for cell surface attachment onto staphylococci.

LysGH15B, the SH3b domain of staphylococcal phage endolysin LysGH15, retains high affinity to staphylococci.

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A method for generation phage cocktail with great therapeutic potential.

Complete genome sequence of Staphylococcus aureus bacteriophage GH15.

Genomic characterization of lytic Staphylococcus aureus phage GH15: providing new clues to intron shift in phages.

LysGH15B, the SH3b domain of staphylococcal phage endolysin LysGH15, retains high affinity to staphylococci.