Bookworm_88
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Hi. I'm doing a project on biomining (in space) for science and i want to know what sort of questions I can formulate around it. I'm also wondering whether its a dead end?
THANKS😀
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Bookworm_88
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Im doing a project for science on biomining. I wanted some suggestions on possible questions and whether its a dead end?
Thanks😀
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Bookworm_88
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Im doing a project for science and want to do it on biomining (in space) and I need some help in formulating some research ideas. I also need some advice on whether its a dead end?
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Bookworm_88
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I wanted to do a project on biomining, and maybe its use in space?( not 100% sure on that last bit) and i needed some help on whether this topic would be a dead end and whether there's enough sources to use.
THANKS!😀
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TSR Jessica
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Sorry you've not had any responses about this. Are you sure you've posted in the right place? Here's a link to our subject forum which should help get you more responses if you post there.
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Bookworm_88
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(Original post by TSR Jessica)
Sorry you've not had any responses about this. Are you sure you've posted in the right place? Here's a link to our subject forum which should help get you more responses if you post there.
Thanks!
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Bookworm_88
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Hi. I need some help, I’m doing a project on bacteriophages (viruses that replicate and burst out of bacteria cells, thus killing the bacterial pathogenic cell. Or incorporate its genome with that of the Host cell and take over that host cell)
I need some Reading material upon either bacterio(phages) or phage therapy. THE MORE THE BETTER!
Thanks
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Bookworm_88
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Hi. I need some help, I’m doing a project on bacteriophages (viruses that replicate and burst out of bacteria cells, thus killing the bacterial pathogenic cell. Or incorporate its genome with that of the Host cell and take over that host cell)
I need some Reading material upon either bacterio(phages) or phage therapy. THE MORE THE BETTER!
Thanks
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Bookworm_88
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Hi. I need some help, I’m doing a project on bacteriophages (viruses that replicate and burst out of bacteria cells, thus killing the bacterial pathogenic cell. Or incorporate its genome with that of the Host cell and take over that host cell)
I need some Reading material upon either bacterio(phages) or phage therapy. THE MORE THE BETTER!
Thanks
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whizzer_wins
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Found these on the Internet and they all seem pretty legit and are free to use (I think) - good luck with the project!
https://www.khanacademy.org/science/...bacteriophages
https://www.britannica.com/science/bacteriophage
https://www.frontiersin.org/articles...019.00513/full
https://virologyj.biomedcentral.com/.../1743-422X-9-9
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becausethenight
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Possibly not the most helpful thing ever, but have you already searched pubmed for "bacteriophage"/"phage therapy"? If you apply the free full text and review filter (as here: https://pubmed.ncbi.nlm.nih.gov/term...er=pubt.review) you'll get summary articles that you can read without having journal subscriptions!
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Bookworm_88
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Thank you for the websites, greatly appreciated, as my internet is currently down meaning it’s a nightmare to do research and My research has been not as fruitful as it could’ve been as all of the info I’ve found is too specialist for me
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Bookworm_88
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(Original post by becausethenight)
Possibly not the most helpful thing ever, but have you already searched pubmed for "bacteriophage"/"phage therapy"? If you apply the free full text and review filter (as here: https://pubmed.ncbi.nlm.nih.gov/term...er=pubt.review) you'll get summary articles that you can read without having journal subscriptions!
Thanks for the advice, but the website link doesn’t come up with anything:confused:
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becausethenight
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(Original post by Bookworm_88)
Thanks for the advice, but the website link doesn’t come up with anything:confused:
Sorry, that might be because of the filters. Here's the link to just pubmed: https://pubmed.ncbi.nlm.nih.gov/
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yeahthatonethere
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One of the easiest ways to get this information is using journal articles. Google scholar is a good free search tool for this. If you want popular articles or only recent ones you can search by number of times cited and date published respectively. Additionally, review articles are very good at covering a lot of information in an understandable manner and are often easier to read and understand (especially if you aren't university level yet) and you can find further sources from these.

A good way to find quick sources of information is to go into Wikipedia, read the article until you find bits you're interested in and go into their citations to get more information.

This is a very broad area so it's hard to give specific material and it's not clear what sort of reading you're after (e.g. Journals, text books, websites - what you're allowed to use depends on your level such as school will probably be fine with websites but uni will mainly want journal articles). Hope this has helped anyway!
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macpatgh-Sheldon
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Evolution and the complexity of bacteriophagesPhilip Serwer*Address: Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas78229-3900, USAEmail: Philip Serwer* - [email protected]* Corresponding authorAbstractBackground: The genomes of both long-genome (> 200 Kb) bacteriophages and long-genomeeukaryotic viruses have cellular gene homologs whose selective advantage is not explained. Thesehomologs add genomic and possibly biochemical complexity. Understanding their significancerequires a definition of complexity that is more biochemically oriented than past empirically baseddefinitions.Hypothesis: Initially, I propose two biochemistry-oriented definitions of complexity: eitherdecreased randomness or increased encoded information that does not serve immediate needs.Then, I make the assumption that these two definitions are equivalent. This assumption and recentdata lead to the following four-part hypothesis that explains the presence of cellular gene homologsin long bacteriophage genomes and also provides a pathway for complexity increases in prokaryoticcells: (1) Prokaryotes underwent evolutionary increases in biochemical complexity after theeukaryote/prokaryote splits. (2) Some of the complexity increases occurred via multi-step, weakselection that was both protected from strong selection and accelerated by embedding evolvingcellular genes in the genomes of bacteriophages and, presumably, also archaeal viruses (first tierselection). (3) The mechanisms for retaining cellular genes in viral genomes evolved underadditional, longer-term selection that was stronger (second tier selection). (4) The second tierselection was based on increased access by prokaryotic cells to improved biochemical systems.This access was achieved when DNA transfer moved to prokaryotic cells both the more evolvedgenes and their more competitive and complex biochemical systems.Testing the hypothesis: I propose testing this hypothesis by controlled evolution in microbialcommunities to (1) determine the effects of deleting individual cellular gene homologs on thegrowth and evolution of long genome bacteriophages and hosts, (2) find the environmentalconditions that select for the presence of cellular gene homologs, (3) determine which, if any,bacteriophage genes were selected for maintaining the homologs and (4) determine the dynamicsof homolog evolution.Implications of the hypothesis: This hypothesis is an explanation of evolutionary leaps ingeneral. If accurate, it will assist both understanding and influencing the evolution of microbes andtheir communities. Analysis of evolutionary complexity increase for at least prokaryotes shouldinclude analysis of genomes of long-genome bacteriophages.Published: 13 March 2007Virology Journal 2007, 4:30 doi:10.1186/1743-422X-4-30Received: 22 December 2006Accepted: 13 March 2007This article is available from: http://www.virologyj.com/content/4/1/30© 2007 Serwer; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Virology Journal 2007, 4:30 http://www.virologyj.com/content/4/1/30Page 2 of 9(page number not for citation purposes)1. BackgroundEmpirical studies of the genomes of virusesBacteriophages and eukaryotic viruses with comparativelylong double-stranded DNA genomes have genes homologousto cellular genes. For illustrating the surprising characterof this observation, the shorter viral genomes serveas a baseline. Specifically, the shorter-genome, virulentdouble-stranded DNA bacteriophages, such as φ29(genome length = 19.3 Kb [1]), T3 (genome length = 38.2Kb [2]) and T7 (genome length = 39.9 Kb [2]), have genesmost of which are tightly packed. The φ29, T3 and T7genes with identified functions almost always have a rolein bacteriophage-specific biochemistry [1-3].The virulent bacteriophage T4 has a longer, 168 Kbgenome. The greater length of the T4 genome is explained,in part, by the more numerous components of T4 structure,especially the tail. However, not so easily explainedis the informatics-detected presence in the T4 genome ofhomologs of transfer RNA genes, genes for nucleotidemetabolism, DNA repair enzymes [4] and, in the case of aT4-related bacteriophage, genes for an NAD salvage pathway[5]; all informatics discussed here uses the genomicbase sequence as input. None of the T4 cellular genehomologs are present in the shorter genomes of φ29, T3and T7 [1-3].In an extension of this pattern, a larger collection of cellulargene homologs is informatics-detected in the evenlonger 280 Kb genome of bacteriophage φKZ [6]. Remarkableis the presence of φKZ genes that (a) encode enzymeswith a wide range of metabolic functions and (b) haveclosest homologs that are from bacteria that are not φKZhosts and that sometimes are distantly related to the φKZhost, Pseudomonas aeruginosa. These latter genes encodeseveral RNA polymerases, DNA repair proteins, cell divisionproteins and stringent starvation protein [6,7]. Thepresence of these genes is not well explained by directneed for the gene products in the process of bacteriophagereproduction, though the gene products can assist viruspropagation by assisting the host.The most frequently sequenced long viral genomes arefrom viruses with eukaryotic hosts. Again, the longeukaryotic viral genomes have cellular gene homologswhose presence in a viral genome is unexplained. Forexample, giant (313 – 415 Kb genome) phycodnavirusvirus genomes have informatics-detected, cellular genehomologs that include genes for tRNAs, ubiquitin, UVspecificDNA repair enzyme, transcriptional elongationfactor TFIIS, chitin synthase, RNA polymerase subunits,N-acetylglucosaminyl transferase and multiple enzymesin each of several metabolic pathways, including those forsynthesis of hyaluranan, sphingolipid, fucose, andpolyamines [8-10].The longest viral genome is the 1,200 Kb genome of thephycodnavirus-related mimivirus of Acanthamoeba polyphaga.Mimivirus also has the largest collection of cellulargene homologs. Informatics-detected mimivirus genesinclude homologs for 40 bacterial proteins and 46 eukaryoticcell proteins. The mimivirus genes include genes for 4aminoacyl tRNA synthetases, 33 enzymes of carbohydratemetabolism, 3 signaling receptors and several translationfactors among many other genes whose products mightassist virus propagation by assisting the host, but are notexpected to have virus-specific functions [9,11-14]. Conservationof a putative promoter sequence indicates thatthe gene products are made and functional [15]. The existenceof these genes in viral genomes is currently considereda major mystery because they increase the length ofviral genomes without producing any known selectiveadvantage [12].Theoretical frameworkA selective advantage must exist for the cellular genehomologs of long-genome viruses. To establish a theoreticalframework for determining what this selective advantageis, I make a first assumption that the cellular genehomologs of long-genome viruses introduce increasedcomplexity that is related, in some way, to increased complexityat the level of biochemistry. Next, I will use pastexperimental work to obtain a definition of complexitythat is applicable to biochemistry. This process led to adeparture from past thinking because, in the past, empiricallybased definitions of biological complexity havefocused on those properties of higher eukaryotes that canbe quantified either via length and randomness ofgenome sequence [16] or via simple characteristics ofstructure [17-21]. These latter definitions are not meant tobe fundamental to complexity at the level of biochemistry.In search of a fundamental definition of change in (notabsolute) biochemistry-based complexity, two well-investigatedexamples are considered here. Both examplesinvolve the transfer of genes to bacteria by bacteriophagevectors. The first example is bacterial gene transfer via bacteriophage-based generalized transduction. Generalizedtransduction happens randomly with regard to the genestransferred [22-24].The second example is bacterial gene transfer via bacteriophage-based lysogenic conversion. In contrast to generalizedtransduction, lysogenic conversion happens withspecificity for a specific gene that, based on past selections,will promote future invasion of a host by the convertedbacterial cell. The basis of the specificity includesencoded, evolutionary selection-derived memory of theusefulness of the gene product [25,26]. This encodedmemory-based specificity sometimes occurs by makingVirology Journal 2007, 4:30 http://www.virologyj.com/content/4/1/30Page 3 of 9(page number not for citation purposes)the gene product part of the bacteriophage particle. Examplesinclude hyaluronidase [27], as well as adhesion proteinsfor bacterial host attachment [25]. Thus, the encodedmemory-based specificity is biochemically complex inthat it comes from not only the product of the gene transferred,but also from other, interacting gene products.Note that information about the future is derived fromselection in past circumstances that mimic future circumstances.No other source of information is involved.From the above example, lysogenic conversion is morecomplex than generalized transduction by two definitionsof increased complexity: (a) decreased randomness thatdoes not serve immediate needs and (b) increasedencoded information that does not serve immediateneeds. Though these two definitions are not necessarilycompletely equivalent, the second assumption made hereis that the above two definitions of change in complexityare completely equivalent in content (equivalenceassumption). The second of these two definitions partiallyoverlaps the following previous definition proposed inthe context of the evolution of "digital organisms" [28]:encoded "information about the environment that can beused to make predictions about it".Blood clotting provides an empirical application and testof the equivalence assumption in the case of eukaryotes.Blood clotting is complex by the second definition, basedon the multiple factors and the cascade needed to initiateclotting. Blood clotting is also an event in which randomness(that will cause clotting either too rapid or too slow)is minimized [29,30]. Randomness in blood clotting is amajor selective disadvantage for survival.Late-evolving complexity of bacteriophage biochemistryAlthough the smaller bacteriophage genomes lack cellulargene homologs, some aspects of small bacteriophage multiplicationhave undergone recognizable increase in biochemicalcomplexity by the second definition of theprevious section. One such aspect is DNA packaging. Allknown double-stranded DNA bacteriophages produceprogeny by, first, assembling a DNA-free capsid (procapsid)and, then, binding and packaging the DNA genome.Figure 1a shows the initiation complex for packaging bacteriophageT3 DNA in a simplified in vitro system. In Figure1a, the DNA molecule binds a DNA-binding accessoryprotein (also called gp18) that binds a DNA packagingATPase, also called gp19. The DNA packaging ATPasebinds a 12-fold symmetric ring (connector) with an axialhole. The DNA molecule is subsequently packagedthrough this hole into a cavity of an outer protein shell(capsid) (reviews [1,31,32]). The structure of the capsidchanges during DNA packaging (not shown in Figure 1).Even though T3 in vitro DNA packaging is efficient withthe initiation complex of Figure 1a[31], the initiationcomplex used in vivo by both T3 and its close relative, T7,has more complexity. The additional complexity comesfrom packaging initiation in vivo that depends on transcriptionby a bacteriophage-encoded RNA polymerase(also called gp1 [33-35]) (illustrated in Figure 1b). At leastthree proteins (gp1, gp18, gp19) have encoded informationfor this interaction. Based on the equivalenceassumption, the additional complexity at the initiation ofpackaging (second definition of complexity) should providedecrease in the randomness of an event of the subsequentprocess of DNA packaging (first definition ofcomplexity).In this case, the literature already supports the equivalenceassumption by describing two possibilities for what thisevent is (both possibilities can be correct): (a) The firstpossibility is entry of the DNA molecule into the cavity ofthe capsid. The selective advantage is controlled (less random)initiation of entry so that entry events are not sonumerous that ATP is consumed to the point that nogenome completes packaging [32]. Evidence also existsfor complexity of this type at the level of the T7 DNA packagingprocess itself [32]. (b) The second possibility is terminationof packaging, an event that includes bothselective replication of a terminally repeated DNAsequence and cleavage of the genome from a longer, concatemericDNA molecule. The selective advantage is that agenome is not cleaved from a concatemer until replicationof its terminal repeat is completed [35].Furthermore, the complexity added by RNA polymerasedependenceof the initiation of T3/T7 DNA packaging wasa product of comparatively recent evolution, based on thefollowing two observations: (a) T3/T7 relatives exist thatdo not have the RNA polymerase in their genomes. Theserelatives are thought to be less evolved in their transcription[36,37]. (b) RNA polymerase-dependence of the initiationof DNA packaging has not yet been found in aeukaryotic virus, even though eukaryotic viruses havecommon ancestors with bacteriophages (below) and aremore intensely studied than bacteriophages. Thus, thechance is high that transcription dependence of T3/T7DNA packaging evolved after the split between bacteriaand eukarya, i.e., after about 1.6 billion years ago (review[38]). The data support the same conclusion for transferto archaea of bacterial chaperonin, hsp70. These datainclude the absence of hsp70 from many archaea (review[39]).Post-split evolution of prokaryotic complexity is a phenomenonoften overlooked during analysis focused oneukaryotes (see, for example ref. [21]). One reasonappears to be that non-adaptive expansion of genome sizeVirology Journal 2007, 4:30 http://www.virologyj.com/content/4/1/30Page 4 of 9(page number not for citation purposes)IFniigtiuatrieo n1 of DNA packaging by the closely related bacteriophages, T3 and T7Initiation of DNA packaging by the closely related bacteriophages, T3 and T7. (a) Initiation is illustrated for the simplest DNApackaging. This packaging has a monomeric DNA substrate and was demonstrated for T3 and assumed for T7. Packaging of thistype occurs only in vitro, as far as is known (review [31,32]). (b) Initiation is illustrated for the more complex DNA packagingthat occurs in vivo for both T3 and T7 (review [31,32]). In (b), the DNA substrate is an end-to-end joined concatemeric DNAfor which only one monomer is completely shown. Dashed lines in (b) indicate part of another monomer within the concatemer.The following details of the concatemer are omitted for simplicity: replication forks and interaction among different procapsids(review [32]). The various proteins and protein assemblies of the initiation complex, including the connector and DNApackaging ATPase, are identified in the rectangular box. Proteins have both descriptive names and names based on genenumber [2], preceded by gp. The letter, R, indicates the right end of the mature DNA molecule; the letter, L, indicates the leftend.Virology Journal 2007, 4:30 http://www.virologyj.com/content/4/1/30Page 5 of 9(page number not for citation purposes)is thought to be the dominant genome length-determiningtheme in eukaryotes [16]. This expansion is an entropicresponse to a low population density-inducedreduction of competition. Environmental populationdensities are not known for most bacteriophage strains.But, the number of bacteriophages produced per cell (typicallyover 100 [40]) and the total environmental bacteriophageconcentrations (108 – 109 per gm in soil [41,42])indicate that this type of non-adaptive genome expansionis unlikely in the case of bacteriophages. In support, longgenome bacteriophages, such as φKZ [6,7], have openreading frames highly compacted, as though under constantselection.Neither the evolution of post-split complexity nor thepresence of cellular gene homologs in the genomes oflong genome viruses is currently explained with a hypothesisthat can be tested. The hypothesis of the next sectionfills this intellectual gap. This hypothesis can be testedbecause of both short life cycles of bacteriophages andrecent advances in isolation and sequencing bacteriophagegenomes.2. A hypothesis for the selective advantage ofcellular gene homologs in long bacteriophagegenomesAlthough the above observations indicate that some postsplitincrease in biochemical complexity has occurred forbacteriophages, the following observations indicate thatsome basics evolved pre-split: structural similaritiesamong the outer shell proteins of bacteriophages,archaeal viruses and eukaryotic viruses [43-48]. The structuralsimilarity extends to the DNA packaging ATPases[49,50]. From these data, viral identity (also called viralself) is based on the secondary/tertiary/quaternary structureof the proteins that constitute the viral particle [44-46,51].Thus, the data indicate that post-split viruses are independentlyevolving and not simply post-split breakawaysfrom their hosts. Furthermore, the data indicate a predominantlyprokaryotic gene pool worldwide with more(about 10 ×) bacteriophages than bacteria (reviews [52-55]). Thus, the bacteriophage cellular gene homologsexist in the context of viral evolution that has the potentialfor major impact on prokaryotic cells.Together with the above data, the equivalence assumptionis used here to derive a hypothesis to explain the selectiveadvantage of the genomic complexity introduced by thepresence of cellular gene homologs in bacteriophages(second definition of complexity). The equivalenceassumption produces the conclusion that the selectiveadvantage is reduction of randomness (first definition ofcomplexity) of an event that both has and will occur forall of the wide-ranging host-like biochemical systemsencoded by these genes. The most fundamental aspect ofthe hypothesis presented here is that this event is proposedto be evolution itself, i.e., evolution of biochemicalsystems encoded by the genes of host bacteria and possiblyother bacteria that exchange DNA with the host. Thefollowing are the details of the hypothesis1) Increase in the biochemical complexity of prokaryoticcells and their viruses occurred after the eukaryote/prokaryote splits (support is above).(2) In the case of prokaryotic cells, the coding for at leastsome of this increase initially evolved not via genes in thecellular genome, but via host cellular gene homologs inthe genomes of long-genome, rapidly evolving prokaryoticviruses. The products of the host cellular genehomologs involved were not participants in bacteriophage-specific events, but did assist bacteriophage infectionby assisting the host. Thus, direct selective pressureoccurred for these genes to evolve, though the genes werenon-essential. The result was multi-step evolution inwhich intermediate steps did not necessarily provideenough selective advantage to survive life or death situations(first tier selection). However, the end products ofsome multi-step selections did provide this type of advantage,as discussed further in the next two paragraphs. Thecellular gene homologs of today's long-genome bacteriophagesare descendants of these earlier homologs. Apotential (not proven) ongoing example of an infectionassisting,viral genome-encoded cellular gene homolog isthe host photosynthetic gene, psbA, present in thegenomes of 8 of 9 sequenced cyanophages. The hostencodedpsbA gene product is subjected to rapid turnoverduring infection. The assumption is that expression of thebacteriophage gene compensates for the rapid turnover[56].(3) In addition to the multi-step first tier selection undergoneby the bacteriophage-associated cellular genehomologs, additional selection and evolution occurredfor the genes whose products maintained cellular genehomologs within a bacteriophage genome (second tierselection). The second tier selection caused the retentionand improvement of the first tier selection because of thelong-term selective advantage of multi-step evolution ofcomplex biochemical systems when transferred (ultimately)to the host. That is to say, selection for complexsystems was two-tiered. The first tier was based on immediate(classical), though potentially minor, short-rangeselective advantage at each step. The second tier was basedon long-range, major selective advantage that arose fromretaining and improving the first tier.Virology Journal 2007, 4:30 http://www.virologyj.com/content/4/1/30Page 6 of 9(page number not for citation purposes)(4) DNA exchange moved to prokaryotic hosts the biochemicalsystems encoded by cellular gene homologs inbacteriophage genomes. This exchange occurred repeatedlyand in both directions. The host cell occasionallyreceived a biochemical system of either (a) immediatemajor selective advantage or (b) major selective advantageafter additional mutation. In either case, introduction orreplacement of a major pathway in the host cell occurredand bacteriophage-associated host gene evolution hadprovided a major competitive advantage.The advantages of bacteriophage-based host evolutionwere the following: (a) Each bacteriophage gene duplicatedand, therefore, evolved at comparatively high ratewhen under selective pressure. Bacteriophages typicallyhave (and presumably had) a burst size of over 100 infectiveparticles produced in a time span of 0.5 – 2.0 hr [40].(b) Bacteriophages engaged in horizontal gene transferamong different hosts within microbial communities,thereby increasing the rate of evolution via geneticexchange [54,57]. (c) Since at least some cellular genehomologs were non-essential, multi-step evolutionary"leaps" in complexity occurred even if some of a leap'scomponent steps provided either no or only minor selectiveadvantage. This aspect resolves a vexing problem inconsidering evolutionary leaps in general. Computer-simulationhas shown the evolution of complex features viadigital mutations that produce intermediates that aresometimes neutral or even detrimental [58].3. Testing and feasibility of the prokaryotic viruscomplexity hypothesisFeasibilityThe prokaryotic virus complexity hypothesis is distinguishedby its second tier evolutionary selection that (a)yields bacteriophage-encoded biochemical systems thatfunction to retain non-essential genes for the first tier and(b) does so with a time delay because of the gene transferand possibly gene transformation events that occur beforethe selective advantage is realized. Although retention ofnon-essential genes initially might seem unlikely, bacteriophagesare already known to have systems to retain genesthat, while not cellular gene homologs, are non-essentialfor growth on laboratory host strains. Presumably, theselatter genes are essential for growth on other strains andwill be called conditionally non-essential genes. Forexample, the virulent bacteriophage, T7, has several genesthat encode functional proteins (ligase, protein kinase,host restriction blocking protein) that can be artificiallydeleted while maintaining T7 viability on laboratory hoststrains [59].The same is true of the lysogenic bacteriophage λ [60].However, progressive deletion of these genes causes a progressiveloss of DNA packaging efficiency for λ and presumablyother bacteriophages with unique DNA ends,because of a "partially full capsid" requirement for DNApackaging [60]. A result is a gene-retaining selective pressurethat is independent of what the genes encode. Thispressure is used in the design of bacteriophage-based genecloning vectors (review [60]). Other mechanisms for thenon-gene-specific retention of genes may exist. Possibilitiesinclude the embedding of promoters in DNA packagingrecognition sites, a phenomenon that is alreadyknown for T3 and T7 [33-35] (Legend to Figure 1).Although the two-tiered selection of the prokaryotic viruscomplexity hypothesis is a new concept for prokaryoticevolution, two-tiered selection is not a concept that contradictsthe fundamentals of previous thinking about evolution.All events proposed in the prokaryotic viruscomplexity hypothesis are based on random mutationand selection. No external guidance is proposed. Similarly,production of antibodies is also two tiered, in thatthe first tier selection produces antibodies in an immunesystem that itself is the product of second tier selection[61]. The second tier genes of the prokaryotic virus complexityhypothesis have been selected to reduce the randomnessof evolution via retention of the first tier. Acomplete, mathematical description (statistical mechanicswith an extended treatment of time?) might introducesome determinism into analysis of evolution. But, at thispoint, the theory and data are not sufficient to say howmuch determinism would be introduced.The departure from past thought is illustrated by comparingcellular gene homologs to known conditionally nonessentialgenes, including the non-essential bacteriophagegenes described above. Conditionally non-essential genesare non-essential only in the short term. They are essentialin the long term because of fluctuations in either the externalenvironment or the interior of the host cell. In the caseof both bacteria and also higher organisms, numerousdocumented examples exist of genetically programmedadaptation to environmental fluctuations. These includeadaptations to (a) utilize thermal fluctuations to obtainvariable outcome, such as a variable lysogenic response,(b) introduce environmentally modulated morphogenesis,(c) introduce environmentally-stimulated increase inmutation rate and (d) introduce cyclic changes in genomeorganization, such as those responsible for phase variationin bacteria (review [62]). Importantly, these previouslystudied adaptations to environmental fluctuationsoccur via genes that encode systems perfected by extensivemutation and selection in the past [62]. In the case of thecellular gene homolog evolution proposed here, the sameis true of the second tier genes that encode components ofthe biochemical systems that maintain genes that evolvein the first tier. But, in contrast to what occurs in the caseof previously described adaptation to fluctuations in theVirology Journal 2007, 4:30 http://www.virologyj.com/content/4/1/30Page 7 of 9(page number not for citation purposes)environment, the first tier homolog mutation and selectionoccurs de novo, i.e., without information from pastselections.When viewed from the perspective of genomics, theprokaryotic virus complexity hypothesis is feasible basedon the following evidence of DNA exchange: (a) ancientand ongoing bacteriophage origin of initially high AT bacterialgenes called ORFans, including genes for somestress-induced proteins and primosome assembly proteins[63], (b) bacteriophage origin of bacterial geneislands, defined by known sequence characteristics(including dinucleotide bias), but also containing novelgenes in comparatively high concentration [64] and (c)bacterial origin of bacteriophage genes (called morons)that arrive by non-homologous recombination in a contextforeign by both base composition and gene expression-controlling elements [54,65].TestingStudies of evolution are plagued by both absence of directobservations and presence of primarily indirect observationsof nonliving fossils. In the case of bacteriophages,however, the potential exists for genome sequencing andhomology-based informatic analysis of the equivalent ofliving fossils, i.e., comparatively un-evolved viruses. Isolationof bacteriophages in this class, including the longgenomeversions, has only just begun. Almost by definition,comparatively un-evolved bacteriophages will notcompete well in most circumstances. The expectation isthat these bacteriophages will be found in niches (probablynot in water; more likely in soil; see [66,67] for examples)that are either isolated from or hostile to the moreevolved and competitive bacteriophages.The potential also exists for further analysis by experimentally(a) determining via gene deletion the extent to whichthe cellular gene homologs assist the growth of longgenomebacteriophages, (b) determining via controlledevolution the external conditions (presence or absence ofa microbial community, for example) in which the cellulargene homologs are retained, (c) determining via genedeletion and mutation, followed by controlled evolution,which (second tier) genes are needed to retain the cellulargene homologs and (d) measuring via controlled evolutionthe extent to which the cellular gene homologsevolve, if they are retained. If, for example, the cellulargene homologs provide advantage only when a virus iswithin a microbial community, then the cellular genehomologs should eventually be lost during propagationin a single host that is not interacting with other microbes.Experiments of this type differ from previous experiments[68-70] in which controlled evolution was performed inthe absence of any aspect of a microbial community andalso without any focus on the cellular gene homologs.Also, experiments of this type should be performed withnewly isolated bacteriophages (certainly not T4) that havenot already evolved during propagation in the laboratory.Informatic analysis of the DNA sequence of bacteriophageliving fossils (if they are found) is also expected to be productivebased on the following characteristics of bacteriophages:large number, small genome and gene diversity.These characteristics have been previously reviewed[52,53,71]. The strategy is to (a) trace via sequence similaritythe past history of homologous viral genes (see, forexample, [50]), (b) integrate this knowledge with knowledgeof the biochemistry and (c) integrate the virussequence similarity-based gene trees with those ofprokaryotes and, eventually with at least the organelleassociatedgenomes [72,73] of eukaryotes. Eventually, thetrees will become unambiguous and detailed enough totrace the sequence of gene evolution, though evolutionarytime will remain to be specified. Comparatively unevolvedviruses potentially will be useful for the analysisof pre-split [74], as well as post-split, evolution.4. Implications of the hypothesisThe prokaryotic virus complexity hypothesis extends themore general concept of reticulate evolution, i.e., evolutionwith hybridization among different species (reticulateevolution is reviewed in [39]). Reticulation has beenproposed to explain the origin of eukaryotes [75]. Thepossibility exists that reticulation subsequently occurredfrom prokaryotes to eukaryotes (see [76], for example)and that both eukaryotic virus cellular gene homologs andsome (not all) of the "junk" DNA eukaryotes [77-80] havea function similar to that of the cellular gene homologs oflong-genome bacteriophages. Thus, if accurate andextendable to eukaryotes, the prokaryotic virus complexityhypothesis will also explain the function of at leastsome eukaryotic junk DNA.The two-tiered aspect of the hypothesis is a new concept,but is related to the concept of hierarchical evolution thathas previously been applied to eukaryotes and their communities[81]. This aspect of the hypothesis is a foundationfor producing evolutionary leaps in complexity and,if found to be accurate, would be an explanation of thephenomenon of punctuated equilibrium (review [21]).In the case of prokaryotes and their communities, theprokaryotic virus complexity hypothesis provides an intellectualframework for both understanding and influencingevolution. For example, desired changes in microbialcommunities might be introduced via long-genomeviruses, rather than via microbial cells.Virology Journal 2007, 4:30 http://www.virologyj.com/content/4/1/30Page 8 of 9(page number not for citation purposes)Competing interestsThe author(s) declare that they have no competing interests.Authors' contributionsBoth the ideas presented here and articulation of theseideas are the product of the author's work.AcknowledgementsThe author thanks Gary A. Griess, Stephen C. Hardies, John C. Lee andRichard Luduena for helpful comments on drafts of this manuscript. Supportwas received from the National Institutes of Health (GM24365), TheRobert J. Kleberg Jr. and Helen C. Kleberg Foundation and The WelchFoundation (AQ-764). Funding bodies were not involved in either thedesign of ideas or the writing of this manuscript.References1. Jardine PJ, Anderson DL: DNA packaging in dsDNA bacteriophages.In The Bacteriophages Edited by: Calendar R. New York:Oxford University Press; 2006 in press.2. Pajunen MI, Elizondo MR, Skurnik M, Kieleczawa J, Molineux IJ: Completenucleotide sequence and likely recombinatorial originof bacteriophage T3. J Mol Biol 2003, 319:1115-1132.3. Meijer WJ, Horcajadas JA, Salas M: φ 29 family of phages. MicrobiolMol Biol Rev 2001, 65:261-287.4. Kutter E, Stidham T, Guttman B, Kutter E, Batts D, Peterson S, DjavakhishviliT, Arisaka F, Mesyanzhinov V, Rüger W, Mosig G:Genomic map of bacteriophage T4. In Molecular Biology of BacteriophageT4 Edited by: Karam JD. Washington, DC: ASM Press;1994:491-519.5. Miller ES, Heidelberg JF, Eisen JA, Nelson WC, Durkin AS, Ciecko A,Feldblyum TV, White O, Paulsen IT, Nierman WC, Lee J, SzczypinskiB, Fraser CM: Complete genome sequence of the broad-hostrangevibriophage KVP40: comparative genomics of a T4-related bacteriophage. J Bacteriol 2003, 185:5220-5233.6. Mesyanzhinov VV, Robben J, Grymonprez B, Kostyuchenko VA,Bourkaltseva MV, Sykilinda NN, Krylov VN, Volckaert G: Thegenome of bacteriophage φ KZ of Pseudomonas aeruginosa. JMol Biol 2002, 317:1-19.7. Krylov V, Pleteneva E, Bourkaltseva M, Shaburova O, Volckaert G,Sykilinda N, Kurochkina L, Mesyanzhinov V: Myoviridae bacteriophagesof Pseudomonas aeruginosa : a long and complex evolutionarypathway. Res Microbiol 2003, 154:269-275.8. Dunigan DD, Fitzgerald LA, Van Etten JL: Phycodnaviruses: a peekat genetic diversity. Virus Res 2006, 117:119-132.9. Iyer LM, Balaji S, Koonin EV, Aravind L: Evolutionary genomics ofnucleo-cytoplasmic large DNA viruses. Virus Res 2006,117:156-184.10. Van Etten JL: Unusual life style of giant chlorella viruses. AnnRev Genet 2003, 37:153-195.11. Claverie JM: Fewer genes, more noncoding RNA. Science 2005,309:1529-1530.12. Claverie JM, Ogata H, Audic S, Abergel C, Suhre K, Fournier PE:Mimivirus and the emerging concept of "giant" virus. VirusRes 2006, 117:133-144.13. Ghedin E, Fraser CM: A virus with big ambitions. Trends Microbiol2005, 13:56-57.14. Raoult D, Audic S, Robert C, Abergel C, Renesto P, Ogata H, La ScolaB, Suzan M, Claverie JM: The 1.2-megabase genome sequence ofMimivirus. Science 2004, 306:1344-1350.15. Suhre K, Audic S, Claverie JM: Mimivirus gene promoters exhibitan unprecedented conservation among all eukaryotes. ProcNatl Acad Sci USA 2005, 102:14689-14693.16. Koonin EV: A non-adaptationist perspective on evolution ofgenomic complexity or the continued dethroning of man.Cell Cycle 2004, 3:280-285.17. Boyajian G, Lutz T: Evolution of biological complexity and itsrelation to taxonomic longevity in the Ammonoidea. Geology1992, 20:983-986.18. McShea DW: Evolutionary change in the morphological complexityof the mammalian vertebral column. Evolution 1993,47:730-740.19. McShea DW: The evolution of complexity without naturalselection, a possible large-scale trend of the fourth kind. Paleobiology(Supplement) 2005, 31:146-156.20. Gould SJ: The evolution of life on the earth. Scientific American1994, 271:84-91.21. Gould SJ: Full House New York: Three Rivers Press; 1996.22. Budzik JM, Rosche WA, Rietsch A, O'Toole GA: Isolation andcharacterization of a generalized transducing phage forPseudomonas aeruginosa strains PAO1 and PA14. J Bact 2004,186:3270-3273.23. Beumer A, Robinson JB: A Broad-Host-Range, GeneralizedTransducing Phage (SN-T) Acquires 16 S rRNA Genes fromDifferent Genera of Bacteria. Appl Environ Microbiol 2005,71:8301-8304.24. Matson EG, Thompson MG, Humphrey SB, Zuerner RL, Stanton TB:Identification of genes of VSH-1, a prophage-like gene transferagent of Brachyspira hyodysenteriae. J Bact 2005,187:5885-5892.25. Brüssow H, Canchaya C, Hardt WD: Phages and the evolution ofbacterial pathogens: from genomic rearrangements to lysogenicconversion. Microbiol Mol Biol Rev 2004, 68:560-602.26. Waldor MK, Friedman DI: Phage regulatory circuits and virulencegene expression. Curr Opin Microbiol 2005, 8:459-465.27. Smith NL, Taylor EJ, Lindsay A-M, Charnock SJ, Turkenburg JP, DodsonEJ, Davies GJ, Black GW: Structure of a group A streptococcalphage-encoded virulence factor reveals a catalyticallyactive triple-stranded β-helix. Proc Natl Acad Sci, USA 2005,102:17652-17657.28. Adami C: What is complexity? BioEssays 2002, 24:1085-1094.29. Ataullakhanov FI, Panteleev MA: Mathematical modeling andcomputer simulation in blood coagulation. Pathophysiol HaemostasisThrombosis 2005, 34:60-70.30. Jesty J, Beltrami E: Positive feedbacks of coagulation: their rolein threshold regulation. Arteriosclerosis, Thrombosis Vascular Biol2005, 25:2463-2469.31. Fujisawa H, Morita M: Phage DNA packaging. Genes to Cells 1997,2:537-545.32. Serwer P: T3/T7 DNA packaging. In Viral Genome PackagingMachines: Genetics, Structure, and Mechanism Edited by: Catalano CE.Georgetown, Texas: Landes Publishing; 2004:59-79.33. Hashimoto C, Fujisawa H: Transcription dependence of DNApackaging of bacteriophages T3 and T7. Virology 1992,191:246-250.34. Zhang X, Studier FW: Isolation of transcriptionally activemutants of T7 RNA polymerase that do not support phagegrowth. J Mol Biol 1995, 250:156-168.35. Zhang X, Studier FW: Multiple roles of T7 RNA polymerase andT7 lysozyme during bacteriophage T7 infection. J Mol Biol2004, 340:707-730.36. Hardies SC, Comeau AM, Serwer P, Suttle CA: The completesequence of marine bacteriophage VpV262 infecting Vibrioparahaemolyticus indicates that an ancestral component of aT7 viral supergroup is widespread in the marine environment.Virology 2003, 310:359-371.37. Rohwer F, Segall A, Steward G, Seguritan V, Breitbart M, Wolven F,Azam F: The complete genomic sequence of the marinephage Roseophage SIO1 shares homology with nonmarinephages. Limnol Oceanogr 2000, 45:408-418.38. Kutschera U, Niklas KJ: The modern theory of biological evolution:an expanded synthesis. Naturwissenschaften 2004,91:255-276.39. Gogarten JP, Townsend JP: Horizontal gene transfer, genomeinnovation and evolution. Nature Rev Microbiol 2005, 3:679-687.40. Carlson K: Appendix: Working with bacteriophages: Commontechniques and methodological appproaches. In Bacteriophages:Biology and Applications Edited by: Kutter E, Sulakvelidze A.Boca Raton: CRC Press; 2005:437-494.41. Ashelford KE, Day MJ, Fry JC: Elevated abundance of bacteriophageinfecting bacteria in soil. Appl Environ Microbiol 2003,69:285-289.42. Williamson KE, Radosevich M, Wommack KE: Abundance anddiversity of viruses in six Delaware soils. Appl Environ Microbiol2005, 71:3119-3125.
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ReviewPhage Therapy: What Have We Learned?Andrzej Górski 1,2,3,* ID , Ryszard Mi˛edzybrodzki 1,2,3 ID , Małgorzata Łobocka 4,5,Aleksandra Głowacka-Rutkowska 4, Agnieszka Bednarek 4, Jan Borysowski 3,Ewa Jon´ czyk-Matysiak 1, Marzanna Łusiak-Szelachowska 1, BeataWeber-Da˛browska 1,2,Natalia Bagin´ ska 1, Sławomir Letkiewicz 2,6, Krystyna Da˛browska 1,7 and Jacques Scheres 8,†1 Bacteriophage Laboratory, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, PolishAcademy of Sciences, Rudolfa Weigla Street 12, 53-114 Wroclaw, Poland; [email protected] (R.M.);[email protected] (E.J.-M.); [email protected] (M.Ł.-S.);[email protected] (B.W.-D.); [email protected] (N.B.); [email protected] (K.D.)2 Phage Therapy Unit, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, PolishAcademy of Sciences, Rudolfa Weigla Street 12, 53-114Wroclaw, Poland; [email protected]3 Department of Clinical Immunology, Transplantation Institute, Medical University ofWarsaw,Nowogrodzka Street 59, 02-006Warsaw, Poland; [email protected]4 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi´ nskiego Street 5 A,02-106Warsaw, Poland; [email protected] (M.Ł.); [email protected] (A.G.-R.);[email protected] (A.B.)5 Autonomous Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw University ofLife Sciences, Nowoursynowska Street 159, 02-776Warsaw, Poland6 Medical Sciences Institute, Katowice School of Economics, Harcerzy Wrze´snia Street 3,40-659 Katowice, Poland7 Research and Development Center, Regional Specialized Hospital, Kamie´ nskiego 73a,51-124 Wrocław, Poland8 National Institute of Public Health NIZP, Chocimska Street 24, 00-971Warsaw, Poland; [email protected]* Correspondence: [email protected]; Tel.: +48-71-370-99-05† Current Address: Department of Medical Microbiology, University Medical Centre Groningen, Hanzeplein1, 9713 GZ Groningen, The NetherlandsReceived: 18 April 2018; Accepted: 22 May 2018; Published: 28 May 2018Abstract: In this article we explain how current events in the field of phage therapy may positivelyinfluence its future development. We discuss the shift in position of the authorities, academia, media,non-governmental organizations, regulatory agencies, patients, and doctors which could enablefurther advances in the research and application of the therapy. In addition, we discuss methods toobtain optimal phage preparations and suggest the potential of novel applications of phage therapyextending beyond its anti-bacterial action.Keywords: phage therapy; experimental therapy; phage cocktails; anti-phage antibodies;prophage; immunomodulationThe intention of this article is to highlight the current events and issues related to phage therapy(PT) which seem to be most relevant for its further progress. These issues correspond to two main topicsaddressed in our article: the regulatory/ethical/awareness raising topic, which will subsequentlyyield to the topic of lysogeny/immunity/optimal use of phage preparations. These issues appear to beespecially timely and relevant from the perspective of our team with leading expertise in PT amongthe EU countries.1. More Room for Phage Therapy on the Horizon?After decades of being kept out of the mainstream infectious disease armamentarium of theWestern world, there now appears to be a silver lining on the horizon for phage therapy. PT isViruses 2018, 10, 288; doi:10.3390/v10060288 www.mdpi.com/journal/virusesViruses 2018, 10, 288 2 of 28shedding its dubious associations with alternative and fringe medicine. Triggered by the growingthreat of antibiotic resistance, there is a slow but substantial change in the appreciation of PT and amore permissive attitude of the main stakeholders in the infectious disease arena. Reviews on PTcovered by PubMed appear almost every month. According toWeb of Science, their average citationnumber per annum in recent years has been around 1100, and increased to approximately 1400 in2017. There is also a growing understanding of the ethical, legal, and administrative rules relevantto experimental therapy which currently allow such treatment to be provided to patients for whomall other available therapies have failed. Below are some observations and reflections on the presentattitudes of doctors, patients, academia, policymakers, media, and industry towards PT.2. Doctors, Pharmacists, and AcademiaThe professionals in the fight against serious infections are doctors, general practitioners,infectiologists or medical microbiologists, and pharmacists. In the case of a serious infection, they haveto choose the most appropriate remedy. From the plenitude of available antibiotics, they selectthose for which the pathogen in question tests sensitive, and standard application protocols arefollowed. However, almost every day doctors and pharmacists are confronted with pathogens that areincreasingly resistant to certain or even a long list of antibiotics. More and more they feel the urgentneed for new antibiotics or other instruments to help them improve or even save the lives of theircritically ill patients. Without effective antibiotics (and thus effectively standing helpless), doctorseagerly look for alternatives. Phage therapy might represent such an alternative, at least in certain cases.In the last decade, many publications on bacteriophages and their possible applicability have appearedregularly in the clinical, applied, and fundamental scientific microbiological literature [1–3]. PT is oftena specific subject on the programme of clinical and fundamental microbiological conferences, and issometimes even the sole focus of dedicated PT symposia. As a result, a growing group of physiciansand pharmacists inWestern countries are acquainted with the potency and the pros and cons of PTas a possible alternative or an auxiliary therapy in cases of untreatable antibiotic-resistant infections,which is applied in neighbouring non-EU countries on the continent. Publications of successful andsometimes spectacular phage therapy cases trigger this interest, and in their aftermath often leadto a flow of requests by doctors to phage laboratories for help in analogous cases. Such requestskeep coming, even from places where phages are not officially registered medical products, and PTstill is generally not available in most of theWest, although it is sometimes available experimentally.Therefore, in some cases doctors refer or mediate their patients to recognized PT centres elsewhere,such as in Poland (Wroclaw) and Georgia (Tbilisi).In general, Western medical professionals show signs of increased openness towards PT as apossibly valuable additional tool in the fight against resistant and seriously threatening or disablinginfections. At the same time, quite a long road of broad basic research, robust clinical trials, adjustmentof the regulatory systems, education, and training still lies ahead before PT becomes practical, optimallyeffective, and compatible with the rules. Nevertheless, it may be advisable for doctors and medicalstudents, pharmacists and pharmaceutical students to inform themselves in anticipation of the possiblerole of bacteriophages in infectious disease treatment. Is it not amazing that most medical professionalsdo not know about bacteriophages, these evolutionarily important creatures which are at least tentimes more frequent in the microbiome than all bacteria and also greatly outnumber them withinour body?In this field, certain non-governmental organizations (NGOs) of professionals sometimes ariseand intend to fulfil a role in closing the existing knowledge gap and building the bridge to formalrecognition of PT. For instance, P.H.A.G.E. (Phages for Human Applications Europe Group) is amultidisciplinary group of doctors with practical experience or strong interest in PT, basic and appliedphage researchers, and policymakers [4]. The exchange of phages, knowledge, and technology,participation in projects, organizing conferences and presentations all over Europe, publication,and education are among its main activities.Viruses 2018, 10, 288 3 of 28In 2015, a number of attendants of a bacteriophage conference in Tbilisi (Georgia) composed amultidisciplinary and intercontinental expert panel to establish an academic and medical initiative forthe re-implementation of PT. The papers on the “Silk Route to the acceptance and re-implementation ofbacteriophage therapy” which have recently been produced by this expert round-table are a significantcontribution to the development of international guidelines and frameworks which are needed for alegal and effective application of bacteriophage therapy by physicians and the receiving patients [5,6].Phages for Global Health is another very interesting multidisciplinary organization. Its missionis “to bring phage expertise to the developing world”. Developing countries are disproportionallyimpacted by infectious diseases (e.g., Campylobacter infection has a fatality rate of about 0.1% inwealthy countries, but 8.8% in Kenya, mostly children) [7]. Phages for Global Health provideslaboratory training workshops, teaching phage biology to scientists on location in developingcountries where the need for alternatives to antibiotics (e.g., PT) is felt especially [8]. In addition,product development projects are performed in which international multidisciplinary teams are builtthat co-develop phage products for specific applications in developing countries [9]. In June andJuly 2018, the Second East African PhageWorkshop will be held at Pwani University in Kilifi, Kenya.The participants will learn how to isolate and characterize phages as antibiotic alternatives for useagainst antibiotic-resistant bacteria.3. CRISPR-Cas: From Phages to EukaryotesAn additional important referral should be made to the recent development of simplified methodsfor high-efficiency gene-editing. This spectacular innovative technology is based on the CRISPR-Casmechanisms which bacteria developed during their evolution in order to protect themselves againstinfections by phages. This has once again made clear how interesting and important the study of thevery old relationship between phages and bacteria can be, and that it can lead to unexpected benefitsand great leaps forward for science and its practical applications, including great promises for theprevention or treatment of genetic and complex diseases [10–12].4. Patients, the Media, and PTThe patient, not the doctor, is the primary stakeholder in health and health care. Stimulatingpatient empowerment, health literacy, shared decision-making, and personal responsibility are coreelements of health policy in almost all countries. Especially when the doctors can neither heal nor helpwith the existing medical means (e.g., in cases of incurable cancer), it is often the patient who opens thequestion of alternative therapies and asks for a referral to any other centre that might be able to helpthem, wherever on Earth, with whatever therapy, and at whatever costs. Sometimes the patient or theirrelatives are, via the internet, well-informed about possible alternatives. Asking for a second opinionhas become the generally accepted standard. This pattern also applies to phage therapy. Though stillquite exceptional, there are patients with chronic untreatable threatening resistant infections whoindeed know about the option of bacteriophages, and ask their doctor to try phage therapy or to referthem for it. A growing number of patients find their way to bacteriophage centres abroad, stayingthere several weeks for phage selection and initial therapy, and are willing to bear the total costs oftreatment, travel, and accommodation themselves. This medical tourism for phage therapy has grownespecially since the media have taken their own responsibility in the national campaigns against theinappropriate use of antibiotics and have also informed the general public about PT as an alternative.They often mention PT as being applied in Central and Eastern European countries, and have reportedspectacular cases of wound healing and the prevention of diabetic limb amputations with phages.Phage stories with basic information and successful cases of PT including the places where and howyou can access it appear on TV [13,14] and in a broad range of societal magazines, ranging fromknowledge magazines such as Der SpiegelWissen in Germany [15] or ElsevierWeekblad [16] to thepopular women’s magazine Libelle [17] in the Netherlands. So, thanks to some pioneering patientsViruses 2018, 10, 288 4 of 28and with the help of the media, PT has gained a place on the stage for the general public in theWesternworld—almost a century later than in the East.5. Industry and SMEsTo make its way from the experimental level towards registration for safe application in humanmedicine, PT needs the engagement of a dedicated industry which is willing to produce phagesfollowing the safety and quality requirements [18], requiring high investments. So far, very few firms,usually SMEs, have chosen to engage in the production of phages ready for use in clinical trialsand human application, usually in the context of developmental projects performed in cooperationwith research institutes, academia, and or state laboratories. This contrasts somewhat with the food,disinfection, cosmetic, and veterinary sector, where phages and phage products (lysins) have alreadyreached consumers. The US Food and Drug Administration (FDA) has approved a small number ofproducts for these markets, and several applications are in the pipeline for approval. Very recently,the phage-producing SME, Phage Technology Center GmbH [19], was present at the internationalAnuga FoodTec International Food Technology Fair (Cologne, Germany, March 2018), presenting itsphages against Salmonella and E. coli for various food applications. According to its Senior ManagerResearch & Development, the market for phages is going to boom in this sector, which is certainly notyet the case in human medicine.6. Authorities and PTGlobally, national authorities consider antibiotic resistance to be a profound threat to health.Their national strategies, action plans, and preventive campaigns focus on a more appropriate useof antibiotics and the search for alternatives. The development of vaccines, innovative diagnostictests, and novel interventions are usually mentioned as alternatives. Only very exceptionally arethe words bacteriophage, PT, or phage products (lysins or endolysins) found in the action plans.The main reason is the current lack of positive clinical trials with PT. The reputation and successesof PT in countries with longstanding application of PT are distrusted and considered to be poorlydocumented, not convincing and not proven, and serious adverse effects of PT are feared or at leastnot to be excluded. The dictum primum non nocere (first do not harm) and quality assurance, bothbased on solid clinical trials according to the standard rules, are indeed strong pillars of drug policy.For similar reasons, there is no mention of PT in the five-year action plan of the European Commissionagainst antibiotic resistance launched in 2012 and updated in 2017 [20]. The words “phage therapy”and “phages” are also lacking in the global action plan to tackle antimicrobial resistance which wasendorsed in May 2015 by the World Health Assembly in Geneva [21]. This action of the Assembly wastruly a unique one, showing the United Nations’ serious concern that antibiotic resistance “threatensthe very core of modern medicine and the sustainability of an effective, global public health responseto the enduring threat from infectious diseases”. Is this emergency situation still not serious enough toallow a little more place for phage therapy, a method which a century ago was effectively applied andappreciated in curative care and public health, in the East and in the West before we used antibiotics?Fortunately, the position of the authorities appears to be shifting, albeit slowly, towards morelatitude for phage therapy. This may be illustrated by the following selection of interesting formalactions and documents of authorities in the US and/or the EU:In mid-2017, the U.S. Food and Drug Agency (FDA), the National Institutes of Health, the NationalInstitute of Allergy and Infectious Diseases, and the Center for Biologics Evaluation and Researchco-organized a two-day workshop to facilitate the development of a rigorous clinical assessment ofbacteriophage therapy [22].In late 2017, the FDA also gave the status of Emergency Investigational New Drug to phagesspecifically active against a multidrug-resistant Acinetobacter baumannii, which were applied in apatient with septic shock who improved within days and survived, being the first case of intravenousViruses 2018, 10, 288 5 of 28use for systemic infection. The phages were obtained from the US Navy and Texas A&M University,in combination with the San Diego biotech firm AmpliPhi [23].Later, the FDA gave its seal of approval to a new phase I/II clinical trial in humans at Mount SinaiHospital in New York City to test a new bacteriophage treatment for Crohn’s disease [24].In 2013, the European Commission funded the PHAGOBURN project co-ordinated by the FrenchMinistry of Defence, with partners from France, Belgium, and Switzerland. The main objective of theproject is “to assess the safety, effectiveness and pharmacodynamics of two therapeutic phage cocktailsto treat either E. coli or P. aeruginosa burn wound infections” [25].In 2015, the White House National Action Plan for combating Antibiotic-Resistant Bacterialaunched by the White House in 2015 listed “the use of phage and phage derived lysins to kill specificbacteria while preserving the microbiota” among the non-traditional therapeutics which should befurther developed [26].The Transatlantic Taskforce on Antimicrobial Resistance (TATFAR) was created in 2009 toenhance synergy and communication between government agencies on both sides of the AtlanticOcean. The first partners were the US and the EU, and Canada and Norway joined later . Actionno. 3.6 of TATFAR’s updated action plan is: “Exchange information on possible regulatoryapproaches to development of alternative approaches for managing bacterial infections, such asbacteriophage therapy and vaccines for health care associated infections (joint action by FDA, EMA,HC, and NMA)”[27]. In a message from the recent TATFAR meeting (Atlanta CDC 7–9 of March 2018),according to Marco Cavaleri from EMA (personal communication, March 12, 2018) it was reiteratedthat in the discussion on the alternatives to antibiotics, phages should be on the radar as an optionthat deserves to be discussed across the Atlantic. The biggest problem is that not many companies areinterested in discussing the topic or in considering how to approach clinical development.In its German Antimicrobial Resistance Strategy entitled “DART 2020: Fighting antibioticresistance for the good of both humans and animals”, the Federal Ministry of Food and Agricultureannounced plans to assess the “Possible positive effects of bacteriophages and other substances toreduce or eliminate bacteria on carcasses as a supplement to process hygiene”. Though this action isclearly meant to improve food hygiene and not as phage therapy for human patients, it is neverthelessnoted here because it is one of the very few governmental documents mentioning bacteriophages as ameans to fight antibiotic resistance, for the good of humanity and animals [28].The same action point was proposed by the Federal Government of Germany in the report“Combating Antimicrobial Resistance. Examples of Best-Practices of the G7 countries” of the G7GERMANY 2015 meeting in Berlin [29].Very recently, a major, hope-giving and possibly historical step for the applicability of PT wastaken by the Belgian Federal Government (January 2018) [30]. In cooperation with academia (includingethicists), researchers and experts from the care sector the Federal Agency for Medicines and HealthProducts succeeded in developing a regulation for phage production and the clinical application ofPT. The procedure, which obtained its legal approval at the end of January 2018, is based on thelegal possibilities in Belgium for a pharmacist to prepare a medical product (including phages) foran individual patient. The active ingredients used in this so-called magistral preparation (in the US,“compound prescription drug preparation”) must meet the requirements of the European, Belgian,or another official Pharmacopoeia. If this magistral route would be copied mutatis mutandis by othercountries, it would truly represent a breakthrough for the application of PT, especially in individuallife-threatening situations (based on the Declaration of Helsinki, WMA, 1964) [31]. In fact, a similarapproach has long been in use in Poland at the Phage Therapy Center of the Institute of Immunologyand Experimental Therapy [32,33].7. National Regulations Enabling Experimental Therapy (Including PT)In view of these developments, it might be useful to summarize the current status of experimentaltherapy in Europe and elsewhere.Viruses 2018, 10, 288 6 of 28Generally, every medicinal product must be approved by a relevant regulatory agency before itcan be used in clinical practice. However, in response to the needs of patients who cannot be treatedsatisfactorily with authorized drugs, many countries have introduced regulations which enable doctorsto use experimental treatments.In the European Union (EU), the legal framework for treatment with unauthorized medicinalproducts (termed compassionate use—CU) was introduced by Article 83 [34] of Regulation (EC) No.726/2004 of the European Parliament and of the Council. This article permits the use of unauthorizedmedicinal products in groups of patients, provided that two main requirements are met: (1) the patienthas a chronically or seriously debilitating disease, or a life-threatening disease which cannot be treatedsatisfactorily with an authorized medicinal product; and (2) the medicinal product must be either thesubject of an application for a centralized marketing authorization or be undergoing clinical trials.Specific CU programs are to be implemented and governed by individual Member States (MSs) [34].As of 2016, 18 out of 28 MSs had specific CU regulations and 20 had implemented CU programmes [35].Moreover, Article 5 of Directive 2001/83/EC of the European Parliament and of the Council allowsthe use of unauthorized medicinal products in individual patients under the direct responsibility of ahealthcare professional (i.e., named-patient basis treatment) [36].In the US, according to the terminology adopted by the FDA, the use of unauthorized drugsoutside of clinical trials is called expanded access (EA). General requirements for EA include thefollowing: (1) a serious or immediately life-threatening disease where no comparable or satisfactoryalternative therapy is available; (2) the potential benefits justify the potential risks and the potentialrisks are not unreasonable in the context of the disease; (3) there is no threat to the initiation, conduct,or completion of clinical trials; (4) informed consent of the patient; (5) Institutional Review Board (IRB)review [37,38]. Independently of the existing FDA regulations, 38 states have recently introducedso-called right-to-try laws which are to facilitate access of terminally ill patients to investigationaldrugs that have completed phase I of a clinical trial. However, these laws have been heavily criticizedby experts for offering “false hope” to patients without providing any actual improvements in access toinvestigational drugs [39]. Nevertheless, at the time of this writing, US Congress has passed a relevantbill which has been a priority of President Trump [40]. If approved by the US Senate, the law wouldallow patients to sidestep FDA approval once they have received permission from a company [41].In Canada, the use of unauthorized drugs is legally permissible in Special Access Programmes(SAPs). Basic information about these programmes is available in the Guidance Document for Industryand Practitioners—Special Access Programme for Drugs developed by the Canadian regulatoryagency Health Canada [42]. Under SAP rules, an unauthorized drug can be used in patients withserious or life-threatening diseases, especially in emergency cases when conventional therapies havefailed, are unsuitable, or are unavailable. The use of an unauthorized drug must be supported bysome credible evidence of its safety and efficacy, and a doctor should obtain informed consent fromthe patient.In Australia, there are two schemes that enable doctors to use unauthorized drugs: the AuthorizedPrescriber Scheme (APS) and the Special Access Scheme (SAS) [43]. In APS, an application for the useof an unauthorized drug needs to be approved by a bioethics committee or endorsed by a specialistin a discipline relevant to the proposed treatment. Important issues that are evaluated include thequalifications and experiences of the doctor, access to facilities necessary to perform the treatment,evidence to support the proposed treatment, clinical justification including whether other therapeuticalternatives have been tried, and an explanation of why the unauthorized drug is proposed. In addition,informed consent of the patient is required. Under this scheme, the doctor can be granted permissionto prescribe a specified unauthorized drug to specific patients (or groups of patients) with a particulardisease. In the other major Australian scheme (the SAS), unauthorized drugs can be used in singlepatients on a case-by-case basis. It is expected that before use of an unauthorized drug, all authorizedtreatment options will be considered. The doctor must also obtain informed consent from the patient.Moreover, in cases when the treated disease is not life-threatening and the unauthorized drug does notViruses 2018, 10, 288 7 of 28have an established history of use, clinical justification for the use of an unauthorized drug must alsobe provided.8. Important Issues Which Need Addressing to Enable Further Progress and Optimization of PTand Relevant Clinical TrialsIn this part of our article, we wish to briefly discuss the issues pertinent to PT that have notbeen dealt with adequately so far, and where the advancement of our knowledge may lead to a fasterintroduction of phages to the health market.The long-lasting effects of PT confirm its safety. Even though the therapeutic value of PT stillawaits confirmation by clinical trials—in line with the requirements of evidence-based medicine—aspointed out by a former FDA commissioner: “Although randomized trials perform an essential role inthe development of therapies, we should not neglect the crucial and complementary role than can beplayed by high-quality observational studies” [44]. In this regard, our results of suggested PT efficacyappear to be quite encouraging (>50% success rate using purified phage preparations), while thesafety of the therapy is remarkable [32]. This has been confirmed by our recent preliminary analysis ofremote observations in a group of 33 patients who completed PT up to 7 years ago. When questioned,two-thirds of those patients were satisfied with therapy results and, importantly, none of them reportedany complications that could be related to PT [45].9. PT and Antibody Responses against PhagesOur studies in animals and patients have provided interesting and potentially useful informationon anti-phage antibody responses during PT. Among healthy donors, 29–82% may be positive forserum anti-phage antibodies depending on phage type (anti-T4 coliphage antibodies being mostcommon) [46]. Antibody responses during PT have been described by us in detail. In patients awaitingPT, very low levels of anti-phage antibodies were detectable (mean K index in 60 patients was 0.17),while the index could reach values as high as 200 during PT. Furthermore, purified phage preparationsseem to induce higher antibody responses than do the lysates. In addition, identical phages canelicit different levels of antibody responses in patients, which may depend on the immune reactivityof those patients. The most important finding has been that a good clinical outcome of PT may beobserved in patients with high antibody responses [47]. Our recent analysis suggests that there isan association between the duration of therapy and antibody responses (for Staphylococcus phages,the Spearman correlation was 0.856, p < 0.0001). Similar data were obtained in mice [46]. While highantibody responses do not appear to affect the outcome of PT, we prefer to terminate the therapy ifthe antibody levels are high to avoid possible complications in the future (e.g., the unknown effect ofphage–antibody complexes).10. Monotherapy vs. Phage CocktailsThe issue of phage cocktails vs. monovalent phage preparations remains undecided: ourpreliminary data might suggest that there is no significant difference in the therapeutic efficacybetween these preparations, while the frequency of high antibody responses was higher in patientstreated with cocktails compared to those on monotherapy [48].11. Optimal Clinical Models for PT and Prognosis of TherapyOne of the key questions asked by the Guest Editor of this volume, Prof. H. Brüssow, was:is it possible to formulate a set of rules with respect to infection type, which predict successfulinterventions? [49] Our experience so far suggests that intrarectal PT of chronic bacterial prostatitisoffers the highest success rate [50]. Several factors could be responsible for those results, among thempossible good penetration of phages from the rectum to the prostatic tissue (phage ability to penetratecell layers has recently been demonstrated) [51,52], eradication of rectal carriage of a pathogen, as wellas low anti-phage antibody responses elicited by this mode of phage administration [47]. Our data onViruses 2018, 10, 288 8 of 28patients’ immunomonitoring suggest that an increase in phagocytosis may be a good prognostic signof PT success [53].12. Mouse Model of Acute Urinary Tract Infection Confirms Neutrophil–Phage SynergyThe value of this parameter has been confirmed by an experimental study in mice.The experiments were performed on a mouse model of acute urinary tract infection [54] causedby transurethral bacterial inoculation with uropathogenic strain isolated from patients: E. faecalis 15/Por P. aeruginosa 119. Spleen mononuclear cells were isolated according to the method describedby Kruisbeck (2000) [55] using a density gradient (Histopaque-1083, Sigma-Aldrich, St. Louis, MO,USA). Intracellular killing of bacteria by splenic macrophages was tested according to the methoddescribed by Buisman et al. (1991) and Leijh et al. (1982) [56,57]. The obtained value correspondedto the percentage of killed phagocytosed bacteria, and it was examined both 3 and 6 days after theinfection. In the infected group of DBA1/LAC J mice (n = 6) (without phage treatment), significantlylower (Mann–Whitney U-test, p = 0.004) intracellular killing of a pathogenic bacterial strain (the sameas the cause of infection) by splenic mononuclear cells (63.2% 7.1 for mice infected with (P. aeruginosa)was observed when compared to the bactericidal capacity of healthy animals (82.8% 8.0). Reducedintracellular killing was observed in infected mice on days 3 and 6 after the infection, regardless ofthe uropathogenic strain used. Importantly, the intraperitoneal administration of the phage lysate(at a concentration of 5 1010 pfu/mL) exerted a stimulatory effect on the spleen phagocytes in thegroup of mice with experimentally-induced infection by E. faecalis 6 days after sequential applicationof three doses (1 h, 24 h, and 48 h after bacterial inoculation) of specific enterococcal phage lysateEnt 15/P (86.7% 3.8) when compared to non-treated mice (74.1% 9.2) (Mann–Whitney U-test,p = 0.014). An improvement in bactericidal activity of splenic mononuclear cells was also obtained fora group of mice treated with three doses of the phage lysate (86.7% 3.8) after 6 days of infection whencompared to the same group tested 3 days after bacterial inoculation (72.2% 6.7, Mann–WhitneyU-test, p = 0.004). The improvement of splenic macrophage anti-bacterial function was paralleled bya significant fall of bacteria counts in liver, kidneys, and urinary bladder of phage-treated mice [58].Recent data fully confirm this assumption by showing that neutrophil–phage synergy is needed forsuccessful PT of experimental pneumonia in mice [59].13. Prophages in Bacterial Strains Used for Therapeutic Phage Propagation: Their Significance,Detection, and EliminationBacterial Strains for the Propagation of Therapeutic PhagesSources of phages for therapeutic use are lysates of cells that serve for the propagation of thosephages. In addition to the desired phage, they contain bacterial cell components and may containcontaminating phages that are produced as a result of prophage induction if the phage propagationstrain is a lysogen [60,61]. Genome analysis of bacterial strains used for phage propagation revealsnot only genes that encode toxins or other virulence determinants, but also mobile genetic elements,including plasmids, transposons, and prophages. The presence of toxins in lysates increases the costof lysate purification. The presence of mobile genetic elements poses a risk of uncontrolled spreadof bacterial virulence or antibiotic-resistance genes. The most problematic lysate contaminants aretemperate phages. Due to the physico-chemical similarity of contaminating temperate phages and lyticphages, the former are practically inseparable from the main phage population in a lysate. Despite thepossibilities of their detection in lysates and even the estimation of what fraction of the total phagepopulation is represented by them [61], the only way to eliminate them is the construction of phagepropagation strains that are depleted of prophages [60,62].A key argument for the removal of active prophages from the genomes of bacteria that serveas therapeutic phage propagation strains is the prophage genetic load. Temperate phages are majordriving forces of horizontal gene transfer and bacterial evolution [63–65]. They typically carry genesViruses 2018, 10, 288 9 of 28that encode functions which are adaptive for their bacterial hosts, and in that way decrease theprobability of overgrowth of the bacterial population by cells that have lost them. In the case ofprophages and plasmids of bacterial pathogens, the adaptive functions encoded by these elementsare nearly always associated with better adaptation of the bacteria to pathogenicity [63,65–72].In addition to virulence factors, certain prophages encode homologs of error-prone DNA polymeraseV subunits [73,74], and were proposed to play roles in the diversification of bacterial strains (e.g.,by facilitating the acquisition of resistance to toxins or antimicrobials by mutations) [75]. Temperatephage virions that contaminate therapeutic phage preparations act not only as vectors of theirown DNA, but can also act as vectors of bacterial, plasmid, or pathogenicity island DNA [76–83].For instance, a spontaneous intraspecies transfer of the blaNDM-1 carbapenemase gene from acarbapenem-resistant strain containing two active prophages to a carbapenem-sensitive Acinetobacterbaumannii strain was attributed to the transduction mediated by a prophage-derived temperatephage [84]. Undoubtedly, the release to the environment of temperate phages containing their ownDNA or sometimes even the DNA of plasmids or bacteria derived from contaminated therapeuticphage preparations can contribute to the spread of virulence or antibiotic-resistance genes. In theworst-case scenario, the contaminating phages could be acquired by the infecting bacteria duringphage therapy and make these bacteria more pathogenic, negatively influencing the treatment outcome.Although incidents of adverse effects of phage therapy have been surprisingly rare, the possibility ofsuch a scenario should be taken into consideration and avoided when possible, especially in view ofthe emergence of strains resistant to certain therapeutic phages in the course of phage therapy [32].Although only about a half of the sequenced bacteria are lysogens, prophages are more frequent inpathogens [85–87]. Their abundance varies among different species of pathogenic bacteria. However,bacteria that are known for especially good adaptation to pathogenicity and for their fast acquisitionof antibiotic resistance, including ESKAPE pathogens (Escherichia coli, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus spp.), are often or evenin most cases polylysogens [86,88–103]. Active and defective prophages in the genomes of certainpathogenic bacterial strains (e.g., E. coli O1577 strain Sakai, or highly virulent S. pyogenes strainMGAS315) can occupy as much as about 15% of total genomic DNA [104,105].The ubiquity of lysogeny among bacterial pathogens makes the selection of non-lysogenicbacteria for phage propagation from environmental samples either difficult or impossible. Hence,the identification of active prophages in the genomes of efficient phage propagation strains and theirsubsequent removal is a strategy of choice in ensuring the monoclonality and safety of therapeuticphage preparations, as well as in decreasing the cost of their production and the evaluation oftheir purity [60].Prophage-free strains may be acquired from among natural isolates of a given bacterial species orselected from laboratory cultures of prophage-carrying phage propagation strains upon the inductionof prophage lytic development and the selection of surviving cells, as reviewed by [60]. Which of thesestrategies may be optimal depends on several factors. The task may not be simple, as a propagationstrain should have all the features of the target bacteria that allow a phage released from this strain toinfect the target pathogenic bacterial strain efficiently.The stability of lysogeny is associated with numerous factors. In general, the rate of prophageloss by induction increases under conditions of decreased host viability, such as upon exposure toUV, reactive oxygen species, or other mutagenic factors that trigger the SOS response (for reviewsee [106–113]), under high temperatures [114], as well as in the response to certain bacteriocins [115],certain antibiotics that block the action of essential enzymes [93,116] or interfere with intracellularregulatory processes [117,118] or to quorum-sensing signalling molecules [119–121]. Typically,induction also occurs spontaneously in a variable fraction of a population of cells [122–129], beingresponsible for the presence of relevant free temperate phages in the cultures of lysogens [62,130,131].Thus, derivatives of lysogens that are depleted of certain prophages are expected to occur in natureViruses 2018, 10, 288 10 of 28and in laboratory cultures, although their number may be low, as together with the prophage they losethe prophage-mediated immunity to the infection by the relevant phage.14. Prophage Detection MethodsSeveral bioinformatic methods have been developed to identify prophages in bacterial genomes.Programs that implement them can be downloaded from internet resources or are accessible online(e.g., PHAST, PHASTER and PHASTEST [132,133]; Prophinder [134]; Phage_Finder [105]; ProphageFinder [135]; PhiSpy [136]; VirSorter [137]). Their performance is in the range 64–85% for sensitivity and74–93% for precision when tested with known prophage sequences in complete bacterial genomes [137].Prophages in the phage propagation strain of a known sequence can also be identified by comparingthe sequence of this strain with sequences of other species representatives and by the identification ofgenome regions that are interrupted by insertions of prophage-size elements [60]. Prophages in thegenomes of S. aureus or Salmonella enterica serovar Typhimurium can be detected by the analysis ofPCR reaction products with total genomic DNA of these bacteria and pairs of primers complementaryto the conserved DNA regions of their species-specific prophages [94,138–140]. The main disadvantageof the aforementioned methods is the distinction of active and defective prophages, which is notalways accurate. While defective prophages may be a source of toxins or virulence factors, theyare unable to contaminate therapeutic phage preparations in a phage form unless their DNA is notpacked into capsids of other phages. To detect active prophages, one should design pairs of primerscomplementary to the prophage sequences identified in a given strain and use them to amplify therelevant temperate phage DNA with the total virion DNA of a lysate as a template. In our hands,this method works sufficiently well to quickly distinguish active prophages from prophages thatcannot produce viable progeny [62]. A necessary condition is to degrade host DNA in a lysate prior tothe amplification experiments.The sensitivity of contaminating phage detection may be increased by inducing prophage lyticdevelopment, with the most commonly used inducing factors such as mitomycin C or UV light.Upon treatment with these factors, bacteria can be grown in a liquid medium until signs of lysis (if any)are observable. Lysate that has been treated with DNase can be used as a source of phages to preparephage DNA for PCR amplification with prophage-specific primer pairs. The inducible factor-treatedcells can also be streaked on a soft agar medium with suspended phage-sensitive cells (in a Petri dish).If the prophage was induced, the lysis zone in the underlying sensitive cell layer should surroundeach growing colony of lysogen. However, a limitation of the latter method is often the lack of aprophage-free strain able to serve as an indicator.15. Elimination of Prophages from Phage Propagation StrainsTraditional phage curing methods have been based on the selection of bacteria that have lostthe prophage spontaneously or in response to inducing factors. If the prophage excision systemis functional, prophage induction can be used to cure bacteria from that prophage [60]. Followingprophage induction, cells are plated on a solid medium and tested for lysogeny. Prophage insertion in achromosome may be associated with a specific phenotype, if it interrupts a gene of easily recognizablefunction. Curing from such prophages is associated with recovery of the wild-type strain phenotype,which may help to recognize prophage-free cells [62,94,141]. However, of the approximately 60% ofphages that use intragenic regions as their attachment sites, over half have the attachment sites intRNA encoding genes [105]. Additionally, other genes interrupted by prophages rarely have an easilyrecognizable phenotype. An additional difficulty may be “prophage jumping”—certain prophagesexcised from the primary attachment site can temporarily integrate into a secondary attachment sitein the same cell, and thus the loss of phage conversion phenotype is not always associated withphage loss [94]. In such cases, the loss of prophage can be verified by testing cells’ sensitivity toa parental strain phage or by PCR with a prophage-specific primer pair. If factors that induce theexcision and lytic development of a given prophage cannot be identified, one can search for coloniesViruses 2018, 10, 288 11 of 28of spontaneously cured cells in a population of lysogens by plating lysogen culture cells onto a solidmedium, growing them, and testing by colony blot for the presence of prophage [142]. An amplicon ofany prophage-specific gene can serve as a probe in blotting tests.The overexpression of a cloned prophage excisionase gene in a respective lysogen can increasethe frequency of prophage cured cell formation, as was shown in the case of lambda or KplE1 phagelysogens [143,144]. In certain cases, one prophage supports the excision of another prophage inthe same cell by providing a helper function [145]. The removal of all active prophages from suchcells using traditional methods is impossible. Thus, more reliable methods of prophage-free bacteriaconstruction rely on recombineering techniques. For example, the S. aureus strain Newman wascured of four prophages by recombinational replacements of prophage-containing regions withthe prophage-free regions of attachment sites for these phages cloned in temperature-sensitivereplicon-based suicidal plasmids [146]. A curable plasmid expressing phage Red recombinationsystem genes was used to replace four prophages in the E. coli chromosome with a PCR-amplifiedantibiotic resistance cassette, which was then eliminated with the help of another curable plasmid [128].16. Future Possibilities to Produce Industrial Phage Propagation StrainsThe construction of new phage propagation hosts using traditional approaches might be anever-ending story possibly requiring hundreds of strains to be cured of plasmids, active prophages,and possibly other mobile genetic elements. However, taking into account recent achievements insynthetic biology as well as the progress in recombineering and genome editing methods, this neednot be the case.Whether a given phage infects a given bacterial strain from a susceptible species depends on thefeatures of the bacterium and the phage. Metabolic compatibility of a bacterium with a phage to supportthe phage propagation in already-established infection appears to be species-specific, but sometimes itis extended to more than one bacterial species of the same or different genera [147,148]. Differentialphage susceptibility determinants that are encoded by various strains of the same species include genesencoding phage receptors or pathways of their synthesis and phage-compatible restriction-modificationsystems [149–155]. Additionally, bacteria encode phage defence mechanisms, but these mechanismsprotect the bacterium by itself either from infection with certain phages or from phage propagation,or induce apoptosis to protect the population from spread of the infection [156–163]. The differentialphage susceptibility determinants are exchangeable between strains of a given species. Bacteria cangain or lose sensitivity to a given phage or the ability to support this phage development by mutation-,recombination-, or horizontal gene transfer-driven changes in their phage susceptibility or phagedefence determinants [151,164–175]. Several genes associated with phage resistance or susceptibilityare carried by mobile genetic elements [120,158,175–187].Phage features important for the successful infection of a metabolically-compatible host includethe compatibility of phage receptor binding proteins with receptors at the surface of a bacterialcell, the compatibility of phage genome modifications with the restriction-modification systemof a bacterium, or the ability to prevent the action of bacterial restriction-modification systemseither by avoiding sites that are recognized by the bacterial restriction-modification systems orby encoding efficient anti-restriction mechanisms [149,188]. Additionally, to productively infectbacteria, phages encode proteins that allow them to overcome bacterial phage resistance mechanisms,such as anti-CRISPR proteins and proteins that prevent the action of bacterial Abi or toxin–antitoxin(TA) systems [189,190].The structure of each phage and its infectivity for particular hosts are determined by thegenome of this phage. The only host-determined features of a phage seem to be certain epigeneticmodifications, namely host-specific DNA methylation patterns [191,192]. They strongly influence theefficiency of infection of new hosts by a phage, being responsible for the limitations of horizontalgene transfer by bacteriophages [86,191,193,194]. Thus, in addition to species-specific basic metabolicpathways supporting the efficient propagation of a given phage, a phage propagation strain shouldViruses 2018, 10, 288 12 of 28be equipped with surface receptors for this phage attachment, cell envelope structures susceptibleto the action of given phage lytic proteins, and a restriction-modification system that will allow thephage released from this strain to infect a desired set of clinical strains. The removal from such astrain of genetic determinants of other phage defence mechanisms (e.g., CRISPR/Cas, Abi, or TAloci), if any are encoded by its genome, could extend the number of phages able to propagate in itscells to phages infecting strains of the same species and using the same host receptors, but unableto overcome the respective phage-defence mechanisms. The acquisition of sensitivity to certainphages upon the abolishment of various bacterial phage defence systems has been demonstrated inseveral cases [120,195–198].An optimal future strategy to acquire therapeutic phage propagation strains of desired propertiesmay be the construction of a bacterial chassis of selected clinically relevant pathogenic species.In synthetic biology, a chassis refers to the organism serving as a foundation to physically house geneticcomponents and support them by providing the resources for basic functions, such as replication,transcription, and translation machinery [199]. The bacterial chassis strains to serve as basic platformsfor the construction of industrial phage propagation strains should have genomes reduced in theircomplexity and the content of undesired genes by the depletion of most of the mobile genetic elementsas well as virulence and phage resistance determinants—a procedure that is known as a top-downstrategy of the genome reduction process [200]. Additionally, they should be ready for the introductionor exchange of genomic modules (e.g., an appropriate restriction-modification system or phagereceptors determining gene cassettes), enabling these strains to serve as microbial cell factories for thepropagation of selected therapeutic phages. Methodologies enabling the abolishment of mobile geneticelements and other genome fragments using genome shuffling, recombineering, oligo-mediated allelicreplacement, or genome editing using CRISPR/Cas-assisted selection of desired clones have beendeveloped for model bacteria, even on a genome-wide scale [201–209]. The repertoire of geneticengineering tools that extend the ability of genomic manipulations to bacteria other than E. coli usingthe newest strategies has been constantly increasing, providing means to edit genomes belonging togenera represented by the most problematic bacterial pathogens, including potential phage propagationstrains [210–218].The results of studies on bacteria that were cured of some or most of the recombinogenic ormobile genetic elements (including prophages) indicate that they have several advantages. For instance,Escherichia coli K-12 with a genome reduced by 15% by the removal of mobileDNAand cryptic virulencegenes preserved good growth profiles and protein production as well as the accurate propagation ofrecombinant genes and plasmids that could not be stably propagated in other strains [219]. The growthproperties and endurance of environmental stresses of a Pseudomonas putida KT2440 derivative whichwas cured of prophages, some transposons, and some restriction-modification cassettes was found tobe superior to its wild-type parent [220,221]. Curing a Corynebacterium glutamicum industrial strain ofprophages caused an increase of strain fitness, stress tolerance, transformability, and protein productionyield [222]. Thus, in our opinion, the construction for the propagation of therapeutic phages, of chassisstrains equipped with certain phage susceptibility determinants and depleted of phage resistancedeterminants as well as certain mobile genetic elements or virulence determinants will not onlyensure the safety of therapeutic phage preparations, but will also reduce the cost of phage productionsubstantially. This reduction will be a result of: (i) minimizing the number of strains required forthe production of different phages; (ii) eliminating the need of evaluating phage preparations forthe content of undesired elements, including temperate phages and toxins; and (iii) increasing thefitness and stability of such strains in the industrial production of therapeutic phages. Additionally,one foundation strain constructed for a bacterial species can serve as a platform for the enrichmentof its genome with various gene cassettes required for the propagation of various phages. We havealready constructed basic prophage- or plasmid-free strains to start the development of a chassis of S.aureus and E. faecalis strains. They serve for the production of monoclonal preparations of certain S.Viruses 2018, 10, 288 13 of 28aureus and E. faecalis phages [62,223]. Further work to remove additional undesired genomic elementsfrom the genomes of these strains is in progress.17. Surrogate Hosts for the Propagation of Therapeutic PhagesThe use of non-pathogenic relatives of pathogenic strains enabling therapeutic phage propagationwas proposed to eliminate the problem of phage preparations’ contaminants derived from virulentphage propagation hosts [224,225]. Unfortunately, suitable “surrogate” hosts can be found onlyin a limited number of cases, and not all of them enable the efficient propagation of therapeuticphages [226–231]. Additionally, long-term effects of the enrichment of a pathogenic strain populationwith prophages released from strains believed to be non-pathogenic are impossible to predict, especiallyin view of documented cases of infections caused by certain strains belonging to the surrogatehost species [232–239] and cross-species transfer of mobile genetic elements between representativesof surrogate host species and their pathogenic relatives [240–246]. Moreover, genomic analysis ofpathogenic strains of certain species and their relatives representing non-pathogenic species indicatesthat the latter may function as reservoirs of accessory genes for the former [103]. Thus, even when usingsurrogate non-pathogenic hosts for the propagation of therapeutic phages, the removal of prophagesfrom such hosts may be a wise strategy to avoid unpredicted problems in the future.18. Economic Aspects of the Industrial Construction of Phage Propagation StrainsIn nature, prophages are temporary components of bacterial genomes which can enter, exit,or change their location in the genome. Their loss is a natural process that occurs with variousfrequencies, as long as the mobility of a prophage is not abolished by deletions or other rearrangementsthat make the prophage remnants a permanent part of the genome. Thus, in most cases, themajor cost of acquiring cells that are depleted of active prophages is the cost of screening (labour,media, and blotting or PCR reactions), and sometimes the cost of recombineering and genomeediting techniques, provided the availability of tools. Economic aspects argue for going furtherand constructing species-specific bacterial chassis for the production of therapeutic phages by theremoval of plasmids, if any, and chromosomal elements that cause genome mutability, phage resistance,or encode virulence factors. The construction of such strains could be done based on recombineeringand genome editing methods analogous to those that have been used in the process of modification ofbacterial producers of various compounds for industry [89,222,247–255]. Subsequently, such a chassisstrain could be used as a platform for the exchange of particular phage-sensitivity determinants in itsgenome with selected strains sensitive to certain phages. The economic benefits of such an approachwould be associated not only with the increased safety of phage preparations produced with the useof these strains, but also with a switch from many different strains of various properties to fewerstrains of the same core genome and only a few gene cassettes to be exchanged. Results of studieson certain model or industrially-applicable bacteria that were depleted of prophages and certainother mobile elements as well as certain determinants of mutability indicate that such strains have abetter genomic stability and are more efficient producers of certain compounds than their wild-typeparents [89,199,219,252,254,255]. Engineering of their genomes does not need to be associated withthe permanent presence of heterologous DNA, as markerless gene knock-out or gene replacementsystems have been developed for a number of pathogenic bacterial species and are in constant furtherdevelopment [254–276].19. PT: Beyond the Antibacterial ActionIn recent years, data have been accumulating indicating that phages may also interact withmammalian cells, thus “crossing the border to eukaryotic cells”—binding to their surface receptorsand penetrating into them. Phages can therefore pass across confluent epithelial cell layers andmigrate to blood, lymph, and other tissues [51]. These findings essentially confirm our hypothesisof “phage translocation” from the intestines [277] extended by Barr, who used the term “journey”Viruses 2018, 10, 288 14 of 28to suggest that phages travel through the human body [278]. Phages have been shown to mediateanti-inflammatory and immunomodulating properties [279]; therefore, such phenomena may berelevant for the maintenance of immunological homeostasis. Consequently, we recently hypothesizedthat phage therapy may be considered for treating disorders such as inflammatory bowel disease,autoimmune hepatitis, allergy, as well as some viral infections [280–283]. Evidently, this requiresfurther work and confirmation by relevant clinical trials. While the most trustworthy advancescome through the performance of well-designed trials, sometimes experimental treatments based ontheoretical considerations alone may lead to major breakthroughs [284]. As stated, “the potential forbroader application of phage therapy is evident and it is certainly worthy of further studies” [285].20. ConclusionsAlmost a century after its consolidation in Eastern countries, a silver lining is appearing on thehorizon for phage therapy in the Western world. The increased threat of antibiotic resistance makes allstakeholders in the sector of infectious disease feel a high pressure to find new antibiotics and search forsafe alternatives. In this situation, phage therapy is increasingly considered as a potential alternativeor auxiliary tool. More and more patients, doctors, pharmacists, media, authorities, and industryshow their active interest and signs of a more open mind to assess the possible benefits of phagetherapy. This is especially triggered by an increasing number of publications of patient cases wherespectacular results were achieved with bacteriophages. It is now essential that the efficacy and safetyof phage application be demonstrated in rigorous clinical trials. National and international authoritiesare opening their doors to such trials, and are prone to regulate phage therapy if it is found to beeffective and safe. Furthermore, progress in research on phage biology suggests that other applicationsof phages unrelated to their anti-bacterial action may be on the horizon.Author Contributions: A.G., M.Ł., J.B., J.S., and R.M. wrote the manuscript; A.G, A.G.-R., A.B., E.J.-M., M.Ł.-S.,B.W.-D., N.B., S.L., and K.D. contributed to the design of the work, acquisition and interpretation of data.All authors have approved the submitted version.Funding: This work was supported by statutory funds from the Ludwik Hirszfeld Institute of Immunology andExperimental Therapy of the Polish Academy of Sciences,Warsaw Medical University, and statutory funds fromthe Institute of Biochemistry and Biophysics of the Polish Academy of Sciences.Acknowledgments: We thank A. Ajdukiewicz-Tarkowska, Head of Scientific Information, Main Library of theMedical University ofWarsaw for her help in accessing information from the Web of Science.Conflicts of Interest: A.G., R.M., M.Ł., A.G.-R , J.B., B.W.-D. and K.D. are co-inventors of patents owned by theInstitute and covering phage preparations. Other authors declare that they have no conflict of interest.References1. Borysowski, J.; Mi˛edzybrodzki, R.; Górski, A. Phage Therapy: Current Research and Application; CaisterAcademic Press: Norfolk, UK, 2014.2. Azeredo, J.; Sillankorva, J. (Eds.) Bacteriophage Therapy: From Lab to Clinical Practice; Springer Nature, HumanaPress: New York, NY, USA, 2018; ISBN 978-1-4939-7395-8.3. Alvarez, D.R.; Abedon, S.T. An online phage therapy bibliography: Separating under-indexed wheat fromoverly indexed chaff. AIMS Microbiol. 2017, 3, 525–528.4. P.H.A.G.E. Phages for Human Applications Europe Group. Available online: www.p-h-a-g-e.org (accessedon 5 April 2018).5. 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[CrossRef] [PubMed]98. Huang,W.;Wang, G.; Sebra, R.; Zhuge, J.; Yin, C.; Aguero-Rosenfeld, M.E.; Schuetz, A.N.; Dimitrova, N.;Fallon, J.T. Emergence and Evolution of Multidrug-Resistant Klebsiella pneumoniae with both bla(KPC) andbla(CTX-M) Integrated in the Chromosome. Antimicrob. Agents Chemother. 2017, 61, e00076-17. [CrossRef][PubMed]99. Wang, X.; Xie, Y.; Li, G.; Liu, J.; Li, X.; Tian, L.; Sun, J.; Ou, H.Y.; Qu, H. Whole-Genome-Sequencingcharacterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotypeK2 and ST374. Virulence 2018, 1, 510–521. [CrossRef] [PubMed]100. Chen, L.; Chavda, K.D.; DeLeo, F.R.; Bryant, K.A.; Jacobs, M.R.; Bonomo, R.A.; Kreiswirth, B.N. GenomeSequence of a Klebsiella pneumoniae Sequence Type 258 Isolate with Prophage-Encoded K. pneumoniaeCarbapenemase. Genome Announc. 2015, 3, e00659-15. [CrossRef] [PubMed]101. Bi, D.; Jiang, X.; Sheng, Z.K.; Ngmenterebo, D.; Tai, C.;Wang, M.; Deng, Z.; Rajakumar, K.; Ou, H.Y. Mappingthe resistance-associated mobilome of a carbapenem-resistant Klebsiella pneumoniae strain reveals insightsinto factors shaping these regions and facilitates generation of a ‘resistance-disarmed’ model organism.J. Antimicrob. Chemother. 2015, 10, 2770–2774. [CrossRef] [PubMed]Viruses 2018, 10, 288 20 of 28102. Zautner, A.E.; Bunk, B.; Pfeifer, Y.; Spröer, C.; Reichard, U.; Eiffert, H.; Scheithauer, S.; Groß, U.; Overmann, J.;Bohne, W. Monitoring microevolution of OXA-48-producing Klebsiella pneumoniae ST147 in a hospital settingby SMRT sequencing. J. Antimicrob. Chemother. 2017, 72, 2737–2744. [CrossRef] [PubMed]103. Di Nocera, P.P.; Rocco, F.; Giannouli, M.; Triassi, M.; Zarrilli, R. Genome organization of epidemicAcinetobacter baumannii strains. BMC Microbiol. 2011, 11, 224. [CrossRef] [PubMed]104. Ohnishi, M.; Kurokawa, K.; Hayashi, T. Diversification of Escherichia coli genomes: Are bacteriophages themajor contributors? Trends Microbiol. 2001, 10, 481–485. [CrossRef]105. Fouts, D.E. Phage_Finder: Automated identification and classification of prophage regions in completebacterial genome sequences. Nucleic Acids Res. 2006, 34, 5839–5851. [CrossRef] [PubMed]106. Ptashne, M. Genetic Switch: Phage Lambda and Higher Organisms, 2nd ed.; Blackwell: Cambridge, MA, USA,1992.107. Cavalcanti, S.M.; Siqueira, J.P., Jr. Cure of prophage in Staphylococcus aureus by furocoumarin photoadditions.Microbios 1995, 327, 85–91.108. Duval-Iflah, Y. Lysogenic conversion of the lipase gene in Staphylococcus pyogenes group III strains.Can. J. Microbiol. 1972, 18, 1491–1497. [CrossRef] [PubMed]109. Gasson, M.J.; Davies, F.L. Prophage-cured derivatives of Streptococcus lactis and Streptococcus cremoris.Appl. Environ. Microbiol. 1980, 40, 964–966. [PubMed]110. Waldor, M.K.; Friedma, D.I. Phage regulatory circuits and virulence gene expression. Curr. Opin. Microbiol.2005, 8, 459–465. [CrossRef] [PubMed]111. Raya, R.R.; H’bert, E.M. Isolation of phage via induction of lysogens. Methods Mol. Biol. 2009, 501, 23–32.[PubMed]112. Selva, L.; Viana, D.; Regev-Yochay, G.; Trzcinski, K.; Corpa, J.M.; Lasa, I.; Novick, R.P.; Penadés, J.R. Killingniche competitors by remote-control bacteriophage induction. Proc. Natl. Acad. Sci. USA 2009, 106, 1234–1238.[CrossRef] [PubMed]113. Banks, D.J.; Lei, B.; Musser, J.M. Prophage induction and expression of prophage-encoded virulence factorsin group A Streptococcus serotype M3 strain MGAS315. Infect. Immun. 2003, 71, 7079–7086. [CrossRef][PubMed]114. Bertani, G. Studies on lysogenesis. III. Superinfection of lysogenic Shigella dysenteriae with temperatemutants of the carried phage. J. Bacteriol. 1954, 67, 696–707. [PubMed]115. Madera, C.; García, P.; Rodríguez, A.; Suárez, J.E.; Martínez, B. Prophage induction in Lactococcus lactis bythe bacteriocin Lactococcin 972. Int. J. Food Microbiol. 2009, 129, 99–102. [CrossRef] [PubMed]116. Affolter, M.; Parent-Vaugeois, C.; Anderson, A. Curing and induction of the Fels 1 and Fels 2 prophages inthe Ames mutagen tester strains of Salmonella typhimurium. Mutat. Res. 1983, 110, 243–262. [CrossRef]117. Menouni, R.; Champ, S.; Espinosa, L.; Boudvillain, M.; Ansaldi, M. Transcription termination controlsprophage maintenance in Escherichia coli genomes. Proc. Natl. Acad. Sci. USA 2013, 110, 14414–14419.[CrossRef] [PubMed]118. Allen, H.K.; Looft, T.; Bayles, D.O.; Humphrey, S.; Levine, U.Y.; Alt, D.; Stanton, T.B. Antibiotics in feedinduce prophages in swine fecal microbiomes. mBio 2011, 6, e00260-11. [CrossRef] [PubMed]119. Ghosh, D.; Roy, K.; Williamson, K.E.; Srinivasiah, S.; Wommack, K.E.; Radosevich, M. Acyl-homoserinelactones can induce virus production in lysogenic bacteria: An alternative paradigm for prophage induction.Appl. Environ. Microbiol. 2009, 75, 7142–7152. [CrossRef] [PubMed]120. Miller, S.T.; Xavier, K.B.; Campagna, S.R.; Taga, M.E.; Semmelhack, M.F.; Bassler, B.L.; Hughson, F.M.Salmonella typhimurium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2.Mol. Cell 2004, 15, 677–687. [CrossRef] [PubMed]121. Rossmann, F.S.; Racek, T.;Wobser, D.; Puchalka, J.; Rabener, E.M.; Reiger, M.; Hendrickx, A.P.; Diederich, A.K.;Jung, K.; Klein, C.; et al. Phage-mediated dispersal of biofilm and distribution of bacterial virulence genes isinduced by quorum sensing. PLoS Pathog. 2015, 11, e1004653. [CrossRef] [PubMed]122. Lwoff, A. Lysogeny. Bacteriol. Rev. 1953, 17, 269–337. [PubMed]123. Birdsell, D.C.; Hathaway, G.M.; Rutberg, L. Characterization of Temperate Bacillus Bacteriophage phi105.J. Virol. 1969, 4, 264–270.

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ReviewPhage Therapy: What Have We Learned?Andrzej Górski 1,2,3,* ID , Ryszard Mi˛edzybrodzki 1,2,3 ID , Małgorzata Łobocka 4,5,Aleksandra Głowacka-Rutkowska 4, Agnieszka Bednarek 4, Jan Borysowski 3,Ewa Jon´ czyk-Matysiak 1, Marzanna Łusiak-Szelachowska 1, BeataWeber-Da˛browska 1,2,Natalia Bagin´ ska 1, Sławomir Letkiewicz 2,6, Krystyna Da˛browska 1,7 and Jacques Scheres 8,†1 Bacteriophage Laboratory, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, PolishAcademy of Sciences, Rudolfa Weigla Street 12, 53-114 Wroclaw, Poland; [email protected] (R.M.);[email protected] (E.J.-M.); [email protected] (M.Ł.-S.);[email protected] (B.W.-D.); [email protected] (N.B.); [email protected] (K.D.)2 Phage Therapy Unit, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, PolishAcademy of Sciences, Rudolfa Weigla Street 12, 53-114Wroclaw, Poland; [email protected]3 Department of Clinical Immunology, Transplantation Institute, Medical University ofWarsaw,Nowogrodzka Street 59, 02-006Warsaw, Poland; [email protected]4 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawi´ nskiego Street 5 A,02-106Warsaw, Poland; [email protected] (M.Ł.); [email protected] (A.G.-R.);[email protected] (A.B.)5 Autonomous Department of Microbial Biology, Faculty of Agriculture and Biology, Warsaw University ofLife Sciences, Nowoursynowska Street 159, 02-776Warsaw, Poland6 Medical Sciences Institute, Katowice School of Economics, Harcerzy Wrze´snia Street 3,40-659 Katowice, Poland7 Research and Development Center, Regional Specialized Hospital, Kamie´ nskiego 73a,51-124 Wrocław, Poland8 National Institute of Public Health NIZP, Chocimska Street 24, 00-971Warsaw, Poland; [email protected]* Correspondence: [email protected]; Tel.: +48-71-370-99-05† Current Address: Department of Medical Microbiology, University Medical Centre Groningen, Hanzeplein1, 9713 GZ Groningen, The NetherlandsReceived: 18 April 2018; Accepted: 22 May 2018; Published: 28 May 2018Abstract: In this article we explain how current events in the field of phage therapy may positivelyinfluence its future development. We discuss the shift in position of the authorities, academia, media,non-governmental organizations, regulatory agencies, patients, and doctors which could enablefurther advances in the research and application of the therapy. In addition, we discuss methods toobtain optimal phage preparations and suggest the potential of novel applications of phage therapyextending beyond its anti-bacterial action.Keywords: phage therapy; experimental therapy; phage cocktails; anti-phage antibodies;prophage; immunomodulationThe intention of this article is to highlight the current events and issues related to phage therapy(PT) which seem to be most relevant for its further progress. These issues correspond to two main topicsaddressed in our article: the regulatory/ethical/awareness raising topic, which will subsequentlyyield to the topic of lysogeny/immunity/optimal use of phage preparations. These issues appear to beespecially timely and relevant from the perspective of our team with leading expertise in PT amongthe EU countries.1. More Room for Phage Therapy on the Horizon?After decades of being kept out of the mainstream infectious disease armamentarium of theWestern world, there now appears to be a silver lining on the horizon for phage therapy. PT isViruses 2018, 10, 288; doi:10.3390/v10060288 www.mdpi.com/journal/virusesViruses 2018, 10, 288 2 of 28shedding its dubious associations with alternative and fringe medicine. Triggered by the growingthreat of antibiotic resistance, there is a slow but substantial change in the appreciation of PT and amore permissive attitude of the main stakeholders in the infectious disease arena. Reviews on PTcovered by PubMed appear almost every month. According toWeb of Science, their average citationnumber per annum in recent years has been around 1100, and increased to approximately 1400 in2017. There is also a growing understanding of the ethical, legal, and administrative rules relevantto experimental therapy which currently allow such treatment to be provided to patients for whomall other available therapies have failed. Below are some observations and reflections on the presentattitudes of doctors, patients, academia, policymakers, media, and industry towards PT.2. Doctors, Pharmacists, and AcademiaThe professionals in the fight against serious infections are doctors, general practitioners,infectiologists or medical microbiologists, and pharmacists. In the case of a serious infection, they haveto choose the most appropriate remedy. From the plenitude of available antibiotics, they selectthose for which the pathogen in question tests sensitive, and standard application protocols arefollowed. However, almost every day doctors and pharmacists are confronted with pathogens that areincreasingly resistant to certain or even a long list of antibiotics. More and more they feel the urgentneed for new antibiotics or other instruments to help them improve or even save the lives of theircritically ill patients. Without effective antibiotics (and thus effectively standing helpless), doctorseagerly look for alternatives. Phage therapy might represent such an alternative, at least in certain cases.In the last decade, many publications on bacteriophages and their possible applicability have appearedregularly in the clinical, applied, and fundamental scientific microbiological literature [1–3]. PT is oftena specific subject on the programme of clinical and fundamental microbiological conferences, and issometimes even the sole focus of dedicated PT symposia. As a result, a growing group of physiciansand pharmacists inWestern countries are acquainted with the potency and the pros and cons of PTas a possible alternative or an auxiliary therapy in cases of untreatable antibiotic-resistant infections,which is applied in neighbouring non-EU countries on the continent. Publications of successful andsometimes spectacular phage therapy cases trigger this interest, and in their aftermath often leadto a flow of requests by doctors to phage laboratories for help in analogous cases. Such requestskeep coming, even from places where phages are not officially registered medical products, and PTstill is generally not available in most of theWest, although it is sometimes available experimentally.Therefore, in some cases doctors refer or mediate their patients to recognized PT centres elsewhere,such as in Poland (Wroclaw) and Georgia (Tbilisi).In general, Western medical professionals show signs of increased openness towards PT as apossibly valuable additional tool in the fight against resistant and seriously threatening or disablinginfections. At the same time, quite a long road of broad basic research, robust clinical trials, adjustmentof the regulatory systems, education, and training still lies ahead before PT becomes practical, optimallyeffective, and compatible with the rules. Nevertheless, it may be advisable for doctors and medicalstudents, pharmacists and pharmaceutical students to inform themselves in anticipation of the possiblerole of bacteriophages in infectious disease treatment. Is it not amazing that most medical professionalsdo not know about bacteriophages, these evolutionarily important creatures which are at least tentimes more frequent in the microbiome than all bacteria and also greatly outnumber them withinour body?In this field, certain non-governmental organizations (NGOs) of professionals sometimes ariseand intend to fulfil a role in closing the existing knowledge gap and building the bridge to formalrecognition of PT. For instance, P.H.A.G.E. (Phages for Human Applications Europe Group) is amultidisciplinary group of doctors with practical experience or strong interest in PT, basic and appliedphage researchers, and policymakers [4]. The exchange of phages, knowledge, and technology,participation in projects, organizing conferences and presentations all over Europe, publication,and education are among its main activities.Viruses 2018, 10, 288 3 of 28In 2015, a number of attendants of a bacteriophage conference in Tbilisi (Georgia) composed amultidisciplinary and intercontinental expert panel to establish an academic and medical initiative forthe re-implementation of PT. The papers on the “Silk Route to the acceptance and re-implementation ofbacteriophage therapy” which have recently been produced by this expert round-table are a significantcontribution to the development of international guidelines and frameworks which are needed for alegal and effective application of bacteriophage therapy by physicians and the receiving patients [5,6].Phages for Global Health is another very interesting multidisciplinary organization. Its missionis “to bring phage expertise to the developing world”. Developing countries are disproportionallyimpacted by infectious diseases (e.g., Campylobacter infection has a fatality rate of about 0.1% inwealthy countries, but 8.8% in Kenya, mostly children) [7]. Phages for Global Health provideslaboratory training workshops, teaching phage biology to scientists on location in developingcountries where the need for alternatives to antibiotics (e.g., PT) is felt especially [8]. In addition,product development projects are performed in which international multidisciplinary teams are builtthat co-develop phage products for specific applications in developing countries [9]. In June andJuly 2018, the Second East African PhageWorkshop will be held at Pwani University in Kilifi, Kenya.The participants will learn how to isolate and characterize phages as antibiotic alternatives for useagainst antibiotic-resistant bacteria.3. CRISPR-Cas: From Phages to EukaryotesAn additional important referral should be made to the recent development of simplified methodsfor high-efficiency gene-editing. This spectacular innovative technology is based on the CRISPR-Casmechanisms which bacteria developed during their evolution in order to protect themselves againstinfections by phages. This has once again made clear how interesting and important the study of thevery old relationship between phages and bacteria can be, and that it can lead to unexpected benefitsand great leaps forward for science and its practical applications, including great promises for theprevention or treatment of genetic and complex diseases [10–12].4. Patients, the Media, and PTThe patient, not the doctor, is the primary stakeholder in health and health care. Stimulatingpatient empowerment, health literacy, shared decision-making, and personal responsibility are coreelements of health policy in almost all countries. Especially when the doctors can neither heal nor helpwith the existing medical means (e.g., in cases of incurable cancer), it is often the patient who opens thequestion of alternative therapies and asks for a referral to any other centre that might be able to helpthem, wherever on Earth, with whatever therapy, and at whatever costs. Sometimes the patient or theirrelatives are, via the internet, well-informed about possible alternatives. Asking for a second opinionhas become the generally accepted standard. This pattern also applies to phage therapy. Though stillquite exceptional, there are patients with chronic untreatable threatening resistant infections whoindeed know about the option of bacteriophages, and ask their doctor to try phage therapy or to referthem for it. A growing number of patients find their way to bacteriophage centres abroad, stayingthere several weeks for phage selection and initial therapy, and are willing to bear the total costs oftreatment, travel, and accommodation themselves. This medical tourism for phage therapy has grownespecially since the media have taken their own responsibility in the national campaigns against theinappropriate use of antibiotics and have also informed the general public about PT as an alternative.They often mention PT as being applied in Central and Eastern European countries, and have reportedspectacular cases of wound healing and the prevention of diabetic limb amputations with phages.Phage stories with basic information and successful cases of PT including the places where and howyou can access it appear on TV [13,14] and in a broad range of societal magazines, ranging fromknowledge magazines such as Der SpiegelWissen in Germany [15] or ElsevierWeekblad [16] to thepopular women’s magazine Libelle [17] in the Netherlands. So, thanks to some pioneering patientsViruses 2018, 10, 288 4 of 28and with the help of the media, PT has gained a place on the stage for the general public in theWesternworld—almost a century later than in the East.5. Industry and SMEsTo make its way from the experimental level towards registration for safe application in humanmedicine, PT needs the engagement of a dedicated industry which is willing to produce phagesfollowing the safety and quality requirements [18], requiring high investments. So far, very few firms,usually SMEs, have chosen to engage in the production of phages ready for use in clinical trialsand human application, usually in the context of developmental projects performed in cooperationwith research institutes, academia, and or state laboratories. This contrasts somewhat with the food,disinfection, cosmetic, and veterinary sector, where phages and phage products (lysins) have alreadyreached consumers. The US Food and Drug Administration (FDA) has approved a small number ofproducts for these markets, and several applications are in the pipeline for approval. Very recently,the phage-producing SME, Phage Technology Center GmbH [19], was present at the internationalAnuga FoodTec International Food Technology Fair (Cologne, Germany, March 2018), presenting itsphages against Salmonella and E. coli for various food applications. According to its Senior ManagerResearch & Development, the market for phages is going to boom in this sector, which is certainly notyet the case in human medicine.6. Authorities and PTGlobally, national authorities consider antibiotic resistance to be a profound threat to health.Their national strategies, action plans, and preventive campaigns focus on a more appropriate useof antibiotics and the search for alternatives. The development of vaccines, innovative diagnostictests, and novel interventions are usually mentioned as alternatives. Only very exceptionally arethe words bacteriophage, PT, or phage products (lysins or endolysins) found in the action plans.The main reason is the current lack of positive clinical trials with PT. The reputation and successesof PT in countries with longstanding application of PT are distrusted and considered to be poorlydocumented, not convincing and not proven, and serious adverse effects of PT are feared or at leastnot to be excluded. The dictum primum non nocere (first do not harm) and quality assurance, bothbased on solid clinical trials according to the standard rules, are indeed strong pillars of drug policy.For similar reasons, there is no mention of PT in the five-year action plan of the European Commissionagainst antibiotic resistance launched in 2012 and updated in 2017 [20]. The words “phage therapy”and “phages” are also lacking in the global action plan to tackle antimicrobial resistance which wasendorsed in May 2015 by the World Health Assembly in Geneva [21]. This action of the Assembly wastruly a unique one, showing the United Nations’ serious concern that antibiotic resistance “threatensthe very core of modern medicine and the sustainability of an effective, global public health responseto the enduring threat from infectious diseases”. Is this emergency situation still not serious enough toallow a little more place for phage therapy, a method which a century ago was effectively applied andappreciated in curative care and public health, in the East and in the West before we used antibiotics?Fortunately, the position of the authorities appears to be shifting, albeit slowly, towards morelatitude for phage therapy. This may be illustrated by the following selection of interesting formalactions and documents of authorities in the US and/or the EU:In mid-2017, the U.S. Food and Drug Agency (FDA), the National Institutes of Health, the NationalInstitute of Allergy and Infectious Diseases, and the Center for Biologics Evaluation and Researchco-organized a two-day workshop to facilitate the development of a rigorous clinical assessment ofbacteriophage therapy [22].In late 2017, the FDA also gave the status of Emergency Investigational New Drug to phagesspecifically active against a multidrug-resistant Acinetobacter baumannii, which were applied in apatient with septic shock who improved within days and survived, being the first case of intravenousViruses 2018, 10, 288 5 of 28use for systemic infection. The phages were obtained from the US Navy and Texas A&M University,in combination with the San Diego biotech firm AmpliPhi [23].Later, the FDA gave its seal of approval to a new phase I/II clinical trial in humans at Mount SinaiHospital in New York City to test a new bacteriophage treatment for Crohn’s disease [24].In 2013, the European Commission funded the PHAGOBURN project co-ordinated by the FrenchMinistry of Defence, with partners from France, Belgium, and Switzerland. The main objective of theproject is “to assess the safety, effectiveness and pharmacodynamics of two therapeutic phage cocktailsto treat either E. coli or P. aeruginosa burn wound infections” [25].In 2015, the White House National Action Plan for combating Antibiotic-Resistant Bacterialaunched by the White House in 2015 listed “the use of phage and phage derived lysins to kill specificbacteria while preserving the microbiota” among the non-traditional therapeutics which should befurther developed [26].The Transatlantic Taskforce on Antimicrobial Resistance (TATFAR) was created in 2009 toenhance synergy and communication between government agencies on both sides of the AtlanticOcean. The first partners were the US and the EU, and Canada and Norway joined later . Actionno. 3.6 of TATFAR’s updated action plan is: “Exchange information on possible regulatoryapproaches to development of alternative approaches for managing bacterial infections, such asbacteriophage therapy and vaccines for health care associated infections (joint action by FDA, EMA,HC, and NMA)”[27]. In a message from the recent TATFAR meeting (Atlanta CDC 7–9 of March 2018),according to Marco Cavaleri from EMA (personal communication, March 12, 2018) it was reiteratedthat in the discussion on the alternatives to antibiotics, phages should be on the radar as an optionthat deserves to be discussed across the Atlantic. The biggest problem is that not many companies areinterested in discussing the topic or in considering how to approach clinical development.In its German Antimicrobial Resistance Strategy entitled “DART 2020: Fighting antibioticresistance for the good of both humans and animals”, the Federal Ministry of Food and Agricultureannounced plans to assess the “Possible positive effects of bacteriophages and other substances toreduce or eliminate bacteria on carcasses as a supplement to process hygiene”. Though this action isclearly meant to improve food hygiene and not as phage therapy for human patients, it is neverthelessnoted here because it is one of the very few governmental documents mentioning bacteriophages as ameans to fight antibiotic resistance, for the good of humanity and animals [28].The same action point was proposed by the Federal Government of Germany in the report“Combating Antimicrobial Resistance. Examples of Best-Practices of the G7 countries” of the G7GERMANY 2015 meeting in Berlin [29].Very recently, a major, hope-giving and possibly historical step for the applicability of PT wastaken by the Belgian Federal Government (January 2018) [30]. In cooperation with academia (includingethicists), researchers and experts from the care sector the Federal Agency for Medicines and HealthProducts succeeded in developing a regulation for phage production and the clinical application ofPT. The procedure, which obtained its legal approval at the end of January 2018, is based on thelegal possibilities in Belgium for a pharmacist to prepare a medical product (including phages) foran individual patient. The active ingredients used in this so-called magistral preparation (in the US,“compound prescription drug preparation”) must meet the requirements of the European, Belgian,or another official Pharmacopoeia. If this magistral route would be copied mutatis mutandis by othercountries, it would truly represent a breakthrough for the application of PT, especially in individuallife-threatening situations (based on the Declaration of Helsinki, WMA, 1964) [31]. In fact, a similarapproach has long been in use in Poland at the Phage Therapy Center of the Institute of Immunologyand Experimental Therapy [32,33].7. National Regulations Enabling Experimental Therapy (Including PT)In view of these developments, it might be useful to summarize the current status of experimentaltherapy in Europe and elsewhere.Viruses 2018, 10, 288 6 of 28Generally, every medicinal product must be approved by a relevant regulatory agency before itcan be used in clinical practice. However, in response to the needs of patients who cannot be treatedsatisfactorily with authorized drugs, many countries have introduced regulations which enable doctorsto use experimental treatments.In the European Union (EU), the legal framework for treatment with unauthorized medicinalproducts (termed compassionate use—CU) was introduced by Article 83 [34] of Regulation (EC) No.726/2004 of the European Parliament and of the Council. This article permits the use of unauthorizedmedicinal products in groups of patients, provided that two main requirements are met: (1) the patienthas a chronically or seriously debilitating disease, or a life-threatening disease which cannot be treatedsatisfactorily with an authorized medicinal product; and (2) the medicinal product must be either thesubject of an application for a centralized marketing authorization or be undergoing clinical trials.Specific CU programs are to be implemented and governed by individual Member States (MSs) [34].As of 2016, 18 out of 28 MSs had specific CU regulations and 20 had implemented CU programmes [35].Moreover, Article 5 of Directive 2001/83/EC of the European Parliament and of the Council allowsthe use of unauthorized medicinal products in individual patients under the direct responsibility of ahealthcare professional (i.e., named-patient basis treatment) [36].In the US, according to the terminology adopted by the FDA, the use of unauthorized drugsoutside of clinical trials is called expanded access (EA). General requirements for EA include thefollowing: (1) a serious or immediately life-threatening disease where no comparable or satisfactoryalternative therapy is available; (2) the potential benefits justify the potential risks and the potentialrisks are not unreasonable in the context of the disease; (3) there is no threat to the initiation, conduct,or completion of clinical trials; (4) informed consent of the patient; (5) Institutional Review Board (IRB)review [37,38]. Independently of the existing FDA regulations, 38 states have recently introducedso-called right-to-try laws which are to facilitate access of terminally ill patients to investigationaldrugs that have completed phase I of a clinical trial. However, these laws have been heavily criticizedby experts for offering “false hope” to patients without providing any actual improvements in access toinvestigational drugs [39]. Nevertheless, at the time of this writing, US Congress has passed a relevantbill which has been a priority of President Trump [40]. If approved by the US Senate, the law wouldallow patients to sidestep FDA approval once they have received permission from a company [41].In Canada, the use of unauthorized drugs is legally permissible in Special Access Programmes(SAPs). Basic information about these programmes is available in the Guidance Document for Industryand Practitioners—Special Access Programme for Drugs developed by the Canadian regulatoryagency Health Canada [42]. Under SAP rules, an unauthorized drug can be used in patients withserious or life-threatening diseases, especially in emergency cases when conventional therapies havefailed, are unsuitable, or are unavailable. The use of an unauthorized drug must be supported bysome credible evidence of its safety and efficacy, and a doctor should obtain informed consent fromthe patient.In Australia, there are two schemes that enable doctors to use unauthorized drugs: the AuthorizedPrescriber Scheme (APS) and the Special Access Scheme (SAS) [43]. In APS, an application for the useof an unauthorized drug needs to be approved by a bioethics committee or endorsed by a specialistin a discipline relevant to the proposed treatment. Important issues that are evaluated include thequalifications and experiences of the doctor, access to facilities necessary to perform the treatment,evidence to support the proposed treatment, clinical justification including whether other therapeuticalternatives have been tried, and an explanation of why the unauthorized drug is proposed. In addition,informed consent of the patient is required. Under this scheme, the doctor can be granted permissionto prescribe a specified unauthorized drug to specific patients (or groups of patients) with a particulardisease. In the other major Australian scheme (the SAS), unauthorized drugs can be used in singlepatients on a case-by-case basis. It is expected that before use of an unauthorized drug, all authorizedtreatment options will be considered. The doctor must also obtain informed consent from the patient.Moreover, in cases when the treated disease is not life-threatening and the unauthorized drug does notViruses 2018, 10, 288 7 of 28have an established history of use, clinical justification for the use of an unauthorized drug must alsobe provided.8. Important Issues Which Need Addressing to Enable Further Progress and Optimization of PTand Relevant Clinical TrialsIn this part of our article, we wish to briefly discuss the issues pertinent to PT that have notbeen dealt with adequately so far, and where the advancement of our knowledge may lead to a fasterintroduction of phages to the health market.The long-lasting effects of PT confirm its safety. Even though the therapeutic value of PT stillawaits confirmation by clinical trials—in line with the requirements of evidence-based medicine—aspointed out by a former FDA commissioner: “Although randomized trials perform an essential role inthe development of therapies, we should not neglect the crucial and complementary role than can beplayed by high-quality observational studies” [44]. In this regard, our results of suggested PT efficacyappear to be quite encouraging (>50% success rate using purified phage preparations), while thesafety of the therapy is remarkable [32]. This has been confirmed by our recent preliminary analysis ofremote observations in a group of 33 patients who completed PT up to 7 years ago. When questioned,two-thirds of those patients were satisfied with therapy results and, importantly, none of them reportedany complications that could be related to PT [45].9. PT and Antibody Responses against PhagesOur studies in animals and patients have provided interesting and potentially useful informationon anti-phage antibody responses during PT. Among healthy donors, 29–82% may be positive forserum anti-phage antibodies depending on phage type (anti-T4 coliphage antibodies being mostcommon) [46]. Antibody responses during PT have been described by us in detail. In patients awaitingPT, very low levels of anti-phage antibodies were detectable (mean K index in 60 patients was 0.17),while the index could reach values as high as 200 during PT. Furthermore, purified phage preparationsseem to induce higher antibody responses than do the lysates. In addition, identical phages canelicit different levels of antibody responses in patients, which may depend on the immune reactivityof those patients. The most important finding has been that a good clinical outcome of PT may beobserved in patients with high antibody responses [47]. Our recent analysis suggests that there isan association between the duration of therapy and antibody responses (for Staphylococcus phages,the Spearman correlation was 0.856, p < 0.0001). Similar data were obtained in mice [46]. While highantibody responses do not appear to affect the outcome of PT, we prefer to terminate the therapy ifthe antibody levels are high to avoid possible complications in the future (e.g., the unknown effect ofphage–antibody complexes).10. Monotherapy vs. Phage CocktailsThe issue of phage cocktails vs. monovalent phage preparations remains undecided: ourpreliminary data might suggest that there is no significant difference in the therapeutic efficacybetween these preparations, while the frequency of high antibody responses was higher in patientstreated with cocktails compared to those on monotherapy [48].11. Optimal Clinical Models for PT and Prognosis of TherapyOne of the key questions asked by the Guest Editor of this volume, Prof. H. Brüssow, was:is it possible to formulate a set of rules with respect to infection type, which predict successfulinterventions? [49] Our experience so far suggests that intrarectal PT of chronic bacterial prostatitisoffers the highest success rate [50]. Several factors could be responsible for those results, among thempossible good penetration of phages from the rectum to the prostatic tissue (phage ability to penetratecell layers has recently been demonstrated) [51,52], eradication of rectal carriage of a pathogen, as wellas low anti-phage antibody responses elicited by this mode of phage administration [47]. Our data onViruses 2018, 10, 288 8 of 28patients’ immunomonitoring suggest that an increase in phagocytosis may be a good prognostic signof PT success [53].12. Mouse Model of Acute Urinary Tract Infection Confirms Neutrophil–Phage SynergyThe value of this parameter has been confirmed by an experimental study in mice.The experiments were performed on a mouse model of acute urinary tract infection [54] causedby transurethral bacterial inoculation with uropathogenic strain isolated from patients: E. faecalis 15/Por P. aeruginosa 119. Spleen mononuclear cells were isolated according to the method describedby Kruisbeck (2000) [55] using a density gradient (Histopaque-1083, Sigma-Aldrich, St. Louis, MO,USA). Intracellular killing of bacteria by splenic macrophages was tested according to the methoddescribed by Buisman et al. (1991) and Leijh et al. (1982) [56,57]. The obtained value correspondedto the percentage of killed phagocytosed bacteria, and it was examined both 3 and 6 days after theinfection. In the infected group of DBA1/LAC J mice (n = 6) (without phage treatment), significantlylower (Mann–Whitney U-test, p = 0.004) intracellular killing of a pathogenic bacterial strain (the sameas the cause of infection) by splenic mononuclear cells (63.2% 7.1 for mice infected with (P. aeruginosa)was observed when compared to the bactericidal capacity of healthy animals (82.8% 8.0). Reducedintracellular killing was observed in infected mice on days 3 and 6 after the infection, regardless ofthe uropathogenic strain used. Importantly, the intraperitoneal administration of the phage lysate(at a concentration of 5 1010 pfu/mL) exerted a stimulatory effect on the spleen phagocytes in thegroup of mice with experimentally-induced infection by E. faecalis 6 days after sequential applicationof three doses (1 h, 24 h, and 48 h after bacterial inoculation) of specific enterococcal phage lysateEnt 15/P (86.7% 3.8) when compared to non-treated mice (74.1% 9.2) (Mann–Whitney U-test,p = 0.014). An improvement in bactericidal activity of splenic mononuclear cells was also obtained fora group of mice treated with three doses of the phage lysate (86.7% 3.8) after 6 days of infection whencompared to the same group tested 3 days after bacterial inoculation (72.2% 6.7, Mann–WhitneyU-test, p = 0.004). The improvement of splenic macrophage anti-bacterial function was paralleled bya significant fall of bacteria counts in liver, kidneys, and urinary bladder of phage-treated mice [58].Recent data fully confirm this assumption by showing that neutrophil–phage synergy is needed forsuccessful PT of experimental pneumonia in mice [59].13. Prophages in Bacterial Strains Used for Therapeutic Phage Propagation: Their Significance,Detection, and EliminationBacterial Strains for the Propagation of Therapeutic PhagesSources of phages for therapeutic use are lysates of cells that serve for the propagation of thosephages. In addition to the desired phage, they contain bacterial cell components and may containcontaminating phages that are produced as a result of prophage induction if the phage propagationstrain is a lysogen [60,61]. Genome analysis of bacterial strains used for phage propagation revealsnot only genes that encode toxins or other virulence determinants, but also mobile genetic elements,including plasmids, transposons, and prophages. The presence of toxins in lysates increases the costof lysate purification. The presence of mobile genetic elements poses a risk of uncontrolled spreadof bacterial virulence or antibiotic-resistance genes. The most problematic lysate contaminants aretemperate phages. Due to the physico-chemical similarity of contaminating temperate phages and lyticphages, the former are practically inseparable from the main phage population in a lysate. Despite thepossibilities of their detection in lysates and even the estimation of what fraction of the total phagepopulation is represented by them [61], the only way to eliminate them is the construction of phagepropagation strains that are depleted of prophages [60,62].A key argument for the removal of active prophages from the genomes of bacteria that serveas therapeutic phage propagation strains is the prophage genetic load. Temperate phages are majordriving forces of horizontal gene transfer and bacterial evolution [63–65]. They typically carry genesViruses 2018, 10, 288 9 of 28that encode functions which are adaptive for their bacterial hosts, and in that way decrease theprobability of overgrowth of the bacterial population by cells that have lost them. In the case ofprophages and plasmids of bacterial pathogens, the adaptive functions encoded by these elementsare nearly always associated with better adaptation of the bacteria to pathogenicity [63,65–72].In addition to virulence factors, certain prophages encode homologs of error-prone DNA polymeraseV subunits [73,74], and were proposed to play roles in the diversification of bacterial strains (e.g.,by facilitating the acquisition of resistance to toxins or antimicrobials by mutations) [75]. Temperatephage virions that contaminate therapeutic phage preparations act not only as vectors of theirown DNA, but can also act as vectors of bacterial, plasmid, or pathogenicity island DNA [76–83].For instance, a spontaneous intraspecies transfer of the blaNDM-1 carbapenemase gene from acarbapenem-resistant strain containing two active prophages to a carbapenem-sensitive Acinetobacterbaumannii strain was attributed to the transduction mediated by a prophage-derived temperatephage [84]. Undoubtedly, the release to the environment of temperate phages containing their ownDNA or sometimes even the DNA of plasmids or bacteria derived from contaminated therapeuticphage preparations can contribute to the spread of virulence or antibiotic-resistance genes. In theworst-case scenario, the contaminating phages could be acquired by the infecting bacteria duringphage therapy and make these bacteria more pathogenic, negatively influencing the treatment outcome.Although incidents of adverse effects of phage therapy have been surprisingly rare, the possibility ofsuch a scenario should be taken into consideration and avoided when possible, especially in view ofthe emergence of strains resistant to certain therapeutic phages in the course of phage therapy [32].Although only about a half of the sequenced bacteria are lysogens, prophages are more frequent inpathogens [85–87]. Their abundance varies among different species of pathogenic bacteria. However,bacteria that are known for especially good adaptation to pathogenicity and for their fast acquisitionof antibiotic resistance, including ESKAPE pathogens (Escherichia coli, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus spp.), are often or evenin most cases polylysogens [86,88–103]. Active and defective prophages in the genomes of certainpathogenic bacterial strains (e.g., E. coli O1577 strain Sakai, or highly virulent S. pyogenes strainMGAS315) can occupy as much as about 15% of total genomic DNA [104,105].The ubiquity of lysogeny among bacterial pathogens makes the selection of non-lysogenicbacteria for phage propagation from environmental samples either difficult or impossible. Hence,the identification of active prophages in the genomes of efficient phage propagation strains and theirsubsequent removal is a strategy of choice in ensuring the monoclonality and safety of therapeuticphage preparations, as well as in decreasing the cost of their production and the evaluation oftheir purity [60].Prophage-free strains may be acquired from among natural isolates of a given bacterial species orselected from laboratory cultures of prophage-carrying phage propagation strains upon the inductionof prophage lytic development and the selection of surviving cells, as reviewed by [60]. Which of thesestrategies may be optimal depends on several factors. The task may not be simple, as a propagationstrain should have all the features of the target bacteria that allow a phage released from this strain toinfect the target pathogenic bacterial strain efficiently.The stability of lysogeny is associated with numerous factors. In general, the rate of prophageloss by induction increases under conditions of decreased host viability, such as upon exposure toUV, reactive oxygen species, or other mutagenic factors that trigger the SOS response (for reviewsee [106–113]), under high temperatures [114], as well as in the response to certain bacteriocins [115],certain antibiotics that block the action of essential enzymes [93,116] or interfere with intracellularregulatory processes [117,118] or to quorum-sensing signalling molecules [119–121]. Typically,induction also occurs spontaneously in a variable fraction of a population of cells [122–129], beingresponsible for the presence of relevant free temperate phages in the cultures of lysogens [62,130,131].Thus, derivatives of lysogens that are depleted of certain prophages are expected to occur in natureViruses 2018, 10, 288 10 of 28and in laboratory cultures, although their number may be low, as together with the prophage they losethe prophage-mediated immunity to the infection by the relevant phage.14. Prophage Detection MethodsSeveral bioinformatic methods have been developed to identify prophages in bacterial genomes.Programs that implement them can be downloaded from internet resources or are accessible online(e.g., PHAST, PHASTER and PHASTEST [132,133]; Prophinder [134]; Phage_Finder [105]; ProphageFinder [135]; PhiSpy [136]; VirSorter [137]). Their performance is in the range 64–85% for sensitivity and74–93% for precision when tested with known prophage sequences in complete bacterial genomes [137].Prophages in the phage propagation strain of a known sequence can also be identified by comparingthe sequence of this strain with sequences of other species representatives and by the identification ofgenome regions that are interrupted by insertions of prophage-size elements [60]. Prophages in thegenomes of S. aureus or Salmonella enterica serovar Typhimurium can be detected by the analysis ofPCR reaction products with total genomic DNA of these bacteria and pairs of primers complementaryto the conserved DNA regions of their species-specific prophages [94,138–140]. The main disadvantageof the aforementioned methods is the distinction of active and defective prophages, which is notalways accurate. While defective prophages may be a source of toxins or virulence factors, theyare unable to contaminate therapeutic phage preparations in a phage form unless their DNA is notpacked into capsids of other phages. To detect active prophages, one should design pairs of primerscomplementary to the prophage sequences identified in a given strain and use them to amplify therelevant temperate phage DNA with the total virion DNA of a lysate as a template. In our hands,this method works sufficiently well to quickly distinguish active prophages from prophages thatcannot produce viable progeny [62]. A necessary condition is to degrade host DNA in a lysate prior tothe amplification experiments.The sensitivity of contaminating phage detection may be increased by inducing prophage lyticdevelopment, with the most commonly used inducing factors such as mitomycin C or UV light.Upon treatment with these factors, bacteria can be grown in a liquid medium until signs of lysis (if any)are observable. Lysate that has been treated with DNase can be used as a source of phages to preparephage DNA for PCR amplification with prophage-specific primer pairs. The inducible factor-treatedcells can also be streaked on a soft agar medium with suspended phage-sensitive cells (in a Petri dish).If the prophage was induced, the lysis zone in the underlying sensitive cell layer should surroundeach growing colony of lysogen. However, a limitation of the latter method is often the lack of aprophage-free strain able to serve as an indicator.15. Elimination of Prophages from Phage Propagation StrainsTraditional phage curing methods have been based on the selection of bacteria that have lostthe prophage spontaneously or in response to inducing factors. If the prophage excision systemis functional, prophage induction can be used to cure bacteria from that prophage [60]. Followingprophage induction, cells are plated on a solid medium and tested for lysogeny. Prophage insertion in achromosome may be associated with a specific phenotype, if it interrupts a gene of easily recognizablefunction. Curing from such prophages is associated with recovery of the wild-type strain phenotype,which may help to recognize prophage-free cells [62,94,141]. However, of the approximately 60% ofphages that use intragenic regions as their attachment sites, over half have the attachment sites intRNA encoding genes [105]. Additionally, other genes interrupted by prophages rarely have an easilyrecognizable phenotype. An additional difficulty may be “prophage jumping”—certain prophagesexcised from the primary attachment site can temporarily integrate into a secondary attachment sitein the same cell, and thus the loss of phage conversion phenotype is not always associated withphage loss [94]. In such cases, the loss of prophage can be verified by testing cells’ sensitivity toa parental strain phage or by PCR with a prophage-specific primer pair. If factors that induce theexcision and lytic development of a given prophage cannot be identified, one can search for coloniesViruses 2018, 10, 288 11 of 28of spontaneously cured cells in a population of lysogens by plating lysogen culture cells onto a solidmedium, growing them, and testing by colony blot for the presence of prophage [142]. An amplicon ofany prophage-specific gene can serve as a probe in blotting tests.The overexpression of a cloned prophage excisionase gene in a respective lysogen can increasethe frequency of prophage cured cell formation, as was shown in the case of lambda or KplE1 phagelysogens [143,144]. In certain cases, one prophage supports the excision of another prophage inthe same cell by providing a helper function [145]. The removal of all active prophages from suchcells using traditional methods is impossible. Thus, more reliable methods of prophage-free bacteriaconstruction rely on recombineering techniques. For example, the S. aureus strain Newman wascured of four prophages by recombinational replacements of prophage-containing regions withthe prophage-free regions of attachment sites for these phages cloned in temperature-sensitivereplicon-based suicidal plasmids [146]. A curable plasmid expressing phage Red recombinationsystem genes was used to replace four prophages in the E. coli chromosome with a PCR-amplifiedantibiotic resistance cassette, which was then eliminated with the help of another curable plasmid [128].16. Future Possibilities to Produce Industrial Phage Propagation StrainsThe construction of new phage propagation hosts using traditional approaches might be anever-ending story possibly requiring hundreds of strains to be cured of plasmids, active prophages,and possibly other mobile genetic elements. However, taking into account recent achievements insynthetic biology as well as the progress in recombineering and genome editing methods, this neednot be the case.Whether a given phage infects a given bacterial strain from a susceptible species depends on thefeatures of the bacterium and the phage. Metabolic compatibility of a bacterium with a phage to supportthe phage propagation in already-established infection appears to be species-specific, but sometimes itis extended to more than one bacterial species of the same or different genera [147,148]. Differentialphage susceptibility determinants that are encoded by various strains of the same species include genesencoding phage receptors or pathways of their synthesis and phage-compatible restriction-modificationsystems [149–155]. Additionally, bacteria encode phage defence mechanisms, but these mechanismsprotect the bacterium by itself either from infection with certain phages or from phage propagation,or induce apoptosis to protect the population from spread of the infection [156–163]. The differentialphage susceptibility determinants are exchangeable between strains of a given species. Bacteria cangain or lose sensitivity to a given phage or the ability to support this phage development by mutation-,recombination-, or horizontal gene transfer-driven changes in their phage susceptibility or phagedefence determinants [151,164–175]. Several genes associated with phage resistance or susceptibilityare carried by mobile genetic elements [120,158,175–187].Phage features important for the successful infection of a metabolically-compatible host includethe compatibility of phage receptor binding proteins with receptors at the surface of a bacterialcell, the compatibility of phage genome modifications with the restriction-modification systemof a bacterium, or the ability to prevent the action of bacterial restriction-modification systemseither by avoiding sites that are recognized by the bacterial restriction-modification systems orby encoding efficient anti-restriction mechanisms [149,188]. Additionally, to productively infectbacteria, phages encode proteins that allow them to overcome bacterial phage resistance mechanisms,such as anti-CRISPR proteins and proteins that prevent the action of bacterial Abi or toxin–antitoxin(TA) systems [189,190].The structure of each phage and its infectivity for particular hosts are determined by thegenome of this phage. The only host-determined features of a phage seem to be certain epigeneticmodifications, namely host-specific DNA methylation patterns [191,192]. They strongly influence theefficiency of infection of new hosts by a phage, being responsible for the limitations of horizontalgene transfer by bacteriophages [86,191,193,194]. Thus, in addition to species-specific basic metabolicpathways supporting the efficient propagation of a given phage, a phage propagation strain shouldViruses 2018, 10, 288 12 of 28be equipped with surface receptors for this phage attachment, cell envelope structures susceptibleto the action of given phage lytic proteins, and a restriction-modification system that will allow thephage released from this strain to infect a desired set of clinical strains. The removal from such astrain of genetic determinants of other phage defence mechanisms (e.g., CRISPR/Cas, Abi, or TAloci), if any are encoded by its genome, could extend the number of phages able to propagate in itscells to phages infecting strains of the same species and using the same host receptors, but unableto overcome the respective phage-defence mechanisms. The acquisition of sensitivity to certainphages upon the abolishment of various bacterial phage defence systems has been demonstrated inseveral cases [120,195–198].An optimal future strategy to acquire therapeutic phage propagation strains of desired propertiesmay be the construction of a bacterial chassis of selected clinically relevant pathogenic species.In synthetic biology, a chassis refers to the organism serving as a foundation to physically house geneticcomponents and support them by providing the resources for basic functions, such as replication,transcription, and translation machinery [199]. The bacterial chassis strains to serve as basic platformsfor the construction of industrial phage propagation strains should have genomes reduced in theircomplexity and the content of undesired genes by the depletion of most of the mobile genetic elementsas well as virulence and phage resistance determinants—a procedure that is known as a top-downstrategy of the genome reduction process [200]. Additionally, they should be ready for the introductionor exchange of genomic modules (e.g., an appropriate restriction-modification system or phagereceptors determining gene cassettes), enabling these strains to serve as microbial cell factories for thepropagation of selected therapeutic phages. Methodologies enabling the abolishment of mobile geneticelements and other genome fragments using genome shuffling, recombineering, oligo-mediated allelicreplacement, or genome editing using CRISPR/Cas-assisted selection of desired clones have beendeveloped for model bacteria, even on a genome-wide scale [201–209]. The repertoire of geneticengineering tools that extend the ability of genomic manipulations to bacteria other than E. coli usingthe newest strategies has been constantly increasing, providing means to edit genomes belonging togenera represented by the most problematic bacterial pathogens, including potential phage propagationstrains [210–218].The results of studies on bacteria that were cured of some or most of the recombinogenic ormobile genetic elements (including prophages) indicate that they have several advantages. For instance,Escherichia coli K-12 with a genome reduced by 15% by the removal of mobileDNAand cryptic virulencegenes preserved good growth profiles and protein production as well as the accurate propagation ofrecombinant genes and plasmids that could not be stably propagated in other strains [219]. The growthproperties and endurance of environmental stresses of a Pseudomonas putida KT2440 derivative whichwas cured of prophages, some transposons, and some restriction-modification cassettes was found tobe superior to its wild-type parent [220,221]. Curing a Corynebacterium glutamicum industrial strain ofprophages caused an increase of strain fitness, stress tolerance, transformability, and protein productionyield [222]. Thus, in our opinion, the construction for the propagation of therapeutic phages, of chassisstrains equipped with certain phage susceptibility determinants and depleted of phage resistancedeterminants as well as certain mobile genetic elements or virulence determinants will not onlyensure the safety of therapeutic phage preparations, but will also reduce the cost of phage productionsubstantially. This reduction will be a result of: (i) minimizing the number of strains required forthe production of different phages; (ii) eliminating the need of evaluating phage preparations forthe content of undesired elements, including temperate phages and toxins; and (iii) increasing thefitness and stability of such strains in the industrial production of therapeutic phages. Additionally,one foundation strain constructed for a bacterial species can serve as a platform for the enrichmentof its genome with various gene cassettes required for the propagation of various phages. We havealready constructed basic prophage- or plasmid-free strains to start the development of a chassis of S.aureus and E. faecalis strains. They serve for the production of monoclonal preparations of certain S.Viruses 2018, 10, 288 13 of 28aureus and E. faecalis phages [62,223]. Further work to remove additional undesired genomic elementsfrom the genomes of these strains is in progress.17. Surrogate Hosts for the Propagation of Therapeutic PhagesThe use of non-pathogenic relatives of pathogenic strains enabling therapeutic phage propagationwas proposed to eliminate the problem of phage preparations’ contaminants derived from virulentphage propagation hosts [224,225]. Unfortunately, suitable “surrogate” hosts can be found onlyin a limited number of cases, and not all of them enable the efficient propagation of therapeuticphages [226–231]. Additionally, long-term effects of the enrichment of a pathogenic strain populationwith prophages released from strains believed to be non-pathogenic are impossible to predict, especiallyin view of documented cases of infections caused by certain strains belonging to the surrogatehost species [232–239] and cross-species transfer of mobile genetic elements between representativesof surrogate host species and their pathogenic relatives [240–246]. Moreover, genomic analysis ofpathogenic strains of certain species and their relatives representing non-pathogenic species indicatesthat the latter may function as reservoirs of accessory genes for the former [103]. Thus, even when usingsurrogate non-pathogenic hosts for the propagation of therapeutic phages, the removal of prophagesfrom such hosts may be a wise strategy to avoid unpredicted problems in the future.18. Economic Aspects of the Industrial Construction of Phage Propagation StrainsIn nature, prophages are temporary components of bacterial genomes which can enter, exit,or change their location in the genome. Their loss is a natural process that occurs with variousfrequencies, as long as the mobility of a prophage is not abolished by deletions or other rearrangementsthat make the prophage remnants a permanent part of the genome. Thus, in most cases, themajor cost of acquiring cells that are depleted of active prophages is the cost of screening (labour,media, and blotting or PCR reactions), and sometimes the cost of recombineering and genomeediting techniques, provided the availability of tools. Economic aspects argue for going furtherand constructing species-specific bacterial chassis for the production of therapeutic phages by theremoval of plasmids, if any, and chromosomal elements that cause genome mutability, phage resistance,or encode virulence factors. The construction of such strains could be done based on recombineeringand genome editing methods analogous to those that have been used in the process of modification ofbacterial producers of various compounds for industry [89,222,247–255]. Subsequently, such a chassisstrain could be used as a platform for the exchange of particular phage-sensitivity determinants in itsgenome with selected strains sensitive to certain phages. The economic benefits of such an approachwould be associated not only with the increased safety of phage preparations produced with the useof these strains, but also with a switch from many different strains of various properties to fewerstrains of the same core genome and only a few gene cassettes to be exchanged. Results of studieson certain model or industrially-applicable bacteria that were depleted of prophages and certainother mobile elements as well as certain determinants of mutability indicate that such strains have abetter genomic stability and are more efficient producers of certain compounds than their wild-typeparents [89,199,219,252,254,255]. Engineering of their genomes does not need to be associated withthe permanent presence of heterologous DNA, as markerless gene knock-out or gene replacementsystems have been developed for a number of pathogenic bacterial species and are in constant furtherdevelopment [254–276].19. PT: Beyond the Antibacterial ActionIn recent years, data have been accumulating indicating that phages may also interact withmammalian cells, thus “crossing the border to eukaryotic cells”—binding to their surface receptorsand penetrating into them. Phages can therefore pass across confluent epithelial cell layers andmigrate to blood, lymph, and other tissues [51]. These findings essentially confirm our hypothesisof “phage translocation” from the intestines [277] extended by Barr, who used the term “journey”Viruses 2018, 10, 288 14 of 28to suggest that phages travel through the human body [278]. Phages have been shown to mediateanti-inflammatory and immunomodulating properties [279]; therefore, such phenomena may berelevant for the maintenance of immunological homeostasis. Consequently, we recently hypothesizedthat phage therapy may be considered for treating disorders such as inflammatory bowel disease,autoimmune hepatitis, allergy, as well as some viral infections [280–283]. Evidently, this requiresfurther work and confirmation by relevant clinical trials. While the most trustworthy advancescome through the performance of well-designed trials, sometimes experimental treatments based ontheoretical considerations alone may lead to major breakthroughs [284]. As stated, “the potential forbroader application of phage therapy is evident and it is certainly worthy of further studies” [285].20. ConclusionsAlmost a century after its consolidation in Eastern countries, a silver lining is appearing on thehorizon for phage therapy in the Western world. The increased threat of antibiotic resistance makes allstakeholders in the sector of infectious disease feel a high pressure to find new antibiotics and search forsafe alternatives. In this situation, phage therapy is increasingly considered as a potential alternativeor auxiliary tool. More and more patients, doctors, pharmacists, media, authorities, and industryshow their active interest and signs of a more open mind to assess the possible benefits of phagetherapy. This is especially triggered by an increasing number of publications of patient cases wherespectacular results were achieved with bacteriophages. It is now essential that the efficacy and safetyof phage application be demonstrated in rigorous clinical trials. National and international authoritiesare opening their doors to such trials, and are prone to regulate phage therapy if it is found to beeffective and safe. Furthermore, progress in research on phage biology suggests that other applicationsof phages unrelated to their anti-bacterial action may be on the horizon.Author Contributions: A.G., M.Ł., J.B., J.S., and R.M. wrote the manuscript; A.G, A.G.-R., A.B., E.J.-M., M.Ł.-S.,B.W.-D., N.B., S.L., and K.D. contributed to the design of the work, acquisition and interpretation of data.All authors have approved the submitted version.Funding: This work was supported by statutory funds from the Ludwik Hirszfeld Institute of Immunology andExperimental Therapy of the Polish Academy of Sciences,Warsaw Medical University, and statutory funds fromthe Institute of Biochemistry and Biophysics of the Polish Academy of Sciences.Acknowledgments: We thank A. Ajdukiewicz-Tarkowska, Head of Scientific Information, Main Library of theMedical University ofWarsaw for her help in accessing information from the Web of Science.Conflicts of Interest: A.G., R.M., M.Ł., A.G.-R , J.B., B.W.-D. and K.D. are co-inventors of patents owned by theInstitute and covering phage preparations. Other authors declare that they have no conflict of interest.References1. Borysowski, J.; Mi˛edzybrodzki, R.; Górski, A. Phage Therapy: Current Research and Application; CaisterAcademic Press: Norfolk, UK, 2014.2. Azeredo, J.; Sillankorva, J. (Eds.) Bacteriophage Therapy: From Lab to Clinical Practice; Springer Nature, HumanaPress: New York, NY, USA, 2018; ISBN 978-1-4939-7395-8.3. Alvarez, D.R.; Abedon, S.T. An online phage therapy bibliography: Separating under-indexed wheat fromoverly indexed chaff. AIMS Microbiol. 2017, 3, 525–528.4. P.H.A.G.E. Phages for Human Applications Europe Group. Available online: www.p-h-a-g-e.org (accessedon 5 April 2018).5. 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Hi "bookworm" - is this enough for you? - Sheldon.
Good luck and get some thick spectacles
Wow! That’s all I can say- This definitely is going to be a tough one.
Yes, I do think that I will have to get a special set of glasses for this!
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Faadhilagoro
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(Original post by Bookworm_88)
Hi. I need some help, I’m doing a project on bacteriophages (viruses that replicate and burst out of bacteria cells, thus killing the bacterial pathogenic cell. Or incorporate its genome with that of the Host cell and take over that host cell)
I need some Reading material upon either bacterio(phages) or phage therapy. THE MORE THE BETTER!
Thanks
Hi, I’m interested in bacteriophages as well, I just finished reading the perfect predator by steffanie strathdee, it’s about a personal account of an epidemiologist who’s husband got a bacterial infection and how bacteriophages were used to cure him, I would strongly advise reading it.
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Bookworm_88
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Does it look at it from a scientific perspective or a more personal outlook?
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