Virologica Sinica special issue on “Phages and Therapy”

Note: Open access to this special issue no longer appears to be available…

2015, Volume 30, Issue 1

EDITORIAL

Bacteriophages, revitalized after 100 years in the shadow of antibiotics [pubmed]
Hongping Wei
In this issue, readers will not only find that bacteriophage research is a booming field but also learn about the diverse applications currently being explored for bacteriophages. The biggest driving force behind these applications is the serious threat of bacterial antibiotic resistance that is emerging in the current era.

REVIEWS

Bacteriophage secondary infection [pubmed]
Stephen T Abedon
Phages are credited with having been first described in what we now, officially, are commemorating as the 100th. anniversary of their discovery. Those one-hundred years of phage history have not been lacking in excitement, controversy, and occasional convolution. One such complication is the concept of secondary infection, which can take on multiple forms with myriad consequences. The terms secondary infection and secondary adsorption, for example, can be used almost synonymously to describe virion interaction with already phage-infected bacteria, and which can result in what are described as superinfection exclusion or superinfection immunity. The phrase secondary infection also may be used equivalently to superinfection or coinfection, with each of these terms borrowed from medical microbiology, and can result in genetic exchange between phages, phage-on-phage parasitism, and various partial reductions in phage productivity that have been termed mutual exclusion, partial exclusion, or the depressor effect. Alternatively, and drawing from epidemiology, secondary infection has been used to describe phage population growth as that can occur during active phage therapy as well as upon phage contamination of industrial ferments. Here primary infections represent initial bacterial population exposure to phages while consequent phage replication can lead to additional, that is, secondary infections of what otherwise are not yet phage-infected bacteria. Here I explore the varying meanings and resultant ambiguity that has been associated with the term secondary infection. I suggest in particular that secondary infection, as distinctly different phenomena, can in multiple ways infl uence the success of phage-mediated biocontrol of bacteria, also known as, phage therapy.

Bacteriophage therapy against Enterobacteriaceae [pubmed]
Youqiang Xu, Yong Liu, Yang Liu, Jiangsen Pei, Su Yao, Chi Cheng
The Enterobacteriaceae are a class of gram-negative facultative anaerobic rods, which can cause a variety of diseases, such as bacteremia, septic arthritis, endocarditis, osteomyelitis, lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections, intra-abdominal infections and ophthalmic infections, in humans, poultry, animals and fi sh. Disease caused by Enterobacteriaceae cause the deaths of millions of people every year, resulting in enormous economic loss. Drug treatment is a useful and effi cient way to control Enterobacteriaceae infections. However, with the abuse of antibiotics, drug resistance has been found in growing number of Enterobacteriaceae infections and, as such, there is an urgent need to find new methods of control. Bacteriophage therapy is an efficient alternative to antibiotics as it employs a different antibacterial mechanism. This paper summarizes the history of bacteriophage therapy, its bacterial lytic mechanisms, and the studies that have focused on Enterobacteriaceae and bacteriophage therapy.

Survival and proliferation of the lysogenic bacteriophage CTXΦ in Vibrio cholerae [pubmed]
Fenxia Fan, Biao Kan
The lysogenic phage CTXΦ of Vibrio cholerae can transfer the cholera toxin gene both horizontally (inter-strain) and vertically (cell proliferation). Due to its diversity in form and species, the complexity of regulatory mechanisms, and the important role of the infection mechanism in the production of new virulent strains of V. cholerae, the study of the lysogenic phage CTXΦ has attracted much attention. Based on the progress of current research, the genomic features and their arrangement, the host-dependent regulatory mechanisms of CTXΦ phage survival, proliferation and propagation were reviewed to further understand the phage’s role in the evolutionary and epidemiological mechanisms of V. cholerae.

Phage lytic enzymes: a history [pubmed]
David Trudil
There are many recent studies regarding the efficacy of bacteriophage-related lytic enzymes: the enzymes of ‘bacteria-eaters’ or viruses that infect bacteria. By degrading the cell wall of the targeted bacteria, these lytic enzymes have been shown to efficiently lyse Gram-positive bacteria without affecting normal fl ora and non-related bacteria. Recent studies have suggested approaches for lysing Gram-negative bacteria as well (Briersa Y, et al., 2014). These enzymes include: phage-lysozyme, endolysin, lysozyme, lysin, phage lysin, phage lytic enzymes, phageassociated enzymes, enzybiotics, muralysin, muramidase, virolysin and designations such as Ply, PAE and others. Bacteriophages are viruses that kill bacteria, do not contribute to antimicrobial resistance, are easy to develop, inexpensive to manufacture and safe for humans, animals and the environment. The current focus on lytic enzymes has been on their use as anti-infectives in humans and more recently in agricultural research models. The initial translational application of lytic enzymes, however, was not associated with treating or preventing a specifi c disease but rather as an extraction method to be incorporated in a rapid bacterial detection assay (Bernstein D, 1997).The current review traces the translational history of phage lytic enzymes-from their initial discovery in 1986 for the rapid detection of group A streptococcus in clinical specimens to evolving applications in the detection and prevention of disease in humans and in agriculture.

RESEARCH ARTICLES

Selection of phages and conditions for the safe phage therapy against Pseudomonas aeruginosa infections [pubmed]
Victor Krylov, Olga Shaburova, Elena Pleteneva, Sergey Krylov, Alla Kaplan, Maria Burkaltseva, Olga Polygach, Elena Chesnokova
The emergence of multidrug-resistant bacterial pathogens forced us to consider the phage therapy as one of the possible alternative approaches to treatment. The purpose of this paper is to consider the conditions for the safe, long-term use of phage therapy against various infections caused by Pseudomonas aeruginosa. We describe the selection of the most suitable phages, their most effective combinations and some approaches for the rapid recognition of phages unsuitable for use in therapy. The benefi ts and disadvantages of the various different approaches to the preparation of phage mixtures are considered, together with the specifi c conditions that are required for the safe application of phage therapy in general hospitals and the possibilities for the development of personalized phage therapy.

Molecular dissection of phage lysin PlySs2: integrity of the catalytic and cell wall binding domains is essential for its broad lytic activity [pubmed]
Yanling Huang, Hang Yang, Junping Yu, Hongping Wei
The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain to be established. In the present study, the lytic activity of the catalytic domain (CD, PlySc) and binding specificity of the cell wall binding domain (CBD, PlySb) of PlySs2 were examined. Our results showed that PlySc alone maintains very limited lytic activity. Enhanced green fluorescent protein (EGFP)-fused PlySb displayed high binding affinity to the streptococcal strains tested, including S. suis, S. dysgalactiae, and S. agalactiae, but not staphylococci, supporting its utility as a good CBD donor for streptococcal-targeted lysin engineering. EGFP-fused intact PlySs2 similarly displayed high affinity for streptococci, but not staphylococci. Notably, four truncated PlySb fragments showed no binding capacity. These fi ndings collectively indicate that integrity of the PlySc and PlySb domains is an essential determinant of the broad lytic activity of PlySs2.

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus [pubmed]
Xiuling Ji, Chunjing Zhang, Yuan Fang, Qi Zhang, Lianbing Lin, Bing Tang, Yunlin Wei
As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a “living fossil” for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head (59.2 nm in length, 31.9 nm in width) and a tail (43.2 nm in length). Bacteriophage VMY22 was classifi ed as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at pH 5.0-9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.

LETTERS

Variation of resistance and infectivity between Pseudomonas fluorescens SBW25 and bacteriophage Ф2 and its therapeutic implications [pubmed]
Hanchen Chen, Guohua Chen
Studies of the coevolutionary dynamics between Pseudomonas fluorescens SBW25 and bacteriophage Ф can explore host resistance and parasite infectivity with applications in the ecological and therapeutic fields.Coevolutionary dynamics determine the efficacy of phage-based therapy. In the study described here, bacterial resistance and phage infectivity fluctuated with culturetime, perhaps resulting from random mutation and temporaladaptation, which reminds us of the necessity toconsider evolutionary mechanisms when applying phageto treat bacterial infections.

A novel transposable Mu-like prophage in Bacillus alcalophilus CGMCC 1.3604 (ATCC 27647) [pubmed]
Junjie Yang, Yimeng Kong, Xuan Li, Sheng Yang
In this letter, we provide evidence for the first transposable prophage BalMu-1 in Bacilli. The transposable prophage (BalMu-1, Genbank No. KP063902 and KP063903) was identified in Bacillus alcalophilus CGMCC 1.3604(ATCC 27647) through high throughput genome sequencing and PCR-dideoxy chain-termination(Sanger) sequencing.

Isolation and characterization of a lytic bacteriophage φKp-lyy15 of Klebsiella pneumoniae [pubmed]
Yinyin Lu, Hongyan Shi, Zhe Zhang, Fang Han, Jinghua Li, Yanbo Sun
In conclusion, the lytic bacteriophage φKp-lyy belonging to the Siphoviridae family specific for K. pneumonia was isolated and characterized. φKp-lyy displayed a short latent period, stability to a wide pH rang, high thermal resistance, and lytic activity toward a relatively broad range of K. pneumonia isolates. Thus, phage φKplyy should be considered as a candidate for inclusion in phage cocktails to control K. pneumoniae-associated nosocomial infections.

Expression and purification of recombinant lyase gp17 from the LSB-1 phage in Escherichia coli [pubmed]
Taiwu Wang, Hui Lin, Lu Zhang, Guorong Huang, Long Wu, Lei Yu, Hongyan Xiong
In this study, we successfully expressed and purified the recombinant gp17 protein from the LSB-1 phage and also confirmed its bacteriostatic effect. Assays also showed that the recombinant enzyme was soluble and had signifi cant lyase effects on the host bacterium, EIEC8401. A preliminary study demonstrated that the enzyme did not have inhibitory effects on other strains (unpublished data), which might indicate that the exclusive antibacterial effect of gp17 on EIEC8401 could have a special significance in practical application in bacterial therapy.

T4-like coliphage φKAZ14 virulent to pathogenic and extended spectrum β-lactamase-producing Escherichia coli of poultry origin [pubmed]
Kaikabo Adamu Ahmad, Abdulkarim Sabo Mohanmmed, Faridah Abas, Sieo Chin Chin
The aim of the present study was to isolate bacteriophages for the pre-harvest biocontrol of APEC 01 and ESBL-producing E. coli in chicken, in order to mitigate the risk of these pathogens to the food chain. Isolation and characterization of the T4-like coliphage KAZ14, lytic to APEC 01 and ESBL-producing E. coli, is reported and discussed.

Isolation and complete genome sequence of a novel virulent mycobacteriophage, CASbig [pubmed]
Tieshan Teng, Junping Yu, Hang Yang, Hongping Wei
In this study, we report the isolation and the complete genome of a novel mycobacteriophage, CASbig, which has an icosahedral head (diameter 50 ± 2 nm) and a long, non-contractile tail (length 160 ± 5 nm) with transverse striations, ending in a small knob. The length of the tail includes the middle of the baseplate, and the head measurements were taken between opposite apices. These characteristics indicate that the phage belongs to the family Siphoviridae morphotypes.

INSIGHT

Experience of the Eliava Institute in bacteriophage therapy [pubmed]
Mzia Kutateladze
The rapid propagation of multidrug resistant bacterial strains is leading to renewed interest in bacteriophage therapy. With challenges in the treatment of bacterial infections, it is essential for people worldwide to understand how alternative approaches, such as bacteriophages, could be used to combat antibiotic resistant bacteria. The Eliava Institute of Bacteriophages, Microbiology and Virology (Tbilisi, Georgia) is arguably the most famous institution in the world focused on the isolation, study, and selection of phages active against a variety of bacterial pathogens.

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E. coli, CRISPR, Biases in Our Understanding of Phage Ecology, and Possible Implications for Phage Therapy

Stephen T. Abedon

Department of Microbiology – The Ohio State University

phage.org – phage-therapy.org – biologyaspoetry.org


 

We’re all biased by what we know best and the link below discusses why, historically as well as microbiologically, we all “grew up” with the notion that envelope mutations are the primary means by which phage resistance evolves in bacteria. So thank you E. coli (I state with sarcasm):

http://schaechter.asmblog.org/schaechter/2014/11/why-crispr-doesnt-work-in-.html

What, I ask, are the implications for phage therapy of resistance mechanisms to specific phages that are essentially cost free and, at least arguably, Lamarckian as well, i.e., as due to CRISPR? For well-trained phage-therapy teams, I suspect not much. This is because, whether employing cocktails or monophages, the intention generally will be to hit bacterial targets hard and with whatever it takes to clear the infection, such as to switching phages during monophage therapy if resistance is noted.

But for monophages in the hands of less well-trained individuals, e.g., over-the-counter phage formulations or in the hands of poorly trained or regulated clinicians, the potential for development and then transmission of fully fit pathogens that nonetheless are fully resistant to a specific monophage could be fairly high. Importantly, and as relevant to the cited E. coi-CRISPR story, this issue may be more relevant for some pathogens, i.e., those with intact CRISPR systems, than it is for others.

So perhaps we can add inhibition of the potential for therapy-induced evolution of phage resistance among pathogens – as could then be transmitted across affected human communities – as an additional advantage of  prêt-à-porter (phage cocktails) versus sur-mesure (monophage therapy), while still retaining an argument for sur-mesure particularly among highly experienced phage therapists.

As we note in Chan and Abedon (2012), I nevertheless don’t buy arguments that spontaneously occurring phage host range mutations can be counted on in situ to counter bacterial evolution to phage resistance whether in the context of phage cocktails or instead monotherapy. From p. 19 of that publication:

A further consideration is that just as cocktails of phages may be thwarted in their ability to target low densities of phage-resistant bacteria, particularly given active treatment, these concerns should be even greater if one is relying on in situ phage evolution to supply resistance-countering phages… The reason for this is that the necessary host-range mutant phage types will be present in even lower densities than the phages explicitly found in cocktails. These same concerns may also be seen even in the absence of spatial structure so long as those phages within a cocktail that are amplified in situ, that is, in the course of active treatment, are not the same phages to which bacterial phage-resistant mutants are sensitive… Active therapy even with phage cocktails thus may be inherently incompatible with early interference with the evolution of bacterial resistance to phages.

 

Phage cocktails nevertheless should be better suited than monophages for dealing with evolving bacterial resistance to phages simply because cocktails inherently possess greater total numbers of phage particles that display divergent host ranges. On the other hand, the generation of cocktails of phages that display divergent host ranges – but where those phages nevertheless have been derived from a common genetic “platform” – might be expected to perform little better than monophages in the face of CRISPR-mediated phage resistance in target bacteria.

Further (Phage Therapy) Reading:

Chan, B. K., S. T. Abedon, and C. Loc-Carrillo. 2013. Phage cocktails and the future of phage therapy. Future.Microbiol. 8:769-783. [PubMed]

Chan, B. K. and S. T. Abedon. 2012. Phage therapy pharmacology: phage cocktails. Adv.Appl.Microbiol.  78:1-23. [PubMed]

Pirnay, J. P., V. D. De, G. Verbeken, M. Merabishvili, N. Chanishvili, M. Vaneechoutte, M. Zizi, G. Laire, R. Lavigne, I. Huys, G. Van den Mooter, A. Buckling, L. Debarbieux, F. Pouillot, J. Azeredo, E. Kutter, A. Dublanchet, A. Gorski, and R. Adamia. 2011. The phage therapy paradigm: prêt-à-porter or sur-mesure? Pharm.Res 28:934-937. [PubMed]

crAssphage

A new study led by researchers at San Diego State University has found that more than half the world’s population is host to a newly described virus, named crAssphage, which infects one of the most
common gut bacterial species, Bacteroides. This bacterium is thought to be connected with obesity, diabetes and other gut-related diseases.

The origin of virulence, and why its important.

Virulence is an abstraction of the harm caused to hosts by a pathogen, and explaining the paradox of virulence has been an active field of study in evolution for a while. In general the harm caused to the hosts of pathogens is not great for the pathogen, after all, why hurt or lose a useful host? However, in studying the abstraction with basic research, we’ve found that virulence is almost always is part of helping the pathogen find a new host. Thus the generalized answer to the paradox is that so long as the harm to the host causes the parasite to spread effectively enough, it doesn’t really matter how much harm is caused to the host – as the parasite will have already found new hosts to spread from. At the same time, helpful bacteria don’t have nearly the same need to spread as pathogenic ones, as they keep their hosts happy and alive and can stick around for longer.

Here I’ll introduce two papers demonstrating this model and try to convince you of how important it is.

The spectrum between virulence and mutualism can be seen as a trade off between two strategies, as well as of course often a mix between the two. A critter existing in community with another one can care little for its host and work to be as infectious as possible at the host’s expense, thus increasing virulence. In this strategy it doesn’t matter so much that the host becomes quickly unsuitable so long as the parasite has already found replacement hosts sneezed on, or transmitted to, by the time that happens. Or it can do the opposite and try its best to reduce impact on the host, spread infectious particles slowly or even not at all, and thus not need to spread too quickly because it will last a while in each host. Most of the critters that live in our guts and on our skin are at this end of the spectrum, and have become so adept at not messing up their host as to actually benefit us in some way. On the other end of the spectrum are parasitoids. These are the parasites that not only destroy their host in their race to infect as many more hosts as possible, but spend the majority of their life cycle doing so and ultimately sterilize or kill, and sometimes consume the host in the process. The Xenomorphs from the movie Alien are a beautiful example of a bunch of these sorts of parasitiod strategies, each inspired by real terrifying stuff in nature. This might all seem uselessly theoretical, but the implications it has for public health are really cool.

Before the advent of antibiotics, we lived with Staphylococcus aureus strains on our skin that existed in a complex mixture of commensal and virulent strategies, but antibiotics suddenly applied very strong selective pressure against any vaguely virulent strategy. Thus, following the model, the observed sudden decrease in both virulence and transmissibility of virulent strains makes a lot of sense. However, the sudden increase in both virulence and transmissibility of virulent strains that we’ve seen in multi-drug resistant (MRSA) strains also makes sense. Indeed, if you look back far enough in the literature all of the crazy new and terrible virulence factors we are now seeing in MRSA strains all existed before the 1930s. For example, while the pyomyositis and necrotizing pneumonia we are now seeing is commonly associated with poverty, tropical climates and HIV, ie: things which didn’t get much attention prior to 1935, it was described. (At lest with this source you’ll need to wade your way past the kinds of phrases that start with “Africans are not different from any other humans, however, …” to page 1214) Until recently it would not be terribly remarkable, being easily addressed with a simple round of I.V. antibiotics.  Additionally, the PVL toxin which that first paper describes as now being found in pneumonia was initially discovered by Van deVelde in 1894 and was named after Sir Philip Noel Panton and Francis Valentine when they associated it with soft tissue infections in 1932. All of this makes logical sense anyhow, the mechanisms of antibiotic resistance are not associated with pathogenesis.

Timing of transmission and the evolution of virulence of an insect virus.

JC de Roode, AJ Yates, & S Altizer. Published 2002 in Proc. R. Soc. Lond. B doi:10.1098/rspb.2002.1976

We used the nuclear polyhedrosis virus of the gypsy moth, Lymantria dispar, to investigate whether the timing of transmission influences the evolution of virulence. In theory, early transmission should favour rapid replication and increase virulence, while late transmission should favour slower replication and reduce virulence. We tested this prediction by subjecting one set of 10 virus lineages to early transmission (Early viruses) and another set to late transmission (Late viruses). Each lineage of virus underwent nine cycles of transmission. Virulence assays on these lineages indicated that viruses transmitted early were significantly more lethal than those transmitted late. Increased exploitation of the host appears to come at a cost, however. While Early viruses initially produced more progeny, Late viruses were ultimately more productive over the entire duration of the infection. These results illustrate fitness trade-offs associated with the evolution of virulence and indicate that milder viruses can obtain a numerical advantage when mild and harmful strains tend to infect separate hosts.

Virulence-transmission trade-offs and population divergence in virulence in a naturally occurring butterfly parasite (PDF).

VS Cooper, MH Reiskind, et al. Published 2002 in PNAS doi:10.1073/pnas.0710909105

Why do parasites harm their hosts? Conventional wisdom holds that because parasites depend on their hosts for survival and transmission, they should evolve to become benign, yet many parasites cause harm. Theory predicts that parasites could evolve virulence (i.e., parasite-induced reductions in host fitness) by balancing the transmission benefits of parasite replication with the costs of host death. This idea has led researchers to predict how human interventions—such as vaccines—may alter virulence evolution, yet empirical support is critically lacking. We studied a protozoan parasite of monarch butterflies and found that higher levels of within-host replication resulted in both higher virulence and greater transmission, thus lending support to the idea that selection for parasite transmission can favor parasite genotypes that cause substantial harm. Parasite fitness was maximized at an intermediate level of parasite replication, beyond which the cost of increased host mortality outweighed the benefit of increased transmission. A separate experiment confirmed genetic relationships between parasite replication and virulence, and showed that parasite genotypes from two monarch populations caused different virulence. These results show that selection on parasite transmission can explain why parasites harm their hosts, and suggest that constraints imposed by host ecology can lead to population divergence in parasite virulence.