Many publications list advantages and disadvantages associated with phage therapy, which is the use of bacterial viruses to combat populations of nuisance or pathogenic bacteria. The goal of this commentary is to discuss many of those issues in a single location. In terms of “Pros,” for example, phages can be bactericidal, can increase in number over the course of treatment, tend to only minimally disrupt normal flora, are equally effective against antibiotic-sensitive and antibiotic-resistant bacteria, often are easily discovered, seem to be capable of disrupting bacterial biofilms, and can have low inherent toxicities. In addition to these assets, we consider aspects of phage therapy that can contribute to its safety, economics, or convenience, but in ways that are perhaps less essential to the phage potential to combat bacteria. For example, autonomous phage transfer between animals during veterinary application could provide convenience or economic advantages by decreasing the need for repeated phage application, but is not necessarily crucial to therapeutic success. We also consider possible disadvantages to phage use as antibacterial agents. These “Cons,” however, tend to be relatively minor.
alternative medicine; antibiotics; antimicrobial drugs; biocontrol; phage therapy
The spread of natural or weaponized drug-resistant plague among humans is a credible high consequence threat to public health that demands the prompt introduction of alternatives to antibiotics such as bacteriophage. Early attempts to treat plague with phages in the 1920s–1930s were sometimes promising but mostly failed, purportedly due to insufficient knowledge of phage biology and poor experimental design. We recently reported the striking stability of plague diagnostic bacteriophages, their safety for animal use, propagation in vivo and partial protection of mice from deadly plague after a single injection of phage. In this addendum we reflect on that article, other recent publications and our unpublished data, and discuss the prospects of phage therapy against plague.
Yersinia pestis; multidrug-resistant plague; bacteriophages; phage safety; pharmacokinetics and pharmacodynamics; plague therapy
Bacteriophage therapy, the use of viruses that infect bacteria as antimicrobials, has been championed as a promising alternative to conventional antibiotics. Although in the laboratory bacterial resistance against phages arises rapidly, resistance so far has been an only minor problem for the effectiveness of phage therapy. Resistance to antibiotics, however, has become a major issue after decades of extensive use. Should we expect similar problems after long-term use of phages as antimicrobials? Like antibiotics, phages are often noted to be drivers of bacterial evolution. Should we expect phage-treated pathogens to develop a general resistance to phages over time, a resistance against which only, for example, hypothetically co-evolved phages might be infective? Here we argue that the global infection patterns of phages suggest that this is not necessarily a concern as environmental phages often can infect bacteria with which those phages lack any recent co-evolutionary history.
antibiotic resistance; ecology; evolution; phage resistance; phage-therapy
Interest in using bacteriophages to control the growth and spread of bacterial pathogens is being revived in the wake of widespread antibiotic resistance. However, little is known about the ecological effects that high concentrations of phages in the environment might have on natural microbial communities. We review the current evidence suggesting phage-mediated environmental perturbation, with a focus on agricultural examples, and describe the potential implications for human health and agriculture. Specifically, we examine the known and potential consequences of phage application in certain agricultural practices, discuss the risks of evolved bacterial resistance to phages, and question whether the future of phage therapy will emulate that of antibiotic treatment in terms of widespread resistance. Finally, we propose some basic precautions that could preclude such phenomena and highlight existing methods for tracking bacterial resistance to phage therapeutic agents.
phage therapy; antibiotic resistance; coevolution; phage resistance; microbial communities
Interest in using bacteriophages to treat bacterial infections (phage therapy) is growing, but there have been few experiments comparing the effects of different treatment strategies on both bacterial densities and resistance evolution. While it is established that multiphage therapy is typically more effective than the application of a single phage type, it is not clear if it is best to apply phages simultaneously or sequentially. We tried single- and multiphage therapy against Pseudomonas aeruginosa PAO1 in vitro, using different combinations of phages either simultaneously or sequentially. Across different phage combinations, simultaneous application was consistently equal or superior to sequential application in terms of reducing bacterial population density, and there was no difference (on average) in terms of minimizing resistance. Phage-resistant bacteria emerged in all experimental treatments and incurred significant fitness costs, expressed as reduced growth rate in the absence of phages. Finally, phage therapy increased the life span of wax moth larvae infected with P. aeruginosa, and a phage cocktail was the most effective short-term treatment. When the ratio of phages to bacteria was very high, phage cocktails cured otherwise lethal infections. These results suggest that while adding all available phages simultaneously tends to be the most successful short-term strategy, there are sequential strategies that are equally effective and potentially better over longer time scales.
Nowadays the most difficult problem in treatment of bacterial infections is the appearance of resistant bacteria to the antimicrobial agents so that the attention is being drawn to other potential targets. In view of the positive findings of phage therapy, many advantages have been mentioned which utilizes phage therapy over chemotherapy and it seems to be a promising agent to replace the antibiotics. This review focuses on an understanding of phages for the treatment of bacterial infectious diseases as a new alternative treatment of infections caused by multiple antibiotic resistant bacteria. Therefore, utilizing bacteriophage may be accounted as an alternative therapy. It is appropriate time to re-evaluate the potential of phage therapy as an effective bactericidal and a promising agent to control multidrug-resistant bacteria.
Bacteria; Bacteriophages; Drug resistance; Therapeutics
Evidence has been presented that the burst size in the Group A and Group C streptococcal phage-host systems are in general similar producing approximately 13 phage particles per infected coccus. The exception was the C1 phage which produced 10 times more virus particles than all the other phages tested. The eclipse period for the A25 phage-host system was found to extend for 34 min, while the C1 phage were found as early as 10 min after infection. Conclusive evidence has been presented indicating that mercuric ions at 2.5 x 10–6 M concentration have the ability to halt intracellular phage production at any point during the infective cycle of A25 bacteriophages. This blocking action can then be quickly reversed with the addition of reduced glutathione with subsequent completion of the viral cycle.
In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn2+ ions for its activity, contrary to the bioinformatics prediction of a Zn2+ ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.
Pseudomonas aeruginosa is a frequent participant in wound infections. Emergence of multiple antibiotic resistant strains has created significant problems in the treatment of infected wounds. Phage therapy (PT) has been proposed as a possible alternative approach. Infected wounds are the perfect place for PT applications, since the basic condition for PT is ensured; namely, the direct contact of bacteria and their viruses. Plenty of virulent (“lytic”) and temperate (“lysogenic”) bacteriophages are known in P. aeruginosa. However, the number of virulent phage species acceptable for PT and their mutability are limited. Besides, there are different deviations in the behavior of virulent (and temperate) phages from their expected canonical models of development. We consider some examples of non-canonical phage-bacterium interactions and the possibility of their use in PT. In addition, some optimal approaches to the development of phage therapy will be discussed from the point of view of a biologist, considering the danger of phage-assisted horizontal gene transfer (HGT), and from the point of view of a surgeon who has accepted the Hippocrates Oath to cure patients by all possible means. It is also time now to discuss the possible approaches in international cooperation for the development of PT. We think it would be advantageous to make phage therapy a kind of personalized medicine.
Pseudomonas aeruginosa bacteriophages diversity; noncanonical relations of phages and bacteria; phage’s migrations; phage genomes instability; pseudolysogeny and pseudovirulence; phage therapy as a kind of personalized medicine
The study of bacterial viruses (bacteriophages or phages) proved pivotal in the nascence of the disciplines of molecular biology and microbial genetics, providing important information on the central processes of the bacterial cell (DNA replication, transcription and translation) and on how DNA can be transferred from one cell to another. As a result of the pioneering genetics studies and modern genomics, it is now known that phages have contributed to the evolution of the microbial cell and to its pathogenic potential. Because of their ability to transmit genes, phages have been exploited to develop cloning vector systems. They also provide a plethora of enzymes for the modern molecular biologist. Until the introduction of antibiotics, phages were used to treat bacterial infections (with variable success). Western science is now having to re-evaluate the application of phage therapy – a therapeutic modality that never went out of vogue in Eastern Europe – because of the emergence of an alarming number of antibiotic-resistant bacteria. The present article introduces the reader to phage biology, and the benefits and pitfalls of phage therapy in humans and animals.
Bacterial virus; Bacteriophage; Diagnostic tools; Human and animal studies; Novel therapies; Phage therapy; Phage typing; Phagotherapy
Bacterial resistance to antibiotics poses a serious health threat. Since research into new antibiotics is not progressing at the same rate as the development of bacterial resistance, widespread calls for alternatives to antibiotics have been made. Phage therapy is an ideal alternative candidate to be investigated. However the success of phage therapy may be hampered by a lack of investment support from large pharmaceutical companies, due to their narrow spectrum of activity in antibiotics, very large costs associated with clinical trials of the variety of phages needed, and regulatory requirements remaining unclear. Intellectual property is difficult to secure for therapeutic phage products for a variety of reasons, and patenting procedures vary widely between the US and the EU. Consequently, companies are more likely to invest in phage products for decontamination or veterinary use, rather than clinical use in humans. Some still raise questions as to the safety of phage therapy overall, suggesting the possibility of cytotoxicity and immunogenicity, depending on the phage preparation and route. On the other hand, with patients dying because of infections untreatable with conventional antibiotics, the question arises as to whether it is ethical not to pursue phage therapy more diligently. A paradigm shift about how phage therapy is perceived is required, as well as more rigorous proof of efficacy in the form of clinical trials of existing medicinal phage products. Phage therapy potential may be fulfilled in the meantime by allowing individual preparations to be used on a named-patient basis, with extensive monitoring and multidisciplinary team input. The National Health Service and academia have a role in carrying out clinical phage research, which would be beneficial to public health, but not necessarily financially rewarding.
phage therapy; limitations; safety; ethics; paradigm changes; clinical use
Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages.
We isolated and characterized a phage, named JG024, which infects a broad range of clinical and environmental P. aeruginosa strains. Sequencing of the phage genome revealed that the phage JG024 is highly related to the ubiquitous and conserved PB1-like phages. The receptor of phage JG024 was determined as lipopolysaccharide. We used an artificial sputum medium to study phage infection under conditions similar to a chronic lung infection. Alginate production was identified as a factor reducing phage infectivity.
Phage JG024 is a suitable broad host range phage which could be used in phage therapy. Phage infection experiments under simulated chronic lung infection conditions showed that alginate production reduces phage infection efficiency.
Understanding the mutual interactions of bacterial and phage populations in the environment of
a human or animal body is essential in any attempt to influence these complex processes,
particularly for rational phage therapy. Current knowledge on the impact of naturally occurring
bacteriophages on the populations of their host bacteria, and their role in the homeostasis
maintenance of a macro host, is still sketchy. The existing data suggest that different
mechanisms stabilize phage–bacteria coexistence in different animal species or different
body sites. The defining set of parameters governing phage infection includes specific
physical, chemical, and biological conditions, such as pH, nutrient densities, host prevalence,
relation to mucosa and other surfaces, the presence of phage inhibiting substances, etc. Phage
therapy is also an ecological process that always implies three components that form a complex
pattern of interactions: populations of the pathogen, the bacteriophages used as antibacterial
agents, and the macroorganism. We present a review of contemporary data on natural
bacteriophages occuring in human– and animal–body associated microbial communities,
and analyze ecological and physiological considerations that determine the success of phage
therapy in mammals.
bacteriophages; phage therapy; human body microbiota; animal body microbiota; bacteriophage ecology
The complete genome sequences of over 220 mycobacteriophages reveal them to be highly diverse, with numerous types sharing little or no nucleotide sequence identity with each other. We have determined the preferences of these phages for M. tuberculosis and for other strains of M. smegmatis, and find there is a correlation between genome type (cluster, subcluster, singleton) and host range. For many of the phages, expansion of host range occurs at relatively high frequencies, and we describe several examples in which host constraints occur at early stages of infection (adsorption or DNA injection), and phages have the ability to expand their host range through mutations in tail genes. We present a model in which phage diversity is a function of both the ability of phages to rapidly adapt to new hosts and the richness of the diversity of the bacterial population from which those phages are isolated.
Bacteriophage; Mycobacteriophage; Host Range; Genome
Flavobacteria and their phages were isolated from Finnish freshwaters and fish farms. Emphasis was placed on finding phages infecting the fish pathogen Flavobacterium columnare for use as phage therapy agents. The host ranges of the flavobacterial phages varied, phages infecting F. columnare being more host specific than the other phages.
The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.
Two Aeromonas hydrophila bacteriophages, Aeh1 and Aeh2, were isolated from sewage. Both phages showed binal symmetry. The dimensions of A. hydrophila phages Aeh1 and Aeh2 differed from those of the other Aeromonas phages. Also, phage Aeh2 was the largest Aeromonas phage studied to date. Phage Aeh1 formed small, clear plaques, and phage Aeh2 formed turbid plaques with clear centers. Both phages were sensitive to chloroform treatment, being totally inactivated after treatment for 1 h at 60°C at pH 3 and 11. However, the infectivity of Aeh1 phage stocks increased by approximately fivefold after they were treated at pH 10 for 1 h at 22°C. Phages Aeh1 and Aeh2 were serologically unrelated and had latent periods of 39 and 52 min, respectively. The average burst sizes of phages Aeh1 and Aeh2 were 17 and 92 PFU per cell, respectively. Phage Aeh1 infected 13 of 22 A. hydrophila strains tested, whereas phage Aeh2 infected only its original host. Phage Aeh1 infected some A. hydrophila strains only at or below 37°C. Neither phage infected the two A. (Plesiomonas) shigelloides strains used in this study.
Knowledge of phage-host interactions at a fundamental level is central to the design of rational strategies for the development of phage-resistant strains that may be applied in industrial settings. Phages infecting lactic acid bacteria, in particular Lactococcus lactis and Streptococcus thermophilus, negatively impact on dairy fermentation processes with serious economic implications. In recent years a wealth of information on structural protein assembly and topology has become available relating to phages infecting Escherichia coli, Bacillus subtilis and Lactococcus lactis, which act as models for structural analyses of dairy phages. In this review, we explore the role of model tailed phages, such as T4 and SPP1, in advancing our knowledge regarding interactions between dairy phages and their hosts. Furthermore, the potential of currently investigated dairy phages to in turn serve as model systems for this particular group of phages is discussed.
bacteriophage; milk fermentation; receptor
Along with the increasing threat of nosocomial infections by vancomycin-resistant Enterococcus faecalis, bacteriophage (phage) therapy has been expected as an alternative therapy against infectious disease. Although genome information and proof of applicability are prerequisites for a modern therapeutic phage, E. faecalis phage has not been analyzed in terms of these aspects. Previously, we reported a novel virulent phage, φEF24C, and its biology indicated its therapeutic potential against E. faecalis infection. In this study, the φEF24C genome was analyzed and the in vivo therapeutic applicability of φEF24C was also briefly assessed. Its complete genome (142,072 bp) was predicted to have 221 open reading frames (ORFs) and five tRNA genes. In our functional analysis of the ORFs by use of a public database, no proteins undesirable in phage therapy, such as pathogenic and integration-related proteins, were predicted. The noncompetitive directions of replication and transcription and the host-adapted translation of the phage were deduced bioinformatically. Its genomic features indicated that φEF24C is a member of the SPO1-like phage genus and especially that it has a close relationship to the Listeria phage P100, which is authorized for prophylactic use. Thus, these bioinformatics analyses rationalized the therapeutic eligibility of φEF24C. Moreover, the in vivo therapeutic potential of φEF24C, which was effective at a low concentration and was not affected by host sensitivity to the phage, was proven by use of sepsis BALB/c mouse models. Furthermore, no change in mouse lethality was observed under either single or repeated phage exposures. Although further study is required, φEF24C can be a promising therapeutic phage against E. faecalis infections.
In this commentary I consider use of the term “lysis from without” (LO) along with the phenomenon's biological relevance. LO originally described an early bacterial lysis induced by high-multiplicity virion adsorption and that occurs without phage production (here indicated as LOV). Notably, this is more than just high phage multiplicities of adsorption leading to bacterial killing. The action on bacteria of exogenously supplied phage lysin, too, has been described as a form of LO (here, LOL). LOV has been somewhat worked out mechanistically for T4 phages, has been used to elucidate various phage-associated phenomena including discovery of the phage eclipse, may be relevant to phage ecology, and, with resistance to LO (LOR), is blocked by certain phage gene products. Speculation as to the impact of LOV on phage therapy also is fairly common. Since LOV assays are relatively easily performed and not all phages are able to induce LOV, a phage's potential to lyse bacteria without first infecting should be subject to at least in vitro experimental confirmation before the LOV label is applied. The term “abortive infection” may be used more generally to describe non-productive phage infections that kill bacteria.
abortive infection; bacteriophage; lysin; lysis; lysis from without; phage therapy
Induction of the synthesis of the receptor for phage lambda is obtained by adding maltose and adenosine 3'-5'-cyclic monophosphate to glucose grown cells of Escherichia coli. Bacteria induced for a short period of time were infected with a high multiplicity of phage lambda , and examined under the electron microscope. Only a fraction of the bacteria were seen to have adsorbed a large number of phage particles. The majority of such bacteria had a constriction indicating formation of a septum, and, in this case, the density of adsorbed particles was highest in the vicinity of the constriction. When found on bacteria showing no sign of septum formation, the adsorbed particles were asymmetrically distributed, one pole of the bacteria being more heavily covered with phage particles than the other. Such asymetrically covered bacteria are believed to have originated from cells which divided during the induction period. The results suggest that the receptor for phage lambda, a protein of the outer membrane, is integrated in the cell envelope during the last quarter of each generation and that the integration process is initiated in the vicinity of the forming septum.
Human dental plaque samples were screened for the presence of bacteriophage for Actinomyces viscosus and Streptococcus sanguis. None of the 336 samples yielded phage for S. sanguis, but 10 contained virulent actinomyces phage. A high host cell specificity was observed in that one phage isolate infected only A. viscosus T14V, eight phage isolates infected only A. viscosus MG-1, and one infected both strains. None was capable of productively infecting various other actinomyces strains that represented the six actinomyces coaggregation groups. Because phage-containing samples occurred randomly in this survey, no correlation between the individual collecting the samples, dental clinic, or type of patient and the presence of phage in the sample was noted. Examination of one of the samples that yielded phage for the presence of a natural host strain for that particular phage resulted in the isolation of two strains which were identified as A. viscosus serotype II and Actinomyces naeslundii serotype I. This is the first report of an A. naeslundii host strain and actinomyces bacteriophage of human dental plaque origin. The finding of both phage and host strains in the same dental plaque sample along with the observation of high host cell specificity by these phage provide indicators that support an active role for actinomyces bacteriophage in oral microbial ecology. The use of these freshly isolated phage as probes to study actinomyces coaggregation properties is discussed.
Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.
For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. Theory suggests that higher bacterial densities should select for shorter phage latent periods. Consistently, we have found that higher host densities (≥∼107 bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type. One such variant, dubbed sta5, displays a latent period that is ∼70 to 80% of that of the wild type—which is nearly as short as the RB69 eclipse period—and which has a corresponding burst size that is ∼30% of that of the wild type. We show that at higher host densities (≥∼107 bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition. We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth. The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (≤∼106 bacteria/ml). From these observations we suggest that phage displaying very short latent periods may be viewed as specialists for propagation when bacteria within cultures are highly prevalent and transmission between cultures is easily accomplished.
A study was undertaken to examine the feasibility of using naturally-occurring bacteriophages to assess the impact of re-laying on levels of viral contamination in Crassostrea gigas, the Pacific oyster. Two phages were chosen. One, male-specific (F+), was enumerated using Salmonella typhimurium. The other, a somatic phage, was detected using an, as yet, uncharacterized Escherichia coli. Investigations, using a variety of re-laying sites, demonstrated that numbers of F+ phage in oyster tissue declined more rapidly than those of somatic phage. For example, in oysters placed in commercially-used sea water ponds, F+ phage reached undetectable levels within 2-3 weeks, whereas somatic phage could still be detected 5 weeks after re-laying. The studies suggest that F+ phage may not be a suitable indicator for virus removal and that somatic phage may be better suited to this role.