Phagotherapy in terms of bacteriophage genetics: hopes, perspectives, safety, limitations

The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine. In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections. Constructs obtained by gene engineering are increasingly used to change some bacteriophage: properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages. In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics. Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harmless phage therapy.     

Phage Therapy in Terms of Bacteriophage Genetics: Hopes, Prospects, Safety, Limitations

The appearance and spreading of multidrug-resistant bacterial pathogens is a consequence of the large-scale use of antibiotics in medicine. In view of this, claims for the phage therapy were renewed: in recent studies, the natural phages and their products neutralizing various proteins, as well as the bacterial products often controlled by defective prophages (bacteriocins) were applied for treatment of bacterial infections. Constructs obtained by gene engineering are increasingly used to change bacteriophage properties to expand the spectrum of their lytic activity and to eliminate therapeutic drawbacks of some natural phages. In this review, the problem of phage therapy is discussed in general with respect to bacteriophage properties, their genetics, structure, evolution, taking into account long-term experience of the author in the field of bacteriophage genetics. Note that the general concept of phage therapy should be developed to ensure long-term, efficient and harmless phage therapy.     

PEGylation of bacteriophages increases blood circulation time and reduces T‐helper type 1 immune response

The increasing occurrence of antibiotic‐resistant pathogens is of growing concern, and must be counteracted by alternative antimicrobial treatments. Bacteriophages represent the natural enemies of bacteria. However, the strong immune response following application of phages and rapid clearance from the blood stream are hurdles which need to be overcome. Towards our goal to render phages less immunogenic and prolong blood circulation time, we have chemically modified intact bacteriophages by conjugation of the non‐immunogenic polymer monomethoxy‐polyethylene glycol (mPEG) to virus proteins. As a proof of concept, we have used two different polyvalent and strictly virulent phages of the Myoviridae, representing typical candidates for therapeutical approaches: Felix‐O1 (infects Salmonella) and A511 (infects Listeria). Loss of phage infectivity after PEGylation was found to be proportional to the degree of modification, and could be conveniently controlled by adjusting the PEG concentration. When injected into naïve mice, PEGylated phages showed a strong increase in circulation half‐life, whereas challenge of immunized mice did not reveal a significant difference. Our results suggest that the prolonged half‐life is due to decreased susceptibility to innate immunity as well as avoidance of cellular defence mechanisms. PEGylated viruses elicited significantly reduced levels of T‐helper type 1‐associated cytokine release (IFN‐γ and IL‐6), in both naïve and immunized mice. This is the first study demonstrating that PEGylation can increases survival of infective phage by delaying immune responses, and indicates that this approach can increase efficacy of bacteriophage therapy.     

Wastewater as a fertility source for novel bacteriophages against multi-drug resistant bacteria

Antibiotic resistance is a common and serious public health worldwide. As an alternative to antibiotics, bacteriophage (phage) therapy offers one of the best solutions to antibiotic resistance. Bacteriophages survive where their bacterial hosts are found; thus, they exist in almost all environments and their applications are quite varied in the medical, environmental, and industrial fields. Moreover, a single phage or a mixture of phages can be used in phage therapy; mixed phages tend to be more effective in reducing the number and/or activity of pathogenic bacteria than that of a single phage.     

Improved method for selecting RNA-binding activities in vivo.

RNA challenge phages are modified versions of bacteriophage P22 that allow one to select directly for a specific RNA-protein interaction in vivo. The original construction method for generating a bacteriophage that encodes a specific RNA target requires two homologous recombination reactions between plasmids and phages in bacteria. An improved method is described that enables one to readily construct RNA challenge phages through a single homologous recombination reaction in vivo. We have applied the new method to construct a derivative of P22R17, an RNA challenge phage that undergoes lysogenic development in bacterial cells that express the bacteriophage R17/MS2 coat protein.     

Successful eradication of methicillin-resistantStaphylococcus aureus (MRSA) intestinal carrier status in a healthcare worker — Case report

We describe bacteriophage therapy in the case of a healthcare worker whose gastrointestinal tract was colonized by methicillin-resistant Staphylococcus aureus (MRSA) with subsequent urinary tract infection caused by the same pathogen. Oral treatment with anti-MRSA phages resulted in eradication of the carrier status.     

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage‐related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.     

The impact of bacteriophage genomics

The discovery of (bacterio)phages revolutionised microbiology and genetics, while phage research has been integral to answering some of the most fundamental biological questions of the twentieth century. The susceptibility of bacteria to bacteriophage attack can be undesirable in some cases, especially in the dairy industry, but can be desirable in others, for example, the use of bacteriophage therapy to eliminate pathogenic bacteria. The relative ease with which entire bacteriophage genome sequences can now be elucidated has had a profound impact on the study of these bacterial parasites.     

Development and validation of a qPCR‐based method for quantifying Shiga toxin‐encoding and other lambdoid bacteriophages

To address whether seasonal variability exists among Shiga toxin‐encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 10³¹ tailed‐bacteriophages are estimated to populate the globe. To address this discrepancy a culture‐independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 × 10⁴ to 6.5 × 10⁶ ml^?1, with one in 10³ free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation‐dependent techniques, which grossly underestimate the abundance of viral entities.     

The next generation of bacteriophage therapy

Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings.     

T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by 'Dickeya solani'

The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.     

Pre‐adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates

Recent years have seen renewed interest in phage therapy – the use of viruses to specifically kill disease‐causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre‐adapted all phage strains against all bacterial strains and then compared the efficacy of pre‐adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre‐adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.     

Identification and comparative analysis of the structural proteomes of ϕKZ and EL,two giant Pseudomonas aeruginosa bacteriophages

Giant bacteriophages ?KZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI‐MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless ?KZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The ?KZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201?2‐1. Phage EL is more distantly related, shown by its 26 non‐conserved structural proteins and the presence of genomic inversions.     

Bacteriophage contamination: is there a simple method to reduce its deleterious effects in laboratory cultures and biotechnological factories?

Infection of bacterial cultures by bacteriophages as well as prophage induction in the host cells are serious problems in both research and biotechnological laboratories. Generally, prevention strategies (like good laboratory/factory hygiene, sterilisation, decontamination and disinfection) are necessary to avoid bacteriophage contamination. However, it is well known that no matter how good the laboratory/factory practice and hygiene are, bacteriophage infections occur from time to time. The use of immunised or resistant bacterial strains against specific phages may be helpful, but properties of the genetically modified strains resistant to phages are often worse (from the point of view of a researcher or a biotechnological company) than those of the parental, phage-sensitive strains. In this article we review recent results that may provide a simple way to minimise deleterious effects of bacteriophage infection and prophage induction. It appears that low bacterial growth rates result in a significant inhibition of lytic development of various bacteriophages. Moreover, spontaneous prophage induction is less frequent in slowly growing bacteria.     

快速从医院废水中大规模分离铜绿假单胞杆菌噬菌体

目的:从医院废水中快速分离多株不同的铜绿假单胞杆菌噬菌体,研究其生物学特性,为建立铜绿假单胞杆菌噬菌体库做准备。方法:利用噬菌斑法从未经处理的医院污水中分离和鉴定铜绿假单胞杆菌噬菌体,根据感染谱的不同确定它们为不同的铜绿假单胞杆菌噬菌体;重点研究其中一株宿主谱较广的噬菌体的生物学特性,采用负染法电镜观察噬菌体的形态和大小,提取该噬菌体的基因组并进行酶切电泳分析,测定噬菌体感染复数并观察其一步生长曲线。结果:通过噬菌斑法分离出90株铜绿假单胞杆菌噬菌体。电镜观察显示,噬菌体Pa27P1头部呈立体对称,有一长尾;酶切结果显示,噬菌体Pa27P1的基因组为双链DNA;生长曲线表明噬菌体Pa27P1感染宿主菌的潜伏期为25 min,爆发时间为25 min,裂解量为514。结论:90株铜绿假单胞杆菌噬菌体中有5株具有较广的噬菌谱,其组合能裂解所有18株铜绿假单胞杆菌,为深入研究铜绿假单胞杆菌噬菌体的生物学特性及其功能提供了依据。     

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice

Background

Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy.

Methodology/Principal Findings

The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence.

Conclusions/Significance

We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.     

Evolution of mutualism from parasitism in experimental virus populations

While theory suggests conditions under which mutualism may evolve from parasitism, few studies have observed this transition empirically. Previously, we evolved Escherichia coli and the filamentous bacteriophage M13 in 96‐well microplates, an environment in which the ancestral phage increased the growth rate and yield of the ancestral bacteria. In the majority of populations, mutualism was maintained or even enhanced between phages and coevolving bacteria; however, these same phages evolved traits that harmed the ancestral E. coli genotype. Here, we set out to determine if mutualism could evolve from this new parasitic interaction. To do so, we chose six evolved phage populations from the original experiment and used them to establish new infections of the ancestral bacteria. After 20 passages, mutualism evolved in almost all replicates, with the remainder growing commensally. Many phage populations also evolved to benefit both their local, evolving bacteria and the ancestral bacteria, though these phages were less beneficial to their co‐occurring hosts than phages that harm the ancestral bacteria. These results demonstrate the rapid recovery of mutualism from parasitism, and we discuss how our findings relate to the evolution of phages that enhance the virulence of bacterial pathogens.     

Isolation and characterization of a Lactobacillus fermentum temperate bacteriophage from Chinese yogurt

AIMS: The aim of this study was to investigate the properties of temperate bacteriophage of Lactobacillus fermentum, based on its morphology, restriction patterns, protein profile and the impact on the growth of host strain. METHODS AND RESULTS: With Mitomycin C, seven temperate phages were induced from Lactobacilli derived from Chinese yogurt. The temperate phages induced belong to the most common Bradley's group B, having hexagonal head and long, noncontractile tail. They were furthermore confirmed to be the same bacteriophage by identical restriction patterns. SDS-PAGE profile showed that the phage studied had one major structure protein about 31.9 kDa. The presence of the prophage influenced the cell shape and colony size of its lysogenic strain. CONCLUSIONS: The phage obtained had similar, but not complete identical properties with other L. fermentum phages reported. It influenced the growth behaviour of its lysogenic strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some information about bacteriophages occurring in the Chinese yoghurt manufacture and contributes to our knowledge on the bacteriophage diversity in the dairy industry.     

Genomic characterization of ϕRS603, a filamentous bacteriophage that is infectious to the phytopathogen Ralstonia solanacearum

A filamentous bacteriophage (?), ?RS603, which is infectious to the phytopathogen Ralstonia solanacearum was isolated. ?RS603 was found to have a circular single‐stranded DNA genome composed of 7679 nucleotides and to contain 13 putative open reading frames (ORFs). The ?RS603 genome showed strong similarity with those of Ralstonia phages ?RSM1 and ?RSM3, as reported by Askora et al. The ?RS603 genome had no ORFs corresponding to ORFs 2, 3, 13 and 14 (integrase) of ?RSM3. ?RS603 had an ORF that was homologous to other Ralstonia phages ?RSS0 and ?RSS1; however, ?RSM1 and ?RSM3 did not.

     

Effects of antagonistic coevolution on parasite‐mediated host coexistence

Parasites can promote diversity by mediating coexistence between a poorer and superior competitor, if the superior competitor is more susceptible to parasitism. However, hosts and parasites frequently undergo antagonistic coevolution. This process may result in the accumulation of pleiotropic fitness costs associated with host resistance, and could breakdown coexistence. We experimentally investigated parasite‐mediated coexistence of two genotypes of the bacterium Pseudomonas fluorescens, where one genotype underwent coevolution with a parasite (a virulent bacteriophage), whereas the other genotype was resistant to the evolving phages at all time points, but a poorer competitor. In the absence of phages, the resistant genotype was rapidly driven extinct in all populations. In the presence of the phages, the resistant genotype persisted in four of six populations and eventually reached higher frequencies than the sensitive genotype. The coevolving genotype showed a reduction in the growth rate, consistent with a cost of resistance, which may be responsible for a decline in its relative fitness. These results demonstrate that the stability of parasite‐mediated coexistence of resistant and susceptible species or genotypes is likely to be affected if parasites and susceptible hosts coevolve.