Outside-host phage therapy as a biological control against environmental infectious diseases

Background

Environmentally growing pathogens present an increasing threat for human health, wildlife and food production. Treating the hosts with antibiotics or parasitic bacteriophages fail to eliminate diseases that grow also in the outside-host environment. However, bacteriophages could be utilized to suppress the pathogen population sizes in the outside-host environment in order to prevent disease outbreaks. Here, we introduce a novel epidemiological model to assess how the phage infections of the bacterial pathogens affect epidemiological dynamics of the environmentally growing pathogens. We assess whether the phage therapy in the outside-host environment could be utilized as a biological control method against these diseases. We also consider how phage-resistant competitors affect the outcome, a common problem in phage therapy. The models give predictions for the scenarios where the outside-host phage therapy will work and where it will fail to control the disease. Parameterization of the model is based on the fish columnaris disease that causes significant economic losses to aquaculture worldwide. However, the model is also suitable for other environmentally growing bacterial diseases.

Results

Transmission rates of the phage determine the success of infectious disease control, with high-transmission phage enabling the recovery of the host population that would in the absence of the phage go asymptotically extinct due to the disease. In the presence of outside-host bacterial competition between the pathogen and phage-resistant strain, the trade-off between the pathogen infectivity and the phage resistance determines phage therapy outcome from stable coexistence to local host extinction.

Conclusions

We propose that the success of phage therapy strongly depends on the underlying biology, such as the strength of trade-off between the pathogen infectivity and the phage-resistance, as well as on the rate that the phages infect the bacteria. Our results indicate that phage therapy can fail if there are phage-resistant bacteria and the trade-off between pathogen infectivity and phage resistance does not completely inhibit the pathogen infectivity. Also, the rate that the phages infect the bacteria should be sufficiently high for phage-therapy to succeed.

     

Evaluation of the passage of Lactobacillus gasseri K7 and bifidobacteria from the stomach to intestines using a single reactor model

Background

The antibacterial activity of bacteriophages has been described rather well. However, knowledge about the direct interactions of bacteriophages with mammalian organisms and their other, i.e. non-antibacterial, activities in mammalian systems is quite scarce. It must be emphasised that bacteriophages are natural parasites of bacteria, which in turn are parasites or symbionts of mammals (including humans). Bacteriophages are constantly present in mammalian bodies and the environment in great amounts. On the other hand, the perspective of the possible use of bacteriophage preparations for antibacterial therapies in cancer patients generates a substantial need to investigate the effects of phages on cancer processes.

Results

In these studies the migration of human and mouse melanoma on fibronectin was inhibited by purified T4 and HAP1 bacteriophage preparations. The migration of human melanoma was also inhibited by the HAP1 phage preparation on matrigel. No response of either melanoma cell line to lipopolysaccharide was observed. Therefore the effect of the phage preparations cannot be attributed to lipopolysaccharide. No differences in the effects of T4 and HAP1 on melanoma migration were observed.

Conclusion

We believe that these observations are of importance for any further attempts to use bacteriophage preparations in antibacterial treatment. The risk of antibiotic-resistant hospital infections strongly affects cancer patients and these results suggest the possibility of beneficial phage treatment. We also believe that they will contribute to the general understanding of bacteriophage biology, as bacteriophages, extremely ubiquitous entities, are in permanent contact with human organisms.     

<Emphasis Type="Italic">Bordetella bronchiseptica</Emphasis> bateriophage suppresses <Emphasis Type="Italic">B. bronchiseptica</Emphasis>-induced inflammation in swine nasal turbinate cells

The development of therapeutic bacteriophages will provide several benefits based on an understanding the basic physiological dynamics of phage and bacteria interactions for therapeutic use in light of the results of antibiotic abuse. However, studies on bacteriophage therapeutics against microbes are very limited, because of lack of phage stability and an incomplete understanding of the physiological intracellular mechanisms of phage. The major objective of this investigation was to provide opportunity for development of a novel therapeutic treatment to control respiratory diseases in swine. The cytokine array system was used to identify the secreted cytokines/chemokines after Bordetella bronchiseptica infection into swine nasal turbinate cells (PT-K75). We also performed the real-time quantitative PCR method to investigate the gene expression regulated by B. bronchiseptica infection or bacteriophage treatment. We found that B. bronchiseptica infection of PT-K75 induces secretion of many cytokines/chemokines to regulate airway inflammation. Of them, secretion and expression of IL-1β and IL-6 are increased in a dose-dependent manner. Interestingly, membrane-bound mucin production via expression of the Muc1 gene is increased in B. bronchiseptica-infected PT-K75 cells. However, cytokine production and Muc1 gene expression are dramatically inhibited by treatment with a specific B. bronchiseptica bacteriophage (Bor-BRP-1). The regulation of cytokine profiles in B. bronchiseptica-induced inflammation by B. bronchiseptica bacteriophage is essential for avoiding inappropriate inflammatory responses. The ability of bacteriophages to downregulate the immune response by inhibiting bacterial infection emphasizes the possibility of bacteriophage-based therapies as a novel anti-inflammatory therapeutic strategy in swine respiratory tracts.     

Effect of nucleases on bacteria infected with bacteriophages

The effect of bacterial nucleases on bacteria infected by DNA- or RNA-containing bacteriophages with different serogroups was studied. Bacillary RNases have a strong inhibitory effect on RNA-containing bacteriophages. It was shown that nucleases suppressed the infection process of bacteria by bacteriophages M12, f2, PP7, and QB. The minimal inhibitory concentration ranged from 0.6 to 6 μg/mL. Bacterial ribonucleases have no impact on the development of DNA-containing bacteriophages PZ-A, PZ-B, P₃k, P118, and a lysogenic culture of Escherichia coli (λ) and Bacillus subtilis 168 (phi105). RNase from Bacillus pumilus did not inactivate bacteriophages Qβ and f2 in vitro and did not influence the adsorption on bacteriophages on the cell wall of the bacteria host E. coli AB301. The enzyme effect was shown at the level of bacteriophage infection of the host bacteria. Presumably, the phase between the adsorption and penetration of phage RNA into bacterial pili is the most sensitive to the effect of RNases.     

Characterization of a <Emphasis Type="Italic">Salmonella</Emphasis> Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria

In this study, we sought to isolate Salmonella Enteritidis-specific lytic bacteriophages (phages), and we found a lytic phage that could lyse not only S. Enteritidis but also other Gramnegative foodborne pathogens. This lytic phage, SS3e, could lyse almost all tested Salmonella enterica serovars as well as other enteric pathogenic bacteria including Escherichia coli, Shigella sonnei, Enterobacter cloacae, and Serratia marcescens. This SS3e phage has an icosahedral head and a long tail, indicating belong to the Siphoviridae. The genome was 40,793 base pairs, containing 58 theoretically determined open reading frames (ORFs). Among the 58 ORFs, ORF49, and ORF25 showed high sequence similarity with tail spike protein and lysozyme-like protein of Salmonella phage SE2, respectively, which are critical proteins recognizing and lysing host bacteria. Unlike SE2 phage whose host restricted to Salmonella enterica serovars Enteritidis and Gallinarum, SS3e showed broader host specificity against Gram-negative enteric bacteria; thus, it could be a promising candidate for the phage utilization against various Gram-negative bacterial infection including foodborne pathogens.     

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains

ResultsEight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 ({"type":"entrez-nucleotide","attrs":{"text":"DQ426904","term_id":"90110542","term_text":"DQ426904"}}DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence {"type":"entrez-nucleotide","attrs":{"text":"DQ426904","term_id":"90110542","term_text":"DQ426904"}}DQ426904 of reference phage PHL101.ConclusionsThe results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.     

The Autolysin LytA Contributes to Efficient Bacteriophage Progeny Release in Streptococcus pneumoniae

Most bacteriophages (phages) release their progeny through the action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. Although the function of each protein is well established in phages infecting Streptococcus pneumoniae, the role—if any—of the powerful bacterial autolysin LytA in virion release is currently unknown. In this study, deletions of the bacterial and phage lysins were done in lysogenic S. pneumoniae strains, allowing the evaluation of the contribution of each lytic enzyme to phage release through the monitoring of bacterial-culture lysis and phage plaque assays. In addition, we assessed membrane integrity during phage-mediated lysis using flow cytometry to evaluate the regulatory role of holins over the lytic activities. Our data show that LytA is activated at the end of the lytic cycle and that its triggering results from holin-induced membrane permeabilization. In the absence of phage lysin, LytA is able to mediate bacterial lysis and phage release, although exclusive dependence on the autolysin results in reduced virion egress and altered kinetics that may impair phage fitness. Under normal conditions, activation of bacterial LytA, together with the phage lysin, leads to greater phage progeny release. Our findings demonstrate that S. pneumoniae phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy.Streptococcus pneumoniae (pneumococcus), a common and important human pathogen, is characterized by the high incidence of lysogeny in isolates associated with infection (34, 44). Pneumococcal bacteriophages (phages) share with the majority of bacteriophages infecting other bacterial species the “holin-lysin” system to lyse the host cell and release their progeny at the end of the lytic cycle. Genes encoding both holins and lysins (historically termed “endolysins”) are indeed found in the genomes of all known pneumococcal phages (8, 28, 31, 37). Supporting this mechanism, a lytic phenotype in the heterologous Escherichia coli system was achieved only by the simultaneous expression of the Ejh holin and the Ejl endolysin of pneumococcal phage EJ-1 (8). When these proteins were independently expressed, cellular lysis was not perceived. Similar results were shown for pneumococcal phage Cp-1, not only in E. coli, but also in the pneumococcus itself (28).Phage lysins destroy the pneumococcal peptidoglycan network due to their muralytic activity, whereas holins have been shown in S. pneumoniae to form nonspecific lesions (8), most likely upon a process of oligomerization in the cytoplasmic membrane, as observed for the E. coli phage λ (13, 14, 43). It was generally proposed that holin lesions allow access of phage lysins to the cell wall (52, 54), as the majority of phage lysins, including the pneumococcal endolysins, lack a typical N-terminal secretory signal sequence and transmembrane domains (8). However, recent evidence also highlights the possibility for a holin-independent targeting of phage lysins to the cell wall, where holin lesions seem to be crucial for the activation of the already externalized phage lysins (42, 50, 51). Regardless of the mechanism operating in S. pneumoniae to activate phage lysins, holin activity compromises membrane integrity.Pneumococcal cells present their own autolytic activity, mainly due to the presence of a powerful bacterial cell wall hydrolase, LytA (an N-acetylmuramoyl-l-alanine-amidase), responsible for bacterial lysis under certain physiological conditions (47). Although other bacterial species also encode peptidoglycan hydrolases, the extensive lysis shortly after entering stationary phase caused by LytA is a unique feature of S. pneumoniae. Interestingly, LytA is translocated across the cytoplasmic membrane to the cell wall—where it remains inactive—in spite of the absence of a canonical N-terminal sequence signal (7). In the cell wall, autolysin activities are tightly regulated by mechanisms that seem to be related to the energized state of the cell membrane. In fact, depolarizing agents are able to trigger autolysis in Bacillus subtilis (16, 17), and bacteriocin-induced depletion of membrane potential triggers autolysis of some species of the genera Lactococcus and Lactobacillus, closely related to streptococci (29). It is therefore possible that the holin-inflicted perturbations of the S. pneumoniae cytoplasmic membrane upon the induction of the lytic cycle may trigger not only the lytic activity of the phage lysin, but also that of inactive LytA located in the cell wall. Accordingly, LytA could also participate in the release of phage particles at the end of the infectious cycle, especially considering its powerful autolytic activity. Previous studies have suggested a role for the host autolytic enzyme in the release of phage progeny (11, 38), but in fact, the evidence is unclear and dubious, considering that the existence of phage-encoded lysins was unknown or very poorly understood and some of the experimental conditions used to show a role of LytA could have also affected the activity of the phage lysin (38).To clarify the possible role of the bacterial autolysin in host lysis, we used the S. pneumoniae strain SVMC28, lysogenic for the SV1 prophage (34), which contains a typical “holin-lysin” cassette, and a different host strain lysogenized with the same SV1 phage. Our results show that LytA is activated by the holin-induced membrane disruption, just like the phage endolysin. In the absence of the endolysin, LytA is capable of mediating host lysis, releasing functional phage particles able to complete their life cycle. Still, sole dependence on LytA results in an altered pattern of phage release that may reduce phage fitness. Importantly, we also show that, together with the endolysin, the concurrent LytA activation is critical for optimal phage progeny release.     

Bacteriophage Diversity in the North Sea

In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10⁴ to 10⁷ particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the γ subdivision of the class Proteobacteria.The marine bacterial community is responsible for a considerable portion of primary production and regeneration of nutrients in the microbial loop and is associated with a great variety of marine bacteriophages (5, 12). These phages are capable of infecting a large portion of the bacterioplankton (32, 34). It is assumed that as part of the marine food web, bacteriophages play important quantitative and qualitative roles in controlling marine bacterial populations (8, 24, 34, 39, 45). The phenotypic diversity and genotypic diversity of the phage populations are related to the interaction between phages and their host organisms, which provides a tool for understanding the interaction itself (13). To estimate the influence of marine bacteriophages on the diversity of bacterioplankton, we investigated phage diversity. The virus species concept proposed by Murphy et al. (37) delineates seven different families of bacteriophages based on morphological criteria and provides criteria for new phage species based on several traits, such as DNA homologies, serological data, protein profiles, and host ranges.In this paper, we describe the diversity and genetic relationships of marine phages based on investigations of 22 representatives from 85 phage-host systems (35, 36) collected between 1988 and 1992 from waters around an island, Helgoland, located in the North Sea. All of the phages were virulent and formed plaques on their host bacteria. We assigned the phages to different virus families, species, and strains based on morphology, DNA homology, and host range. Furthermore, we characterized the phenotypic and genotypic features of the host bacteria.     

Atomic structure of bacteriophage Sf6 tail needle knob

Podoviridae are double-stranded DNA bacteriophages that use short, non-contractile tails to adsorb to the host cell surface. Within the tail apparatus of P22-like phages, a dedicated fiber known as the “tail needle” likely functions as a cell envelope-penetrating device to promote ejection of viral DNA inside the host. In Sf6, a P22-like phage that infects Shigella flexneri, the tail needle presents a C-terminal globular knob. This knob, absent in phage P22 but shared in other members of the P22-like genus, represents the outermost exposed tip of the virion that contacts the host cell surface. Here, we report a crystal structure of the Sf6 tail needle knob determined at 1.0 Å resolution. The structure reveals a trimeric globular domain of the TNF fold structurally superimposable with that of the tail-less phage PRD1 spike protein P5 and the adenovirus knob, domains that in both viruses function in receptor binding. However, P22-like phages are not known to utilize a protein receptor and are thought to directly penetrate the host surface. At 1.0 Å resolution, we identified three equivalents of l-glutamic acid (l-Glu) bound to each subunit interface. Although intimately bound to the protein, l-Glu does not increase the structural stability of the trimer nor it affects its ability to self-trimerize in vitro. In analogy to P22 gp26, we suggest the tail needle of phage Sf6 is ejected through the bacterial cell envelope during infection and its C-terminal knob is threaded through peptidoglycan pores formed by glycan strands.     

Use of Logistic Regression for Prediction of the Fate of Staphylococcus aureus in Pasteurized Milk in the Presence of Two Lytic Phages

The use of bacteriophages provides an attractive approach to the fight against food-borne pathogenic bacteria, since they can be found in different environments and are unable to infect humans, both characteristics of which support their use as biocontrol agents. Two lytic bacteriophages, vB_SauS-phiIPLA35 (phiIPLA35) and vB_SauS-phiIPLA88 (phiIPLA88), previously isolated from the dairy environment inhibited the growth of Staphylococcus aureus. To facilitate the successful application of both bacteriophages as biocontrol agents, probabilistic models for predicting S. aureus inactivation by the phages in pasteurized milk were developed. A linear logistic regression procedure was used to describe the survival/death interface of S. aureus after 8 h of storage as a function of the initial phage titer (2 to 8 log₁₀ PFU/ml), initial bacterial contamination (2 to 6 log₁₀ CFU/ml), and temperature (15 to 37°C). Two successive models were built, with the first including only data from the experimental design and a global one in which results derived from the validation experiments were also included. The temperature, interaction temperature-initial level of bacterial contamination, and initial level of bacterial contamination-phage titer contributed significantly to the first model prediction. However, only the phage titer and temperature were significantly involved in the global model prediction. The predictions of both models were fail-safe and highly consistent with the observed S. aureus responses. Nevertheless, the global model, deduced from a higher number of experiments (with a higher degree of freedom), was dependent on a lower number of variables and had an apparent better fit. Therefore, it can be considered a convenient evolution of the first model. Besides, the global model provides the minimum phage concentration (about 2 × 10⁸ PFU/ml) required to inactivate S. aureus in milk at different temperatures, irrespective of the bacterial contamination level.Staphylococcus aureus is one of the pathogenic bacteria considered a threat to food safety. Worldwide, it has a particular relevance to the food-processing industry because of the ability of some strains to produce heat-stable enterotoxins and other virulence factors responsible for staphylococcal food poisoning (14, 18). The ability of S. aureus to grow in different foodstuffs over a wide range of temperatures (10 to 45°C), pHs (4.5 to 9.3), and NaCl concentrations (up to 15%) explains its incidence in foods subjected to manipulation throughout the manufacturing process (1). Milk and dairy products have often been involved in several episodes of staphylococcal food poisoning (6). Food handlers and cattle are usually the main source of dairy product contamination, as humans and animals are the primary reservoirs for staphylococci (28).S. aureus contamination can be avoided by heat treatment of food, but recontamination postpasteurization can occur if the hygienic conditions are inadequate. Alternative approaches to control S. aureus populations in dairy products, such as the use of bacteriocinogenic strains (24) and, more recently, bacteriophages (8), have been tested. Bacteriophages, as well as bacteriocins, are regarded as natural antibacterial agents since they are able to infect and lyse specific undesired target bacteria without disturbing the normal microbiota (19). Among the advantages of using phages as biocontrol strategies in foods, their ubiquity in the environment, their history of safe use, and their high host specificity can be quoted (10). Likewise, their potential use as biopreservatives in several food systems has recently been reviewed (13).Factors affecting S. aureus growth and survival in foods (physicochemical characteristics of the products and conditions associated with food processing, storage, and distribution) have been studied, and mathematical models have been developed to estimate the microorganism''s response (5, 30). However, the effect of lytic phages on S. aureus growth in milk has not been described by predictive models so far. There are several physical parameters which may influence bacterial inhibition by phages in milk. Among them, temperature is the first choice to be studied, as this parameter is always involved in milk processing and can easily be subjected to manipulation without markedly affecting the typical organoleptic characteristics of milk.In this regard, probabilistic models have been widely used to describe the survival/death interface as a function of environmental hurdles (22, 23), and the positions of these limits are of interest in establishing conditions for product stabilization. A procedure of forward or backward stepwise regression with some criteria to include or reject dummy or quantitative explanatory variables, their quadratic terms, or their interactions is included in most of the standard statistical software packages (11, 12).The aim of the present work was to determine the effectiveness of a cocktail of two lytic phages, selected according to its wide host range (9), at reducing S. aureus contamination in pasteurized milk, using different environmental conditions, such as different temperatures, initial bacterial viable counts, and initial phage titers. For this purpose, the survival/death interface was deduced using a logistic regression model.     

Effects of bacteriophages on biofilm formation by strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

The effects of two Pseudomonas aeruginosa bacteriophages, vB-Pa 4 and vB-Pa 5, on the formation and development of biofilms of six polyresistant hospital strains of P. aeruginosa have been investigated. Pretreatment of bacteriophages prevented the formation or almost completely prevented the growth of adequate biofilms. The biofilms that had already formed were partially or completely destroyed after phage treatment. The results demonstrate the prospects of using isolated bacteriophages of P. aeruginosa to destroy biofilms and prevent their formation.     

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt

Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt (caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying. However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected, suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophagebased biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

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Antibacterial efficacy of lytic <Emphasis Type="Italic">Pseudomonas</Emphasis> bacteriophage in normal and neutropenic mice models

Recently, lytic bacteriophages (phages) have been focused on treating bacterial infectious diseases. We investigated the protective efficacy of a novel Pseudomonas aeruginosa phage, PA1Ø, in normal and neutropenic mice. A lethal dose of P. aeruginosa PAO1 was administered via the intraperitoneal route and a single dose of PA1Ø with different multiplicities of infection (MOI) was treated into infected mice. Immunocompetent mice infected with P. aeruginosa PAO1 were successfully protected by PA1Ø of 1 MOI, 10 MOI or 100 MOI with 80% to 100% survival rate. No viable bacteria were found in organ samples after 48 h of the phage treatment. Phage clearing patterns were different in the presence or absence of host bacteria but PA1Ø disappeared from all organs after 72 h except spleen in the presence of host bacteria. On the contrary, PA1Ø treatment could not protect neutropenic mice infected with P. aeruginosa PAO1 even though could extend their lives for a short time. In in vitro phage-neutrophil bactericidal test, a stronger bactericidal effect was observed in phage-neutrophil co-treatment than in phage single treatment without neutrophils, suggesting phage-neutrophil co-work is essential for the efficient killing of bacteria in the mouse model. In conclusion, PA1Ø can be possibly utilized in future phage therapy endeavors since it exhibited strong protective effects against virulent P. aeruginosa infection.     

The return of the phage

Phages have been used to treat infectious diseases since their discovery nearly a century ago. Modern sequencing and genetic engineering technologies now enable researchers to vastly expand the use of phages as general drug delivery vehicles....it is only in the past five years that the regulatory guidelines for the approval of phage products—both in therapy and food safety—have been createdOver the past decade, bacteriophages have occasionally stirred public and media interest because of their potential as biological weapons against bacterial infections. Such reports have tended to come from Russian or Georgian laboratories, whereas Western research institutes and companies have usually found that phages do not live up to their promise. More than a decade later, however, the view of bacteriophages is set to change. Spurred on by advances in sequencing and other molecular techniques, research into phages has yielded its first applications. Not only are phages proving effective as therapeutic agents, but they are also playing a role in food safety and as delivery vehicles for drugs against a wide range of diseases.Interest in phages as therapeutic agents emerged almost immediately after their discovery nearly a century ago (Twort, 1915; d''Hérelle, 1917). This interest evaporated quickly in the West after the discovery of penicillin, but phage research was kept alive in the old Soviet Union and continued after its collapse in the 1990s. Ongoing studies there, although not always conforming to the most rigorous standards, provided the only evidence of the therapeutic potential of phages.Eventually, especially in the light of the increasing threat from drug-resistant bacteria, Western researchers turned to exploring phages again. However, it is only in the past five years that the regulatory guidelines for the approval of phage products—in both therapy and food safety—have been created. Previously, the US Food and Drug Administration (FDA) had lacked the appropriate regulatory measures; it took them four years to approve the first phage product for use in food safety in 2006. ListShield^TM is a cocktail of several phages that target Listeria monocytogenes, contaminants in meat and poultry products. Approvals for other food safety products have followed with greater speed (Sulakvelidze, 2011). Moreover, in 2008, the FDA approved the first phase 1 clinical trial of phages. This again involved a cocktail of eight phages to target various bacteria including Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, in venous leg ulcers. This trial eventually established the safety of the phage preparation and cleared the way for more phage therapy trials (www.clinicaltrials.gov).The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria...The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria, according to Martin Loessner from the Institute of Food, Nutrition and Health in Zürich, Switzerland. “It then became possible to [...] harness the specificity of phage for applications such as recognition of the host cell, and also for reporter phage, which is a genetically modified phage with a gene so [you] can easily see the phage''s impact on the target cell,” he explained. “Later on we figured why not go and revisit the idea of using phages against pathogens.”This approach turned out to be highly successful against key food pathogens, Loessner said, because of the way phages work: “[T]he phage has been very finely tuned through zillions of generations in the evolutionary arms race, and is highly specific.” This specificity is important for targeting the few bacteria that cause food poisoning while sparing the bacteria in fermented food—such as soft cheeses—that are harmless and contribute flavour. “The phage is also immune to development of resistance by the host bacteria, because if not it would have become extinct a long time ago,” Loessner said.It is bacterial toxins that cause food poisoning rather than bacteria themselves, so phages are used as a preventive measure to stop the growth of bacteria such as Listeria in the first place. As such, it is important to bombard food products with a large number of phages to ensure that virtually all target bacteria are eradicated. “I always have this magic number of 10⁸, or 100 million per gram of food,” Loessner said. “In 1 g of food there are often only 500 target bacteria, so there is not enough to amplify the phage and you need really high numbers to kill the bacteria in one round of infection.” He added that, in his view, phages would soon become the main treatment for preventing bacterial contamination. “Phage in the near future will be the number one [treatment against] Listeria and Salmonella. It''s becoming number one already, especially in the US.”In Europe, the use of phages in food safety therapy is being held back by the requirement that foods treated with them are labelled as containing viruses, which means they are likely to meet consumer resistance, as happened with foods containing or made from genetically modified organisms. Loessner commented that education is required to raise awareness that the properly controlled use of phages involves minimal risk and could greatly enhance food safety. However, he also emphasized that the use of phages should represent an extra level of protection, not replace existing quality control measures....because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteriaThe ability of phages to target specific bacteria while leaving others alone also has great potential for treating bacterial infections, particularly in the light of increasing antibiotic resistance. Such treatments would not necessarily involve the phage themselves, but rather the use of their lysins—the enzymes that weaken the bacterial cell wall to allow newly formed viruses to exit the host cell. Lysins can be administered as antibiotics, at least for gram-positive bacteria that lack a separate outer membrane around the cell wall. Moreover, because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteria. “The fact that phage lysins leave the commensal microflora undisturbed is particularly significant,” commented Olivia McAuliffe, Senior Research Officer at the Teagasc Food Research Centre in Cork, Ireland. “Most of the antibiotics used clinically have broad-activity spectra and treatment with these antibiotics can have devastating effects on the normal flora, in particular for those taking long-term antibiotic courses.”Phages also have another great advantage over most conventional antibiotics in being potent against both dividing and non-dividing cells. “Because most antibiotics target pathways such as protein synthesis, DNA replication, and cell wall biosynthesis, they can only act when the cells are actively growing,” McAuliffe added. “Because lysins are enzymes, they will chew away the peptidoglycan in both viable and non-viable cells, dividing and non-dividing cells. This would be particularly important in the case of slow-growing organisms that cause infection, an example being Mycobacterium species.”This specificity of phages and their lysins is particularly important for treating chronic conditions resulting from persistent bacterial infection, particularly in the respiratory system or digestive tract. Broad-spectrum antibiotics also attack harmless and beneficial commensal bacteria, and can even worsen the condition by encouraging the growth of resistant bacteria. This is the case with Clostridium difficile, a cause of secondary infections and a major nosocomial (hospital-acquired) antibiotic-resistant pathogen, according to McAuliffe. It is a Gram-positive, rod-shaped, spore-forming bacterium that is the most serious and common cause of diarrhoea and other intestinal disease when competing bacteria in the gut flora have been wiped out by antibiotics. The bacterium and its spores, which form in aerobic conditions outside the body, are widespread in the environment and are present in the guts of 3% of healthy individuals and 66% of infants, according to the UK''s Health Protection Agency. Clostridium spreads readily on the hands of healthcare staff and visitors in hospitals. The ability of the bacteria to form spores resistant to heat, drying and disinfectants, which then adhere to surfaces, enables them to persist in the hospital environment.Because Clostridium is resistant to most conventional antibiotics, it has for some years usually been treated with metronidazole, which exploits the fact that Clostridium is anaerobic during infection. Metronidazole has proven particularly appealing as it has relatively little impact on human cells or commensal aerobic bacteria in the gut as it does not work in the presence of oxygen. But metronidazole does not always work, and physicians have therefore been using vancomycin, a stronger but more toxic antibiotic, as a last resort. Moreover, even in cases where antibiotics seem to eliminate Clostridium and cure the associated diarrhoea, infection recurs in as many as 20% of hospital patients (Kelly & LaMont, 2008). About one-fifth of these 20%, or 4% of the total number of patients succumbing to Clostridium, end up with a long-term infection that at present is difficult to eradicate.This is where phages step in, because they are well tolerated by patients and their specificity means that they will not target other gut bacteria. Clostridium phages have already been demonstrated to work selectively and there is the possibility of extracting lysins against Clostridium from the phage itself; an avenue being pursued by Aidan Coffey''s group at the Department of Biological Sciences at the Cork Institute of Technology in Bishoptown, Ireland.There is also growing interest in using phages to tackle various other infections that are resistant to existing drugs—for example, in wounds that fail to heal, which are a major risk for diabetics. The application of phages in such cases is not new—before penicillin it was often the only option—but the difference now is that modern molecular techniques for isolating bacterial strains from biopsies and matching them to phages greatly increases efficiency. One clinical trial, organized by the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, is currently recruiting patients to evaluate the use of phage preparations against a range of drug-resistant bacteria, including MRSA (methicillin-resistant Staphylococcus aureus), Enterococcus, Escherichia, Citrobacter, Enterobacter, Klebsiella, Shigella and Salmonella. The intention is to isolate bacterial strains from each patient and to identify matching phages from the Institute''s bacteriophage collection in Wrocław.Although the potential of phages or their lysins to combat bacterial pathogens, whether in food or those causing infectious diseases, has long been recognized, more recent work has identified new applications as delivery vehicles for vaccines or cytotoxic drugs to treat cancer. These applications do not exploit the phage''s natural targeting of bacteria, but make use of their ability to carry surface ligands that attract them to specific host cells.Even though phages do not attack human cells, they elicit an immune response and can be used as vectors to carry an engineered antigen on their surface to vaccinate against viral or bacterial disease. This approach has been tested in rabbits with a DNA vaccine against hepatitis B (Clark et al, 2011). The study compared the phage DNA vaccine with Engerix B—a commercially available vaccine based on a homologous recombinant protein—and found that the phage vaccine produced a significantly higher antibody response more quickly, as well as being potentially cheaper to produce and stable at a wider range of temperatures. This hepatitis B vaccine is now being developed by the UK biotech firm BigDNA in Edinburgh, Scotland, which has been granted a European patent, pending future clinical trials in humans.Modified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cellsModified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cells. Phages are a promising candidate vehicle because they can be readily engineered both to display appropriate ligands for targeting tumour cells specifically, and to carry a cytotoxic payload that is only released inside the target. One Israeli group has developed a technology for manufacturing phage nanoparticles that in principle can be used to target drugs to either tumour cells or pathogens (Bar et al, 2008). The group chose one particular phage family, known as filamentous phages, because of their small size and the relative ease of engineering them. Filamentous phages comprise just 10 genes with a sheath of several thousand identical α-helical coat proteins in a helical array assembled around a single-stranded circular DNA molecule. The Israeli scientists combine genetic modification and chemical engineering to create a phage that is able to attach to its target cell and release cytotoxic molecules. “Genetic engineering makes it possible to convert the phage to a targeted particle by displaying a target-specifying molecule on the phage coat,” explained Itai Benhar from Tel-Aviv University, the lead author of the paper. “Genetic engineering also makes it possible to design a drug-release mechanism. Finally chemical engineering makes it possible to load the particle with a large payload of cargo.”The group has used the same approach to target two bacteria species, Staphylococcus aureus and Escherichia coli, with the antibiotic chloramphenicol, which was first developed in 1949 but has raised concerns over its toxicity. According to the Israeli group, the phage nanoparticle loaded with the drug was 20,000 times more potent against both bacteria than the drug administered on its own. Just as importantly, the phage particles do not affect other cells. The overall advantage of the phage-based delivery approach is that it can deliver highly effective and toxic drugs in a safe way. The other point is that this and other methods in which phages are engineered to reach specific targets have nothing directly to do with the natural ability of phage viruses to attack bacteria. “The phage''s natural ability to infect bacteria is totally irrelevant to their application for targeting non-bacterial cells,” said Benhar. “In fact, they are not relevant for targeting bacteria either in this case, since the chemical modification we subject the phages to renders them non-infective.”However, the phage nanoparticles retain their immunogenic effect, which is a problem if the objective is merely to deliver a drug to the target while minimizing all other impacts. “Phages are immunogenic, and although we found a way to reduce their immunogenicity we did not totally eliminate it,” Benhar said. The other challenge is that, as the particles carry the payload drug on their surface, the physical and chemical properties change every time a new drug is loaded. Although the payload itself is inert until it reaches the target, the varying characteristics could alter the host response and therefore affect regulatory approval for each new phage construct, as safety would have to be demonstarted in each case.The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackledNevertheless, this approach holds great promise as a novel way of delivering not just new drugs but also existing ones that are effective but too toxic for healthy cells. This is exactly the most exciting aspect of recent therapeutic phage research. The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackled.     

Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum

Fusobacterium nucleatum is a periodontal pathogen that has been directly associated with the development and progression of periodontal disease, a widespread pathology that affects the support tissues of the tooth. We isolated a new bacteriophage (FnpΦ02) that specifically infects this bacterium. Transmission electron microscopy showed that the virion is composed of an icosahedral head and a segmented tail. The size of the phage genome was estimated to be approximately 59 kbp of double-stranded DNA. The morphological features and the genetic characteristics suggest that FnpΦ02 is part of the Siphoviridae family. Using one-step growth and adsorption experiments, the latent period, burst size, and adsorption rate were estimated to be 15 h, 100 infectious units per cell, and 7.5 × 10⁻¹⁰ ml min⁻¹, respectively. A small fragment of phage DNA was cloned and sequenced, showing 93% nucleotide identity with the phage PA6 of Propionibacterium acnes and amino acid identity with fragments of two proteins (Gp3 and Gp4) of this phage. To our knowledge, FnpΦ02 is the first phage described to infect Fusobacterium nucleatum and provides the base for future exploration of phages in the control of periodontal disease.The term “periodontal disease” refers to a wide set of pathological alterations of the periodontal tissue. The most common clinical manifestations are known as gingivitis and periodontitis, and both are widely distributed around the world (18). Periodontitis is a multifactorial inflammatory-based infection of the supporting tissues of the tooth. It is essentially characterized by the progressive destruction of the periodontal ligament and the alveolar bone, leading to the loss of the affected tooth (2). Periodontitis is caused by bacteria or bacterial groups embedded in a biofilm or dental plaque that protects them against antimicrobial agents (18). The bacterial species involved in periodontal disease are predominantly Gram-negative anaerobes, and although they are usually isolated from affected patients, they are also isolated from healthy individuals, but in a lesser proportion and frequency (26).Fusobacterium nucleatum is an anaerobic, Gram-negative, long bacillus and a member of the microflora in the oral cavity. F. nucleatum is considered a periodontal pathogen because it is frequently isolated from lesions, produces a high number of tissue irritants, and has the ability to form coaggregates with other periodontal pathogens, acting as a bridge between early and late colonizers in the surface of the enamel (4, 11). Three different subspecies of F. nucleatum have been related to the pathology of periodontal disease, F. nucleatum subsp. nucleatum, subsp. polymorphum, and subsp. vincentii, all of which have been associated with lesions of periodontitis but also have been isolated in high numbers from successfully treated patients (9).Bacteriophages are viruses that can infect and kill only bacteria and have been used for many years as powerful tools for the study of bacterial genetics and, given their specificity, used in the identification and characterization of microorganisms (phage typing). Nevertheless, phages were originally described as therapeutic elements to treat human and animal infections (34). This application, known as phage therapy, has regained interest in the past years, particularly in an era when antibiotic resistance and biofilm-based infections are permanent issues (25). Bacteriophages are denominated “temperate” when their genetic material is integrated within the bacterial genome with no immediate lysis of the bacterium until, under certain conditions, the expression of the viral genome is induced and the production of new virus particles lyses the host cell; they are called “lytic” or “virulent” when, immediately after the infection, they redirect the bacterial metabolism to the production of new phages, which are released during the bacterial lysis (22, 36). There are many examples of the use of bacteriophages at a clinical (14, 32) and commercial level (20). Specifically in the dentistry area, several bacteriophages that infect diverse oral bacteria have been isolated from saliva and dental plaque (12, 13, 23, 37).Although F. nucleatum is an important periodontal pathogen, reports of bacteriophages for this microorganism do not exist. In this work, we isolated and characterized a new bacteriophage for F. nucleatum from a saliva sample, designated FnpΦ02, and to our knowledge this is the first bacteriophage for this bacterium.     

A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes

Background

Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naïve antibody repertoire of the chicken can easily be accessed using only two sets of primers.

Results

With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA.

Conclusion

The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naïve phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist.

     

Complete WO Phage Sequences Reveal Their Dynamic Evolutionary Trajectories and Putative Functional Elements Required for Integration into the Wolbachia Genome

Wolbachia endosymbionts are ubiquitously found in diverse insects including many medical and hygienic pests, causing a variety of reproductive phenotypes, such as cytoplasmic incompatibility, and thereby efficiently spreading in host insect populations. Recently, Wolbachia-mediated approaches to pest control and management have been proposed, but the application of these approaches has been hindered by the lack of genetic transformation techniques for symbiotic bacteria. Here, we report the genome and structure of active bacteriophages from a Wolbachia endosymbiont. From the Wolbachia strain wCauB infecting the moth Ephestia kuehniella two closely related WO prophages, WOcauB2 of 43,016 bp with 47 open reading frames (ORFs) and WOcauB3 of 45,078 bp with 46 ORFs, were characterized. In each of the prophage genomes, an integrase gene and an attachment site core sequence were identified, which are putatively involved in integration and excision of the mobile genetic elements. The 3′ region of the prophages encoded genes with sequence motifs related to bacterial virulence and protein-protein interactions, which might represent effector molecules that affect cellular processes and functions of their host bacterium and/or insect. Database searches and phylogenetic analyses revealed that the prophage genes have experienced dynamic evolutionary trajectories. Genes similar to the prophage genes were found across divergent bacterial phyla, highlighting the active and mobile nature of the genetic elements. We suggest that the active WO prophage genomes and their constituent sequence elements would provide a clue to development of a genetic transformation vector for Wolbachia endosymbionts.Members of the genus Wolbachia are endosymbiotic bacteria belonging to the Alphaproteobacteria and infecting a wide range of arthropods, including over 60% of insect species, and some filarial nematodes. They are vertically transmitted through the maternal germ line of their host and are known to distort host reproduction by causing cytoplasmic incompatibility (CI), parthenogenesis, male killing, or feminization. The ability of Wolbachia to cause these reproductive phenotypes is thought to be responsible for their efficient and rapid spread into host populations (5, 21, 35, 51).Recently, Wolbachia-mediated pest control approaches have been proposed. A number of insect pests that have important medical and hygienic consequences, such as tsetse flies and mosquitoes that vector devastating human pathogens including African sleeping disease trypanosomes, malaria plasmodia, dengue viruses, Japanese encephalitis viruses, and others, often also carry Wolbachia infections (8, 24, 25, 34). In theory, if maternally transmitted genetic elements coinherited with a CI-inducing Wolbachia, such as mitochondria, the Wolbachia itself, or other coinfecting endosymbionts, are transformed with a gene of interest (like a gene that confers resistance of the vector insect against the pathogen infection), the genetic trait is expected to be spread and fixed in the host insect population, driven by the symbiont-induced reproductive phenotype (1, 2, 10, 11, 13, 32, 43, 44). The paratransgenesis and Wolbachia-driven population replacement approaches are, although potentially promising in controlling such insect-borne diseases, still at a conceptual stage mainly because no technique has been available for Wolbachia transformation.For genetic transformation of bacteria, mobile genetic elements such as plasmids, bacteriophages, and transposons have been used successfully. For example, pUC plasmids, λ phages, and transposons have been widely utilized for transforming Escherichia coli and other model bacterial species (38). While few plasmids and transposons have been reported from Wolbachia, a family of bacteriophages, called WO phages, has been detected from a diverse array of Wolbachia strains (3, 6, 7, 12, 17, 18, 31, 39, 49). For example, in the genomes of the Wolbachia strains wMel from the fruit fly Drosophila melanogaster and wPip from the mosquito Culex quinquefasciatus, three and five WO prophages are present, respectively (26, 52). Many of the prophages are pseudogenized and inactive while some are active and capable of producing phage particles (4, 7, 15, 17, 30, 40). Such active WO phage elements may provide tools for genetic transformation of Wolbachia endosymbionts.λ phage and many other temperate bacteriophages alternate between lytic phase and lysogenic phase in their life cycles. In the lytic phase, phage particles are produced and released via host cell lysis for infection to new host cells. In the lysogenic phase, the phage genome is integrated into the host genome via a site-specific recombination process, and the integrated phage genome, called prophage, is maintained in the host genome and multiplies together with the host DNA replication (38). Upon infection and lysogenic integration of λ phage, both ends of the linear phage genomic DNA are connected by DNA ligase, and the resultant circular phage genome is inserted into the E. coli genome by site-specific recombination at a region containing a core sequence of an attachment (att) site (28). att sites on the phage genome and the bacterial genome are called attP (phage att site) and attB (bacterial att site), respectively. After integration, attP and attB are located on both ends of the prophage, called attL (left prophage att site) and attR (right prophage att site), respectively. The integration and excision processes are mediated by a site-specific recombinase, called λ integrase, encoded in the phage genome (see Fig. S1 in the supplemental material) (27, 50). Hence, the att site and the integrase are the pivotal functional elements that mediate site-specific integration and excision of λ phage. Considering the structural similarity between λ phage and WO phage (31), identification of the att site and integrase from WO phage is of interest in that these elements could be utilized for delivering foreign genes into the Wolbachia genome.In order to identify a functional att site and integrase of WO phage, the complete genome sequences of active prophage elements producing phage particles should be determined. Here, the Wolbachia strain wCauB derived from the almond moth Cadra cautella was investigated because wCauB was reported to actively produce phage particles, and a partial genome sequence of its WO phage has been determined (15). In the original host insect, C. cautella, wCauB coexists with another Wolbachia strain wCauA, and both cause CI phenotypes and produce phage particles (15, 41). Not to be confounded by the coinfecting Wolbachia strains, we used a transfected line of the Mediterranean flour moth Ephestia kuehniella infected with wCauB only, which was generated by interspecific ooplasm transfer (42). It should be noted that a mass preparation procedure for WO phage particles by centrifugation has been established for the wCauB-infected E. kuehniella (15).In this study, we determined the complete genome sequences of two active WO prophages, named WOcauB2 and WOcauB3, that are capable of producing phage particles and that are located on the genome of the Wolbachia strain wCauB. Furthermore, we identified core sequences of att sites and integrase genes of these WO phages that are putatively involved in integration of the genetic elements into the Wolbachia genome.     

The Role of Horizontal Gene Transfer by Bacteriophages in the Origin of Pathogenic Bacteria

The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent KZ-like phages) of conditional pathogen Pseudomonas aeruginosa.Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches.     

Comparative Genomic Analysis of Ten Streptococcus pneumoniae Temperate Bacteriophages

Streptococcus pneumoniae is an important human pathogen that often carries temperate bacteriophages. As part of a program to characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, we performed comparative genomic analysis of 10 temperate pneumococcal phages. All of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis clusters. All of the phage particles observed showed a Siphoviridae morphology. The only genes that are well conserved in all the genomes studied are those involved in the integration and the lysis of the host in addition to two genes, of unknown function, within the replication module. We observed that a high percentage of the open reading frames contained no similarities to any sequences catalogued in public databases; however, genes that were homologous to known phage virulence genes, including the pblB gene of Streptococcus mitis and the vapE gene of Dichelobacter nodosus, were also identified. Interestingly, bioinformatic tools showed the presence of a toxin-antitoxin system in the phage φSpn_6, and this represents the first time that an addition system in a pneumophage has been identified. Collectively, the temperate pneumophages contain a diverse set of genes with various levels of similarity among them.Streptococcus pneumoniae (the pneumococcus) is an important human pathogen and a major etiological agent of pneumonia, bacteremia, and meningitis in adults and of otitis media in children. The casualties due to the pneumococcus are estimated to be over 1.6 million deaths per year, and most of these deaths are of young children in developing countries (40). S. pneumoniae is also a human commensal that resides in the upper respiratory tract, and it is asymptomatically carried in the nasopharynx of up to 60% of the normal population (48).Bacteriophages of S. pneumoniae (pneumophages) were first identified in 1975 from samples isolated from throat swabs of healthy children by two independent groups (46, 65). Since then, pneumophages have been identified from different sources and a variety of locations (44). The abundance of temperate bacteriophages in S. pneumoniae has been reported in different studies in the past (6, 54). Up to 76% of clinical isolates have been showed to contain prophages (or prophage remnants) when studied with a DNA probe specific for the major autolysin gene, lytA, which hybridizes with many of the endolysin genes of temperate pneumococcal phages (54). Hybridization analyses have identified highly similar prophages among pneumococcal clinical isolates even of different capsular serotypes, a result which indicates the widespread distribution of these mobile genetic elements among virulent strains (26).Only three S. pneumoniae bacteriophage genomes have been characterized in detail, and their sequences have been determined. Dp-1 and Cp-1 are lytic bacteriophages, whereas MM1 is a temperate pneumophage (45, 50, 52). Genes coding for virulence factors such as toxins or secreted enzymes have been associated with the presence of prophages in both gram-negative (67) and gram-positive bacteria, such as Streptococcus pyogenes (7) and Staphylococcus aureus (23). Because a considerable number of toxin genes are located in prophages, phage dynamics are of apparent importance for bacterial pathogenesis. Unfortunately, the role of temperate bacteriophages in the virulence of S. pneumoniae remains mostly unknown.Recently, the availability of relatively inexpensive next-generation sequencing technologies has permitted the complete genomic analysis of dozens of genomes of pneumococcal clinical isolates. In this report, we present a comparative genomic analysis of 10 pneumophages identified in the genomes of newly sequenced S. pneumoniae strains. The proteome of these phages has been predicted and annotated by comparative sequence analyses by using the available databases at the National Center for Biotechnological Information website (http://www.ncbi.nlm.nih.gov/). This systematic characterization of pneumophage genomes provides for a substantial increase in our knowledge of the global proteome and the overall genetic diversity of this important human pathogen. The comparative analysis of multiple temperate bacteriophages from a single species offers a unique opportunity to study one of the mechanisms of lateral gene transfer that drive prokaryotic genetic diversity.