Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Jul 2008; 74(13): 4149–4163.
Published online May 2, 2008. doi:  10.1128/AEM.02371-07
PMCID: PMC2446516

In Silico and In Vivo Evaluation of Bacteriophage [var phi]EF24C, a Candidate for Treatment of Enterococcus faecalis Infections[down-pointing small open triangle]

Abstract

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, [var phi]EF24C, and its biology indicated its therapeutic potential against E. faecalis infection. In this study, the [var phi]EF24C genome was analyzed and the in vivo therapeutic applicability of [var phi]EF24C 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 [var phi]EF24C 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 [var phi]EF24C. Moreover, the in vivo therapeutic potential of [var phi]EF24C, 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, [var phi]EF24C can be a promising therapeutic phage against E. faecalis infections.

Enterococcus species can be found in the environment and normal microflora of large mammals, and some of these bacterial species occasionally cause a variety of diseases (30). The emergence of vancomycin-resistant Enterococcus (VRE) disrupts effective conventional chemotherapy and causes fatal infections in nosocomial settings. An increase in the number of VRE cases has been reported worldwide (5, 9, 14). Among the enterococcal infections, E. faecalis is the most clinically isolated species (19, 30).

Bacteriophage (phage) therapy harnesses a live prokaryotic virus as a bioagent to target and destroy disease-causing bacteria (7, 15). Phage therapy has a long history of successful use in the former Eastern bloc countries, whereas it has almost no such history in the West (15, 27). The recent increase in the number of multiple-drug-resistant bacteria including VRE has renewed the interest of the Western scientific community in phage therapy (15, 27). However, because the past failures in phage therapy resulted from a lack of scientific knowledge of phage biology, this therapeutic approach needs to be scientifically rationalized (27). Hence, each therapeutic phage needs to be well characterized, like other approved drugs.

Genome analysis and proof of applicability are simple and effective methods for the primary evaluation of each phage. First, the phage genome reflects biological information such as morphology (e.g., drug formulation) and life cycle (e.g., propagation mechanism and drug efficacy), enabling the elucidation of phage drug features. In addition, genome analysis allows us to examine the safety of the approach by determining the presence or absence of undesirable genes such as pathogenic and integration-related genes (36, 43). Moreover, the lytic activity and intrinsic effects of each phage in vivo are usually unknown, so that in vivo therapeutic effectiveness and phage toxicity must also be examined. Unfortunately, no therapeutic phage with such evaluation is currently available against E. faecalis infections.

Previously, [var phi]EF24C was isolated and its biology was characterized briefly. [var phi]EF24C, which was classified in the family Myoviridae morphotype A1, has a broad host specificity with strong virulence against E. faecalis, including the VRE strains (47). The morphology of [var phi]EF24C, together with the N-terminal sequences of its structural proteins, implies a relationship to members of the SPO1-like phage genus (28, 47). Some of these members are considered to be therapeutic and prophylactic phage candidates (e.g., Staphylococcus phages K and 812 and Listeria phage P100) (8, 20, 29, 40). Consequently, [var phi]EF24C was proposed as a putative therapeutic candidate. In the present study, the [var phi]EF24C genome was analyzed for the first time. Next, in vivo therapeutic effectiveness was evaluated using severe sepsis mouse models infected with an E. faecalis strain having either high or low phage sensitivity. In addition, phage toxicity was briefly examined in vivo.

MATERIALS AND METHODS

Culture media.

Both tryptic soy broth (TSB) and heart infusion broth were obtained from Becton, Dickinson and Company (Sparks, MD). Constituents of culture media used were purchased from Nacalai Tesque (Kyoto, Japan) unless otherwise stated. Heart infusion broth supplemented with 20 mM MgCl2 and 20 mM CaCl2 (HIMC) was prepared. For the phage plaque formation assay, TSB-based solid media containing 1.5% and 0.5% agar were used for the lower and upper layers, respectively.

Bacterial strains.

E. faecalis strain EF24 was employed as a host for the phage propagation and phage plaque formation assay. E. faecalis strains EF14 and VRE2 were employed in the animal experiments. These bacterial strains were described previously (47).

Phage purification.

Phage purification was carried out as described previously (47). Briefly, phage was propagated with host strain EF24 in 1 liter of TSB medium. After the removal of bacterial debris by centrifugation (10,000 × g, 4°C, 10 min) and supplementation of the lysate with polyethylene glycol 6000 (Sigma-Aldrich Co., MI) and NaCl (final concentrations of 10% and 0.5 M, respectively), phage was precipitated by centrifugation (10,000 × g, 4°C, 30 min). Phage precipitate was treated with DNase I (type II; Sigma-Aldrich) and RNase A (type IA; Sigma-Aldrich) (both 50 μg/ml). Finally, phage was sequentially purified by CsCl step gradient ultracentrifugation (50,000 × g, 4°C, 2 h) twice. An S80AT3 rotor and a GX series Himac CS 100GX microultracentrifuge (Hitachi Ltd., Tokyo, Japan) were used for ultracentrifugation. After the phage band was collected, the purified phage was treated differently for each experimental purpose.

Genome sequencing.

The extraction of purified phage DNA was carried out as described previously (47). Briefly, the purified phage suspension containing CsCl was diluted with AAS (0.1 M ammonium acetate, 10 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.2) four times, and phage was pelleted by ultracentrifugation (100,000 × g, 4°C, 1 h). The phage pellet was incubated with proteinase K (Takara Bio, Kyoto, Japan), and phenol extraction and ethanol precipitation were conducted. Finally, the genome DNA was solubilized in water.

The phage DNA was digested by restriction endonuclease HindIII (50 ng DNA/U in 50 μl, 37°C, 4 h) (Takara Bio) and then electrophoresed in 0.8% agarose. After visualization with ethidium bromide (1 μg/ml), the DNA fragments were excised and extracted from the gel. The DNA fragments were cloned into pUC19 vectors and transformed into competent Escherichia coli DH5α.

Sequencing of each cloned fragment was performed by PCR, using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, CA). The PCR products were purified through a Sephadex G-50 column (Sigma-Aldrich) and were then analyzed using an ABI Prism 3100-Avant genetic analyzer (Applied Biosystems). Cloned fragments were sequenced by primer walking. The regions uncovered by the cloned fragments were amplified by PCR with primers based on the cloned fragments, and then the PCR products were purified by LaboPass PCR (Hokkaido System Science, Sapporo, Japan) and sequenced by primer walking as described above. Both strands were sequenced, and the sequence data were connected using the Genetyx-Mac ATSQ program, version 4.2.1 (Genetyx Co., Tokyo, Japan). The sequence coverage redundancy was at least double.

Genome analysis.

The potential open reading frames (ORFs) that possibly encode the gene products were first predicted by the following gene predication tools: GeneMark VIORIN (http://opal.biology.gatech.edu/GeneMark/) (3), FGENESB (http://www.softberry.com/berry.phtml), and Microbial Genome Annotation Tools (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/glimmer_3.cgi) (12, 46). ATG, TTG, and GTG were considered to be start codons, and TAA, TGA, and TAG were considered to be stop codons. The ORFs were then determined from the program-predicted ORFs based on the criteria of a length of more than 72 nucleotides and a maximum length equivalent to that of the program-predicted ORFs with stop codons at the same locations. To examine the therapeutic eligibility of [var phi]EF24C strictly, such criteria were purposely used to determine the ORFs in this study. To increase the possibility of identifying protein-coding sequences, the ribosomal binding site (RBS) sequence of each ORF was also subsequently investigated. Moreover, tRNA genes were predicted using the tRNAscan-SE program (http://lowelab.ucsc.edu/tRNAscan-SE/) (34).

The putative products of the ORFs were analyzed by BLASTP at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and by InterProScan with BlastProDom, FPrintScan, HMMPIR, HMMPfam, HMMSmart, HMMTigr, ProfileScan, ScanRegExp, SuperFamily, HMMPanther, and Gene3D at the European Bioinformatics Institute (http://www.ebi.ac.uk/InterProScan/) (38). Transmembrane domains and signal peptides were also predicted by TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and SignalIP 3.0 (http://www.cbs.dtu.dk/services/SignalP/), respectively (2, 31). Using an E value threshold of 0.1 for both BLASTP and InterProScan, the functions of the putative gene products were specified.

The following genome features were also analyzed using in silico molecular cloning genomics edition (In Silico Biology, Inc., Yokohama, Japan): GC content, GC scanning, GC skew, cumulative GC skew, and codon usage.

An unrooted phylogenic tree was constructed by the neighbor-joining method, using DNASIS Pro (Hitachi Software Engineering Co., Ltd., Tokyo, Japan). Bootstrap analysis was performed by resampling the data sets 10,000 times. Bootstrap values of greater than 95% were considered to be statistically significant for the grouping.

The gene order was compared with the most related phage genome; this comparison was manually performed by in-house BLAST search, using in silico molecular cloning genomics edition (In Silico Biology, Inc.). An E value of less than 0.1 was considered as indicative of homology.

Comparative data for the following were retrieved from the GenBank database: bacteriophage G1 (accession number AY954969), Staphylococcus phage K (accession number AY176327), Staphylococcus phage Twort (accession number AY954970), Listeria bacteriophage P100 (accession number DQ004855), Lactobacillus plantarum bacteriophage LP65 (accession number AY682195), and E. faecalis V583 (accession number AE016830.1).

Phage preparation for animal experiments.

For the purpose of a mouse rescue experiment, the purified phage sample was continuously dialyzed against SMC (saline with 20 mM MgCl2 and 20 mM CaCl2) (4°C, 30 min) and HIMC (4°C, 30 min). On the other hand, the purified phage sample was dialyzed against SMC (4°C, 60 min) for the repeated administration. The titers (PFU/ml) of the phage were then determined by inoculation into bacterial strain EF24. The phage was stored at 4°C until use.

Animal experiments.

All animal experiments were conducted with the approval of the Animal Experiment Committee of Kochi Medical School. Female 6- to 8-week-old BALB/c mice (weighing up to 18 g) were used in the following experiments.

E. faecalis cells (either strain EF14 or strain VRE2) were grown in 300 ml of TSB medium at 37°C until the early stationary phase (up to ca. 200 Klett units) and were then centrifuged (10,000 × g, 4°C, 10 min). The cell pellet was washed with 300 ml of saline, centrifuged (10,000 × g, 4°C, 10 min), and finally resuspended in 3 ml of saline. After appropriate dilution using saline, the bacterial concentration (bacteria/ml) was determined by turbidity (in Klett units), measured with a Klett-Summerson photoelectric colorimeter (Klett Mfg. Co., NY). EF14 was prepared to be at 1.0 ×1010, 2.0 × 1010, 5.0 × 1010, 1.0 × 1011, 2.0 × 1011, and 5.0 ×1011 bacteria/ml. VRE2 was prepared to be at 5.0 × 1010, 1.0 × 1010, 2.1 × 1010, 5.0 × 1010, 1.0 × 1011, and 2.1 × 1011 bacteria/ml. Saline (0.2 ml; control) or bacterial suspension at different concentrations was injected into the peritoneal cavities of 5 mice through the left side of the abdomen (in total, 70 mice were used). The survival rates for and activities of all tested animals were observed for 7 days. The minimum bacterial concentration showing 100% lethality was determined as the minimum lethal bacterial dosage.

For the mouse rescue experiment by phage, the phage was diluted in HIMC to the following concentrations (expressed in multiplicities of infection [MOI]): 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001. Mice were inoculated on the left side of the abdomen with 0.2 ml of the minimum lethal bacterial dose (in total, 70 mice). About 20 min after the inoculation of the lethal bacterial dose, 0.2 ml of phage solution at different concentrations (in MOI), HIMC, or saline was administered to five mice on the right side of the abdomen. The data on the survival rates of the mice were analyzed statistically with a two-tailed Fisher's exact test. Moreover, to measure the intrinsic effects of phage alone and buffers, 0.5-ml aliquots of HIMC, saline, or phage suspension (total, 1.0 × 1012 PFU) alone was administered into the abdominal cavities of 5 mice (a total of 15 mice). The survival rates for and activities of all tested animals were observed for 7 days.

To examine the effects of repeated phage exposure, 0.5 ml of an SMC phage suspension (in total, 3.5 × 1010 PFU) or SMC alone was intraperitoneally administered seven times at 4-day intervals into the abdominal cavities of 10 mice (a total of 20 mice). The mouse survival rate was recorded for 2 months from the initial phage administration.

Nucleotide sequence accession number.

The genome data of phage [var phi]EF24C was deposited to GenBank (accession number AP009390).

RESULTS AND DISCUSSION

General genome description.

The [var phi]EF24C genome was determined to be 142,072 bp. Also, the genome sequence was circularly permuted because no definite terminal ends were identified by genomic sequencing. The GC content was 35.7%. Two hundred twenty-one ORFs and five tRNA genes were determined. When we checked for the presence of an RBS upstream from each ORF, most ORFs seemed to have a typical RBS (see the supplemental material). In the following text, ORFs considered to have a functional putative product were defined as putative genes and are designated using an “orf” prefix. The putative gene (i.e., orf) product is likewise shown using “Orf.”

Bioinformatic analysis revealed that 45.2% (100/221) of the putative ORF products were assumed to have either protein functional domains or similarity to other phage gene products or both. Overall, 20.8% (46/221) of the ORFs were deduced to encode functional proteins. No genes coding for site-specific integrase, toxin and antibiotic resistance genes, or other pathogenic factors were predicted. A BLASTP search on the hypothetical ORF-encoded proteins showed similarities with the gene products of the other large virulent phages, including Staphylococcus phage K, G1, Twort, Lactobacillus phage LP65, and Listeria phage P100. The genomes of these phages are well characterized (8, 10, 32, 39). The genome map of [var phi]EF24C is shown in Fig. Fig.1.1. The annotation of the genome is also shown in Table Table11.

FIG. 1.
Genome map of phage [var phi]EF24C. Arrows indicate putative ORFs and tRNA genes, along with their orientations. Functionally assigned genes are differently colored (blue, structural gene; red, lysis gene; green, DNA-associated gene; violet, tRNA gene). ...
TABLE 1.
Features of phage [var phi]EF24C gene products and their functional assignments

The N-terminal amino acid sequences of the products of the orf16, orf23, orf24, orf40, orf69, and orf219 genes precisely corresponded to those of six virion proteins (42, 62, 16, 26, 21, and 12.6 kDa, respectively) of [var phi]EF24C as reported in our previous paper (47). Only Orf16 and Orf23 were bioinformatically specified as major capsid protein (MCP) and tail sheath protein, respectively; the others were unknown. Except for that of Orf219, the N-terminal ends were considered to be processed. During morphogenesis, the N-terminal 20 amino acid residues of the MCP (Orf16) were considered to be removed, which was assumed to be mediated by the putative prohead protease (Orf14). The other structural proteins (Orf23, Orf24, Orf40, and Orf69) were assumed to be similarly digested between methionine and alanine.

In general, functionally relevant genes are clustered as a module in phage (6, 11). Three modules were speculated to range from 1 bp to 71 kbp in [var phi]EF24C, according to genome annotation and structural protein identification (Fig. (Fig.1).1). The large structural module seemed to be associated with head and tail components (1 bp to 46 kbp). The replication-associated genes were clustered as a DNA replication module (46 kbp to 68 kbp). The small structural module included at least orf68 and orf69 (69 kbp to 71 kbp) due to an immunoglobulin (Ig)-like domain specified on Orf68 (an Ig-like domain is typically found in a virion protein of phage) and the identification of Orf69 as a structural protein from the proteomic analysis (16, 17).

In the functionally uncategorized region, some putative genes associated with de novo synthesis of nucleic acid precursors were speculated. These included the genes for Orf103 (cytidine deaminase), Orf203 (thymidylate synthase), and Orf208, Orf209, and Orf210 (ribonucleotide diphosphate reductase). Like T4 phage, [var phi]EF24C may produce its own modified base from host DNA breakdown (22, 37).

At the final stage of the latent period, progeny phages within a bacterial cell were released by the degradation of the cell wall (25, 27). This cell wall lysis is typically induced by two phage-encoded proteins called holin and endolysin (25, 27). At the late period of infection, holin forms a hole in the cell membrane, and endolysin passes through the hole and destroys the peptidoglycan structure (25, 27). In the phage [var phi]EF24C genome, the putative genes for endolysin (Orf9 and Orf10) and holin (Orf67) are thought to be distantly positioned, as occurs in phage T4 (37). orf9 and orf10 are thought to be located in one structural module, and orf67 is thought to be located in very close proximity to another structural module. Thus, these genes are possibly expressed late in the period of infection (37). Consequently, Orf9, Orf10, and Orf67 may function as a holin-endolysin system.

Phylogenetic analyses of [var phi]EF24C within the SPO1-like phages.

The [var phi]EF24C genome contains approximately 142 kbp, the GC content of which is 35.7%, as described above. The genome is circularly permuted, and DNA polymerase A (Orf61) has been tentatively identified. These attributes, together with the morphological and biological features of [var phi]EF24C, suggest that [var phi]EF24C is a member of the SPO1-like phage genus (28). Therefore, the phylogenic relationships of [var phi]EF24C to the other SPO1-like phages were examined.

A phylogenic tree based on the MCP is frequently used in phage phylogenic analysis (1, 26). The MCPs of the following phages were obtained from their genome sequences: Staphylococcus phage K, G1, Twort, Lactobacillus phage LP65, and Listeria phage P100. The MCP-based phylogenic analysis showed that [var phi]EF24C is most closely related to Listeria phage P100 among these phages (Fig. (Fig.2A).2A). Next, the gene organization of the [var phi]EF24C genome was also compared with that of Listeria phage P100. Genome synteny was observed, particularly on the predicted structural and DNA replication modules in [var phi]EF24C (ca. 70% of the genome) (Fig. (Fig.2B),2B), whereas the genes on the other region of [var phi]EF24C (the remaining ca. 30% of the genome) are not only functionally unknown but also dissimilar to any genes of Listeria phage P100. By considering the difference in bacterial species and phylogenic relation based on MCP, [var phi]EF24C and Listeria phage P100 were considered to have evolved divergently from the same virus origin (44).

FIG. 2.
Phylogenetic analyses of [var phi]EF24C. (A) Phylogenetic tree based on MCPs. (B) Comparison of the gene order between Enterococcus phage [var phi]EF24C and Listeria phage P100. The ORF of [var phi]EF24C is connected to that of P100 by a black line where ...

Noncompetitive nature between replication and transcription directions.

The origin of replication (ori) and replication terminus (ter) can be deduced by GC skew and cumulative GC skew analyses (23, 24, 45). The cumulative GC skew analysis for Fig. Fig.33 shows the predicted ori and ter, which are the lowest (ca. 1-bp) and highest (108-kbp) regions, respectively. Although the process of [var phi]EF24C genome replication remains unknown, it seems to replicate in a manner slightly different from that of another large phage, T4 of E. coli. The [var phi]EF24C genome seems to have only one ori, whereas phage T4 has multiple oris (37). Moreover, the speculated replication direction (ori to ter) matched with the direction of transcription (direction of genes). According to this mutual correspondence, bimolecular collisions during replication and transcription can be avoided (13, 18). Hence, [var phi]EF24C is assumed to multiply without such mutual interference between replication and transcription.

FIG. 3.
GC-associated genome analysis. In the GC scanning, the low region in GC is indicated by the bar. In the cumulative GC skew, the origin of replication (black arrow) and the changing point of gene direction/termination of DNA replication (white arrow) are ...

Host-adapted translation.

Host and phage codon usage comparisons and phage tRNA analysis implied efficient translation. The codon usages of [var phi]EF24C and E. faecalis V583 were mutually similar (Fig. (Fig.4A),4A), which can indicate overall efficient translation in the host. Moreover, the codon usage of [var phi]EF24C exceeded that of the host on five predicted tRNAs whose genes were carried by the phage (Fig. (Fig.4B).4B). Thus, we can infer that [var phi]EF24C supplies specific tRNAs on its own in case of tRNA deficiency.

FIG. 4.
Optimization of [var phi]EF24C codon usage to its host codon usage and its possible tRNA function. (A) Comparison of codon usage between [var phi]EF24C and E. faecalis V583. (B) Location of tRNA genes (top), predicted secondary structures of tRNAs (middle), ...

E. faecalis sepsis mouse model.

Sepsis mouse models are typically used for the preliminary assessment of phage therapy against nosocomial bacteria (4, 35, 48-50). To examine therapeutic effectiveness and the effect of host sensitivity difference on the phage in vivo, E. faecalis sepsis mouse models using BALB/c mice were set up using two strains, EF14 and VRE2. In the previous study, EF14 had phage sensitivity about 32 times greater than that of VRE2 (efficiency of plating [EOP] against EF14, 1; EOP against VRE2, 0.032) (47). After intraperitoneal bacterial inoculation at different concentrations to mice, the minimum lethal bacterial dosages of EF14 and VRE2 were determined to be 1.0 × 1010 and 4.2 × 109 bacteria, respectively; these dosages resulted in 100% lethality within 2 days. These bacterial dosages were used for the following experiments to assess mouse rescue by phage.

In both cases, inoculation of the minimum lethal bacterial dosages seemed to induce severe sepsis complications. As time elapsed after bacterial inoculation, an increase in the number of bacteria in the blood was also observed (bacteremia). In addition, an increasing frequency of unusual changes in the mice was observed, such as decrease in activity, low body temperature, shivering, blood clotting, and hyperventilation. These abnormal conditions were considered to be typical features of severe sepsis, although histological analyses were not performed (21, 41).

[var phi]EF24C therapeutic effectiveness in vivo.

No abnormal mouse behavior or altered survival rate was observed following the administration of saline, HIMC, or phage alone (1.0 × 1012 PFU). Thus, the phage rescue experiments were considered to be conducted without bias.

The in vivo therapeutic effectiveness of [var phi]EF24C was then examined. After the inoculation of the minimum lethal bacterial dosage, HIMC or [var phi]EF24C at different MOI of 10, 1, 0.1, 0.01, 0.001, and 0.0001 was administered. Figure Figure55 shows the results of mouse rescue experiments using [var phi]EF24C. The dose-dependent effectiveness was observed to be less than MOI of 0.01 and 0.1 in the EF14 and VRE2 mouse sepsis models, respectively. Compared with the control (HIMC or saline treatment), the [var phi]EF24C treatment was significantly effective for both EF14-infected mice at MOI of 10, 1, 0.1, and 0.01 (P < 0.01) and VRE2-infected mice at MOI of 10, 1, and 0.1 (P < 0.01) or 0.01 (P < 0.05). According to these results, [var phi]EF24C can efficiently rescue mice infected with both EF14 and VRE2 at an MOI of 0.01. Under these experimental conditions, the therapeutic efficacy of [var phi]EF24C did not seem to be affected by the sensitivity of the host to the phage. The [var phi]EF24C burst size in vitro was reported as ca. 100 in a previous work (47). Under in vivo conditions, the phage may not infect the bacteria or propagate as efficiently as it does under in vitro conditions. Therefore, these results suggest that the efficient propagation of [var phi]EF24C led to its significant therapeutic effectiveness in vivo.

FIG. 5.
Experiment examining mouse rescue by phage [var phi]EF24C administration. At approximately 20 min after the intraperitoneal inoculation of the minimum lethal bacterial dosage (EF14, 1.0 × 1010 bacteria; VRE2, 4.2 × 109 bacteria) to BALB/c ...

Eligibility of [var phi]EF24C as a potential therapeutic phage.

[var phi]EF24C is considered to be eligible as a therapeutic phage for the following reasons. First, as demonstrated in a previous study, [var phi]EF24C has broad host specificity and strong virulence against E. faecalis strains. Second, undesirable genes for phage therapy such as integration-related and pathogenic (e.g., toxin and antibiotic resistance, etc.) genes have not yet been identified. Third, we can infer from the following features that the biological nature of [var phi]EF24C is appropriate for a therapeutic phage: its de novo nucleic acid synthesis from host DNA breakdown, its holin-endolysin system, its host-adapted translation, and the noncompetitive nature of its transcription and replication. Fourth, its genomic features, together with its morphology, allow [var phi]EF24C to be categorized in the SPO1-like phage genus, some members of which, including phages K and P100, are used or are under consideration for therapy or prophylaxis. Phylogenetic and genome synteny analyses revealed a close relationship between [var phi]EF24C and the Listeria phage P100, which has been approved for prophylactic use and has been commercialized (EBI Food Safety [http://www.ebifoodsafety.com/]) (8, 42). Thus, [var phi]EF24C can be encompassed in the general therapeutic phage group. Fifth, accidental homologous recombination is also not likely in the use of [var phi]EF24C, because similarities between the [var phi]EF24C genome and the host E. faecalis V583 genome were restricted to tRNA genes and a few genes for hypothetical proteins, as for the other therapeutic phages K and P100 (data not shown). This animal experiment showed that a single low-dosage administration of [var phi]EF24C can effectively treat sepsis in mice without the effect of host sensitivity to the phage observed in vitro (i.e., EOP difference between strains EF14 and VRE2). The phage bacteriolytic action is believed to be the primary mechanism of mouse rescue effects. However, [var phi]EF24C efficacy may be altered in different mouse strains and animals. For example, some possible phage-inactivating factors, such as phage-neutralizing antibody and liver or bile acids, may alter phage efficacy, and the phage may not efficiently reach the focus of infection. Therefore, further study is required (i.e., of the pharmacokinetics and pharmacodynamics associated with the compromised route of administration).

Safety issues are of great concern in phage therapy. Surprisingly, no significant side effects of phage therapy have been reported to date in the East (36, 43). In this experiment, phage administration did not cause any lethality or mouse behavior change both in the mouse rescue experiment and in the administration of a high-concentration dosage of phage alone. In addition, repeated exposure to phage (administration seven times at 4-day intervals) did not cause any change in mouse behavior. However, different phages have different molecular features, and different mouse strains have different levels of immunity, so the safety of each phage must be examined in the future (25, 33, 36). In this study, [var phi]EF24C was investigated primarily as a therapeutic phage. Although further development of this phage and other methods is still necessary to address some remaining problems, [var phi]EF24C is a promising therapeutic phage against E. faecalis infections.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Hiromi Kataoka (Clinical Laboratory Centers, Kochi Medical School) for access to DNASIS Pro and Toshimitsu Uchiyama (Toho University, Tokyo, Japan) for helpful scientific advice.

This study was supported by The Special Research Project of Green Science, Kochi University.

Footnotes

[down-pointing small open triangle]Published ahead of print on 2 May 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

1. Bamford, D. H., J. M. Grimes, and D. I. Stuart. 2005. What does structure tell us about virus evolution? Curr. Opin. Struct. Biol. 15:655-663. [PubMed]
2. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783-795. [PubMed]
3. Besemer, J., and M. Borodovsky. 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 33:e451-e454. [PMC free article] [PubMed]
4. Biswas, B., S. Adhya, P. Washart, B. Paul, A. N. Trostel, B. Powell, R. Carlton, and C. R. Merril. 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect. Immun. 70:204-210. [PMC free article] [PubMed]
5. Bonten, M. J., R. Willems, and R. A. Weinstein. 2001. Vancomycin-resistant enterococci: why are they here, and where do they come from? Lancet Infect. Dis. 1:314-325. [PubMed]
6. Botstein, D. 1980. A theory of modular evolution for bacteriophages. Ann. N. Y. Acad. Sci. 354:484-490. [PubMed]
7. Bradley, J. S., R. Guidos, S. Baragona, J. G. Bartlett, E. Rubinstein, G. G. Zhanel, M. D. Tino, D. L. Pompliano, F. Tally, P. Tipirneni, G. S. Tillotson, J. H. Powers, and G. S. Tillotson. 2007. Anti-infective research and development-problems, challenges, and solutions. Lancet Infect. Dis. 7:68-78. [PubMed]
8. Carlton, R. M., W. H. Noordman, B. Biswas, E. D. de Meester, and M. J. Loessner. 2005. Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxicity study, and application. Regul. Toxicol. Pharmacol. 43:301-312. [PubMed]
9. Cetinkaya, Y., P. Falk, and C. G. Mayhall. 2000. Vancomycin-resistant enterococci. Clin. Microbiol. Rev. 13:686-707. [PMC free article] [PubMed]
10. Chibani-Chennoufi, S., M. L. Dillmann, L. Marvin-Guy, S. Rami-Shojaei, and H. Brüssow. 2004. Lactobacillus plantarum bacteriophage LP65: a new member of the SPO1-like genus of the family Myoviridae. J. Bacteriol. 186:7069-7083. [PMC free article] [PubMed]
11. Comeau, A. M., C. Bertrand, A. Letarov, F. Tétart, and H. M. Krisch. 2007. Modular architecture of the T4 phage superfamily: a conserved core genome and a plastic periphery. Virology 362:384-396. [PubMed]
12. Delcher, A. L., K. A. Bratke, E. C. Powers, and S. L. Salzberg. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673-679. [PMC free article] [PubMed]
13. Deshpande, A. M., and C. S. Newlon. 1996. DNA replication fork pause sites dependent on transcription. Science 272:1030-1033. [PubMed]
14. Deshpande, L. M., T. R. Fritsche, G. J. Moet, D. J. Biedenbach, and R. N. Jones. 2007. Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe: a report from the SENTRY antimicrobial surveillance program. Diagn. Microbiol. Infect. Dis. 58:163-170. [PubMed]
15. Fischetti, V. A., D. Nelson, and R. Schuch. 2006. Reinventing phage therapy: are the parts greater than the sum? Nat. Biotechnol. 24:1508-1511. [PubMed]
16. Fraser, J. S., K. L. Maxwell, and A. R. Davidson. 2007. Immunoglobulin-like domains on bacteriophage: weapons of modest damage? Curr. Opin. Microbiol. 10:382-387. [PubMed]
17. Fraser, J. S., Z. Yu, K. L. Maxwell, and A. R. Davidson. 2006. Ig-like domains on bacteriophages: a tale of promiscuity and deceit. J. Mol. Biol. 359:496-507. [PubMed]
18. Freeman, M. J., N. T. Plasterer, F. T. Smith, and C. S. Mohr. 1998. Patterns of genome organization in bacteria. Science 279:1827.
19. Fujita, N. 2005. Vancomycin-resistant enterococci (VRE)-for VRE endemics in Japan. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi 16:1-16. (In Japanese.) [PubMed]
20. Gill, J. J., M. E. Pacan, M. E. Carson, K. E. Leslie, M. W. Griffiths, and P. M. Sabour. 2006. Efficacy and pharmacokinetics of bacteriophage therapy in treatment of subclinical Staphylococcus aureus mastitis in lactating dairy cattle. Antimicrob. Agents Chemother. 50:2912-2918. [PMC free article] [PubMed]
21. Gomi, H. 2007. Sepsis. Nippon Rinsho 65(Suppl. 3):613-616. (In Japanese.) [PubMed]
22. Greenberg, R. G., P. He, J. Hilfinger, and M. J. Tseng. 1994. Deoxyribonucleoside triphosphate synthesis and phage T4 DNA replication, p. 14-27. In J. D. Karam, J. W. Drake, and K. N. Kreuzer (ed.), Molecular biology of bacteriophage T4. ASM Press, Washington, DC.
23. Grigoriev, A. 1998. Analyzing genomes with cumulative skew diagrams. Nucleic Acids Res. 26:2286-2290. [PMC free article] [PubMed]
24. Grigoriev, A. 1999. Strand-specific compositional asymmetries in double-stranded DNA viruses. Virus Res. 60:1-19. [PubMed]
25. Guttman, B., R. Raya, and E. Kutter. 2005. Basic phage biology, p. 29-66. In E. Kutter and A. Sulakvelidze (ed.), Bacteriophages: biology and applications. CRC Press, Boca Raton, FL.
26. Hambly, E., F. Tétart, C. Desplats, W. H. Wilson, H. M. Krisch, and N. H. Mann. 2001. A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2. Proc. Natl. Acad. Sci. USA 98:11411-11416. [PMC free article] [PubMed]
27. Hanlon, G. W. 2007. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Int. J. Antimicrob. Agents 30:118-128. [PubMed]
28. Hendrix, R. W., and S. R. Casjens. 2005. Myoviridae, p. 43-55. In C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (ed.), Virus taxonomy. Eighth report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, CA.
29. Kaspárek, P., R. Pantůcek, J. Kahánková, V. Růzicková, and J. Doskar. 2007. Genome rearrangements in host-range mutants of the polyvalent staphylococcal bacteriophage 812. Folia Microbiol. (Praha) 52:331-338. [PubMed]
30. Kayser, F. H. 2003. Safety aspects of enterococci from the medical point of view. Int. J. Food Microbiol. 88:255-262. [PubMed]
31. Krogh, A., B. Larsson, G. von Heijne, and E. L. Sonnhammer. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305:567-580. [PubMed]
32. Kwan, T., J. Liu, M. DuBow, P. Gros, and J. Pelletier. 2005. The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proc. Natl. Acad. Sci. USA 102:5174-5179. [PMC free article] [PubMed]
33. Lei, H. Y., S. H. Lee, and S. H. Leir. 1996. Antigen-induced anaphylactic death in mice. Int. Arch. Allergy Immunol. 109:407-412. [PubMed]
34. Lowe, T. M., and S. R. Eddy. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955-964. [PMC free article] [PubMed]
35. Matsuzaki, S., M. Yasuda, H. Nishikawa, M. Kuroda, T. Ujihara, T. Shuin, Y. Shen, Z. Jin, S. Fujimoto, M. D. Nasimuzzaman, H. Wakiguchi, S. Sugihara, T. Sugiura, S. Koda, A. Muraoka, and S. Imai. 2003. Experimental protection of mice against lethal Staphylococcus aureus infection by novel bacteriophage [var phi]MR11. J. Infect. Dis. 187:613-624. [PubMed]
36. Merril, R. C., D. Scholl, and S. Adhya. 2006. Phage therapy, p. 725-741. In R. Calender and T. S. Abedon (ed.), The bacteriophages, 2nd ed. Oxford University Press, New York, NY.
37. Miller, E. S., E. Kutter, G. Mosig, F. Arisaka, T. Kunisawa, and W. Ruger. 2003. Bacteriophage T4 genome. Microbiol. Mol. Biol. Rev. 67:86-156. [PMC free article] [PubMed]
38. Mulder, N. J., R. Apweiler, T. K. Attwood, A. Bairoch, A. Bateman, D. Binns, P. Bork, V. Buillard, L. Cerutti, R. Copley, E. Courcelle, U. Das, L. Daugherty, M. Dibley, R. Finn, W. Fleischmann, J. Gough, D. Haft, N. Hulo, S. Hunter, D. Kahn, A. Kanapin, A. Kejariwal, A. Labarga, P. S. Langendijk-Genevaux, D. Lonsdale, R. Lopez, I. Letunic, M. Madera, J. Maslen, C. McAnulla, J. McDowall, J. Mistry, A. Mitchell, A. N. Nikolskaya, S. Orchard, C. Orengo, R. Petryszak, J. D. Selengut, C. J. Sigrist, P. D. Thomas, F. Valentin, D. Wilson, C. H. Wu, and C. Yeats. 2007. New developments in the InterPro database. Nucleic Acids Res. 35:e224-e228. [PMC free article] [PubMed]
39. O'Flaherty, S., A. Coffey, R. Edwards, W. Meaney, G. F. Fitzgerald, and R. P. Ross. 2004. Genome of staphylococcal phage K: a new lineage of Myoviridae infecting gram-positive bacteria with a low G+C content. J. Bacteriol. 186:2862-2871. [PMC free article] [PubMed]
40. O'Flaherty, S., R. P. Ross, W. Meaney, G. F. Fitzgerald, M. F. Elbreki, and A. Coffey. 2005. Potential of the polyvalent anti-Staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals. Appl. Environ. Microbiol. 71:1836-1842. [PMC free article] [PubMed]
41. Okajima, K. 2007. Multiple organ failure associated with severe infection-the molecular mechanism(s) and new therapeutic strategies. Nippon Rinsho 65(Suppl. 3):619-626. (In Japanese.) [PubMed]
42. Petty, N. K., T. J. Evans, P. C. Fineran, and G. P. Salmond. 2007. Biotechnological exploitation of bacteriophage research. Trends Biotechnol. 25:7-15. [PubMed]
43. Sulakvelidze, A., and E. Kutter. 2005. Bacteriophage therapy in humans, p. 381-436. In E. Kutter and A. Sulakevelidze (ed.), Bacteriophages: biology and applications. CRC Press, Boca Raton, FL.
44. Tamames, J. 2001. Evolution of gene order conservation in prokaryotes. Genome Biol. 2:research0020.1-research0020.11. doi:.10.1186/gb-2001-2-6-research0020 [PMC free article] [PubMed] [Cross Ref]
45. Tang, S. L., S. Nuttall, K. Ngui, C. Fisher, P. Lopez, and M. Dyall-Smith. 2002. HF2: a double-stranded DNA tailed haloarchaeal virus with a mosaic genome. Mol. Microbiol. 44:283-296. [PubMed]
46. Tyson, G. W., J. Chapman, P. Hugenholtz, E. E. Allen, R. J. Ram, P. M. Richardson, V. V. Solovyev, E. M. Rubin, D. S. Rokhsar, and J. F. Banfield. 2004. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428:37-43. [PubMed]
47. Uchiyama, J., M. Rashel, Y. Maeda, I. Takemura, S. Sugihara, K. Akechi, A. Muraoka, H. Wakiguchi, and S. Matsuzaki. 2008. Isolation and characterization of a novel Enterococcus faecalis bacteriophage [var phi]EF24C as a candidate therapeutic. FEMS Microbiol. Lett. 278:200-206. [PubMed]
48. Vinodkumar, C. S., Y. F. Neelagund, and S. Kalsurmath. 2005. Bacteriophage in the treatment of experimental septicemic mice from a clinical isolate of multidrug resistant Klebsiella pneumoniae. J. Commun. Dis. 37:18-29. [PubMed]
49. Wang, J., B. Hu, M. Xu, Q. Yan, S. Liu, X. Zhu, Z. Sun, D. Tao, L. Ding, E. Reed, J. Gong, Q. Q. Li, and J. Hu. 2006. Therapeutic effectiveness of bacteriophages in the rescue of mice with extended spectrum beta-lactamase-producing Escherichia coli bacteremia. Int. J. Mol. Med. 17:347-355. [PubMed]
50. Watanabe, R., T. Matsumoto, G. Sano, Y. Ishii, K. Tateda, Y. Sumiyama, J. Uchiyama, S. Sakurai, S. Matsuzaki, S. Imai, and K. Yamaguchi. 2007. Efficacy of bacteriophage therapy against gut-derived sepsis caused by Pseudomonas aeruginosa in mice. Antimicrob. Agents Chemother. 51:446-452. [PMC free article] [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • BioProject
    BioProject
    BioProject links
  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • Gene
    Gene
    Gene links
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...