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. 2008 May 2;74(13):4149–4163. doi: 10.1128/AEM.02371-07

In Silico and In Vivo Evaluation of Bacteriophage φEF24C, a Candidate for Treatment of Enterococcus faecalis Infections▿ †

Jumpei Uchiyama 1,2, Mohammad Rashel 2, Iyo Takemura 2, Hiroshi Wakiguchi 1, Shigenobu Matsuzaki 2,*
PMCID: PMC2446516  PMID: 18456848

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, φEF24C, and its biology indicated its therapeutic potential against E. faecalis infection. In this study, the φEF24C genome was analyzed and the in vivo therapeutic applicability of φEF24C was also briefly assessed. Its complete genome (142,072 bp) was predicted to have 221 open reading frames (ORFs) and five tRNA genes. In our functional analysis of the ORFs by use of a public database, no proteins undesirable in phage therapy, such as pathogenic and integration-related proteins, were predicted. The noncompetitive directions of replication and transcription and the host-adapted translation of the phage were deduced bioinformatically. Its genomic features indicated that φEF24C is a member of the SPO1-like phage genus and especially that it has a close relationship to the Listeria phage P100, which is authorized for prophylactic use. Thus, these bioinformatics analyses rationalized the therapeutic eligibility of φEF24C. Moreover, the in vivo therapeutic potential of φEF24C, which was effective at a low concentration and was not affected by host sensitivity to the phage, was proven by use of sepsis BALB/c mouse models. Furthermore, no change in mouse lethality was observed under either single or repeated phage exposures. Although further study is required, φEF24C can be a promising therapeutic phage against E. faecalis infections.

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, φEF24C was isolated and its biology was characterized briefly. φEF24C, 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 φEF24C, 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, φEF24C was proposed as a putative therapeutic candidate. In the present study, the φEF24C 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 φEF24C 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 φEF24C was deposited to GenBank (accession number AP009390).

RESULTS AND DISCUSSION

General genome description.

The φEF24C 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 φEF24C is shown in Fig. 1. The annotation of the genome is also shown in Table 1.

FIG. 1.

FIG. 1.

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Genome map of phage φEF24C. 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). Speculated modules are enclosed by boxes (black, structural module; pink, DNA replication module). *, gene for structural protein.

TABLE 1.

Features of phage φEF24C gene products and their functional assignments

ORF Position
Stranda Length (nt)b Size (aa)c Mol mass (kDa)d pIe Putative functional assignment NCBI database search result
Protein domain search result
Predicted TMHf and signal peptide
Start End Similarities/homologies to gene products of phages and bacteria Tool Bits E value Predicted domain Tool E value TMHHMg SignalPh
1 114 383 + 270 89 10.1 9.8 2
2 404 682 + 279 92 10.3 9.9 ORF133 (Staphylococcus phage Twort) BLASTP 64.3 9.00E-11
3 692 1114 + 423 140 15.9 4.4 gp3 (Listeria bacteriophage P100) BLASTP 73.2 3.00E-12
4 1114 1482 + 369 122 13.8 9.6 Large terminase Large terminase (Bacillus subtilis phage 1102phil-3) BLASTP 160 2.00E-38
5 1767 3272 + 1,506 501 56.7 6.3 Large terminase gp5 (Listeria bacteriophage P100) BLASTP 633 8.00E-180 Phage terminase large subunit (GpA) RPSBLAST 4.00E-08
6 3372 4160 + 789 262 29.6 7.6 gp6 (Listeria bacteriophage P100) BLASTP 111 4.00E-23
7 4265 4981 + 717 238 27.5 4.7 gp8 (Listeria bacteriophage P100) BLASTP 102 1.00E-20
8 4971 5315 + 345 114 12.8 6.6 gp9 (Listeria bacteriophage P100) BLASTP 35 0.92
9 5409 6278 + 870 289 31.4 7.0 Endolysin-associated protein N-Acetylmuramyl-l-alanine amidase, negative regulator of AmpC, AmpD (Rubrobacter xylanophilus DSM 9941) BLASTP 70.5 9.00E-11 N-Acetylmuramoyl-l-alanine amidase, family 2 RPSBLAST 4.00E-12
10 6445 7083 + 639 212 23.1 4.4 Endolysin-associated protein LysM domain protein (Enterococcus faecalis V583) BLASTP 159 1.00E-37 LysM domain RPSBLAST 6.00E-09 Y
11 7227 7571 + 345 114 13.5 5.9 gp13 (Listeria bacteriophage P100) BLASTP 76.6 3.00E-13
12 7586 9310 + 1,725 574 64.9 5.6 Portal protein gp14 (Listeria bacteriophage P100) BLASTP 644 0 Phage portal protein RPSBLAST 6.00E-08
13 9344 9430 + 87 28 3.1 9.3
14 9417 10208 + 792 263 29.6 5.2 Prohead protease gp15 (Listeria bacteriophage P100) BLASTP 281 2.00E-74 Peptidase U35 HMMPfam 8.10E-06
15 10215 11141 + 927 308 34.8 4.3 IgA-specific metalloendopeptidase (EC 3.4.24.13) type 1 precursor - Haemophilus influenzae (strain HK613) BLASTP 47.8 7.00E-04
16 11282 12676 + 1,395 464 51.2 5.2 MCP Cps (Listeria bacteriophage P100) BLASTP 674 0 1
17 12763 13044 + 282 93 10.3 6.7 gp18 (Listeria bacteriophage P100) BLASTP 29.3 3.4
18 13057 13956 + 900 299 34.0 4.9 gp19 (Listeria bacteriophage P100) BLASTP 379 7.00E-104
19 13976 14845 + 870 289 32.6 5.9 gp20 (Listeria bacteriophage P100) BLASTP 264 5.00E-69
20 14838 15461 + 624 207 23.7 10.5 gp21 (Listeria bacteriophage P100) BLASTP 183 5.00E-45
21 15465 16310 + 846 281 31.8 4.6 ORF6 (Listeria bacteriophage A511) BLASTP 239 1.00E-61
22 16310 16543 + 234 77 9.0 9.3 ORF185 (Staphylococcus phage Twort) BLASTP 66.6 3.00E-10
23 16547 18256 + 1,710 569 62.0 4.9 Tail sheath protein Tsh (Listeria bacteriophage P100) BLASTP 697 0
24 18317 18739 + 423 140 15.5 5.4 Structural protein ORF8 (Listeria bacteriophage A511) BLASTP 205 4.00E-52
25 18863 19699 + 837 278 32.1 9.8
26 19713 19859 + 147 48 5.8 5.0
27 19995 20468 + 474 157 18.3 4.9 gp26 (Listeria bacteriophage P100) BLASTP 128 6.00E-29
28 20536 21111 + 576 191 22.4 4.4 RNA polymarase gp27 (Listeria bacteriophage P100) BLASTP 90.9 3.00E-17 Mitochondrial DNA-directed RNA polymerase RPSBLAST 0.006
29 21156 24800 + 3,645 1214 129.4 8.3 Tail lysin gp28 (Listeria bacteriophage P100) BLASTP 400 2.00E-109 SLT domain proteins RPSBLAST 7.00E-09
30 24839 28024 + 3,186 1061 118.1 5.2 Tail lysin gp29 (Listeria bacteriophage P100) BLASTP 548 4.00E-154 NlpC/P60 family RPSBLAST 0.000002
Peptidase family M23/M37 RPSBLAST 0.0002
31 28108 33585 + 5,478 1825 203.0 4.7 Tail fiber gp30 (Listeria bacteriophage P100) BLASTP 277 3.00E-73 Galactose-binding domain-like SuperFamily 0.00021
LuxS/MPP-like metallohydrolase SuperFamily 0.012
32 33685 36117 + 2,433 810 89.4 9.0 Minor structural protein Prophage LambdaSal, minor structural protein, putative (Streptococcus agalactiae 2603V/R) BLASTP 70.1 5.00E-10 Lipocalin ProfileScan
33 36111 36836 + 726 241 27.9 4.4
34 36868 37014 + 147 48 5.7 4.9
35 37151 37840 + 690 229 25.5 9.1 gp31 (Listeria bacteriophage P100) BLASTP 220 3.00E-56
36 37844 38380 + 537 178 20.2 4.6 gp32 (Listeria bacteriophage P100) BLASTP 146 3.00E-34
37 38367 39071 + 705 234 26.3 4.6 Baseplate gp33 (Listeria bacteriophage P100) BLASTP 243 4.00E-63
38 39087 40139 + 1,053 350 39.6 5.2 Structural protein gp34 (Listeria bacteriophage P100) BLASTP 409 1.00E-112 Uncharacterized homolog of RPSBLAST 0.00003
39 40158 41558 + 1,401 466 53.1 4.7 gp35 (Listeria bacteriophage P100) BLASTP 322 3.00E-86     phage Mu protein gp47
40 41664 42209 + 546 181 20.3 5.9 Structural protein gp36 (Listeria bacteriophage P100) BLASTP 184 2.00E-45
41 42224 45688 + 3,465 1154 128.8 4.9 Adsorption- associated tail protein gp37 (Listeria bacteriophage P100) BLASTP 1464 0 Sialidases (neuraminidases) SuperFamily 0.0037
42 45765 45971 + 207 68 7.8 5.7 Putative transposase (Wolinella succinogenes DSM 1740) BLASTP 32.7 5.4
43 46226 47998 + 1,773 590 67.2 6.4 Helicase gp42 (Listeria bacteriophage P100) BLASTP 706 0 Helicase superfamily C-terminal domain RPSBLAST 2.00E-11
44 48026 49654 + 1,629 542 62.9 8.4 Transcriptional regulator gp43 (Listeria bacteriophage P100) BLASTP 311 7.00E-83 Predicted transcriptional regulator RPSBLAST 0.002
45 49687 51159 + 1,473 490 55.4 4.8 Helicase gp44 (Listeria bacteriophage P100) BLASTP 423 1.00E-116 DnaB helicase C terminal domain RPSBLAST 4.00E-13
46 51159 52214 + 1,056 351 39.8 5.5 Exonuclease gp45 (Listeria bacteriophage P100) BLASTP 259 1.00E-67 SbcD, DNA repair exonuclease RPSBLAST 2.00E-16
47 52330 54222 + 1,893 630 71.2 5.2 Exonuclease Putative exonuclease (Staphylococcus phage K) BLASTP 374 6.00E-102 SbcCD and other Mre11/Rad50 (MR) complexes RPSBLAST 6.00E-09
48 54231 54896 + 666 221 25.9 4.9 ORF065 (Staphylococcus phage Twort) BLASTP 49.7 1.00E-04
49 54897 55955 + 1,059 352 40.4 7.1 Primase gp49 (Listeria bacteriophage P100) BLASTP 336 1.00E-90 DnaG, DNA primase (bacterial type) RPSBLAST 3.00E-13
50 55972 56604 + 633 210 24.2 5.1
51 56630 57514 + 885 294 32.7 6.0 gp50 (Listeria bacteriophage P100) BLASTP 150 7.00E-35
52 57517 57747 + 231 76 9.0 9.0 dUTPase gp51 (Listeria bacteriophage P100) BLASTP 32.3 6.5 dUTPase RPSBLAST 0.0008
53 57749 58057 + 309 102 12.1 8.6
54 58044 58355 + 312 103 12.2 4.6 Phosphotransferase/anion transport protein Phosphotransferase/anion transport protein SuperFamily 0.014
55 58348 58719 + 372 123 14.3 6.2 gp53 (Listeria bacteriophage P100) BLASTP 46.2 4.00E-04
56 58739 59407 + 669 222 25.7 5.0 Resolvase Hypothetical protein KgORF78 (Staphylo-coccus phage K) BLASTP 172 8.00E-42 Holliday junction resolvase, archaeal type RPSBLAST 0.001
57 59409 59714 + 306 101 11.3 5.4
58 59715 60194 + 480 159 18.5 6.3 Hypothetical protein phil2p15 (Staphylococcus aureus phage phi 12) BLASTP 73.6 3.00E-12
59 60287 61081 + 795 264 31.5 7.0 gp66 (Listeria bacteriophage P100) BLASTP 240 4.00E-62
60 61074 61385 + 312 103 11.9 9.7 Integration host factor Putative integration host factor (Staphylococcus phage K) BLASTP 72 7.00E-12 Integration host factor (IHF) and HU RPSBLAST 0.0002
61 61475 64546 + 3072 1023 119.0 6.1 DNA polymerase Putative DNA polymerase (Staphylo- coccus phage K) BLASTP 791 0 DNA polymerase family A RPSBLAST 7.00E-67
62 64649 65191 + 543 180 21.5 5.2 gp70 (Listeria bacteriophage P100) BLASTP 124 1.00E-27
63 65246 66535 + 1,290 429 48.2 4.9 gp71 (Listeria bacteriophage P100) BLASTP 125 6.00E-27
64 66620 67867 + 1,248 415 46.2 5.7 RecA gp72 (Listeria bacteriophage P100) BLASTP 434 5.00E-120 RecA RPSBLAST 7.00E-37
65 67921 68307 + 387 128 14.7 8.6 gp73 (Listeria bacteriophage P100) BLASTP 72.4 6.00E-12
66 68300 68914 + 615 204 23.7 6.2 Sigma factor gp74 (Listeria bacteriophage P100) BLASTP 173 4.00E-42
67 68975 69250 + 276 91 10.2 6.2 Holin Holin-like protein similar to ORF of bacteriophage BK5-T (Bifidobacterium longum NCC2705) BLASTP 43.5 0.002 Holin RPSBLAST 0.0003 2
68 69298 70260 + 963 320 35.2 4.5 Ig-like protein Ig-like, group 2 (Flavobacterium johnsoniae UW101) BLASTP 89 1.00E-17 Bacterial Ig-like domain (group 2) RPSBLAST 3.00E-07
69 70281 70724 + 444 147 16.6 4.5 Structural protein gp77 (Listeria bacteriophage P100) BLASTP 113 3.00E-24
70 70830 71135 + 306 101 12.1 5.2
71 71132 72085 + 954 317 35.9 5.7 gp79 (Listeria bacteriophage P100) BLASTP 138 4.00E-31
72 72139 73422 + 1,284 427 48.8 5.7 gp80 (Listeria bacteriophage P100) BLASTP 434 6.00E-120 Metallophospho-esterase HMMPfam 0.00096
73 73434 73808 + 375 124 14.2 9.6 gp81 (Listeria bacteriophage P100) BLASTP 43.5 0.003 3
74 73847 74467 + 621 206 23.2 5.7 gp82 (Listeria bacteriophage P100) BLASTP 113 4.00E-24
75 74467 75207 + 741 246 28.3 9.3 gp84 (Listeria bacteriophage P100) BLASTP 287 3.00E-76
76 75197 75703 + 507 168 19.1 10.3 gp85 (Listeria bacteriophage P100) BLASTP 94 2.00E-18
77 75717 76067 + 351 116 12.6 3.8
78 76092 76949 + 858 285 31.5 4.9 ORF036 (bacteriophage G1) BLASTP 55.8 2.00E-06
79 77054 77899 + 846 281 32.4 4.8 Nucleotidyltransferase SuperFamily 0.0016
80 77892 79520 + 1,629 542 62.7 7.0 ORF137 (Lactobacillus plantarum bacteriophage LP65) BLASTP 75.5 6.00E-12 YC53_LISIN_Q925W4 BlastProDom 7.00E-13
81 79839 80528 + 690 229 26.3 4.9 gp96 (Listeria bacteriophage P100) BLASTP 203 5.00E-51
82 80539 81006 + 468 155 18.2 4.8 gp97 (Listeria bacteriophage P100) BLASTP 90.9 2.00E-17
83 81106 83385 + 2280 759 86.5 4.6 Hypothetical protein KgORF105 (Staph- ylococcus phage K) BLASTP 71.6 1.00E-10
84 83445 83651 + 207 68 7.5 6.5 2 Y
85 83670 84176 + 507 168 18.6 5.3 2
86 84249 84353 + 105 34 3.9 3.4
87 84355 84525 + 171 56 6.7 4.5
88 84515 84787 + 273 90 10.3 4.4
89 84895 85146 + 252 83 9.6 9.2 Transcriptional regulator DNA-binding protein (Pseudomonas syringae pv. phaseolicola 1448A) BLASTP 46.6 3.00E-04 Helix-turn-helix XRE-family-like proteins RPSBLAST 4.00E-06
90 85159 85446 + 288 95 11.4 4.5
91 85449 86237 + 789 262 29.9 6.7
92 86320 87393 + 1,074 357 38.3 4.3 LPXTG_anchor HMMTigr 0.0047 1
93 87519 87821 + 303 100 11.5 10.3
94 87823 88125 + 303 100 11.7 4.0
95 88128 88421 + 294 97 10.6 9.5 2
96 88418 88597 + 180 59 6.8 4.1
97 88610 88963 + 354 117 13.7 4.1
98 88990 89367 + 378 125 14.3 8.9 2
99 89360 89590 + 231 76 8.8 5.2 2
100 89594 89968 + 375 124 14.2 4.7
101 89965 90192 + 228 75 8.8 4.7
102 90210 90398 + 189 62 7.3 4.9
103 90395 90994 + 600 199 23.0 5.0 Cytidine deaminase Cytidine deaminase RPSBLAST 0.0006
104 91036 91878 + 843 280 32.2 4.8
105 91891 92520 + 630 209 23.9 5.0
106 92598 92840 + 243 80 9.6 4.8
107 92858 92998 + 141 46 5.3 9.0
108 93011 93205 + 195 64 7.6 5.2
109 93221 93862 + 642 213 24.2 5.3
110 93874 94317 + 444 147 16.6 5.2
111 94454 94900 + 447 148 17.0 4.2
112 95256 95555 + 300 99 11.2 8.4
113 95633 95923 + 291 96 11.8 5.3
114 96006 96329 + 324 107 12.3 4.9
115 96426 96566 + 141 46 5.5 10.1
116 96625 96918 + 294 97 11.5 6.9
117 97025 97231 + 207 68 7.6 9.2 2
118 97313 97555 + 243 80 9.4 4.2
119 97632 97877 + 246 81 9.4 7.8 ORF160 (Lactobacillus plantarum bacterio-phage LP65) BLASTP 64.3 1.00E-09
120 97944 98180 + 237 78 9.2 3.8
121 98231 98437 + 207 68 7.9 6.3
122 98459 98626 + 168 55 6.8 10.1
123 98623 98712 + 90 29 3.3 9.8 1
124 98770 99066 + 297 98 11.3 8.9
125 99132 99362 + 231 76 9.0 4.9
126 99481 99912 + 432 143 16.7 4.3
127 99996 100346 + 351 116 12.8 5.4
128 100378 100803 + 426 141 16.4 3.9
129 100797 101009 + 213 70 8.3 7.9
130 101038 101229 + 192 63 7.4 4.6
131 101242 101601 + 360 119 14.0 10.0
132 101606 101707 + 102 33 3.8 8.1 1
133 102621 102875 + 255 84 10.0 4.2
134 102964 103230 + 267 88 9.8 4.8
135 103328 103495 + 168 55 6.1 8.7
136 103580 103756 + 177 58 6.7 4.4
137 103838 104011 + 174 57 6.7 4.3
138 104087 104311 + 225 74 8.4 4.1
139 104414 104722 + 309 102 11.8 4.9
140 104790 105101 + 312 103 11.8 9.2
141 105193 105351 + 159 52 5.9 4.9
142 105485 105724 + 240 79 9.0 4.2
143 105805 106050 + 246 81 9.1 5.7
144 106171 106866 + 696 231 27.0 4.0 Myosin heavy chain RPSBLAST 0.006
145 106973 107221 + 249 82 9.8 4.6
146 107292 107486 + 195 64 7.4 4.9
147 107512 107691 + 180 59 6.9 9.6
148 108021 108281 − 261 86 9.8 8.7
149 108278 108550 − 273 90 9.9 5.4 2
150 108555 108896 − 342 113 13.0 5.2
151 108929 109240 − 312 103 11.3 5.0 3
152 109233 109496 − 264 87 9.9 4.7 2
153 109497 109802 − 306 101 11.9 6.5
154 110021 110212 − 192 63 7.0 6.5
155 110256 110480 − 225 74 8.3 3.8
156 110494 110898 − 405 134 15.4 5.0
157 111288 111485 − 198 65 7.5 4.6
158 111485 112000 − 516 171 19.9 4.9 Protein gp51 (Listeria monocytogenes strain 4b H7858) BLASTP 58.2 2.00E-07 DUF1642 HMMPfam 6.10E-09
159 112013 112261 − 249 82 9.3 5.1
160 112274 112798 − 525 174 20.4 5.3 Hypothetical protein EfaeDRAFT_2183 (Enterococcus faecium DO) BLASTP 72 9.00E-12 DUF1642 HMMPfam 2.70E-30
161 112799 113191 − 393 130 15.7 5.0 Hypothetical protein EF1441 (Enterococcus faecalis V583) BLASTP 109 4.00E-23
162 113184 113543 − 360 119 14.0 4.5 Hypothetical protein EF1441 (Enterococcus faecalis V583) BLASTP 42.7 0.005
163 113547 113717 − 171 56 6.4 3.8 Hypothetical protein Sdys1_01002815 (Shigella dysenteriae 1012) BLASTP 42.7 0.005
164 113705 114247 − 543 180 20.8 6.4 Hypothetical protein EF2118 (Enterococcus faecalis V583) BLASTP 97.8 2.00E-19
165 114250 114714 − 465 154 18.2 8.7 Hypothetical protein EF0326 (Enterococcus faecalis V583) BLASTP 65.5 6.00E-10
166 114711 114815 − 105 34 4.1 4.3
167 114928 115293 − 366 121 13.9 5.5
168 115290 115469 − 180 59 6.8 3.4
169 115483 116001 − 519 172 20.0 9.3 gp158 (Listeria bacteriophage P100) BLASTP 117 2.00E−26 Conserved hypothetical protein CHP02464 TIGRFAM 3.40E−58
COG3236 RPSBLAST 2.00E−26
170 116002 116475 − 474 157 18.1 6.4
171 116472 117179 − 708 235 26.2 5.3
172 117208 117648 − 441 146 16.9 5.0
173 117698 117886 − 189 62 7.6 5.1
174 117883 118344 − 462 153 16.9 3.9 ABC transporter ProfileScan
175 118358 118534 − 177 58 6.7 3.8
176 118531 118728 − 198 65 7.8 4.8
177 118725 119162 − 438 145 17.5 9.7 1
178 119255 119995 − 741 246 28.0 4.9 Serine threonine-protein phosphatase gp135 (Listeria bacteriophage P100) BLASTP 148 2.00E-34 Serum/threonine-specific protein phosphatase and bis(5-nucleosyl)-tetraphosphatase FPrintScan 1.10E−05
179 119992 120471 − 480 159 18.1 9.4 4
180 120475 120726 − 252 83 10.3 9.8
181 120723 121265 − 543 180 20.8 8.0 Phosphoesterase Phosphoesterase (putative) (Lactobacillus reuteri JCM 1112) BLASTP 120 3.00E−26 Phosphoesterase/ phosphohydrolase RPSBLAST 4.00E−14
182 121345 121695 − 351 116 13.7 6.4 2
183 121692 121808 − 117 38 4.2 3.8
184 121822 122034 − 213 70 8.0 4.5
185 122034 122114 − 81 26 2.8 5.6 1
186 122115 122528 − 414 137 16.0 4.9
187 122533 122916 − 384 127 14.6 4.5
188 122985 123875 − 891 296 34.4 5.1
189 123877 124065 − 189 62 7.5 5.6
190 124156 124329 − 174 57 6.6 9.6 1 Y
191 124319 124663 − 345 114 13.4 5.0
192 124663 124917 − 255 84 9.6 4.8
193 124898 125152 − 255 84 10.0 5.1
194 125163 125498 − 336 111 12.9 4.5
195 125473 125910 − 438 145 17.2 5.3
196 125926 126387 − 462 153 17.5 4.9 Putative TNP-like transposable element [Oryza sativa (japonica cultivar-group)] BLASTP 36.2 0.45
197 126380 126634 − 255 84 9.5 8.9
198 126635 126853 − 219 72 8.2 4.5
199 126841 127272 − 432 143 16.7 4.5
200 127275 127505 − 231 76 8.7 3.9
201 127502 127933 − 432 143 16.5 4.8
202 127937 128089 − 153 50 5.7 9.8
203 128089 129036 − 948 315 36.0 5.7 Thymidylate synthase Thymidylate synthase (Bacillus mojavensis) BLASTP 199 1.00E−49 Thymidylate synthase RPSBLAST 3.00E−50
204 129006 129098 − 96 31 3.6 10.3
205 129089 129475 − 387 128 14.9 9.0 Hypothetical protein F116p23 (Pseudomonas aeruginosa phage F116) BLASTP 83.2 3.00E−15
206 129566 129715 − 150 49 5.7 8.0 1 Y
207 129715 130572 − 858 285 31.1 8.4 Antiproliferative protein Phage-like protein (Bacillus licheniformis ATCC 14580) BLASTP 177 4.00E−43 Band 7 domain of flotillin (reggie)-like protein RPSBLAST 1.00E−13 1 Y
208 130733 131716 − 984 327 37.3 4.6 Ribonucleotide reductase Unknown (bacteriophage SPBc2) BLASTP 253 8.00E−66 Ribonucleotide reductase R2/beta subunit (RNRR2) RPSBLAST 2.00E−63
209 131729 133879 − 2151 716 80.7 5.1 Ribonucleotide reductase Ribonucleotide-diphosphate reductase alpha subunit (Lactobacillus plantarum WCFS1) BLASTP 926 0 Class 1 ribonucleotide reductase (RNR) RPSBLAST 4.00E−119
210 133882 134124 − 243 80 9.0 5.7 Ribonucleotide reductase Ribonucleotide reductase, NrdH-redoxin (Lactobacillus sakei subsp. sakei 23K) BLASTP 68.6 9.00E−11 NrdH-redoxin (NrdH) family RPSBLAST 5.00E−12
211 134251 134517 − 267 88 10.0 4.9
212 134523 134828 − 306 101 11.9 9.0
213 134919 135149 − 231 76 8.9 9.1 Transcriptional regulator ORF187 (bacteriophage G1) BLASTP 59.3 5.00E−08 Helix-helix XRE-family-like proteins RPSBLAST 2.00E−08
214 135213 135437 − 225 74 8.4 9.8 gp166 (Listeria bacteriophage P100) BLASTP 32.7 4.9
215 135519 136610 − 1092 363 41.7 5.0 AAA protein family Hypothetical protein RBTH_07188 (Bacillus thuringiensis serovar israelensis) BLASTP 348 2.00E−94 ATPase family associated with various cellular activities (AAA) RPSBLAST 1.00E−07
tRNA-Met 136981 137054 − 74 Anticodon CAU
216 137370 137636 − 267 88 10.0 4.8
tRNA-Leu 137677 137761 − 85 Anticodon UAG
tRNA-Arg 138483 138556 − 74 Anticodon UCU
217 138698 138769 − 72 23 2.6 9.7
tRNA-Trp 139157 139228 − 72 23 Anticodon CCA
tRNA-Asp 139478 139553 − 76 Anticodon GUC
218 140224 140358 − 135 44 5.0 8.9
219 141028 141366 − 339 112 12.6 4.8 Structural protein gp171 (Listeria bacteriophage P100) BLASTP 37.7 0.14 STAT SUPERFAMILY 0.0072
220 141430 141774 − 345 114 13.3 4.9 gp172 (Listeria bacteriophage P100) BLASTP 92.4 6.00E−18 1
221 141809 142069 − 261 86 9.9 7.7 3
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a

Orientations of the ORFs in the φEF24C genome. + and −, rightward and leftward orientations, respectively, of ORFs in Fig. 2.

b

Sizes of ORFs, including stop codons, in nucleotides (nt).

c

Sizes of ORF products, in amino acids (aa).

d

Predicted molecular masses of ORF products.

e

Predicted pI.

f

TMH, transmembrane helix.

g

TMHHM, the prediction tool of transmembrane helices in proteins; the numbers of transmembrane helices are shown in the column.

h

SignalP, the prediction tool for signal peptide in proteins. “Y” indicates the presence of a signal peptide.

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 φEF24C 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 φEF24C, according to genome annotation and structural protein identification (Fig. 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, φEF24C 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 φEF24C 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 φEF24C within the SPO1-like phages.

The φEF24C 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 φEF24C, suggest that φEF24C is a member of the SPO1-like phage genus (28). Therefore, the phylogenic relationships of φEF24C 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 φEF24C is most closely related to Listeria phage P100 among these phages (Fig. 2A). Next, the gene organization of the φEF24C genome was also compared with that of Listeria phage P100. Genome synteny was observed, particularly on the predicted structural and DNA replication modules in φEF24C (ca. 70% of the genome) (Fig. 2B), whereas the genes on the other region of φEF24C (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, φEF24C and Listeria phage P100 were considered to have evolved divergently from the same virus origin (44).

FIG. 2.

FIG. 2.

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Phylogenetic analyses of φEF24C. (A) Phylogenetic tree based on MCPs. (B) Comparison of the gene order between Enterococcus phage φEF24C and Listeria phage P100. The ORF of φEF24C is connected to that of P100 by a black line where the E value from the in-house BLAST search is less than 0.1.

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. 3 shows the predicted ori and ter, which are the lowest (ca. 1-bp) and highest (108-kbp) regions, respectively. Although the process of φEF24C genome replication remains unknown, it seems to replicate in a manner slightly different from that of another large phage, T4 of E. coli. The φEF24C 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, φEF24C is assumed to multiply without such mutual interference between replication and transcription.

FIG. 3.

FIG. 3.

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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 indicated.

Host-adapted translation.

Host and phage codon usage comparisons and phage tRNA analysis implied efficient translation. The codon usages of φEF24C and E. faecalis V583 were mutually similar (Fig. 4A), which can indicate overall efficient translation in the host. Moreover, the codon usage of φEF24C exceeded that of the host on five predicted tRNAs whose genes were carried by the phage (Fig. 4B). Thus, we can infer that φEF24C supplies specific tRNAs on its own in case of tRNA deficiency.

FIG. 4.

FIG. 4.

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Optimization of φEF24C codon usage to its host codon usage and its possible tRNA function. (A) Comparison of codon usage between φEF24C and E. faecalis V583. (B) Location of tRNA genes (top), predicted secondary structures of tRNAs (middle), and phage and host codon usage comparisons on the tRNA anticodons (bottom). In the bar chart, codons for φEF24C tRNAs are indicated in boldface.

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).

φEF24C 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 φEF24C was then examined. After the inoculation of the minimum lethal bacterial dosage, HIMC or φEF24C at different MOI of 10, 1, 0.1, 0.01, 0.001, and 0.0001 was administered. Figure 5 shows the results of mouse rescue experiments using φEF24C. 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 φEF24C 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, φEF24C can efficiently rescue mice infected with both EF14 and VRE2 at an MOI of 0.01. Under these experimental conditions, the therapeutic efficacy of φEF24C did not seem to be affected by the sensitivity of the host to the phage. The φEF24C 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 φEF24C led to its significant therapeutic effectiveness in vivo.

FIG. 5.

FIG. 5.

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Experiment examining mouse rescue by phage φEF24C 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 mice, different concentrations of phage (MOI of 10, 1, 0.1, 0.01, 0.001, and 0.0001) or HIMC medium (control) were administered to the opposite side of the abdominal cavities of five mice. The survival rate was recorded after 7 days. Values significantly different from the control values (P < 0.05 and P < 0.01) are indicated by asterisks and double asterisks, respectively.

Eligibility of φEF24C as a potential therapeutic phage.

φEF24C is considered to be eligible as a therapeutic phage for the following reasons. First, as demonstrated in a previous study, φEF24C 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 φEF24C 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 φEF24C 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 φEF24C 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, φEF24C can be encompassed in the general therapeutic phage group. Fifth, accidental homologous recombination is also not likely in the use of φEF24C, because similarities between the φEF24C 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 φEF24C 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, φEF24C 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, φEF24C was investigated primarily as a therapeutic phage. Although further development of this phage and other methods is still necessary to address some remaining problems, φEF24C is a promising therapeutic phage against E. faecalis infections.

Supplementary Material

[Supplemental material]
supp_74_13_4149__index.html (918B, html)

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

▿

Published ahead of print on 2 May 2008.

†

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

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