143 3.2.11. Nucleotide accession numbers
The nucleotide sequence data of the RNA polymerase, DNA polymerase, large subunit terminase, tail fiber, muramidase, head portal protein and one hypothetical protein gene in the phage PAS-1 were deposited in the GenBank database under the accession numbers JF342689, JF342683, JF342686, JF342690, JF342687, JF342688 and JF342684, respectively.
144
A. salmonicida strains, and the highest EOP was detected in the multidrug-resistant AS09 strain (Table 3.1). However, PAS-1 was not able to lyse the other 10 bacterial species or the motile Aeromonas spp. used in this study.
3.3.2. One step growth and stability of Aeromonas phage PAS-1
One step growth of PAS-1 was examined to assess the growth pattern and the number of progeny phages released by the lysis of the indicator host strain, AS01.
The latent period and the average burst sizes were estimated to be approximately 40 min and 116.7 PFU/cell, respectively (Figure 3.2). The stability of PAS-1 was assessed by calculating PFU changes under different pH and temperature conditions. Almost no reduction in the PFUs was observed after 1 h incubation at pH 5.0, 7.0, 9.0 and 11.0, whereas considerable reductions were found at pH 3.0. In the thermo-stability tests, the phage was stable at 4°C and 20°C for 1 h but not at 40°C and 55°C (data not shown). These results suggest that low pH and high temperature conditions might affect the stability of PAS-1.
3.3.3. Genomic and proteomic characteristics of Aeromonas phage PAS-1
In general, Myoviridae phages are known to possess double strand (ds) DNA genomes (1). Likewise, the gDNA of PAS-1 was completely digested by DNase I but not by RNase A or Mung bean nuclease; thus, it was presumed to be ds DNA.
In addition, the gDNA was digested by SacII, Sau3AI, MspI, NcoI, HpaII, and its size was estimated by distinct fragments of NcoI as approximately 48 kb (data not shown). For the genome analysis of the PAS-1, a preliminary phage genome database was constructed by random shotgun sequencing. We were able to find 4
145
partial ORF sequences of phage-related proteins (RNA polymerase, DNA polymerase, large subunit terminase, tail fiber) and 3 complete ORF sequences (muramidase, head portal protein, hypothetical protein) by BLASTP searches in the GenBank database, and the amino acid size, identity and E-values are shown in Table 3.2. The predicted RNA polymerase, DNA polymerase, large subunit terminase and one hypothetical protein of PAS-1 showed similarity to those of enterobacteria phage phiEcoM-GJ1 [GenBank accession number NC_010106], and the predicted tail fiber and putative muramidase proteins were homologous with Aeromonas phage phiO18P [GenBank accession number NC_009542] which was classified as a P2-like Myoviridae phage.
To investigate the structural proteins of PAS-1, purified phage particles were subjected to SDS-PAGE and LC-MS/MS analysis. At least 14 distinct protein bands, with molecular masses ranging from 8 to 140 kDa were separated, and 9 major protein bands were subjected to LC-MS/MS for peptide sequencing. From these results, several partial peptide sequences of structural proteins, such as tail sheath (140 and 11 kDa), head morphogenesis (52 kDa), wac fibritin neck whiskers (48 kDa), major capsid (30 kDa) and prohead core (15 kDa) were obtained (Table 3.3).
3.3.4. Selection of a virulent A. salmonicida strain and lysis test
Among the 17 A. salmonicida strains used in this study, the ascV gene was detected in AS03 (subsp. achromogenes), AS05 (subsp. salmonicida) and ATCC 27013 (subsp. masoucida). Therefore, the lytic activity of PAS-1 was tested on early exponential-phase cultures of strain AS05 (Figure 3.3). When the cultures of
146
the AS05 strains were not infected by PAS-1 (MOI: 0), the OD600 value continued to increase during the incubations. In contrast, when they were infected with PAS-1, bacterial growths were apparently retarded at MOI 100 and 10,000 until 48 h after phage infection. However, at MOI of 0.01 and 1, the OD600 values started to increase gradually at 24 h after phage inoculation and nearly reached 0.5 at 48 h.
The presences of viable phage-resistant A. salmonicida was determined by plating the lysates of the 4 phage-inoculated MOI groups (MOI: 0.01, 1, 100 and 10,000), and several colonies were obtained from all the experimental groups regardless of their increase in OD600 values. The appearance ratios of phage-resistant colonies were inversely proportional to the MOI values of the samples, and the resistance was continuously observed during their successive cultures in TSA plates (date not shown).
3.3.5. Fate of Aeromonas phage PAS-1 in fish
For the evaluation of the fate of PAS-1 in rainbow trout, the purified phage was administrated by the IM method to fish (3.0 × 107 PFU/fish). The phage in fish kidneys was detected from 6 to 200 h pa, showing gradual reductions in its PFUs, but not at 240 and 360 h pa. Moreover, the phage was also detected in the aquarium waters of phage-administrated fish from 6 to 360 h pa, showing gradual reductions in its PFUs (Figure 3.4). In addition, significant neutralizing activities against PAS-1 were observed at 10 and 15 days pa (P < 0.01), and declined by 20, 25 and 30 days pa (Figure 3.5).
3.3.6. Protective effects of Aeromonas phage PAS-1 in fish furunculosis model
147
To demonstrate the therapeutic potential of PAS-1, the protective effects of the phage were evaluated using a triploid rainbow trout-furunculosis model. Prior to the experiments, an optimal challenge dose of A. salmonicida subsp. salmonicida AS05 strain was investigated by the IM injection of different numbers of bacterial cells, which ranged from 2.5 × 102 to 2.5 × 104 CFU/fish. The results indicated that 100% of the fish were killed within 2 days after challenge with 2.5 × 103 and 2.5 × 104 CFU/fish. In contrast, when 2.5 × 102 cells were injected, 70% of the fish were killed within 3 days, and the remainder died after 3 to 4 days (data not shown).
Therefore, the lowest dose (2.5 × 102 CFU/fish) was used throughout the experiments for lethal A. salmonicida infection.
To evaluate the protective effects of PAS-1, purified phage was administrated into fish at an MOI of 10,000 immediately after bacterial challenge, and the fish were observed for 14 days. All the fish in control groups, which were administrated SM buffer without PAS-1, died within 3 days (mean time to death: 2.5±0.3 days), showing furuncles in the bacterial injection site. However, we were able to find significant protective effects in phage-administrated groups; the fish in phage- administrated groups showed 26.7±2.9% survival rates from lethal A. salmonicida infection, and the mean time to death (3.3±0.6 days) was also increased as compared to those of control group (Table 3.4). The survived fish did not showed furuncles in the bacterial injection site and remained healthy until 14 days pa. The bacteria were re-isolated from all the dead fish, from phage-administrated groups as well as control groups, thus indicating that the mortalities and protective effects were caused by A. salmonicida and the phage PAS-1, respectively.
148