Morphological of DRL-P1
Isolated phage was screened against P. aeruginosa through spot test. A clear zone over the bacteria lawn was observed due to the lytic activity of phage (Fig. 1a). This lytic phage was named ‘DRLP1’which was further identified and characterized. Further, ‘DRLP1’ produced clear small plaques of 2 mm in diameter of similar morphology indicating lytic activity against P. aeruginosa (Fig. 1b). Further, bacteriophage enrichment was performed by repeated plaque purification method and a stock of 109PFU/ml was prepared for further studies and characterization (Fig. 1c). Purified phages were examined under transmission electron microscopy (TEM) and classified according to the guidelines of the International Committee on Taxonomy of Viruses (ICTV). TEM images revealed the presence of phage belonging to the Family Myoviridae under the Order Caudovirales, signified by a neck, contractile tail, base plate, and the tail fibers (Fig 2. a, b, c, & d). DRLP1 had a capsid of 70 nm diameter and a contractile tail that was about 120 nm long and 20 nm in diameter.
Antibiotic sensitivity and host range
The antibiotic resistance pattern of the isolate is shown in Table (Supplementary data, Table.S1).Resistance was documented against Ceftazidime (CAZ), Nitrofurantoin (NIT), Nalidixic acid (NA), Ampicillin (AMP),Co-Trimoxazole (COT). However, P. aeruginosa isolates were found sensitive to certain antibiotics including Ciprofloxacin (CIP), Amikacin (AK), Amoxyclav (AMC), Cefotaxime (CTX), and Gentamicin (GEN), while intermediate sensitivity was documented against Netillin (NET), Tobramycin (TOB). Moreover, bacteriophages are highly specific, with the majority of them infecting only a single species of bacteria. DRL-P1 did not show lytic activity against other bacteria, including Escherichia coli(443), Vibrio cholera [Classical 01] (3904), Bacillus megaterium (428), Shigella flexneri (1457), P. aeruginosa (1688), Bacillus subtilis (1305), Salmonella typhimurium (1252), Salmonella typhimurium (1251), Streptococcus pyrogenes (442), Klebsiella pneumonia (8911) however, the clear lytic zone was observed on pseudomonas isolates (IS1-IS9) isolated from soil samples of Arunachal Pradesh, India. Their characterization is being done in the laboratory (Supplementary data, Table.S2).
Features of the DRLP1 genome
NGS-based sequencing resulted in the generation of a total of 1,295,948 raw reads (read length 150) amounting to 194.4 Mb bases. After sequence QC, a total of 1,221,536 reads (178.93 Mb bases) were used to assemble a terminally redundant genome of 66,243 nts having GC content of 54.9%, consisting of 22.75% A, 22.31% T, 27.52% G, and 27.40% C. The genome sequence was predicted to be ‘intact’ (completeness score 120) in PHASTER analysis. In Blastn search, the DRL-P1 phage genome sequence was found to be most similar to Pseudomonas phage sequences from the genus Pbunavirus (Order: Caudovirales; Family: Myoviridae), with top 10 hits namely being isolates- DL52 (KR054028), misfit (MT119367), zikora (MW557846), R26 (NC_048663), datas (NC_050143), Epa 14 (NC_050144), billy (MT133563), elmo(MT119364), kraken (KT372692), Jollyroger (KT372691), showing percent nucleotide identity (PNI) ranging from 95.55%-97.77% over 99% query coverage.
A total of 93 phage-hit ORFs were identified, of which 36 were functionally annotated based on homology with similar phage proteins, while 57 were annotated as phage hypothetical proteins. Predicted ORFs were found to encode proteins ranging from 31–1035 aa in length, the largest being the DNA polymerase ORF (Table 1). Identified ORFs included genetic regions, responsible for encoding proteins related to virion structure, genome replication, assembly & packaging, DNA synthesis & repair, regulation of gene expression, host identification & infection, host lysis, and recombination that are essential for the phage cycle. Among the 93 ORFs, 54 (58%) and 39 (42%) ORFs were encoded on each of the strands of the dsDNA, respectively. The strand with most of the ORFs was considered as the plus strand in further analyses. A genome map showing predicted ORFs (with definite phage-related proteins) is presented in (Fig. 3). Together, all the ORFs were encoded within 65,495 bps (from nts 634 to 66,128 nt), resulting in an extremely high coding density of 98.87%. Notably, the start codon of 25 ORFs (26.88%) overlapped with the stop codon of the previous gene, suggesting transcriptional interactions among these neighboring genes. No putative tRNA encoding genes were identified in the genome. A total of 83 promoter regions and 27 Rho-independent terminators across the genome sequence were identified (Supplementary data, Table. S3 & S4 ). No antimicrobial resistance-related gene was predicted in the genome.
Subsequently, phylogenetic trees were reconstructed for Terminase and DNA polymerase III genetic sequences with top 100 BLAST hit sequences (including RefSeq sequences). In the Terminase phylogeny (Fig. 4), DRL-P1 clustered with a Pbunavirus RefSeq (NC_028745, isolate 'DL60' from the UK) and an unclassified Pbunavirus (MW557846, isolate 'zikora', recently isolated from Nigeria). In the DNA polymerase III generated phylogeny (Fig. 5), DRL-P1 clustered with two unclassified Pbunaviruses (MW557846, isolate 'zikora' and MT119364, isolate 'elmo'). However, the DNA polymerase sequence of DRL-P1 was phylogenetically closer to RefSeq NC_050143 (isolate 'datas') and another isolate 'DL52' (KR054028), diverging from the phylogenetic relatedness of the Terminase genetic region with RefSeq NC_028745 (isolate 'DL60'). Therefore, a phylogeny was constructed with whole-genome sequences to resolve this conflict. In the complete genome phylogeny (Fig. 6), DRL-P1 showed the closest phylogenetic relatedness with 'zikora', and relationship with isolate 'datas', corroborating with the DNA polymerase genetic region phylogeny.
The relationship of the DRL-P1 became further complicated in the results of VIRIDIC analyses. When the analysis was performed only with 37 genus Pbunavirus RefSeq genomes, DRL-P1 was included in the species cluster 1 along with RefSeq NC_011810 (isolate ‘PB1’) with a PNI score of 95.34% (Supplementary data, Table S5). However, when analyzed against 100 top BLAST hits including 37 RefSeq and 63 other complete genome sequences, DRL-P1 was placed in a cluster (separately from isolate 'PB1') along with isolates DL52, zikora, elmo, and steven (MT119370), having PNI ranging from 96.0 to 97.5% % (Supplementary data, Table S6 & Table S7). In the VIRIDIC analysis of complete genomes, PNI of DRL-P1 was calculated to be 93.3 and 92.8 with isolates DL60 and datas, respectively, which were found to be most closely related in previous phylogenetic analysis of the Terminase and DNA Polymerase III genetic regions.
Results from the phylogenetic analyses and the VIRIDIC analysis suggested a possibility of horizontal gene transfer or recombination, which is better represented in NeighborNet (NN), as compared to phylogenetic trees. Therefore, a NN was reconstructed with RefSeq and DRL-P1 complete genome sequence (Fig. 7). Extensive reticulation at the base of the NN suggested frequent exchanges of sequences among the ancestral isolates in the evolution and emergence of present isolates. The NN represented the relation of DRL-P1 with various other isolates including datas (closest isolate), PB1, DL60, AB28 (NC_026600), supporting the divergence observed in clustering in the terminase and the DNA polymerase phylogenies. Subsequent analysis of recombination using RDP4 program detected with a high probability value, evidence of large fragment of sequences similar to the isolates datas, PB1, while smaller fragments of sequences from other isolates (Fig. 8 & Table-2). This suggested that the evolution of the DRL-P1 genome involves frequent genetic interaction with different Pbuna viruses.
Phage adsorption and growth kinetics
Effect of Calcium and magnesium ion on Adsorption rate:
Within 5 min approximately 90% of phages were adsorbed in the samples supplemented with MgCl2 and after 15 mins only 4% phages remained unabsorbed. Only 1 % percent of the phages were in free form whereas maximum adsorption was observed at around 20min without adding MgCl2. The study indicates that Mg2+ ions accelerate the phage adsorption by increasing phage infectivity hence resulting ineffective lysis of the host bacterium (Fig 9.a)
Single-step growth curve
A single-step growth curve was calculated for P. aeruginosa phage as shown in (Fig. 9b) The latent period was determined to be about 30 min which signifies the time interval between phage adsorption and the start of the first burst. The duration of the rise period was 40-50 min with a burst size of 100 PFU/infected cells during the experiment.
Stability of phage at different temperatures and pH condition
The temperature vs. phage stability was observed at six different temperatures viz. 4°C, 25°C, 37°C, 40°C, 50°C, 60°C, and 70°C. Results demonstrated that the purified P. aeruginosa phage was considerably stable at 4°C, 25°C, 37°C. Further, phage stability was also noted at 40°C. However, at temperatures above 40°C, stability was found to decrease significantly (Fig.10 a). A decrease in phage titer was noted at 50 and 60°C. Further, at 70°C only 14% phage survivability was documented.
After 18 h, the phage was stable at pH 6, 7, and 8 without any significant loss in the titer. However, beyond pH 10 and below pH 3 very little phage percentage was recorded. Approx. 70% phages were viable between pH 5 and 10. Also, no plaque formation was seen at pH 1, 2, 13, and 14. (Fig.10 b)
Decontamination of fomites through phage preparations
In the present work, we used glass coverslip and surgical blade to represent solid surface and surgical tool, respectively to demonstrate decontamination by application of phages. The ability of phages to decontaminate P. aeruginosa infection was determined according to Jensen et al.9. Reduction in the bacterial count was recorded to be 1.2 logs in glass coverslip and 1 log in surgical blade decontamination, respectively (Fig. 11a & 11b).
Phage action on bacteria:
Phage action on bacteria was observed through a change in OD at 600nm. Bacterial control (MOI:0) showing a sigmoid curve representing a continuous increase in optical density (OD) 600 values during the 8 h of incubation whereas, bacteria mixed with phages at different MOI: 1, 0.1, 0.01, and 0.001, indicates the reduction of bacteria with phage application (Fig.12). At different MOI bacterial growth increased up to 60 min then lysed by phage at an MOI of 1 to 0.001.
Stability of lyophilized phage and after encapsulation on alginate:
Lyophilization of bacteriophage stock (109PFU/ml) in both skim milk and sucrose resulted in a slight drop in the phage titer (108 PFU/ml). Once lyophilized, even after 12 months, the lyophilized sample retains its lytic activity without a further drop in the titer. Samples were reconstituted in 2 ml TM buffer and plaque assay was performed for PFU count. Similarly, phage was adsorbed over sodium alginate beads (5-6 mm), the lytic activity of encapsulated and non-encapsulated bacteriophage was tested against P. aeruginosa by placing a bead over the lawn of P. aeruginosa. The clear zone was reported with the adsorbed phage over the bead.