Isolation And Characterization Of Proteus Mirabilis And Escherichia Coli Bacteriophages
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Date
2015-10
Authors
WIRJON, IRA ARYANI
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Abstract
Malaysia has a huge untapped phage diversity with potential applications, including a phage therapy that treats antibiotic-resistant bacteria. In this study, three phages namely, pPM_01, pEC_02 and pEC_03 specific to Proteus mirabilis, Escherichia coli O157:H7 and Escherichia coli ATCC 13706 respectively, were successfully isolated from a raw sewage treatment facility in Batu Maung, Penang, Malaysia. Transmission electron microscopy (TEM) analysis suggested that phage pPM_01 belongs to the Siphoviridae family while phages pEC_02 and pEC_03 belong to the Myoviridae family. The isolates had narrow host range and double-stranded DNA as genetic material. Besides that, DNA restriction analysis and partial protein profile analysis showed that they were significantly different from one another as well as from the common phages T4, T7 and λ. Phages pPM_01 and pEC_02 were classified as lytic phages whereas pEC_03 was allegedly suspected to be a lysogenic phage. For the next characterization steps, only lytic phages pPM_01 and pEC_02 were selected. The highest infectivity of phage pPM_01 was recorded at pH 6, 37°C and 0.17M of salt concentration, meanwhile for phage pEC_02, the highest infectivity was recorded at pH 7, temperature range from 10°C to 45°C and 0.17M of salt concentration. Single-step growth curve analysis of phage pPM_01 revealed that the eclipse period, latent period and burst size were 20 min, 25 min, and 32 pfu/cell respectively. On the other hand, analysis on phage pEC_02 showed that the eclipse period, latent period and burst size were 25 min, 30 min and 18 pfu/cell, respectively. In the partial genomic study, DNA sequence analysis demonstrated that phage pEC_02 shared identity with Escherichia phage vB_EcoM_PhAPEC2. Whole genome sequence annotation and functional analysis of phage pPM_01 revealed that the phage is evolutionarily closely related to Enterobacter phage Enc34 with 67% identity of 14% query coverage. Low identity with the sequences in the database suggested phage
pPM_01 as a potentially new isolate. The whole genome sequence of phage pPM_01 was 58,546 bp in length and the overall guanine-cytosine (GC) base composition was 46.9%. Seventy open reading frames (ORFs) were annotated based on the NCBI database for their functional proteins. Twenty ORFs were designated functions according to their known homologous protein sequences; these annotated ORFs were mainly head and tail structural components and assembly proteins, as well as enzymes that were involved in DNA replication, repairing, packaging, and modification. The remaining ORFs were proteins assigned as hypothetical proteins, which indicate further genomic study of phage pPM_01 is required to elucidate more novel discoveries. The absence of virulent or toxic genes in the whole genome sequence indicated that the lytic phage pPM_01 is safe to proceed with further research involving phage therapy. In vitro testing showed that phage pPM_01 was highly lytic and virulent in reducing the growth of P. mirabilis in broth culture from ~0.51 to ~0.08 OD600 within 250 minutes.
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Keywords
Isolation And Characterization Of Proteus Mirabilis , And Escherichia Coli Bacteriophages