Isolation and characterisation of lytic bacteriophages infecting Shigella spp. / Sudhangshu Kumar Biswas

Sudhangshu Kumar , Biswas (2019) Isolation and characterisation of lytic bacteriophages infecting Shigella spp. / Sudhangshu Kumar Biswas. PhD thesis, Universiti Malaya.

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Abstract

Shigella infected bacillary dysentery is a leading cause of morbidity and mortality worldwide. The gradual emergence of multidrug-resistant Shigella spp. has triggered the search for alternatives to conventional antibiotics. Bacteriophage could be one such suitable alternative for its proven long-term safety profile as well as the rapid expansion of phage therapy research. Hence the general objective of this study was to isolate and characterize different Shigella strains from clinical and environmental samples Forty-nine Shigella strains [clinical (n=39), environmental (n=10)] were isolated and identified through biochemical test, serotyping and multiplex PCR amplification. Among the strains, one was Shigella dysenteriae, three were Shigella boydii, eight were Shigella sonnei and 37 were Shigella flexneri. Antibiotic profiling of these strains was performed using ten commercially available antibiotics through disc diffusion methods where 98% of the strains were drug-resistant and 59% were multidrug resistant. Ten bacteriophages were isolated and purified against these drug-resistant Shigella spp. through spot plating assay. The genomic content of the isolated phages was extracted through phenol-chloroform-isoamyl alcohol (25:24:1) extraction method and digested with DNase I and RNase to validate that all phages isolated, were DNA phage in nature. The transmission electron microscopy revealed that phage TB002 and TB004 belonged to the family Myoviridae, TB009, TB010 and TB013 belonged to the family Siphoviridae while TB006 and TB014 other belonged to the family Podoviridae. Phage TB007, TB008 and TB011 were tailless bacteriophages and belonged to either group D or E according to Bradley’s classification. The host range of the phages was determined through spot plating assay where two of the phages TB004 and TB002 showed wider host range and lysed 49 and 48 strains out of 49 strains respectively and demonstrated the coverage on all four species of Shigella genus. Therefore, the TB004 phage was selected for sequencing and safety assessment while the whole genome sequencing was performed through massively parallel sequencing technology on the Illumina platform. Sequencing assembly and subsequent analysis were done through different bioinformatics tools. Genomic studies confirmed that the TB004 was a phage of T4 genus under Myoviridae family consisting of 169,988 bp with 35.46% G+C content having 10 tRNA and 5 repeat sequences. Two hundred and seventy three genes were encoded through GeneMarkS of which the functions of 235 genes were annotated through Swiss-Prot where 126 genes had assigned functions and 109 were hypothetical proteins. No toxic or deleterious gene products were found among this annotation. The phylogenetic analyses of five selected proteins also indicated its probability of safety as phage TB004 appeared within the same branch of some other T4 phages and their safety were approved earlier. So, phage TB004 together with other phages isolated in this study could be considered as potential and promising candidates for phage therapy and phage biology research against drug-resistant Shigella spp. due to their extended host range cell lysis capacity and probable safety profile. The outcomes of this study could be considered as a good possibility of using bacteriophages against Shigella spp. in the near future.

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