Division of Electron Microscopy, ICMR-National Institute of Cholera & Enteric Diseases, P-33, C.I.T. Road, Scheme- XM, Beliaghata, Kolkata 700010, India

Enteric diseases such as Shigellosis are one of the leading causes of childhood morbidity and mortality worldwide. To cure bacterial infections, reckless use of antibiotics has gradually made them resistant to them. Antibiotic-resistant bacterial infections cause an impact on healthcare, food security, and different socio-economic condition in many developing and under-developed countries. The unmet needs of developing and underdeveloped countries are clear as the global antibiotic resistance epidemic has now reached a catastrophic level. The increased prevalence of multidrug-resistant Shigella species has revived the importance of bacteriophages as an alternative therapy to antibiotics. Because of the bactericidal properties of lytic bacteriophages, they are considered potential biocontrol and therapeutic agents. As a result, phage research involving isolation, characterization, and applications has recently significantly increased.

In our study, a Shigella bacteriophage Sfk20 was isolated from the environmental water of the diarrheal outbreak area of Kolkata and found to be a novel lytic phage with promising potential against the host bacteria Shigella flexneri 2a. The morphological study (using transmission electron microscopy and scanning electron microscopy) revealed that the bacteriophage had a prolate head 91.08 ± 4.92 nm (length), 62.34 ± 4.82 nm (width), and a long contractile tail 99.59 ± 4.92 nm (length) 18.66 ± 2.52 nm width and belongs to the myoviridae family. Phage Sfk20 showed infectivity against Sh. flexneri, Sh. sonnei, Sh. dysenteriae1 and two non-typhoidal Salmonella strains. The one-step growth curve study of Sfk20 revealed a latent period (20 mins) and large burst size (123 pfu per infected cell). Phage Sfk20 showed high stability at a 4-37ºC temperature range and at a pH range of 7-9. A study on phage stability conducted at different salinity revealed phage Sfk20 remains active within 0-5% salt concentration.  A study on understanding the nature of phage Sfk20 host receptor suggested that the outer membrane LPS of the Sh. flexneri 2a acts as a receptor for the phage Sfk20. The bacteriolytic activity of phage Sfk20 at various MOI studies revealed that at high MOI the growth of the host bacteria became restricted. The whole-genome sequencing study revealed that the bacteriophage Sfk20 contains a linear double-stranded genome that consists of 164878 bp, 35.62% GC content 241 ORF, and 10 tRNA. Genomic analysis also confirmed the presence of lytic genes and the absence of lysogeny, virulent, and toxic genes. The comparative genomic study and phylogenetic analysis suggested that Phage Sfk20 belongs to the T4-like virus genus family of Myoviridae and caudovirales order.  In proteomic analysis, the LC-MS/MS technique used here for detecting and identifying 40 Sfk20 phage proteins helped us to get an initial understanding of the structural landscape of phage Sfk20. Among the identified proteins, six structurally relevant proteins were selected and their structure was predicted using neural network and template-based modeling software. The structural characterization of the phage Sfk20 was done using single-particle cryo-electron microscopy and image processing. Reconstruction of T-4 like myoviridae phage Sfk20 was performed using EMAN 2.9 and Relion 3.1 software. The structural characterization of phage and its proteins further expands our knowledge of phage biology. The attachment of the phage particle to its host and subsequent intracellular development of phage and host cell lysis were visualized in a time-dependent experiment using thin-section transmission electron microscopy (FEI Tecnai 12 BioTwin) and scanning electron microscopy (FEI Quanta 200). This study further confirmed the lytic cycle of phage Sfk20. Bacteriophage Sfk20 showed antibiofilm activity against Shigella bacteria both alone and in combination with ampicillin. The synergistic effect of Sfk20 and ampicillin on the removal of biofilm was visualized by scanning electron microscopy. This study further revealed that the efficacy of the phage could be enhanced by utilizing the antimicrobial synergy between phages and antibiotics. All the results of this study imply that Sfk20 has the potential to be used as a biocontrol agent and phage therapy candidate.

Keywords: Bacteriophage; transmission electron microscopy; scanning electron microscopy; host-phage interaction; biofilm