Genomic and Proteomic Characterizations of Sfin-1, a Novel Lytic Phage Infecting Multidrug-Resistant Shigella spp. and Escherichia coli C

Abstract

Shigellosis is a public health threat in developed as well as developing countries like "India." While antibiotic therapy is the mainstay of treatment for shigellosis, current emergence of multidrug-resistant strains of Shigella spp. has posed the problem more challenging. Lytic bacteriophages which destroy antibiotic resistant Shigella spp. have great potential in this context and hence their identification and detailed characterization is necessary. In this study we presented the isolation and a detailed characterization of a novel bacteriophage Sfin-1, which shows potent lytic activity against multidrug-resistant isolates of Shigella flexneri, Shigella dysenteriae, Shigella sonnei obtained from clinical specimens from shigellosis patients. It is also active against Escherichia coli C. The purified phage is lytic in nature, exhibited absorption within 5-10 min, a latent period of 5-20 min and burst size of ∼28 to ∼146 PFU/cell. The isolated phage shows stability in a broad pH range and survives an hour at 50°C. Genome sequencing and phylogenetic analyses showed that Sfin-1 is a novel bacteriophage, which is very closely related to T1-like phages (89.59% identity with Escherichia virus T1). In silico analysis indicates that Sfin-1 genome consists of double stranded linear DNA of 50,403 bp (GC content of 45.2%) encoding 82 potential coding sequences, several potential promoters and transcriptional terminators. Under electron microscopy, Sfin-1 shows morphology characteristics of the family Siphoviridae with an isometric head (61 nm) and a non-contractile tail (155 nm). This is most likely the first report of a lytic bacteriophage that is active against three of the most virulent multidrug-resistant Shigella species and therefore might have a potential role in phage therapy of patients infected with these organisms.

Keywords: LC-MS/MS; Shigella spp.; bacteriophage; genome sequencing; large terminase; phage therapy.

FIGURE 1

Shgella spp. specific phage Sfin-1 and its morphology. (A) Plaques of Sfin-1 in the lawn of Shigella flexneri 2a. The phage particles were prepared, negatively stained and examined by electron microscope as described in “Materials and Methods” section. (B,C) The electron micrograph presented as broad view of the phage in 100 and 200 nm scale, respectively.

FIGURE 2

Bacterial challenge test of phage Sfin-1 on different clinical isolates of Shigella spp. Clinically isolated species of (A) Shigella flexneri 2a, (B) Shigella dysenteriae 1, and (C) Shigella sonnei were grown (OD600 = 0.3) in 20 mL LB in presence of several antibiotics as described in “Materials and Methods” section, hervested by centrifugation, resuspended in 1 mL LB medium and infected with Sfin-1 at an MOI of 0.1, 0.01, and 0.001. After adsorption, the cultures were diluted 21 fold in LB and incubated for 5 h with shaking at 37°C. At different time intervals, viability of Shigella spp. was determined by spread plate method. As negative control Shigell spp. were grown in absence of Sfin-1 in presence of antibiotics. Two way ANOVA indicated significant difference between control and Sfin-1 infected sets (P < 0.0001, n = 3).

FIGURE 3

Stability of phage Sfin-1 in wide temperature and pH range. (A) Thermal stability of phage Sfin-1 at various temperatures as indicated. Sfin-1 phage particles (16 × 10¹²) were incubated at different temperatures in 1 mL and for each temperature the number of infectious phage particles was determined from 100 μL aliquots from various time points by plaque assay against S. flexineri 2a. Result was plotted as mean ± SD (n = 3). (B) pH stability of phage Sfin-1. In 1 mL of TM buffer having different pH Sfin-1 phage particles (14 × 10¹⁰) were incubated at 37°C for 1 h and the number of infectious phage particles from each sample was determined with 100 μL aliquots by plaque assay against S. flexineri 2a. Result was plotted as mean ± SD (n = 3).

FIGURE 4

One step growth curve of bacteriophage Sfin-1. Shigella flexneri 2a, Shigella dysenteriae 1, Shigella sonnei were infected with Sfin-1 at 37°C at an MOI of 0.01. After phage absorption, the cultures were diluted 10⁴-fold, incubated at 37°C and the titers in PFU per mL of Sfin-1 from the infected cultures at different time points were determined. Result was plotted as mean ± SD (n = 3). (A–C) Present one step growth curves of Sfin-1 in Shigella flexneri 2a, Shigella dysenteriae 1, and Shigella sonnei, respectively.

FIGURE 5

Genome organization of Sfin-1. (A) The Sfin-1 genome map was schematically presented. The predicted CDSs are indicated as arrows, the orientation of which shows the transcription. With different colors predicted molecular function for CDS of virion morphogenesis (green arrows), DNA metabolism and replication (red arrows), DNA packaging (yellow arrows), cell lysis (violet arrows), hypothetical proteins (blue arrows), putative promoters (pink) are denoted. (B) Comparative genomic maps of phage Sfin-1, pSf-2, Shfl1 was constructed using the Mauve progressive alignments to determine conserved sequence regions. This alignment resulted into two large synteny locally collinear blocks (LCBs) with 28,894 bp (red) and 16,173 bp (green), one small LCB with 5,334 bp (sky), indicating DNA regions which are homologous among the genomes. Graphs inside the blocks show high similarity between the genomes. There are some non-identical genome regions which are denoted with white color inside the blocks. Although there seems to be genomic rearrangement, the block sequence remains the same across the genomes of all phages.

FIGURE 6

Cumulative GC skew analysis of Sfin-1 genome sequence. The cumulative graph displays the global minimum and maximum. The window size of 1,000 bp and a step size of 100 bp were used to calculate the global minimum and maximum. The blue and red lines represent the GC-skew and the cumulative GC-skew, respectively. The putative origin of replication (9,401 nt) and the putative terminus location (34,201 nt) can be predicted from the minimum and maximum of a GC-skew.

FIGURE 7

Phylogenetic tree of terminase large subunit. Phages with known packaging mechanisms were only included. Bootstrap analysis was performed with 1,000 repetitions. The terminase large subunits were compared in the MEGA 7.0 version using neighbor-joining method.

FIGURE 8

Enzymatic analysis of Sfin-1 genomic DNA. Phage DNA was completely digested with BglII and MluI and the products were analyzed by 0.8% agarose gel electrophoresis, Lane M indicates the 1 kb Plus DNA Ladder. F and S indicate that the digests were heated to 80°C for 15 min and then cooled fast on ice or slow at room temperature, respectively.

FIGURE 9

Sfin-1 infections on proteinase K and periodate treated host. The effect of proteinase K and sodium periodate on adsorption of phage Sfin-1. Shigella flexneri 2a, Shigella dysenteriae 1, and Shigella sonnei cultures (OD600 = 0.3) were treated with proteinase K (250 mg/mL) or sodium periodate (200 mM NaIO₄) followed by Sfin-1 (MOI 0.0001) infection. Upon centrifugation, the phage titer in supernatant was determined as described in “Materials and Methods” section. Cells suspended in LB, cells incubated at 55°C in LB and cells in acetate buffer were used as control. The results are shown as residual PFU percentages. The phage titer in the control supernatant was set to 100%. Mean ± SD of three independent experiments are indicated. To determine the significance of the differences between group means, unpaired t-tests were performed between the controls and the tests. ^∗Significance level, i.e., P < 0.05, “ns” indicates non-significant. (A–C) Results of S. flexneri 2a, S. dysentariae1, and S. sonnei, respectively.