Vibrio Phage VMJ710 Can Prevent and Treat Disease Caused by Pathogenic MDR V. cholerae O1 in an Infant Mouse Model

Abstract

Cholera, a disease of antiquity, is still festering in developing countries that lack safe drinking water and sewage disposal. Vibrio cholerae, the causative agent of cholera, has developed multi-drug resistance to many antimicrobial agents. In aquatic habitats, phages are known to influence the occurrence and dispersion of pathogenic V. cholerae. We isolated Vibrio phage VMJ710 from a community sewage water sample of Manimajra, Chandigarh, in 2015 during an outbreak of cholera. It lysed 46% of multidrug-resistant V. cholerae O1 strains. It had significantly reduced the bacterial density within the first 4-6 h of treatment at the three multiplicity of infection (MOI 0.01, 0.1, and 1.0) values used. No bacterial resistance was observed against phage VMJ710 for 20 h in the time-kill assay. It is nearest to an ICP1 phage, i.e., Vibrio phage ICP1_2012 (MH310936.1), belonging to the class Caudoviricetes. ICP1 phages have been the dominant bacteriophages found in cholera patients' stools since 2001. Comparative genome analysis of phage VMJ710 and related phages indicated a high level of genetic conservation. The phage was stable over a wide range of temperatures and pH, which will be an advantage for applications in different environmental settings. The phage VMJ710 showed a reduction in biofilm mass growth, bacterial dispersal, and a clear disruption of bacterial biofilm structure. We further tested the phage VMJ710 for its potential therapeutic and prophylactic properties using infant BALB/c mice. Bacterial counts were reduced significantly when phages were administered before and after the challenge of orogastric inoculation with V. cholerae serotype O1. A comprehensive whole genome study revealed no indication of lysogenic genes, genes associated with possible virulence factors, or antibiotic resistance. Based on all these properties, phage VMJ710 can be a suitable candidate for oral phage administration and could be a viable method of combatting cholera infection caused by MDR V. cholerae pathogenic strains.

Keywords: antibiotic-resistance; cholera; genome; mice; phage.

Figure 1

Overview of the procedure used to estimate the efficacy of phage VMJ710 against V. cholerae infection using an infant mouse model.

Figure 2

(a) Plaque morphology of phage VMJ710. (b) Transmission electron microscopy phage of VMJ710 with a scale bar (red) of 100 nm.

Figure 3

Vibrio phage VMJ710 stability rate: (a) Thermal stability; (b) pH stability. Each data point represents the mean result of experiments performed in triplicates, while the error bars represent the standard deviation.

Figure 4

Circular genome map of phage VMJ710.

Figure 5

(a) Phylogenetic relationship between Vibrio phage VMJ710 (red sphere) and the other related phages. (b,c) are rectangular and circular phylogenetic trees generated using ViPTree [30]. The external and internal rings are colored according to the host bacterial group and virus family, respectively. Red star represents genome of Vibrio phage VMJ710.

Figure 6

Whole-genome sequence comparison between the genomes of phage VMJ710 and four selected Vibrio phages using Mauve 2.0: Annotated CDS (genes) are depicted as white rectangles, with reverse-strand genes, relocated downward. The height of the similarity profile indicates the degree of genomic sequence similarity between the matched regions. Three different colored local collinear blocks (LCBs) illustrate the homologous areas between VMJ710 and four related phages.

Figure 7

ORF divergence in the core genome. The average pairwise similarity of both DNA nucleotide sequence (yellow triangles and amino acid residue (red dots) alignments are used to organize all ICP1 core-genome ORFs.

Figure 8

Time–kill assay of phage VMJ710 against MDR V. cholerae strains at MOI 1.0, MOI 0.1, and MOI 1. Error bars indicate the standard deviation among triplicate experiments.

Figure 9

(a) Activity of phage VMJ710 at three different titers (10⁶, 10⁷, 10⁸ PFU) on preformed biofilm structure after 24 h incubation in terms of biofilm viable counts. Error bars indicate Mean ± SD. ** indicates a statistically significant difference at the p-value < 0.01. (b) SEM images at 5000× (magnification) (i) Control group (ii) Effect of phage VMJ710 (10⁸ PFU) on biofilm after 24 h of incubation. Scale bars (white)—5 µm.

Figure 10

(a) Vibrio phage number (PFU/g of tissue) retained in the mouse intestine without host bacteria. (b) V. cholerae (CFU/g of tissue) cells recovered when phages were administered before bacterial challenge as follows: Prophylaxis group A (6 h), prophylaxis group B (12 h), and prophylaxis group C (24 h). (c) Bacterial load (CFU/g) recovered from mice intestine tissue after treatment with phage at 12, 24, and 48 h post-infection. (d) Percent survival of mice in different groups. Error bars indicate Mean ± SD. The significant difference indicated by *p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 11

Histopathology of the mouse small intestine: (i) Negative control showing normal villus architecture. (ii) Infection-only control group; disruption of overlying mucosa and inflammatory cell infiltration (blue arrow). (iii) Intestinal architecture after phage treatment at 10⁸ PFU. Scale bar (resolution)-200 µm.