Isolation and Characterisation of Bacteriophages with Activity against Invasive Non-Typhoidal Salmonella Causing Bloodstream Infection in Malawi

Abstract

In recent years, novel lineages of invasive non-typhoidal Salmonella (iNTS) serovars Typhimurium and Enteritidis have been identified in patients with bloodstream infection in Sub-Saharan Africa. Here, we isolated and characterised 32 phages capable of infecting S. Typhimurium and S. Enteritidis, from water sources in Malawi and the UK. The phages were classified in three major phylogenetic clusters that were geographically distributed. In terms of host range, Cluster 1 phages were able to infect all bacterial hosts tested, whereas Clusters 2 and 3 had a more restricted profile. Cluster 3 contained two sub-clusters, and 3.b contained the most novel isolates. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in Sub-Saharan Africa.

Keywords: Enteritidis; Malawi; Typhimurium; environmental phage.

Figure 1

Host range of isolated phages. Host range analysis was determined by spot assay on S. Enteritidis or S. Typhimurium host strains, with phages isolated in the UK (blue) or Malawi (red). Reference phages are displayed in black. Host range of biological replicates was recorded as defined lysis (dark green), turbid edge or smaller plaques (light green), and no lysis (grey). Examples of the different lysis observed are shown in Figure S1. The original Salmonella host used for phage isolation is indicated in purple. NT: not tested.

Figure 2

Phylogenetic and phenotypic characterisation of related isolated phages. Gene alignment of terL gene visualised in iTOL, with numbers indicating bootstraps on branches leading to clusters. The three clusters of related phages identified are presented in lilac (Cluster 1), green (Cluster 2), light blue (Cluster 3.a) and dark blue (Cluster 3.b). The host range for S. Typhimurium and S. Enteritidis is presented as a filled circle (lysis) or a clear circle (no lysis) from Figure 1, genome size (kb), and morphology observed by transmission electron microscopy (TEM) with scale bars of 100 nm. Inferred taxonomy is indicated next to TEM images. Phage names in blue were isolated from the UK and red from Malawi. Names in black indicate reference phages, and stars indicate representative phages used for TEM.

Figure 3

Comparative genomics of phages from Cluster 1. (A) Maximum-likelihood tree of phages in Cluster 1, unrooted and visualised in iTOL with numbers on branches indicating bootstraps. Scale bar represents number of single nucleotide polymorphisms. Phage names in blue were isolated from the UK and black font indicates reference phages. (B) Whole-genome comparison of phages in Cluster 1. The local similarity of each genome at the nucleotide level was calculated based on BLASTn high scoring pairs and plotted against a circular map of BPS6 represented as the inner circle. Blue circles represent reference genomes. Genome annotation is shown as arrows in the inner circle, colour-coded based on gene categories. Full annotation can be found in Table S2.

Figure 4

Comparative genomics of phages from Cluster 2. (A) Maximum-likelihood tree of phages in Cluster 2, unrooted and visualised in iTOL with numbers on branches indicating bootstraps. Scale bar represents number of single nucleotide polymorphisms. Phage names in blue were isolated from the UK and red from Malawi. Names in black font indicate reference phages. (B) Whole-genome comparison of phages in Cluster 2. The local similarity of each genome at the nucleotide level was calculated based on BLASTn high scoring pairs and plotted against a circular map of ER6 represented as the inner circle. Blue circles represent reference genomes. Genome annotation is shown as arrows in the inner circle, colour-coded based on gene categories. Full annotation can be found in Table S3.

Figure 5

Comparative genomics of phages from Cluster 3. (A) Maximum-likelihood tree of phages in Cluster 3, unrooted and visualised in iTOL with numbers on branches indicating bootstraps. Scale bar represents number of SNPs. Phage names in blue were isolated from the UK and red from Malawi. Names in black font indicate reference phages. (B) Whole-genome comparison of phages in Cluster 3.a. The local similarity of each phage genome at the nucleotide level was calculated based on BLASTn high scoring pairs and plotted against a circular map of ER22, represented as the inner circle. Blue circles represent reference genomes. Genome annotation is shown as arrows in the inner circle, colour-coded based on gene categories. Full annotation can be found in Table S4. (C) Whole-genome comparison of phages in Cluster 3.b. The local similarity of each genome was calculated based on BLASTn high scoring pairs and plotted against a circular map of ER20 represented as the inner circle. Blue circles represent reference genomes. Genome annotation is shown as arrows in the inner circle, colour-coded based on gene categories. Full annotation can be found in Table S5.