Pathogen genomics and phage-based solutions for accurately identifying and controlling Salmonella pathogens

Abstract

Salmonella is a food-borne pathogen often linked to poultry sources, causing gastrointestinal infections in humans, with the numbers of multidrug resistant (MDR) isolates increasing globally. To gain insight into the genomic diversity of common serovars and their potential contribution to disease, we characterized antimicrobial resistance genes, and virulence factors encoded in 88 UK and 55 Thai isolates from poultry; the presence of virulence genes was detected through an extensive virulence determinants database compiled in this study. Long-read sequencing of three MDR isolates, each from a different serovar, was used to explore the links between virulence and resistance. To augment current control methods, we determined the sensitivity of isolates to 22 previously characterized Salmonella bacteriophages. Of the 17 serovars included, Salmonella Typhimurium and its monophasic variants were the most common, followed by S. Enteritidis, S. Mbandaka, and S. Virchow. Phylogenetic analysis of Typhumurium and monophasic variants showed poultry isolates were generally distinct from pigs. Resistance to sulfamethoxazole and ciprofloxacin was highest in isolates from the UK and Thailand, respectively, with 14-15% of all isolates being MDR. We noted that >90% of MDR isolates were likely to carry virulence genes as diverse as the srjF, lpfD, fhuA, and stc operons. Long-read sequencing revealed the presence of global epidemic MDR clones in our dataset, indicating they are possibly widespread in poultry. The clones included MDR ST198 S. Kentucky, harboring a Salmonella Genomic Island-1 (SGI)-K, European ST34 S. 1,4,[5],12:i:-, harboring SGI-4 and mercury-resistance genes, and a S. 1,4,12:i:- isolate from the Spanish clone harboring an MDR-plasmid. Testing of all isolates against a panel of bacteriophages showed variable sensitivity to phages, with STW-77 found to be the most effective. STW-77 lysed 37.76% of the isolates, including serovars important for human clinical infections: S. Enteritidis (80.95%), S. Typhimurium (66.67%), S. 1,4,[5],12:i:- (83.3%), and S. 1,4,12: i:- (71.43%). Therefore, our study revealed that combining genomics and phage sensitivity assays is promising for accurately identifying and providing biocontrols for Salmonella to prevent its dissemination in poultry flocks and through the food chain to cause infections in humans.

Keywords: Salmonella; antimicrobial resistance; bacteriophages; genomics; serovar; virulence genes.

Figure 1

Maximum likelihood tree of S. Typhimurium, S. Typhimurium variant Copenhagen, S. 1,4,[5],12:i:-, and S. 1,4,12:i:- isolates based on SNPs in the core-genome. The tree was constructed in RaxML-NG using 17,263 single nucleotide polymorphisms (SNP). The isolate names have been colored based on their country, source, and database of origin. Dark blue corresponds to sequences from the APHA isolate collection belonging to the UK poultry included in this study (n = 21); dark green represents archived APHA UK pig isolates (n = 14); dark orange represents isolates from the study collection belonging to Thai poultry (n = 13); light blue represents Enterobase isolates belonging to UK poultry (n = 64); light green represents Enterobase isolates belonging to UK pigs (n = 96); light orange represents Enterobase isolates belonging to poultry from Thailand (n = 2); and light pink represents Enterobase isolates from pigs in Thailand (n = 57). Branches are colored in black for bootstrap values of >70 to provide confidence. The outer ring indicates the serovar of the isolates.

Figure 2

The percentage of isolates per country harboring AMR genes, grouped according to resistance to different AMR classes. Thai isolates are shown in yellow and UK isolates in blue. The total numbers of UK and Thai isolates were 88 and 55, respectively.

Figure 3

Percentages of UK and Thai MDR, non-MDR, and sensitive isolates harboring virulence determinants. MDR isolates from the UK (green), non-MDR isolates from the UK (grey), sensitive isolates from the UK (blue), MDR isolates from Thailand (pink), non-MDR isolates from Thailand (yellow), and sensitive isolates from Thailand (orange) have been included.

Figure 4

Comparison of SGI1-K (accession number AY463797.8; bottom line) with SGI1-K from BL700 (top line). Genes are represented by arrows pointing in the direction of transcription and different colors based on predicted function; transposase, recombinase, and resolvase are indicated by green arrows; AMR genes are indicated in red; mercury resistance genes are indicated in blue; and open reading frames of unknown function are indicated in grey. The grey shading connecting regions of nucleotides indicates sequence identity, ranging from 80 to 100% (see scale).

Figure 5

Genome comparison of SGI-1-K present in two MDR S. Kentucky isolates through the alignment of SGI-1-K present in BL700 and BL800 to the reference (accession number AY463797.8). Solid lines denote 100% sequence identity, with yellow indicating BL700 and blue indicating BL800; low or no sequence identity are shown in grey or as gaps. Genes flanking SGI-1-K in the reference (grey), AMR (red), mercury resistance (blue), transposon, resolvase, and integron genes (green) are shown.

Figure 6

Comparison of pBL661 with the reference plasmid CP018220. The outer ring shows the annotated genes, with 100% identity (solid brown), as well as low (light brown or grey) or no sequence identity (gaps).