Genetic and Structural Variation in the O-Antigen of Salmonella enterica Serovar Typhimurium Isolates Causing Bloodstream Infections in the Democratic Republic of the Congo

Abstract

Salmonella enterica serovar Typhimurium causes a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. No licensed vaccine is available, but O-antigen-based candidates are in development, as the O-antigen moiety of lipopolysaccharides is the principal target of protective immunity. The vaccines under development are designed based on isolates with O-antigen O-acetylated at position C-2 of abequose, giving the O:5 antigen. Serotyping data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without O:5. The importance and distribution of this loss of O:5 antigen in the population as well as the genetic mechanism responsible for the loss and chemical characteristics of the O-antigen are poorly understood. In this study, we Illumina whole-genome sequenced 354 Salmonella Typhimurium isolates from the DRC, which were isolated between 2002 and 2017. We used genomics and phylogenetics combined with chemical approaches (¹H nuclear magnetic resonance [NMR], high-performance anion-exchange chromatography with pulsed amperometric detection [HPAEC-PAD], high-performance liquid chromatography-PAD [HPLC-PAD], and HPLC-size exclusion chromatography [HPLC-SEC]) to characterize the O-antigen features within the bacterial population. We observed convergent evolution toward the loss of the O:5 epitope predominantly caused by recombination events in a single gene, the O-acetyltransferase gene oafA. In addition, we observe further O-antigen variations, including O-acetylation of the rhamnose residue, different levels of glucosylation, and the absence of O-antigen repeating units. Large recombination events underlying O-antigen variation were resolved using long-read MinION sequencing. Our study suggests evolutionary pressure toward O-antigen variants in a region where invasive disease by Salmonella Typhimurium is highly endemic. This needs to be taken into account when developing O-antigen-based vaccines, as it might impact the breadth of coverage in such regions. IMPORTANCE The bacterium Salmonella Typhimurium forms a devastating burden in sub-Saharan Africa by causing invasive bloodstream infections. Additionally, Salmonella Typhimurium presents high levels of antimicrobial resistance, jeopardizing treatment. No licensed vaccine is available, but candidates are in development, with lipopolysaccharides being the principal target of protective immunity. The vaccines under development are designed based on the O:5 antigen variant of bacterial lipopolysaccharides. Data on recent Salmonella Typhimurium clinical isolates from the Democratic Republic of the Congo (DRC), however, indicate increasing levels of isolates without this O:5 antigen. We studied this loss of O:5 antigen in the population at the genetic and chemical levels. We genome sequenced 354 isolates from the DRC and used advanced bioinformatics and chemical methods to characterize the lipopolysaccharide features within the bacterial population. Our results suggest evolutionary pressure toward O-antigen variants. This needs to be taken into account when developing vaccines, as it might impact vaccine coverage.

Keywords: Democratic Republic of the Congo; O-antigen; Salmonella Typhimurium; genomics; iNTS; surface structures; vaccines; whole-genome sequencing.

FIG 1

Phylogenetic tree of invasive Salmonella Typhimurium isolates from the Democratic Republic of the Congo (DRC) indicating the presence and absence of the O:5 antigen phenotype. The maximum likelihood phylogenetic tree is based on the 354 genome sequences from this study (summarized in Table S1 in the supplemental material). Sequencing reads were mapped to S. Typhimurium ST313 lineage II reference strain D23580 (50). The tree is based on 2,131 chromosomal SNPs. Metadata are visualized on the concentric rings according to the key as follows, from the inside to the outside; O:5 phenotype (ring 1); province of isolation (ring 2); and mutation in the oafA gene, rfb operon, and gtrC gene (rings 3 to 5). Branches are colored by the O:5 phenotype. Isolate DT2 is included as the outgroup to root the tree. Branch lengths represent the number of SNPs, as indicated by the scale bar.

FIG 2

Mutational events in the oafA gene associated with the loss of the O:5 phenotype. (A) Pairwise amino acid (AA) assembly of an intact OafA protein and the OafA protein sequence of an isolate showing a frameshift mutation linked to a 7-nucleotide ATTTTAT deletion in the oafA gene. (B) Pairwise comparison of the oafA gene sequences of D23580 (O:5⁺) and 4701/4 (O:5⁻) presenting an insertion of an IS4 family transposase into oafA, as resolved through MinION long-read sequencing.

FIG 3

Resolved rfb recombinations underlying OAg loss using MinION sequencing. The genome sequences of isolates 11480/3, 3832/3, 6088/3, and 7123/11 are pairwise compared to that of isolate D23580 (GenBank accession number FN424405). Homologous regions between both sequences are colored according to the scale bar. Transposase genes are shown in red. Genes of S. Typhimurium D23580 are annotated with the S. Typhimurium LT homologs (GenBank accession number AE006468.2).

FIG 4

Diverse O-acetylation patterns. (A) ¹H NMR spectra of OAgs extracted from 4 isolates representative of the variability observed. From top to bottom, the spectra are shown from an isolate that (i) lacks OAg repeats, (ii) is O-acetylated (OAc) on both rhamnose (Rha) and abequose (Abe), (iii) is O-acetylated on Rha only, or (iv) is O-acetylated on Abe only. (B) Resolved grtC2 recombination underlying the loss of O-acetylation of rhamnose in isolate 18034/3 compared to isolate D23580. Homologous regions between both sequences are colored according to the scale bar.

FIG 5

Binding capacity and bactericidal activity of commercial anti-OAg antibodies. (A) FACS analysis of S. Typhimurium isolates using a rabbit polyclonal or a mouse monoclonal antibody. The mean fluorescence intensities (MFIs) of 10,000 acquired events are reported. (B) Serum bactericidal activity of commercial antibodies against the selected S. Typhimurium strains. IC₅₀ values, corresponding to 50% bacterial growth inhibition, are reported.

FIG 6

S. Typhimurium O-antigen repeating unit. The observed modifications are highlighted in red: O-acetylation on Abe or Rha residues and glucosylation.