A genomic appraisal of invasive Salmonella Typhimurium and associated antibiotic resistance in sub-Saharan Africa

Abstract

Invasive non-typhoidal Salmonella (iNTS) disease manifesting as bloodstream infection with high mortality is responsible for a huge public health burden in sub-Saharan Africa. Salmonella enterica serovar Typhimurium (S. Typhimurium) is the main cause of iNTS disease in Africa. By analysing whole genome sequence data from 1303 S. Typhimurium isolates originating from 19 African countries and isolated between 1979 and 2017, here we show a thorough scaled appraisal of the population structure of iNTS disease caused by S. Typhimurium across many of Africa's most impacted countries. At least six invasive S. Typhimurium clades have already emerged, with ST313 lineage 2 or ST313-L2 driving the current pandemic. ST313-L2 likely emerged in the Democratic Republic of Congo around 1980 and further spread in the mid 1990s. We observed plasmid-borne as well as chromosomally encoded fluoroquinolone resistance underlying emergences of extensive-drug and pan-drug resistance. Our work provides an overview of the evolution of invasive S. Typhimurium disease, and can be exploited to target control measures.

Fig. 1. The distribution of invasive S. Typhimurium in Africa.

a Maximum likelihood phylogenetic tree of the 1419 S. Typhimurium isolates sequences from this study (summarised in Supplementary Data 1). Sequencing reads were mapped to S. Typhimurium ST313 reference strain D23580. The tree is based on 71521 chromosomal SNPs. Branches are coloured by the country of isolation. Invasive S. Typhimurium clades as identified in this study are annotated. Metadata is visualised on the concentric rings in compliance to the legend, from the inside to outside; (1) Country of origin, (2–5) presence of multidrug resistance markers (MDR; blaTEM, cat, dfrA, sul), (6) invasive S. Typhimurium clades. Branch lengths represent the number of SNPs as indicated in the scale bar. b Distribution of invasive S. Typhimurium clades per country for the studied isolates assigned to an invasive S. Typhimurium clade (1 = ST19-L1, 2 = ST19-L2, 3 = ST19-L3, 4 = ST19-L4, 5 = ST313-L1, 6 = ST313-L2). Bar charts show the number of isolates per clade coloured by the country of isolation. c Distribution of S. Typhimurium isolates over time assigned to an invasive S. Typhimurium clade (1 = ST19-L1, 2 = ST19-L2, 3 = ST19-L3, 4 = ST19-L4, 5 = ST313-L1, 6 = ST313-L2). Historical isolates (older than 1975) were excluded in this representation.

Fig. 2. The distribution of invasive S. Typhimurium clade ST313-L2 in Africa.

a Maximum likelihood phylogenetic tree of ST313-L2 isolates, based on mapping to reference strain D23580 (highlighted in blue in the phylogeny). The tree is based on 5380 chromosomal SNPs. The tree is rooted with S. Typhimurium strain DT2B, a European ST313 strain. Branches are coloured by the country of isolation. ST313-L2 subclades as identified in this study are coloured in the outher circle. Subclade 3 confers with the previously called ST313 sublineage II.1, and reference isolate 10433_3 is highlighted in blue. Metadata are visualised on the concentric rings in compliance to the legend, from the inside to outside; (1) Country of origin, (2–4) antimicrobial resistance markers associated with XDR and PDR outbreaks (ESBL, azithromycin resistance and the GyrA S83Y mutation associated with decreased susceptibility to ciprofloxacin). (5) ST313-L2 subclades. Branch lengths represent the number of SNPs as indicated in the scale bar. b Distribution ST313-L2 subclades per country. Bar charts show the number of isolates per subclade coloured by the country of isolation.

Fig. 3. Estimated spread of ST313-L2 across sub-Saharan Africa.

a Time-tree from BEAST showing phylogeographical reconstruction of ST313-L2. Estimated ages of nodes where transmission between African regions (East, Central and West) occurred are annotated with black circles and predicted years are reported with the 95% HPD interval. Triangles and squares indicate transmission events respectively within the region to a neighbouring country, and outside the continent (travel-associated). Branches and nodes are coloured according to the country with the highest posterior probability. Branch lengths represent the number of years as indicated in the scale bar. Isolate D23580 from Malawi is annotated in the three, as well as all ST313-L2 subclades (black circle at most recent common ancestor). Subclade 3 coincides with the previously identified ST313 sublineage II.1. Supplementary Fig. 8 presents the raw data of (a) with confidence intervals included. b A map showing the transmission events between the African regions (East, Central and West) and the respective predicted years of transmission. Additional cross-country transmission is observed in West Africa after the introduction in the region in 1994, annotated with arrows in the region.

Fig. 4. Continuous emergence of individual genetic events of quinolone resistance-determining regions (QRDR) SNP acquisitions in African invasive S. Typhimurium clades over time.

Events are plotted per year and coloured by the country of isolation. The years of the oldest isolate from a genetically related cluster of isolates presenting the same QRDR SNP are plotted. DRC Democratic Republic of Congo.

Fig. 5. Observed molecular mechanisms of extensive drug resistance (XDR) and pan-drug resistance (PDR) in invasive S. Typhimurium.

XDR in invasive S. Typhimurium presented as either caused by a plasmid carrying genetic markers for extended-spectrum beta-lactamase (ESBL) activity and azithromycin (AZI) resistance or by a plasmid carrying genetic markers for ESBL and fluoroquinolone (FQ) resistance. PDR in invasive S. Typhimurium presented as either caused by a plasmid carrying XDR in an isolate carrying a SNP in a quinolone resistance-determining region (QRDR) or by a plasmid carrying resistance markers for ESBL, AZI and FQ. The plasmid type is annotated per the observed mechanism. The specific plasmids per isolate are listed in Table 2. *Described as part of ref. .

Fig. 6. Related IncHI2 and IncI1 plasmid drive XDR and PDR in African invasive S. Typhimurium.

Pairwise similarity of IncHI2 and IncI1 plasmids of invasive S. Typhimurium isolates. a Pairwise comparison of IncHI2 plasmid pSTM-10530_17 with pSTM-3152_4, pSTM-5390_4, pSTM-7593_12, pSTM-ST313-II.1, pKST313, pSTm-A54650 and Illumina assembled contigs of pSTM-S15BD01306 and pSTM-AA00491. b Pairwise comparison of IncI1 plasmid pSTM-23060_3 with pSTM-22392_3, pSTM-22400_3 and Illumina assembled contigs of pSTM-8892, pSTM-K171 and pSTM2-AA00491.

Fig. 7. Antimicrobial resistance (AMR) hotspots for invasive S. Typhimurium in sub-Saharan Africa.

GPS locations are plotted for isolates showing IncHI2/IncI1 plasmid-mediated pan-drug resistance (PDR), extensively drug resistance (XDR) and extended-spectrum beta-lactamase (ESBL) activity as well as locations with isolates presenting chromosomal quinolone resistance-determining region (QRDR) SNPs. Locations where plasmid-driven PDR, XDR or ESBL isolates co-circulate with chromosomal QRDR SNPs are annotated on the map, presenting a risk for increased AMR through plasmid transfer.

Fig. 8. Phylogenetic relationship of invasive S. Typhimurium clades in the global population structure of S. Typhimurium.

a Maximum likelihood phylogenetic tree containing 10 isolates from each invasive S. Typhimurium clade (blue branches) and a representative collection of 131 global S. Typhimurium isolates (black branches). The tree was constructed using sequence variation (SNPs) in the core genome with reference to S. Typhimurium strain SL1344. The root was identified using S. Heidelberg (accession number NC_011083.1) as the outgroup (not shown). The heatmap to the right of the tree highlights the invasive S. Typhimurium clades and the 19 previously determined population structure groups of the 131 strains (Bawn et al.,). Isolate names are shown as branch labels where high-quality long-read reference sequences, clonal complexes described in literature are also shown. The bifurcations giving rise to S. Typhimurium clade-α and clade-β are annotated. b Box plots of the geometric mean Delta Bitscore (DBS: bitscore SL1344 (FQ312003)—test strain bitscore) of proteomes of the same representative collection of 131 S. Typhimurium isolates are shown. Centres represent the geometric mean DBS, minima and maxima of the boxes represent the first and third quartiles, vertical lines indicate first or second quartile + 1.5× the interquartile range, and outliers > or less than these values plotted as points. The number of genomes (biological replicates) analysed in each group are indicated within the figure (n = ). Boxes are coloured to indicate phylogroups in (a). Basally rooted population structure group 1 not shown.