An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation

Abstract

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum β-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

Fig. 1

Geographical origin of S. Typhimurium showing azithromycin resistance in the Democratic Republic of the Congo (DRC). Red dots with numbers (n) of the included resistant strains in the respective surveillance sites (Kinshasa, Kisangani and Kisantu). The map of DRC was constructed using the maps package in R

Fig. 2

The population structure of S. Typhimurium ST313 with emergence of sublineage II.1. a Maximum likelihood phylogenetic tree based on the 81 genome sequences from this study and 153 African and 42 non-African publicly available S. Typhimurium strains (summarized in Supplementary Data 2). Sequencing reads were mapped to S. Typhimurium ST313 lineage II reference strain D23580. The tree is based on 62,884 chromosomal SNPs. Branches of S. Typhimurium ST313 sublineage II.1 are coloured in red. Metadata is visualized on the concentric rings in compliance to the legend, from the inside to outside; 1. Country of origin, 2. Year of isolation, 3. Presence of IncHI2 replicon, 4–8. Presence of multidrug resistance markers (MDR; bla, cat, sul, dfrA), mphA, blaSHV-2A, qnrS and gyrA antimicrobial resistance markers (AMR). Reference strains A130 (lineage I), D23580 (lineage II) and 10433_3 (sublineage II.1), as well as strain 5390_4, are indicated in blue. Branch lengths represent the number of SNPs as indicated in the scale bar. The tree is publicly available on MicroReact (https://microreact.org/project/xS5Xw6b3A). b Maximum likelihood phylogenetic tree of all S. Typhimurium ST313 lineage II strains included in this study, based on mapping to sublineage II.1 reference strain 10433_3 (this study). The tree is based on 1207 chromosomal SNPs. A collapsed branch is annotated with a grey triangle. The tree is rooted with S. Typhimurium strain DT2B, a European ST313 strain. Branches of S. Typhimurium ST313 sublineage II.1 are coloured in red. Metadata is visualized in lanes in compliance to the legend, from left to right; 1. Country of origin, 2. Year of isolation, 3. Presence of IncHI2 replicon, 4–8. Presence of multidrug resistance markers (MDR; bla, cat, sul, dfrA), mphA, blaSHV-2A, qnrS and gyrA AMR markers, 9. Location in the Democratic Republic of the Congo (DRC). Reference strain 10433_3 (sublineage II.1) is indicated in blue. Branch lengths are indicated and represent the number of SNPs. The map of DRC was constructed using the maps package in R

Fig. 3

Bayesian time-tree of S. Typhimurium ST313 lineage II Bayesian maximum clade credibility phylogeny of African S. Typhimurium ST313 lineage II and sublineage II.1 isolates. The time-tree is based on 1187 SNP differences detected across the core genome of 175 lineage II and II.1 sequenced isolates. The tree was visualized and coloured in Figtree v1.4.2, with the horizontal axis representing the years. A divergence date (median estimate and its respective 95% HPD) is indicated for the ST313 II.1 sublineage. Tree tips are colour coded according to their country of origin (coloured by legend at top)

Fig. 4

IncHI2 resistance plasmid pSTm-ST313-II.1. The genetic makeup of the pSTm-ST313-II.1 resistance plasmid from isolate 10433_3 is given. pSTm-ST313-II.1 is 274,695 nucleotides long and has 290 annotated genes. Annotations are shown on the outer circle and coloured by gene function: resistance genes (red), plasmid functions (blue), transposon related genes (green), metabolism functions (white) and hypothetical genes (grey). The inner five circles show pairwise similarity regions of 100% with previously reported IncHI2 plasmids in S. Typhimurium ST313 (pKST313 from Kenya (LN794248) and pSTm-A54650 from Malawi (LK056646)), a draft assembly from the IncHI2 plasmid of strain 5390_4 from Kisangani (this study) and R478 IncHI2 from Serratia marcescens isolated in the USA (BX664015)

Fig. 5

Genomic changes in S. Typhimurium ST313 sublineage II.1 versus ST313 lineage II. A schematic overview of the genomic differences between S. Typhimurium ST313 sublineage II.1 versus ST313 lineage II is shown. The genetic changes in S. Typhimurium sublineage II.1 include the acquisition of a resistance plasmid, pSTm-ST313-II.1, the loss of the flagellin fljB gene and the acquisition of single nucleotide polymorphisms (SNPs) in the chromosome sequence

Fig. 6

Signatures for host adaptation of S. Typhimurium ST313 Sublineage II.1. a Invasiveness index values for all S. Typhimurium sequences included in this study, grouped into lineage, as calculated by the method of Wheeler et al.. Summary of statistics on different clades: Non-ST313: median = 0.184, standard deviation (SD) = 0.093; Lineage I: median = 0.185, SD = 0.003; Lineage II: median = 0.205, SD = 0.005; Lineage II.1: median = 0.222, SD = 0.004. The groups were compared using a Mann Whitney U-test. Boxplot centre lines represent median values; box limits represent upper and lower quartiles; whiskers represent the 1.5 interquartile range and points represent the outliers. Source data are provided as a Source Data file. b Red, dry and rough (rdar) morphotype of S. Typhimurium ST313 lineage II strain D23580, S. Typhimurium ST313 lineage II.1 strain 10433_3 (this study) and a S. Typhimurium ST313 lineage II D23580 asmA knock-out strain (this study). Source data are provided as a Source Data file