We used a HiSeq WGS platform (Illumina, https://www.illumina.com) to generate 150 bp paired-end reads from Salmonella Typhi isolates collected during 2011–2017 by Chile’s National Typhoid Surveillance System. We assigned sequences to previously defined genotypes and identified 7 genotype 4.3.1 isolates (Appendix 1). Isolates were obtained from clinical cases in the Santiago metropolitan region: 1 in 2012, 5 in 2015, and 1 in 2016. For global context, we analyzed these 7 genomes and 2,386 publicly available sequences (Appendix 2 Table 1). Among publicly available sequences, 2,326 were genotype 4.3.1 and 60 were non–4.3.1 genotypes (Appendix 1 Table 2). We used the non–4.3.1 genotypes and a Salmonella Paratyphi A sequence as an outgroup for phylogenetic tree rooting. We produced clean and filtered SNP alignments (Appendix 1) and used these alignments to infer maximum likelihood phylogenies and specified a generalized time-reversible model and a Gamma distribution to model site-specific rate variation by using GTRGAMMA in RAxML version 8.2.9 (https://github.com/stamatak/standard-RAxML) and 100 bootstrap pseudoreplicates to assess branch support. SNP distances were calculated by using snp-dists (https://github.com/tseemann/snp-dists; Appendix 2). Raw Illumina reads were assembled by using either Velvet version 1.2 (European Bioinformatics Institute, https://www.ebi.ac.uk/~zerbino/velvet) or Unicycler version 0.4.7 (10). Assembled reads were input into Pathogenwatch (https://pathogen.watch) to detect nonsynonymous mutations in the quinolone-resistance determining region of gyrA and parC genes responsible for reduced fluoroquinolone susceptibility. We also used this approach to look for known antimicrobial resistance (AMR) genes. We further screened the sequences from Chile and close genetic relatives (Figure 2) to determine molecular determinants of AMR and known plasmid replicon genes (Appendix 1 Table).

Phylogenomic and SNP analyses confirmed 7 Salmonella Typhi genotype 4.3.1 isolates from Chile. Contact tracing implies that 4/5 isolates from 2015 were part of a localized outbreak. We found that the 7 isolates were members of 2 different sublineages, lineage I (4.3.1.1) and lineage II (4.3.1.2), suggesting multiple introductions into Chile. The 2 isolates of lineage I carried a single gyrA-S83F mutation predicted to confer reduced susceptibility to fluoroquinolones. The 5 lineage II isolates carried 3 quinolone-resistance determining region mutations, 2 in gyrA genes, S83F and D87N, and 1 in parC-S80I. Genotype 4.3.1 triple mutants were predicted to be resistant to fluoroquinolones, and isolates of this sublineage with identical mutations have been observed on the subcontinent of India and have been associated with treatment failure (2,11,12). None of the lineage II triple mutants in Chile carried detectable horizontally acquired AMR genes.

To provide a global contextualization of Salmonella Typhi genotype 4.3.1 in Chile, we analyzed the novel sequences alongside 2,326 existing sequences from 31 countries (Figure 1). The 4.3.1.2 triple mutants from Chile formed a closely related phylogenetic cluster (median distance 2 SNPs) with sequences that have the same antimicrobial susceptibility profile isolated from India during 2012–2014, indicating an introduction from South Asia (Figure 2, panel A).

The two 4.3.1.1 isolates from 2015 and 2016 in Chile were in distinct subclades of the tree and were separated by 19 SNPs, suggestive of 2 separate introductions. Of these, 1 introduction was closely related to a 2015 isolate from India (5 SNPs apart) (Figure 2, panel B) and the other was nested in a cluster of sequences from Southeast and South Asia and most closely related (median distance of 20 SNPs) to sequences from India and Bangladesh (Figure 2, panel C).

Our study confirmed Salmonella Typhi H58 genotype 4.3.1 in South America. Phylogenomic and SNP analyses indicate >3 separate genotype introductions into Chile; 5/7 isolates carried 3 distinct mutations, 2 in the gyrA gene, at D87N and S83F, and 1 in the parC gene at S80I, which are associated with ciprofloxacin resistance. For a high-income country with adequate surveillance, like Chile, the presence of fluoroquinolone-resistant genotype 4.3.1 Salmonella Typhi has no immediate implications. However, if this genotype is transferred to low- or middle-income countries in South America, it could have major consequences. Therefore, these data should be of concern to other countries in the region where potential typhoid fever transmission remains high and adequate sanitation might be lacking (5,6,10). Ciprofloxacin is a first-line drug for typhoid fever in much of Latin America, and fluoroquinolone-resistant genotype 4.3.1 would reduce its long-term efficacy.

Most diagnostic laboratories across South America are using pulsed-field gel electrophoresis to study Salmonella Typhi epidemiology (13), but efforts are underway to implement WGS for epidemiologic surveillance in several countries (14,15). However, WGS-based approaches for detecting genotype 4.3.1 and understanding trends in genotype population, circulating lineages, and AMR dynamics have not been adopted widely across the region. Our work highlights the need for a uniform WGS platform for global Salmonella Typhi monitoring and the need to elucidate the current epidemiology of typhoid fever in South America.

Additional information on methods of detection for multiple introductions of Salmonella Typhi H58 genotype 4.3.1, Chile.