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Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora.

The GenoTyphi genotyping scheme divides theSalmonella enterica serovarTyphi ("Typhi") population into 4 major lineages, and >75 different clades and subclades.The scheme was introduced in the paper"An extended genotyping framework for Salmonella enterica serovar Typhi, the cause of human typhoid", Wong et al, 2016, Nature Communications. Subsequent updates, including new genotypes and mutations conferring resistance to fluoroquinolones and azithromycin, are summarised in"Five years of GenoTyphi: updates to the global Salmonella Typhi genotyping framework", Dyson & Holt, 2021, Journal of Infectious Diseases. The original code that was made available for assigning GenoTyphi genotypes to Typhi genomes, implemented in Python, tookas input BAM or VCF files that the user has already generated by mapping Illumina reads to the reference genome for Typhi CT18. Here we describe a new kmer-based approach to assigning GenoTyphi genotypes to Typhi genomes, implemented in the Mykrobe open-source software platform and taking raw sequencing reads (FASTQ files) as input. This implementation also includes five newly defined genotypes, taking the total to n=87. We tested the new implementation on n=12,848 Typhi genomes, and found near-perfect concordance with the original mapping-based implementation. Code is available athttps://github.com/katholt/genotyphi(v2.0, doi: 10.5281/zenodo.7430538).

Typhoid fever epidemiology was investigated rigorously in Santiago, Chile during the 1980s, when Salmonella enterica serovar Typhi (S. Typhi) caused seasonal, hyperendemic disease. Targeted interventions reduced the annual typhoid incidence rates from 128-220 cases/105 population occurring between 1977-1984 to 5 from 1992 onwards. As such, Santiago represents a contemporary example of the epidemiologic transition of an industrialized city from amplified hyperendemic typhoid fever to a period when typhoid is no longer endemic. We used whole genome sequencing (WGS) and phylogenetic analysis to compare the genotypes of S. Typhi cultured from acute cases of typhoid fever occurring in Santiago during the hyperendemic period of the 1980s (n=74) versus the nonendemic 2010s (n=80) when typhoid fever was rare. The genotype distribution between “historical” (1980s) isolates and “modern” (2011-2016) isolates was similar, with genotypes 3.5 and 2 comprising the majority of isolations, and 73/80 (91.3%) of modern isolates matching a genotype detected in the 1980s. Additionally, phylogenomically ‘ancient’ genotypes 1.1 and 1.2.1, uncommon in the global collections, were also detected in both eras, with a notable rise amongst the modern isolates. Thus, genotypes of S. Typhi causing acute illness in the modern nonendemic era match the genotypes circulating during the hyperendemic 1980s. The persistence of historical genotypes may be explained by chronic typhoid carriers originally infected during or before the 1980s.Author summaryStudies of Salmonella Typhi (the cause of typhoid fever) rarely include isolates collected both before and after the interruption of hyperendemic transmission because this typically occurred decades before modern bacteria preservation methods. After interruption, it was assumed that sporadic cases and infrequent outbreaks were due to either chronic biliary carriers or importations, but this was difficult to characterize with low resolution bacterial typing methods. In Santiago, Chile, typhoid fever persisted at hyperendemic levels through the 1980s until organized control efforts in the 1980s and changes to wastewater policy in 1991 caused annual typhoid incidence to plummet.In this study, we used whole genome sequencing (WGS) to investigate whether recent sporadic cases occurring in Santiago in the 2010s were genomically similar to S. Typhi circulating in the 1980s, or dissimilar, possibly representing importations of S. Typhi from outside of Chile. We found concordance amongst S. Typhi genotypes between the 1980s and 2010s, and differences from genotypes circulating in Southeast Asia and Africa where typhoid remains hyperendemic. Our findings suggest that a proportion of modern, rare typhoid cases in Santiago are autochthonous, and that chronic carriers or another unknown reservoir likely contribute. Broadly, our findings corroborate the epidemiologic importance of long-term reservoirs of typhoid fever decades after typhoid elimination.

Additional file 1: Figure S1. Temporal distribution of typhoidal Salmonella serovars. Representation of typhoidal serovars, treatment status and genotype distribution as per the inset legends between adults and children over the study period. Figure S2. Phylogenetic tree of 4.3.1 (H58) S. Typhi isolates from Bengaluru and a global collection. This tree is made up of all 100 4.3.1 isolates from this study plus 1133 globally representative 4.3.1 isolates, and the rings are coloured according to the inset legend. The intermingling of isolates of children and adults is evident from the coloured branches and ring1. Branch lengths are indicative of the estimated number of substitutions rate per variable site; the tree was outgroup rooted using S. Paratyphi A strain AKU_12601. Figure S3. Radiological imaging of the right hip joint of a patient with septic arthritis due to S. Typhi (red arrows point to regions of interest as mentioned in the description of each panel). Panel A: CT image, transverse section, showing irregularity and erosions due to the infective process with no obvious breach in cortex. Panel B: MRI image, transverse section, showing involvement of the right iliac crest ,acetabular roof, head of the femur (cystic change), greater and lesser trochanter. Panel C: MRI image, T2W coronal view, shows the extent of bony involvement with effusion in the joint space. Panel D: MRI image, T2W, shows inflammation of the surrounding soft tissue including right iliacus muscle. Left hip joint appears normal.

Contaminated water is a major risk factor associated with the transmission of Salmonella enterica serovar Typhi (S. Typhi), the aetiological agent of human typhoid. However, little is known about how this pathogen adapts to living in the aqueous environment. We used transcriptome analysis (RNA-seq) and transposon mutagenesis (TraDIS) to characterize these adaptive changes and identify multiple genes that contribute to survival. Over half of the genes in the S. Typhi genome altered expression level within the first 24 h following transfer from broth culture to water, although relatively few did so in the first 30 min. Genes linked to central metabolism, stress associated with arrested proton motive force and respiratory chain factors changed expression levels. Additionally, motility and chemotaxis genes increased expression, consistent with a scavenging lifestyle. The viaB-associated gene tviC encoding a glcNAc epimerase that is required for Vi polysaccharide biosynthesis was, along with several other genes, shown to contribute to survival in water. Thus, we define regulatory adaptation operating in S. Typhi that facilitates survival in water.