Genomic analysis unveils genome degradation events and gene flux in the emergence and persistence of S. Paratyphi A lineages.

Acknowledgements: We thank Prof. Nicholas Grassly, Imperial College London, for assistance with study design and research proposal development. We gratefully acknowledge Dr. Arif M. Tanmoy, Dr. Senjuti Saha and Dr. Yogesh Hooda (CHRF, Dhaka, Bangladesh) for help with the genotyping analysis. We acknowledge Dr. Duncan Steele, Ms. Megan Carey & Dr. Supriya Kumar, Bill & Melinda Gates Foundation for their technical support throughout the study on behalf of SEFI consortium. We thank all the lab members involved in SEFI reference lab activities, especially Dr. Anushree Amladi, Ms. Baby Abirami S, Ms. Dhanabhagyam K, Ms. Beebi E, Ms. Suganya S, Ms. Udaya and Mr. Ayyanraj N, CMC Vellore implicated in phenotypic testing and stock culture maintenance. We would also like to thank all the members of SEFI consortium, Wellcome Trust Research Laboratory, CMC Vellore and core sequencing teams at the Wellcome Trust Sanger Institute for their contribution to genome sequencing. The authors thank Ms Catherine Trueman (Clinical Pharmacist, CMC Vellore) for helping with language editing.Paratyphoid fever caused by S. Paratyphi A is endemic in parts of South Asia and Southeast Asia. The proportion of enteric fever cases caused by S. Paratyphi A has substantially increased, yet only limited data is available on the population structure and genetic diversity of this serovar. We examined the phylogenetic distribution and evolutionary trajectory of S. Paratyphi A isolates collected as part of the Indian enteric fever surveillance study "Surveillance of Enteric Fever in India (SEFI)." In the study period (2017-2020), S. Paratyphi A comprised 17.6% (441/2503) of total enteric fever cases in India, with the isolates highly susceptible to all the major antibiotics used for treatment except fluoroquinolones. Phylogenetic analysis clustered the global S. Paratyphi A collection into seven lineages (A-G), and the present study isolates were distributed in lineages A, C and F. Our analysis highlights that the genome degradation events and gene acquisitions or losses are key molecular events in the evolution of new S. Paratyphi A lineages/sub-lineages. A total of 10 hypothetically disrupted coding sequences (HDCS) or pseudogenes-forming mutations possibly associated with the emergence of lineages were identified. The pan-genome analysis identified the insertion of P2/PSP3 phage and acquisition of IncX1 plasmid during the selection in 2.3.2/2.3.3 and 1.2.2 genotypes, respectively. We have identified six characteristic missense mutations associated with lipopolysaccharide (LPS) biosynthesis genes of S. Paratyphi A, however, these mutations confer only a low structural impact and possibly have minimal impact on vaccine effectiveness. Since S. Paratyphi A is human-restricted, high levels of genetic drift are not expected unless these bacteria transmit to naive hosts. However, public-health investigation and monitoring by means of genomic surveillance would be constantly needed to avoid S. Paratyphi A serovar becoming a public health threat similar to the S. Typhi of today

Lineage-defining missense mutations in <i>S</i>. Paratyphi A genomes.

Lineage-defining missense mutations in S. Paratyphi A genomes.</p

Antimicrobial susceptibility profile of <i>S</i>. Paratyphi A tested in the present study.

Antimicrobial susceptibility profile of S. Paratyphi A tested in the present study.</p

Distribution of <i>S</i>. Typhi and <i>S</i>. Paratyphi A isolates collected across the participating sites of the SEFI network.

Distribution of S. Typhi and S. Paratyphi A isolates collected across the participating sites of the SEFI network.</p

List of functional gene inactivation mutations identified between phylogenetic lineages.

List of functional gene inactivation mutations identified between phylogenetic lineages.</p

List of whole genome sequenced isolates collected from the participating sites of the SEFI network.

List of whole genome sequenced isolates collected from the participating sites of the SEFI network.</p

List of <i>S</i>. Paratyphi A genomes used in this study with accession IDs and metadata.

List of S. Paratyphi A genomes used in this study with accession IDs and metadata.</p

Rooted maximum likelihood phylogenetic tree of <i>rfb</i> loci of <i>S</i>. Paratyphi A isolates derived from the whole genome alignment by mapping against the reference genome of <i>S</i>. Paratyphi ATCC 9150 (Accession No: CP000026.1) using Snippy.

Lineages and genotypes are labelled as colour strips. Amino acid substitutions in the rfb loci are represented by heat maps. (TIF)</p

Time-calibrated Bayesian phylogeny phylogenetic tree showing the evolutionary events (HDCS forming mutations, insertions and deletions) that define the seven modern lineages and sub-lineages of <i>S</i>. Paratyphi A.

Major lineages/ genotypes were simplified as colored cartoon triangles using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Red arrow represents frameshift mutation/ gene degradation, Black arrow represent acquisition/ gene gain. Grey arrows demarcate nodes of interest, and the accompanying data indicate 95% HPD of node heights.</p

List of lineage-defining mutations in the O-antigen biosynthesis genes (<i>rfb</i> region) of <i>S</i>. Paratyphi A and their predicted impact on protein structures.

List of lineage-defining mutations in the O-antigen biosynthesis genes (rfb region) of S. Paratyphi A and their predicted impact on protein structures.</p