Article Figures & Data
Figures
- FIG 1
Detection of azithromycin resistance and types of AcrB-717 mutation in Salmonella Typhi and Paratyphi A in Bangladesh. (A) Temporal distribution of 3043 Salmonella Typhi isolates identified in this study and by Hooda et al. (14). The total number of isolates tested is shown as the line plot from 2013 to 2018. The numbers of AzmR strains isolated each year is shown in the bar plot. (B) Azithromycin MICs among AcrB-WT and AcrB-R717Q and AcrB-R717QL mutant strains of Salmonella Typhi. (C) Temporal distribution of 587 Salmonella Paratyphi A isolates identified in this study and by Hooda et al. (14). The number of isolates is shown as the line plot from 2013 to 2018. The number of AzmR strains isolated each year is shown in the bar plot. (D) Azithromycin MICs among acrB wild-type and AcrB-R717Q and AcrB-R717QL mutant strains of Salmonella Paratyphi A. ****, P ≤ 0.0001; ***, P ≤ 0.001.
- FIG 2
Spontaneous emergence of AcrB-R717Q/L mutation in five different genotypes of Salmonella Typhi. (A) A whole-genome SNP tree containing 825 Salmonella Typhi strains highlights different azithromycin-resistant Salmonella Typhi genotypes. The inner circle shows the distribution of Salmonella Typhi genotypes, and the outer circle shows the mutations in AcrB-717. (B) Temporal distribution of the acrB mutation in different genotypes between 2013 and 2018.
- FIG 3
Maximum-likelihood tree based on SNP alignment of 141 Salmonella Paratyphi A strains with six azithromycin-resistant isolates. The inner circle depicts the country of isolation, the circle in the middle depicts the year of isolation, and the outer circle depicts the different mutations at AcrB-717.
- FIG 4
Bayesian estimation of the maximum clade credibility tree of genotype 4.3.1.1 azithromycin-resistant and related azithromycin-sensitive Salmonella Typhi isolates. The azithromycin-resistant clade containing the AcrB-R717Q mutation is shaded in gray, and the closely related AcrB-WT strains are outside the gray box. The Salmonella Typhi strain P-stx-12 (AcrB-WT) belonging to 4.3.1.1 was used as a reference strain. The AcrB-R717Q mutation is predicted to have emerged between 2010 and 2012 in Bangladesh. The travel-related Salmonella Typhi isolate from the United Kingdom is highlighted with a red arrow. The scale bar indicates the number of substitutions per variable site per year.
- FIG 5
Design and sensitivity and specificity of PCR-based mismatch amplification mutation assay (MAMA) for detecting AcrB mutations. (A) PCR cycling condition, primer sequences, and amplicon sizes generated by the AcrB MAMA and ParC control primers utilized in this study. (B) MAMA PCR primers for detecting mutation at the nucleotide position 2150 (amino acid 717) of the acrB gene. A single mismatch was incorporated at the conserved nucleotide (A to C; red, AcrB-MAMA-R) to increase allelic discrimination and chain termination in the presence of any mutation (G to A or T; red highlight). Published sequences of typhoidal Salmonella (e.g., Salmonella Typhi 5330M and Salmonella Paratyphi A 3144M [14]) with both Q (glutamine) and L (leucine) mutations were used to design the assay in silico. (C) Interpretation of the bands generated by AcrB MAMA and ParC control primers designed in this study. (D) Sensitivity and specificity of MAMA and WGS. (E) Sensitivity and specificity of MAMA and Etest compared to each other.
Supplemental Material
TABLE S1
Details of 113 Salmonella Typhi and Paratyphi A isolates detected in Bangladesh and used to validate the mismatch amplification mutation assay. Download Table S1, XLSX file, 0.01 MB.
Copyright © 2021 Sajib et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
MICs of the 92 Salmonella Typhi (A) and 21 Salmonella Paratyphi A (B) isolates tested for azithromycin resistance using E-strips. Further details of all tested isolates are provided in Table S1. Download FIG S1, PDF file, 0.4 MB.
Copyright © 2021 Sajib et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
Details of all 835 azithromycin-resistant and azithromycin-sensitive Salmonella Typhi strains used to build a maximum-likelihood phylogenetic tree. Download Table S2, XLSX file, 0.05 MB.
Copyright © 2021 Sajib et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S3
Details of all 141 azithromycin-resistant and azithromycin-sensitive Salmonella Paratyphi A strains used to build a maximum-likelihood phylogenetic tree. Download Table S3, XLSX file, 0.01 MB.
Copyright © 2021 Sajib et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
Gel electrophoresis image of the bands generated by AcrB MAMA and ParC control primers designed in this study. Wild-type strains produce a 397-bp band alongside the ParC internal control band of 758 bp. AcrB-R717Q/L mutants produce no band except the 758-bp ParC internal control. Download FIG S2, PDF file, 0.6 MB.
Copyright © 2021 Sajib et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Spatial distribution of azithromycin-resistant strains in the Dhaka district, Bangladesh. (A) Salmonella Typhi genotypes with AcrB-R717Q/L mutation in Dhaka (n = 42) colored by genotype. (B) Salmonella Typhi with AcrB-R717Q/L mutation in Dhaka from 2013 to 2018 (n = 42) colored by year of isolation. (C) Salmonella Paratyphi A with AcrB-R717Q/L mutation in Dhaka from 2013 to 2018 (n = 6) colored by year of isolation. Download FIG S3, PDF file, 1.5 MB.
Copyright © 2021 Sajib et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S4
Details of 39 azithromycin-resistant and azithromycin-sensitive Salmonella Typhi strains belonging to genotype 4.3.1.1 used for Bayesian analysis. Download Table S4, XLSX file, 0.01 MB.
Copyright © 2021 Sajib et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
