A large outbreak of extensively drug-resistant (XDR) Salmonella enterica serotype Typhi infections is ongoing in Pakistan, predominantly in Sindh Province. Here, we report the sequencing and characterization of five XDR Salmonella Typhi isolates from the Punjab province of Pakistan that are closely related to the outbreak strain and carry the same IncY plasmid.
ABSTRACT
A large outbreak of extensively drug-resistant (XDR) Salmonella enterica serotype Typhi infections is ongoing in Pakistan, predominantly in Sindh Province. Here, we report the sequencing and characterization of five XDR Salmonella Typhi isolates from the Punjab province of Pakistan that are closely related to the outbreak strain and carry the same IncY plasmid.
ANNOUNCEMENT
An outbreak of extensively drug-resistant (XDR) Salmonella enterica serotype Typhi infections is ongoing in Pakistan (1, 2). Cases have been reported from November 2016 until the present, mostly from Sindh Province in the cities of Karachi and Hyderabad (1, 2). The outbreak strain has been genetically characterized previously, including identification of the resistance determinants and plasmids responsible for the XDR phenotype (2). Here, we report the sequencing and characterization of five XDR Salmonella Typhi isolates from patients in the province of Punjab in 2018.
Five isolates (Table 1) identified as XDR Salmonella Typhi at the National Institute of Health in Pakistan were subcultured on blood agar, stored in 40% glycerol Trypticase soy broth, and sent to the U.S. Centers for Disease Control and Prevention. DNA extraction was performed on cultures grown from single-colony frozen stocks on blood agar at 37°C overnight before sequencing on the Illumina MiSeq platform, as previously described (3). Default parameters were used for all software, unless otherwise specified. Lyve-SET v.1.1.4f (4) was used for high-quality single nucleotide polymorphism (hqSNP) analysis with an external reference (GenBank accession number LT882486), using the following options: –min_coverage, 20; –min_alt_frac, 0.95; –allowedFlanking, 5 bp; and –mask-phages. SMALT v.0.7.4 (5) was used for mapping, and SNPs were called with VarScan v.2.3.7 (6). De novo assemblies generated using shovill v.1.0.4 (https://github.com/tseemann/shovill) were analyzed for resistance determinants using the ResFinder database (90% identity, 50% cutoff) and the PointFinder scheme for Salmonella spp. implemented in staramr v.0.4.0. Plasmid replicons were identified using abricate v.0.8.10 and a database adapted from PlasmidFinder (90% identity, 60% cutoff). Antibiotic susceptibility testing was performed at the CDC National Antimicrobial Resistance Monitoring System (NARMS) laboratory, using CLSI interpretative criteria (7). Extracted DNA from one isolate (2018K-0756) from a 29-year-old male resident of Punjab was also sequenced by Pacific Biosciences single-molecule real-time (SMRT) technology. Reads were filtered using RS_Filter from SMRT Analysis v.2.3.0 (minsubreadlength, 1,000) and de novo assembled using FLYE v.2.4.2 (8, 9), followed by initial polishing using multiple rounds of Quiver v.2.3.0, and final polishing with Illumina reads using Pilon v.1.22. Annotation of the final assembled chromosome and plasmid was performed using Prokka v.1.13.3 and Galileo AMR (10) (http://galileoamr.arcbio.com/mara/). Additionally, assembly and annotation were performed through the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) upon the submission of reads to GenBank (11). Alignment of 2018K-0756 to the previously published Sindh Province outbreak strain 22420_1_10_Pak60006_2016 (GenBank accession number LT882486) was performed with Mauve in Geneious v.2019.1.1.
TABLE 1.
BioSample, GenBank, and SRA accession numbers for sequenced isolatesa
| Isolate | BioSample accession no. | GenBank accession no. | SRA accession no. | No. of total reads | Coverage (×) | Plasmid replicon(s) |
|---|---|---|---|---|---|---|
| 2018K-0753 | SAMN13019481 | AALWUW000000000 | SRR10267561 | 759,755 | 36.8 | IncY, ΔIncQ1 |
| 2018K-0754 | SAMN13019479 | AALZFY000000000 | SRR10267562 | 1,141,854 | 56.1 | IncY, ΔIncQ1 |
| 2018K-0755 | SAMN13019474 | AALZFX000000000 | SRR10279247 | 1,015,738 | 49.8 | IncY, ΔIncQ1 |
| 2018K-0756 | SAMN12774580 | CP044007 (chromosome), CP044008 (plasmid) | SRR10279247 | 1,085,924 | 53.3 | IncY |
| 2018K-0757 | SAMN13019471 | AALWUU000000000 | SRR10267564 | 1,106,063 | 53.8 | IncY, ΔIncQ1 |
All five isolates display the same phenotype (ACSSuCxCipNalCot [A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulfisoxazole; Cx, ceftriaxone; Cip, ciprofloxacin; Nal, nalidixic acid; Cot, trimethoprim-sulfamethoxazole]) and carry the same resistance determinants, namely, aph(3″)-Ib, aph(6)-Id, blaCTX-M-15, blaTEM-1B, catA1, dfrA7, qnrS1, sul1, sul2, and S83F(gyrA).
All isolates were within five pairwise hqSNPs of each other and within five SNPs of 22420_1_10_Pak60006_2016 according to Pathogen Detection (SNP cluster PDS000002867.370) (https://www.ncbi.nlm.nih.gov/pathogens/), confirming their relatedness to the outbreak strain. All had an S83F mutation in the quinolone resistance-determining region of gyrA and carried nine resistance genes [aph(3″)-Ib, aph(6)-Id, blaCTX-M-15, blaTEM-1B, catA1, dfrA7, qnrS1, sul1, and sul2]. The XDR phenotype conferred by these resistance determinants was confirmed for all five isolates, namely, resistance to ampicillin, ceftriaxone, chloramphenicol, ciprofloxacin, nalidixic acid, streptomycin, sulfisoxazole, and trimethoprim-sulfamethoxazole. All contained an IncY plasmid replicon and a partial IncQ1 replicon (529 bp of the 796-bp IncQ1 replicon sequence) within the resistance region, except 2018K-0756, which lacked IncQ1. 2018K-0756 carries catA1, dfrA7, and sul1 in a chromosomally integrated IS1-mediated composite transposon at the same location as in 22420_1_10_Pak60006_2016 but lacks a 7,857-bp internal region containing the partial IncQ1 replicon, aph(3″)-Ib, aph(6)-Id, blaTEM-1B, and sul2. The IncY plasmid from 2018K-0756 (p2018K-0756) carries aph(3″)-Ib, aph(6)-Id, blaCTX-M-15, blaTEM-1B, qnrS1, and sul2 and is 99.99% identical (5 SNPs) to that from 22420_1_10_Pak60006_2016 (p60006) (GenBank accession number LT906492).
Data availability.
The sequences discussed here have been deposited in GenBank and SRA under the accession and BioSample numbers listed in Table 1.
ACKNOWLEDGMENTS
This work was supported through the Centers for Disease Control and Prevention.
The findings and conclusions of this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
We acknowledge Gohar Zaman, Summiya Nizamuddin, and Muhammad Usman for the provision of isolates.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The sequences discussed here have been deposited in GenBank and SRA under the accession and BioSample numbers listed in Table 1.