Multilocus sequence typing (MLST) sequence type 1903 (ST1903) is the most common ST of ceftriaxone-resistant Neisseria gonorrhoeae. Here, we report three completed genome sequences of MLST ST1903 N. gonorrhoeae isolates collected from patients at Faculty of Medicine Siriraj Hospital, a university hospital in Bangkok, Thailand, in 2009 to 2011.
ABSTRACT
Multilocus sequence typing (MLST) sequence type 1903 (ST1903) is the most common ST of ceftriaxone-resistant Neisseria gonorrhoeae. Here, we report three completed genome sequences of MLST ST1903 N. gonorrhoeae isolates collected from patients at Faculty of Medicine Siriraj Hospital, a university hospital in Bangkok, Thailand, in 2009 to 2011.
ANNOUNCEMENT
Neisseria gonorrhoeae causes gonorrhea, the second leading sexually transmitted infection worldwide. Antimicrobial-resistant N. gonorrhoeae is considered an urgent threat by the World Health Organization and the U.S. Centers for Disease Control and Prevention (1–5). Ceftriaxone-resistant strains have been identified worldwide, and sequence data are helpful in transmission analysis and tracking (6, 7). We sequenced three sequence type 1903 (ST1903) strains collected at the Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand, in 2009 to 2011.
This study was approved by the institutional review boards (IRBs) of the Faculty of Medicine Siriraj Hospital (FWA number 00002882, IORG number 0002101, IRB number 00005261, certificate of approval number Si479/2015). The National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NHHSTP), U.S. Centers for Disease Control and Prevention (CDC), also reviewed and approved this study protocol and determined it to be research that does not involve identifiable human subjects, using unlinked or anonymous data or specimens; therefore, CDC IRB approval was not required.
All isolates were from routine diagnoses and were resistant to penicillin, tetracycline, and fluoroquinolone (as determined by the disk diffusion method, using Clinical and Laboratory Standards Institute [CLSI] criteria) (8). The isolates possessed the blaTEM gene for β-lactam resistance (9). Bacteria from frozen stocks were subcultured on chocolate agar at 35°C with 5% CO2 and were reidentified by Gram staining, presumptive tests (oxidase and superoxol tests), and biochemical tests using an API NH kit (bioMérieux). MICs for ceftriaxone, cefixime, azithromycin, gentamicin, and tetracycline were determined by Etest (bioMérieux) and interpreted with CLSI standards, except for gentamicin (8). We purposely selected multilocus sequence typing (MLST) ST1903 for genome comparison with reported ceftriaxone-resistant strains (10, 11).
Genomic DNA extracted with the Gentra Puregene yeast/bacteria kit (Qiagen) was sequenced using Oxford Nanopore Technologies (ONT) and Illumina platforms. Library preparation for ONT sequencing followed the rapid barcoding DNA sequencing protocol with the SQK-RBK004 kit without DNA size selection, to preserve the plasmid DNA, and the libraries were sequenced using a single R9.4.1/FLO-MIN106 flow cell on a MinION Mk1B sequencer. ONT raw signals were base called and demultiplexed using Guppy v3.0.3. Quality control of ONT reads was undertaken using a modified pipeline described by Jenjaroenpun et al. (12). The ONT adapters were trimmed by Porechop v0.2.3 (https://github.com/rrwick/Porechop), with default parameters. Only reads with a mean quality score of >8 and a length of >1,000 bases were retained for de novo assembly using NanoFilt v2.5.0 (13) with the following parameters: q, 8; l, 1,000; s, sequencing.summary.txt. For Illumina reads, 100-bp paired-end reads were generated from TruSeq DNA PCR-free libraries. The genomic libraries of the three isolates were sequenced using the Illumina HiSeq platform. The quality filtering of Illumina reads was performed using Fastp v0.19.5 (14) with default parameters. Hybrid assemblies with ONT and Illumina data were performed using Unicycler v0.4.4 (15) with the default settings of genome error correction, circularization, and rotation. The genome sequences were checked for quality using QUAST v5.0.2 (16) with default settings. Genome sequences were submitted to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.9 (17, 18) for annotation, using default parameters.
This study emphasizes the usefulness of combining ONT and Illumina sequencing data for completing circular plasmids. All isolates contain three circular plasmids each. We detected plasmid 1 as a conjugative plasmid and plasmid 3 as a cryptic plasmid. From whole-genome sequencing, antimicrobial resistance determinants were defined (e.g., penA, ponA, blaTEM, tetM, gyrA, parC, parE, rpoB, mtrR, and folP), and the MLST findings indicated ST1903 and type IX nonmosaic penA9.001 with F504L and P551L mutations in their genomes (as determined using the Neisseria MLST database [https://pubmlst.org/neisseria/]). However, the isolates had different N. gonorrhoeae multiantigen sequence typing (NG-MAST) STs.
Data availability.
These sequences have been submitted to GenBank under BioProject accession number PRJNA563548. Accession numbers and assembly statistics are provided in Table 1.
TABLE 1.
Assembly metrics and accession numbers of three Neisseria gonorrhoeae strains isolated from clinical specimens from Faculty of Medicine Siriraj Hospital (Bangkok, Thailand) in 2009 to 2011
| Strain | BioSample accession no. | SRA accession no. | N50 (bp) | Total no. of reads | GenBank accession no. | Type of contig | Total length (bp) | Sequencing depth (×) for: |
GC content (%) | No. of ORFsa | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| ONT sequencing | Illumina sequencing | ||||||||||
| NG196 | SAMN12676038 | SRR10353549 (ONT) | 3,771 | 183,626 | CP043871 | Circular chromosome | 2,218,879 | 56 | 414 | 52.43 | 2,537 |
| CP043872 | Circular plasmid | 41,355 | 107 | 750 | 47.95 | 47 | |||||
| SRR10350533 (Illumina) | 100 | 11,336,944 | CP043873 | Circular plasmid | 5,600 | 28,086 | 24,328 | 39.64 | 6 | ||
| CP043874 | Circular plasmid | 4,207 | 15,131 | 11,153 | 51.60 | 10 | |||||
| NG251 | SAMN12676039 | SRR10353548 (ONT) | 4,362 | 337,281 | CP043816 | Circular chromosome | 2,218,831 | 203 | 583 | 52.43 | 2,527 |
| CP043817 | Circular plasmid | 41,356 | 493 | 1,211 | 47.95 | 47 | |||||
| SRR10350532 (Illumina) | 100 | 15,550,718 | CP043818 | Circular plasmid | 5,600 | 52,030 | 27,068 | 39.63 | 6 | ||
| CP043819 | Circular plasmid | 4,207 | 45,749 | 16,308 | 51.58 | 10 | |||||
| NG290 | SAMN12676040 | SRR10353547 (ONT) | 4,309 | 254,520 | CP043812 | Circular chromosome | 2,218,679 | 121 | 455 | 52.41 | 2,530 |
| CP043813 | Circular plasmid | 41,357 | 245 | 925 | 47.96 | 47 | |||||
| SRR10350531 (Illumina) | 100 | 12,749,420 | CP043814 | Circular plasmid | 5,600 | 42,434 | 28,727 | 39.64 | 6 | ||
| CP043815 | Circular plasmid | 4,207 | 26,307 | 17,090 | 51.58 | 10 | |||||
ORFs, open reading frames.
ACKNOWLEDGMENTS
This work was supported by the Siriraj Foundation for Graduate Students (grant D003474) and the Division of HIV/AIDS Prevention, NHHSTP, CDC.
We thank John R. Papp, Emily Weston, Cau D. Pham, and Sarika Pattanasin for technical advice and support.
The findings and conclusions of this manuscript are those of the authors and do not necessarily represent the official views of the CDC. Use of trade names is for identification only and does not imply endorsement by the CDC or by the U.S. Department of Health and Human Services.
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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
These sequences have been submitted to GenBank under BioProject accession number PRJNA563548. Accession numbers and assembly statistics are provided in Table 1.
TABLE 1.
Assembly metrics and accession numbers of three Neisseria gonorrhoeae strains isolated from clinical specimens from Faculty of Medicine Siriraj Hospital (Bangkok, Thailand) in 2009 to 2011
| Strain | BioSample accession no. | SRA accession no. | N50 (bp) | Total no. of reads | GenBank accession no. | Type of contig | Total length (bp) | Sequencing depth (×) for: |
GC content (%) | No. of ORFsa | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| ONT sequencing | Illumina sequencing | ||||||||||
| NG196 | SAMN12676038 | SRR10353549 (ONT) | 3,771 | 183,626 | CP043871 | Circular chromosome | 2,218,879 | 56 | 414 | 52.43 | 2,537 |
| CP043872 | Circular plasmid | 41,355 | 107 | 750 | 47.95 | 47 | |||||
| SRR10350533 (Illumina) | 100 | 11,336,944 | CP043873 | Circular plasmid | 5,600 | 28,086 | 24,328 | 39.64 | 6 | ||
| CP043874 | Circular plasmid | 4,207 | 15,131 | 11,153 | 51.60 | 10 | |||||
| NG251 | SAMN12676039 | SRR10353548 (ONT) | 4,362 | 337,281 | CP043816 | Circular chromosome | 2,218,831 | 203 | 583 | 52.43 | 2,527 |
| CP043817 | Circular plasmid | 41,356 | 493 | 1,211 | 47.95 | 47 | |||||
| SRR10350532 (Illumina) | 100 | 15,550,718 | CP043818 | Circular plasmid | 5,600 | 52,030 | 27,068 | 39.63 | 6 | ||
| CP043819 | Circular plasmid | 4,207 | 45,749 | 16,308 | 51.58 | 10 | |||||
| NG290 | SAMN12676040 | SRR10353547 (ONT) | 4,309 | 254,520 | CP043812 | Circular chromosome | 2,218,679 | 121 | 455 | 52.41 | 2,530 |
| CP043813 | Circular plasmid | 41,357 | 245 | 925 | 47.96 | 47 | |||||
| SRR10350531 (Illumina) | 100 | 12,749,420 | CP043814 | Circular plasmid | 5,600 | 42,434 | 28,727 | 39.64 | 6 | ||
| CP043815 | Circular plasmid | 4,207 | 26,307 | 17,090 | 51.58 | 10 | |||||
ORFs, open reading frames.