ABSTRACT
Here, we report the draft genome assemblies of 14 azithromycin-resistant Neisseria gonorrhoeae clinical isolates, representing the first such strains identified in Ireland. Among these isolates are the first reported highly resistant strains (MIC >256 mg/liter), which both belonged to the ST1580 sequence type.
GENOME ANNOUNCEMENT
Neisseria gonorrhoeae remains an urgent health care challenge in the face of emerging resistance to first-line therapeutic agents. Resistance to azithromycin (AZT), an antibiotic used in dual treatment regimes, represents a worrying development (1). We have previously described two clinical cases involving highly AZT-resistant N. gonorrhoeae isolates belonging to ST1580—the first such cases identified in Ireland (2, 3). These strains were identified during surveillance of drug-resistant N. gonorrhoeae and noted for their high-level resistance (MIC>256 mg/liter). Here, we report genome assemblies for these and 12 additional resistant strains detected in Ireland (MICs ranging from 1 to 16 mg/liter).
Sequencing libraries of N. gonorrhoeae genomic DNA were generated using the NexteraXT library preparation kit (Illumina, Eindhoven, the Netherlands) and sequenced on an Illumina MiSeq instrument at the TrinSeq sequencing lab (Trinity College Dublin) using MiSeq v3 reagents (300-base paired-end run). Genome sequence assembly, analysis and automated reporting were carried out using the Simplicity analysis platform (NSilico Lifescience Ltd., Ireland)—pipeline v1.4 (4). Genome assemblies are detailed in Table 1. Strain multilocus sequence types (MLST) were determined using pubMLST (http://pubmlst.org/neisseria/—database release 2017/4/10) (5). Sequencing reads were also aligned to the N. gonorrhoeae NCCP11945 genome (CP001050) using the Burrows-Wheeler short-read aligner (BWA, version 0.7.12-r1039) and interstrain genetic variations, including those present in antibiotic resistance genes, were resolved using SAMtools (version 0.1.19-96b5f2294a) (6, 7).
TABLE 1 .
Genomic sequence assembly overview
| Strain | Yr isolated | Specimen type | AZT MIC (mg/liter) | 23S rRNA mutationsa |
MLST | Assembly size (bp) | Fold coverage | % G+C | No. of contigs | N50 (bp) | Size of largest contig (bp) | GenBank accession no. | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A2059G | C2611T | ||||||||||||
| NGSJH7 | 2008 | Urethral swab | >256 | + (3/4) | − | ST1580 | 2,075,384 | 173× | 52.69 | 120 | 33,484 | 68,114 | NAGL00000000 |
| NGSJH11 | 2014 | Urethral swab | >256 | + | − | ST1580 | 2,079,908 | 116× | 52.76 | 145 | 22,826 | 139,128 | NAGP00000000 |
| NGSJH13 | 2008 | Urethral swab | 16 | − | − | Novel STb | 2,139,248 | 147× | 52.63 | 131 | 30,939 | 106,477 | NAGR00000000 |
| NGSJH16 | 2008 | Urethral swab | 16 | − | + | ST7822 | 2,120,180 | 166× | 52.54 | 125 | 35,303 | 108,373 | NAGT00000000 |
| NGSJH5 | 2011 | Urethral swab | 12 | − | + | ST9363 | 2,097,066 | 130× | 52.75 | 147 | 23,449 | 75,854 | NAGK00000000 |
| NGSJH9 | 2014 | Urethral swab | 8 | − | + (2/4) | ST1901 | 2,120,291 | 147× | 52.5 | 122 | 32,984 | 105,727 | NAGN00000000 |
| NGSJH12 | 2014 | Urethral swab | 8 | − | + | ST1587 | 2,131,824 | 114× | 52.62 | 121 | 26,278 | 82,135 | NAGQ00000000 |
| NGSJH8 | 2011 | Urethral swab | 2 | − | − | ST1579 | 2,090,734 | 158× | 52.69 | 135 | 23,447 | 105,772 | NAGM00000000 |
| NGSJH4 | 2012 | Urethral swab | 2 | − | + | ST9363 | 2,053,541 | 130× | 52.83 | 119 | 27,039 | 108,385 | NAGJ00000000 |
| NGSJH1 | 2009 | Urethral swab | 1 | − | − | ST1901 | 2,127,937 | 72× | 52.47 | 131 | 27,371 | 108,554 | NAGH00000000 |
| NGSJH2 | 2009 | Urethral swab | 1 | − | − | ST1582 | 2,108,844 | 186× | 52.65 | 105 | 35,856 | 175,104 | NAGI00000000 |
| NGSJH10 | 2014 | Urethral swab | 1 | − | − | ST9363 | 2,075,749 | 161× | 52.69 | 111 | 34,949 | 106,004 | NAGO00000000 |
| NGSJH14 | 2014 | Urethral swab | 1 | − | − | ST9363 | 2,069,975 | 173× | 52.73 | 113 | 35,282 | 139,973 | NAGS00000000 |
| NGSJH17 | 2014 | Urethral swab | 1 | − | − | ST7363 | 2,125,521 | 146× | 52.54 | 121 | 34,743 | 105,124 | NAGU00000000 |
+, supported by 100% of mutant calls; (3/4), supported by 75% of mutant calls; (2/4), supported by 50% of mutant calls; −, wild type.
Allelic profile: abcZ, 59; adk, 39; aroE, 67; fumC, 156; gdh, 150; pdhC, 153; pgm, 133.
Analysis of the two highly AZT-resistant strains (MIC>256 mg/liter) confirmed the presence the A2059G mutation in the 23s rRNA gene as previously reported (2). Three and four copies of this mutant allele were inferred in the AZT-resistant ST1580 strains from 2008 and 2014, respectively, through assessment of the percentage reads supporting mutant allele calls (ca. 75% versus 100% supporting calls in the 2008 versus 2014 isolate relative to the NCCP11945 23s rRNA sequence). In addition, 355 variant loci differed between the 2008 and 2014 ST1580 isolates. Interestingly, the majority of these variants (314/355) occurred within a 7,715 bp region known to contain phase-variable antigenic factors including pilin (opa) and exopolyphosphatase (ppx) genes in NCCP11945 (NGK_0745-0755) (8). Thus, this putative antigen-switching event differentiated the temporally isolated highly AZT-resistant ST1580 strains, which also differed from each other at 41 other sites broadly distributed throughout the genome. Among five non-ST1580 strains exhibiting lower AZT resistance levels (MIC 50 = 8 mg/liter), an alternative AZT resistance mutation in the 23s rRNA (C2611T) was observed, whereas strains with neither the A2059G nor C2611T mutations were less resistant again (n = 8, MIC 50 = 1 mg/liter). An exception to this trend observed in a single strain (NGSJH13, MIC = 16 mg/liter) lacking any known resistance mutations in the 23s rRNA gene. This strain harbored a distinguishing loss of function mutation in mtrR, which could potentially account for its increased resistance, as functional loss of the MtrR regulator has been linked to AZT resistance in this species (9). These data provide a foundation for future surveillance of resistant N. gonorrhoeae in Ireland and internationally and highlights mechanisms of resistance and antigenic variability among AZT-resistant strains.
Accession number(s).
This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession numbers given in Table 1.
ACKNOWLEDGMENTS
This work was supported by the Department of Clinical Microbiology, Trinity College Dublin. M.M.A. is the recipient of an Irish Research Council fellowship (EPSPD/2015/32).
Footnotes
Citation Mac Aogáin M, Fennelly N, Walsh A, Lynagh Y, Bekaert M, Lawlor B, Walsh P, Kelly B, Rogers TR, Crowley B. 2017. Fourteen draft genome sequences for the first reported cases of azithromycin-resistant Neisseria gonorrhoeae in Ireland. Genome Announc 5:e00403-17. https://doi.org/10.1128/genomeA.00403-17.
REFERENCES
- 1.Unemo M. 2015. Current and future antimicrobial treatment of gonorrhoea—the rapidly evolving Neisseria gonorrhoeae continues to challenge. BMC Infect Dis 15:364. doi: 10.1186/s12879-015-1029-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Lynagh Y, Mac Aogáin M, Walsh A, Rogers TR, Unemo M, Crowley B. 2015. Detailed characterization of the first high-level azithromycin-resistant Neisseria gonorrhoeae cases in Ireland. J Antimicrob Chemother 70:2411–2413. doi: 10.1093/jac/dkv106. [DOI] [PubMed] [Google Scholar]
- 3.Jacobsson S, Golparian D, Cole M, Spiteri G, Martin I, Bergheim T, Borrego MJ, Crowley B, Crucitti T, Van Dam AP, Hoffmann S, Jeverica S, Kohl P, Mlynarczyk-Bonikowska B, Pakarna G, Stary A, Stefanelli P, Pavlik P, Tzelepi E, Abad R, Harris SR, Unemo M. 2016. WGS analysis and molecular resistance mechanisms of azithromycin-resistant (MIC >2 mg/L) Neisseria gonorrhoeae isolates in Europe from 2009 to 2014. J Antimicrob Chemother 71:3109–3116. doi: 10.1093/jac/dkw279. [DOI] [PubMed] [Google Scholar]
- 4.Walsh P, Carroll J, Sleator RD. 2013. Accelerating in silico research with workflows: a lesson in simplicity. Comput Biol Med 43:2028–2035. doi: 10.1016/j.compbiomed.2013.09.011. [DOI] [PubMed] [Google Scholar]
- 5.Jolley KA, Maiden MC. 2010. BIGSdb: scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics 11:595. doi: 10.1186/1471-2105-11-595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup . 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079. doi: 10.1093/bioinformatics/btp352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. doi: 10.1093/bioinformatics/btp324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Zelewska MA, Pulijala M, Spencer-Smith R, Mahmood HA, Norman B, Churchward CP, Calder A, Snyder LAS. 2016. Phase variable DNA repeats in Neisseria gonorrhoeae influence transcription, translation, and protein sequence variation. Microbial Genomics 2:e000078. doi: 10.1099/mgen.0.000078. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Veal WL, Yellen A, Balthazar JT, Pan W, Spratt BG, Shafer WM. 1998. Loss-of-function mutations in the mtr efflux system of Neisseria gonorrhoeae. Microbiology 144:621–627. doi: 10.1099/00221287-144-3-621. [DOI] [PubMed] [Google Scholar]