More Mutation Accumulation in Neisseria gonorrhoeae (NG) with Susceptibility than in NG with Decreased Susceptibility to Ceftriaxone
Introduction
Gonorrhea, caused by Neisseria gonorrhoeae (NG), is a significant public health concern. Ceftriaxone (CRO) was once a key first-line antimicrobial monotherapy, but its efficacy has been challenged as NG strains with decreased susceptibility to CRO (CRO-DS) have spread globally. This has led to changes in treatment strategies, such as the adoption of dual therapy (CRO with azithromycin). Understanding the genetic basis of these changes, especially in terms of mutation accumulation, is crucial.
Methods
Isolate Collection and Susceptibility Testing
NG isolates were collected from patients at sexually transmitted infection clinics in Guangzhou, China, between 2009 and 2013. Antimicrobial susceptibility was determined using the agar dilution method. Twenty-two CRO-DS NG isolates (MIC ≥ 0.25 mg/L) and 22 CRO-S NG isolates (MIC < 0.125 mg/L) were selected. The isolates in the two groups were matched as closely as possible by comparing their MICs for ciprofloxacin, spectinomycin, and azithromycin.
Whole-Genome Sequencing (WGS)
WGS was performed using the Illumina HiSeq 4000 platform. High-quality reads were aligned onto the publicly available reference genome (NG NCCP11945, NC_011035) with Burrows-Wheeler Aligner version 0.5.9-r16. Single-nucleotide variants were identified using Sequence Alignment Map tools (version 0.1.19-44428cd). Gene mutations attributed to CRO-DS, such as mtrR promoter 23 to 35 A deletion, mtrR Gly45, penA Ala501, penA GLy542, penA Pro551, porB1b Gly120, and porB1b Ala121, were identified from the WGS data. A neighbor-joining phylogenetic tree of single-nucleotide variants was generated by MEGAN7.
Results
WGS Data Quality and Mutation Analysis
After filtering low-quality bases and adapter sequences, the abundance of clean WGS reads showed no difference between CRO-S and CRO-DS NG (P > 0.05). However, the point mutations in CRO-S NG strain were significantly more than those in CRO-DS NG isolates. In terms of total mutations (6206.46 ± 776.50 vs. 5420.73 ± 770.68, P < 0.01), homozygous mutations (5694.32 ± 766.86 vs. 4968.59 ± 738.41, P < 0.01), and heterozygous mutations (512.14 ± 61.27 vs. 452.14 ± 69.85, P < 0.01), CRO-S NG had more.
Correlation with CRO MICs
A significant negative correlation was found between CRO MICs (range 0.004–0.500 mg/L) and the total number of point mutations (r = -0.4737, P = 0.0012), homozygous point mutations (r = -0.4631, P = 0.0015), or heterozygous point mutations (r = -0.3348, P = 0.0263).
Mutation Types and Distribution
Point mutation types such as A > G, G > A, C > T, and T > C had high frequency in both homozygous and heterozygous mutations. These types were significantly higher in CRO-S NG compared to CRO-DS NG. The circos plots also showed that CRO-S NG possessed more mutations than CRO-DS NG.
Specific Mutations and Their Distribution
Mutations like mtrR promoter 23 to 35 A deletion, penA (Ala501, GLy542, or Pro551), and porB1b (Gly120 or Ala121), which correlated with CRO-DS, were more frequently observed in CRO-DS NG. However, these mutations were also present in CRO-S NG, although the difference in some cases (except for mtrR Gly45) was not statistically significant.
Discussion
Implications of Mutation Accumulation
This study provides evidence that CRO-S NG has more point mutations (homozygous and/or heterozygous) than CRO-DS NG. One hypothesis is that CRO-S NG evolving under certain selection may need more mutations to confer resistance, while CRO-DS NG may have already acquired some resistance evolution with relatively fewer mutations. Previous studies have suggested a positive relationship between hypermutable bacteria and antibiotic resistance acquisition. Wistrand-Yuen et al. reported that bacteria evolving high-level resistance under lethal selection differ from those under sub-MIC selection. Lethal selection leads to few strong-effect resistance mutations, while sub-MIC selection generates many small-effect mutations that can combine to cause high-level resistance.
Future Directions
Due to the hypermutation and potential for resistance mutations in CRO-S NG, molecular surveillance of both CRO-S and CRO-DS NG should be enhanced in the future. This will help in better understanding the evolution of NG resistance and in developing more effective treatment strategies.
Conclusion
This research using WGS has shown that CRO-S NG has more mutation accumulation compared to CRO-DS NG. This finding has important implications for understanding the evolution of NG resistance and for guiding future surveillance and treatment efforts.
References
- Fifer H, Natarajan U, Jones L, Alexander S, Hughes G, Golparian D, et al. Failure of dual antimicrobial therapy in treatment of gonorrhea. N Engl J Med 2016;374:2504–2506. doi: 10.1056/NEJMc1512757.
- Blazquez J. Hypermutation as a factor contributing to the acquisition of antimicrobial resistance. Clin Infect Dis 2003;37:1201–1209. doi: 10.1086/378810.
- Wistrand-Yuen E, Knopp M, Hjort K, Koskiniemi S, Berg OG, Andersson DI. Evolution of high-level resistance during low-level antibiotic exposure. Nat Commun 2018;9:1599. doi: 10.1038/s41467-018-04059-1.
DOI
doi.org/10.1097/CM9.0000000000000884
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