More Mutation Accumulation in Neisseria gonorrhoeae with Susceptibility than that in Neisseria gonorrhoeae with Decreased Susceptibility to Ceftriaxone
Neisseria gonorrhoeae (NG) is a pathogen responsible for the sexually transmitted infection gonorrhea. The emergence of NG strains with decreased susceptibility to ceftriaxone (CRO), a critical antibiotic used in the treatment of gonorrhea, has become a global health concern. Ceftriaxone, often combined with azithromycin, has been the last option for first-line antimicrobial monotherapy. However, the spread of NG strains with reduced susceptibility to CRO has led to treatment failures, prompting changes in therapeutic strategies. This study investigates the genetic mechanisms underlying the resistance and susceptibility of NG to CRO by comparing the mutation accumulation in CRO-susceptible (CRO-S) and CRO-decreased susceptibility (CRO-DS) NG isolates.
Background and Significance
Ceftriaxone, a third-generation cephalosporin, has been a cornerstone in the treatment of gonorrhea due to its efficacy. However, the emergence of NG strains with decreased susceptibility to CRO has raised alarms. More than ten clinical treatment failures with CRO have been reported, with minimum inhibitory concentrations (MICs) ranging from 0.5 to 2.0 mg/L. Consequently, CRO is no longer recommended as monotherapy for gonorrhea. Instead, dual therapy combining CRO with azithromycin is now widely used. Despite this, the first treatment failure to dual therapy (500 mg CRO plus 1 g azithromycin) was reported in 2014 in London. The development of resistance in NG is attributed to spontaneous mutations and horizontal genetic transfer. Understanding the genetic basis of resistance is crucial for developing effective treatment strategies and preventing the spread of resistant strains.
Study Design and Methodology
This study utilized whole-genome sequencing (WGS) to compare the mutation profiles of CRO-S and CRO-DS NG isolates. NG isolates were collected from patients attending 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 for analysis. The isolates were matched as closely as possible based on their MICs for ciprofloxacin, spectinomycin, and azithromycin to ensure comparability.
WGS was performed using the Illumina HiSeq 4000 platform. High-quality reads were aligned to the reference genome (NG NCCP11945, NC_011035) using the Burrows-Wheeler Aligner (version 0.5.9-r16). Single-nucleotide variants (SNVs) were identified using Sequence Alignment Map tools (version 0.1.19-44428cd). Specific gene mutations associated with 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 SNVs was generated using MEGAN7.
Results
The analysis revealed no significant difference in the abundance of clean WGS reads between CRO-S and CRO-DS NG isolates. However, the number of point mutations was significantly higher in CRO-S NG isolates compared to CRO-DS NG isolates. Specifically, the total number of point mutations was 6206.46 ± 776.50 in CRO-S NG versus 5420.73 ± 770.68 in CRO-DS NG (P < 0.01). Similarly, homozygous mutations were more abundant in CRO-S NG (5694.32 ± 766.86) than in CRO-DS NG (4968.59 ± 738.41, P < 0.01), and heterozygous mutations were also higher in CRO-S NG (512.14 ± 61.27) compared to CRO-DS NG (452.14 ± 69.85, P < 0.01).
A significant negative correlation was observed between CRO MICs and the total number of point mutations (r = -0.4737, P = 0.0012), homozygous point mutations (r = -0.4631, P = 0.0015), and heterozygous point mutations (r = -0.3348, P = 0.0263). The types of point mutations, including A > G, G > A, C > T, and T > C, were more frequent in CRO-S NG isolates. The circos plots further illustrated the difference in point mutations between CRO-S and CRO-DS NG isolates, confirming that CRO-S NG possessed more mutations.
Interestingly, specific mutations associated with CRO-DS, such as mtrR promoter 23 to 35 A deletion, penA Ala501, penA Gly542, penA Pro551, porB1b Gly120, and porB1b Ala121, were slightly more frequent in CRO-DS NG isolates, although the differences were not statistically significant. The phylogenetic analysis based on SNVs also highlighted these mutations in CRO-DS NG isolates.
Discussion
The findings of this study suggest that CRO-S NG isolates accumulate more point mutations compared to CRO-DS NG isolates. This observation may be explained by the hypothesis that CRO-S NG isolates require more mutations to develop resistance under selective pressure, whereas CRO-DS NG isolates have already acquired some degree of resistance and thus require fewer additional mutations. This aligns with previous studies suggesting a positive relationship between hypermutable bacteria and the acquisition of antibiotic resistance.
The study also highlights the importance of monitoring both CRO-S and CRO-DS NG isolates. While CRO-DS NG isolates are of immediate concern due to their reduced susceptibility to CRO, CRO-S NG isolates, with their higher mutation rates, have the potential to develop resistance rapidly. The presence of specific mutations in CRO-DS NG isolates, such as those in the mtrR promoter and penA gene, underscores the genetic basis of resistance and the need for molecular surveillance.
Conclusion
This study provides valuable insights into the genetic mechanisms underlying the susceptibility and resistance of NG to ceftriaxone. The higher mutation accumulation in CRO-S NG isolates compared to CRO-DS NG isolates suggests that CRO-S NG may be under greater selective pressure to develop resistance. The findings emphasize the need for enhanced molecular surveillance of both CRO-S and CRO-DS NG isolates to prevent the spread of resistant strains and inform treatment strategies. Understanding the genetic basis of resistance is crucial for developing effective interventions and ensuring the continued efficacy of antibiotics in the treatment of gonorrhea.
doi.org/10.1097/CM9.0000000000000884
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