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. 2022 Jul 29;17(7):e0271657. doi: 10.1371/journal.pone.0271657

Whole-genome sequence analysis of high-level penicillin-resistant strains and antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from Thailand

Natakorn Nokchan 1, Thidathip Wongsurawat 2,3, Piroon Jenjaroenpun 2,3, Perapon Nitayanon 1, Chanwit Tribuddharat 1,*
Editor: Ayesha Sabah Rahman4
PMCID: PMC9337635  PMID: 35905043

Abstract

Background

The increasing rate of antimicrobial-resistant Neisseria gonorrhoeae poses a considerable public health threat due to the difficulty in treating gonococcal infections. This study examined antimicrobial resistance (AMR) to drugs recommended for gonorrhea treatment between 2015 and 2017, and the AMR determinants and genetic compositions of plasmids in 3 gonococcal strains with high-level penicillin resistance.

Methods

We collected 117 N. gonorrhoeae isolates from patients with gonococcal infections who attended Siriraj Hospital, Bangkok, Thailand, between 2015 and 2017. Minimum inhibitory concentrations (MICs) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method. PCR amplification and sequencing of 23S rRNA and mtrR (a negative regulator of MtrCDE efflux pump) were performed. Whole genomes of 3 PPNG strains with high-level penicillin resistance (MIC ≥ 128 μg/ml) were sequenced using Illumina and Nanopore sequencing platforms.

Results

The proportions of N. gonorrhoeae isolates with resistance were 84.6% for penicillin, 91.5% for tetracycline, and 96.6% for ciprofloxacin. All isolates were susceptible to spectinomycin, azithromycin, cefixime, and ceftriaxone. An adenine deletion within a 13 bp inverted repeat sequence in the mtrR promoter and an H105Y mutation in the mtrR coding region were found in the N. gonorrhoeae isolate with the highest azithromycin MIC value (1 μg/ml). Three high-level penicillin-resistant isolates contained nonmosaic type II penA and had mutations in penB and the mtrR coding region. All isolates with high-level penicillin resistance carried the conjugative plasmids with or without the Dutch type tetM determinant, the beta-lactamase plasmid (Rio/Toronto), and the cryptic plasmid.

Conclusions

The gonococcal population in Thailand showed high susceptibility to ceftriaxone and azithromycin, current dual therapy recommended for gonorrhea treatment. As elevated MIC of azithromycin has been observed in 1 strain of N. gonorrhoeae, expanded and enhanced surveillance of antimicrobial susceptibility and study of genetic resistance determinants are essential to improve treatment guidelines.

Introduction

Gonorrhea is one of the most common sexually transmitted infections globally, with an estimated 86.9 million new gonorrhea cases among adults aged 15 to 49 years annually, according to the World Health Organization (WHO) [1]. The causative agent, Neisseria gonorrhoeae, has become a serious public health problem and is of increasing concern due to its distinct capacity to progressively develop resistance to all known classes of antibiotics recommended for treatment, including sulfonamide, beta-lactams (penicillin and narrow-spectrum cephalosporins), tetracycline, fluoroquinolones, and macrolides [2]. N. gonorrhoeae was therefore classified as an urgent threat by the Centers for Disease Control and Prevention (CDC). The WHO also included it in the global priority pathogen list due to the urgent need for new antibiotics [3]. In the absence of an effective vaccine, patient management is primarily based on preventive measures and effective antibiotic treatment. Currently, the WHO recommends dual antimicrobial therapy (ceftriaxone plus azithromycin) as a first-line empirical treatment for uncomplicated gonorrhea [4]. Nonetheless, it was of great concern when gonococcal strains with reduced susceptibility or resistance to ceftriaxone or azithromycin began to be reported in multiple countries around the world [5, 6], resulting in treatment failures [7]. Gonorrhea may soon become untreatable. In 2016, the first failure of gonorrhea treatment with recommended dual therapy was recorded [8], and in 2018, gonococcal strains exhibiting ceftriaxone resistance and high-level resistance to azithromycin were identified in England and Australia [9]. The continued development of resistance in N. gonorrhoeae emphasizes the urgent need for improved antimicrobial resistance surveillance and new antimicrobials for gonorrhea treatment.

Penicillin, the first effective antibiotic, was officially approved for the treatment of gonococcal urethritis in 1943. Penicillin resistance in N. gonorrhoeae is acquired from stepwise mutations of several determinants of penicillin resistance. These are mutations on chromosomes (chromosomal-mediated resistance) and horizontal gene transfer of the beta-lactamase plasmids. The high-level penicillin resistance is conferred by plasmids, namely, Asian, African, and Rio/Toronto plasmids. The chromosomal mutations in associated penicillin resistance genes are penA (encoding the main lethal target penicillin-binding protein 2 [PBP2]); ponA (encoding PBP1); mtrR (encoding the transcriptional repressor of the mtrCDE efflux pump); penB (encoding a major outer membrane protein); and pilQ (encoding pore-forming secretin PilQ). These mutations were associated with the following AMR mechanisms:

  1. Reducing the target affinity for the drug (penA and ponA mutations)

  2. Increasing the efflux of the drug (mtrR mutation)

  3. Reducing the influx of the drug (penB and pilQ mutations).

The beta-lactamase plasmids contain the blaTEM-1 gene, which encodes a beta-lactamase capable of hydrolyzing the cyclic amide bond of penicillin’s beta-lactam ring [2]. Several variants of blaTEM have been reported in N. gonorrhoeae: blaTEM-135 (M182T substitution), blaTEM-220 (M182T and A185T substitutions), and blaTEM-239 [10, 11]. Only 1 additional amino acid change in blaTEM-135, such as blaTEM-20 (G238S), results in extended-spectrum beta-lactamase (ESBL) evolution, which can destroy the activity of third-generation cephalosporins available for gonorrhea treatment during the study period, including cefixime and ceftriaxone. For extended-spectrum cephalosporins (ESCs), the main mechanisms of reduced susceptibility or resistance were penA mutations (A501V and A501T) and mosaic penA alleles (associated with up to 70 amino acid substitutions relative to wild-type), which are acquired from interspecies recombination between penA genes of N. gonorrhoeae and commensal Neisseria species (N. cinerea and N. perflava) [12]. The elevated minimum inhibitory concentrations (MICs) of azithromycin result from mutations in the 23S rRNA gene (A2058G, A2059G, and C2611T), which contribute to intermediate-level or high-level resistance and, to a lesser extent, in the mtr (multiple transferable resistance) efflux pump locus, causing increased drug efflux [2].

Typically, N. gonorrhoeae carries up to 3 types of plasmids: conjugative, beta-lactamase, and cryptic. The first 2 types contribute to high-level resistance to tetracycline and penicillin, respectively. The 3 types of conjugative plasmids have been described based on tetM resistance determinant and restriction endonuclease mapping, including American, Dutch, and markerless (lacking tetM) types. Furthermore, the difference in nucleotide sequences of the tetM determinants carried by American- and Dutch-type plasmids was identified, resulting in the designations “American” and “Dutch.” The conjugative plasmid enables small non-self-transmissible beta-lactamase plasmids to mobilize into other bacteria, such as Escherichia coli. The small cryptic plasmid was frequently found in N. gonorrhoeae; nevertheless, its role remains unknown [13].

In Thailand, an overall gonorrhea incidence rate of 15.13 per 100 000 population was reported in 2018. However, this incidence was probably underestimated because of suboptimal diagnosis and surveillance and inadequate screening in asymptomatic patients, especially among men who have sex with men [14]. The AMR surveillance was based on the Enhanced Gonococcal Antimicrobial Surveillance Program of the WHO and the Centers for Disease Control and Prevention of the United States [15]. Siriraj Hospital, Bangkok, managed the AMR surveillance within Thailand [16]. Located in the central part of Thailand, the hospital is the largest tertiary-care university hospital in the nation. It services large numbers of patients, many of whom are referred from other hospitals. According to Tribuddharat et al. (2017), all N. gonorrhoeae isolates from Siriraj Hospital and Bangrak Hospital (National Center for Sexually Transmitted Infections) showed susceptibility to spectinomycin, azithromycin, and ceftriaxone, indicating that the recommended treatment of dual antimicrobial therapy for gonorrhea remained effective in Thailand [16]. No ceftriaxone-resistant gonococcal strains have been reported in Thailand to date. However, decreased susceptibility to ceftriaxone has been identified in a few gonococcal isolates, as described elsewhere [17].

Integrons are mobile genetic elements that play a pivotal role in conferring AMR and disseminating AMR determinants, particularly in Gram-negative bacterial pathogens. The fundamental structure of integrons consists of a 5’-conserved segment (5’-CS) with an integrase gene (intI) and an attI site. There is also a 3’-conserved segment (3’-CS) with a quaternary ammonium compound resistance gene (qacEΔ1), a sulfonamide resistance gene (sul), and an open reading frame of unknown function (orf5). Of the 5 reported integron classes, class 1 integrons are very common in isolates from clinical settings. They can capture and express exogenous genes (antibiotic resistance, metabolic, or virulence) embedded as gene cassettes of variable regions between the 2 conserved segments [18]. According to the literature, class 1 integrons have never been examined in clinical isolates of N. gonorrhoeae.

With advances in technology, whole-genome sequencing has become an ideal tool in well-resourced settings as it provides more detailed information and greater accuracy than other methods [19]. It is used to (1) study the molecular epidemiology, genetic relationships, and transmission dynamics of AMR gonococcal strains; (2) predict antimicrobial susceptibilities; and (3) elucidate novel or known AMR determinants.

Relatively limited antimicrobial susceptibility data are available on N. gonorrhoeae in Thailand. In addition, comprehensive characterizations of beta-lactam resistance genes and mutations have not been described. More data on antimicrobial resistance in N. gonorrhoeae clinical isolates from Thailand are needed to inform treatment guidelines. Additional local- and national-level details on the emergence and dissemination of resistant gonococcal strains are essential for international countries to control and prevent these strains. This study had the following objectives:

  1. Determine the antimicrobial susceptibility of N. gonorrhoeae isolates to drugs used for gonorrhea treatment in Thailand between 2015 and 2017

  2. Identify point mutations that confer an elevated MIC of azithromycin in the isolate with the highest azithromycin MIC of the isolates

  3. Investigate the presence of class 1 integrons

  4. Uncover beta-lactam resistance determinants and analyze the plasmid composition of 3 N. gonorrhoeae strains with high-level penicillin resistance

Materials and methods

N. gonorrhoeae isolates and species identification

A total of 117 gonococcal isolates were obtained from Siriraj Hospital patients suspected of having gonococcal infections between January 2015 and December 2017. All isolates were collected from specimens from different anatomical body sites (the vagina, cervix, urethra, penis, fallopian tube, urine, eye, joint, and wrist) and stored in Amies medium to maintain the viability of the isolates during their transportation. N. gonorrhoeae culture was carried out on chocolate agar (Clinical Diagnostics Ltd., Thailand) and incubated at 35°C ± 2°C in a 5% CO2 atmosphere for 20 to 24 hours. The identification of N. gonorrhoeae species was confirmed by the growth of typical colonies on chocolate agar, the presence of Gram-negative, coffee bean-shaped diplococci with polymorphonuclear leukocytes under a microscope, and the VITEK 2 Neisseria-Haemophilus (NH) identification card (bioMérieux Inc., Durham, NC, USA). The pure culture of N. gonorrhoeae isolates was preserved in brain-heart infusion broth supplemented with 30% (volume/volume) glycerol at -80°C. The routine clinical diagnostic laboratory sourced all clinical isolates of N. gonorrhoeae and the N. gonorrhoeae ATCC 49226 used for quality control of the antimicrobial susceptibility testing.

Beta-lactamase and antimicrobial susceptibility testing

Beta-lactamase production of N. gonorrhoeae isolates was tested by a nitrocefin disk (BD Diagnostics, Franklin Lakes, NJ, USA) as per the manufacturer’s instructions. All beta-lactamase-positive isolates were known penicillinase-producing N. gonorrhoeae (PPNG) isolates.

The MICs (μg/ml) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method on GC agar base (Oxoid Ltd., Hampshire, UK) supplemented with 1% IsoVitaleX (BBL Microbiology Systems, Cockeysville, MD, USA). The antibiotic powder was purchased from Sigma–Aldrich (Milwaukee, WI, USA). The practice of conducting the agar dilution method was performed following the Clinical and Laboratory Standards Institute guidelines [20]. The agar dilution was performed in duplicate, and the obtained MIC values were interpreted based on the interpretative criteria of the CLSI guidelines [21]. N. gonorrhoeae ATCC 49226 was used as the quality control strain for the agar dilution test.

Genomic DNA extraction

Total genomic DNA was extracted from N. gonorrhoeae isolates using the Gentra Puregene Yeast/Bacteria Kit, following the manufacturer’s instructions for DNA purification from Gram-negative bacteria (Qiagen, Valencia, CA, USA). DNA concentration and purity were determined with a NanoDrop ND-100 spectrophotometer (Bio-Active Co., Ltd, Bangkok, Thailand), and DNA samples were stored at -20°C until use.

PCR and DNA sequencing of azithromycin resistance genes

The N. gonorrhoeae isolate with the highest azithromycin MIC in this study underwent PCR and DNA sequencing to identify mutations in the 23S rRNA alleles, the mtrR promoter (nucleotide -120 to -1), and coding regions. A two-step PCR method was used to determine mutations in the peptidyl transferase region in domain V of 4 alleles of 23S rRNA, as described previously [22]. Amplification of the mtrR promoter and coding regions was performed using previously described primers and PCR conditions [23]. All PCR products were purified using a NucleoSpin Gel and PCR Clean-up Kit (Macherey–Nagel Inc., Easton, PA, USA) before sending samples to Macrogen Inc. (Seoul, Korea) for DNA sequencing. DNA gene sequences were aligned with their wild-type gene sequences obtained from the pansusceptible N. gonorrhoeae strain FA1090 (GenBank accession number AE004969.1) using BioEdit version 7.2.5 to determine the mutations in these genes.

Detection of class 1 integron-integrase gene

PCR was used to determine the presence of class 1 integron-integrase gene in all N. gonorrhoeae clinical isolates using IntI1F and IntI1R primers as described elsewhere [24].

Whole-genome sequencing

In order to obtain the complete genome sequence, whole-genome sequencing with both Illumina (Illumina Inc., San Diego, CA, USA) and Nanopore (Oxford Nanopore Technologies Ltd. [ONT], Oxford, UK) platforms was performed. Three gonococcal strains were randomly selected based on different N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence types (STs). The strains also exhibited high-level penicillin resistance (MIC ≥ 128 μg/ml). The characteristics of the strains are summarized in Table 1. DNA libraries for Nanopore sequencing were prepared using the rapid barcoding sequencing kit (SQK-RBK004) without DNA size selection and sequenced on a MinION device (version Mk1B; ONT) using a flow cell (FLO-MIN106, v.R9.4, ONT) for 48 h. The NEBNext Ultra II DNA Library Prep Kit (New England Biolabs, Ipswich, USA) was used to generate 150-bp paired-end libraries for the Illumina sequencing, and the libraries were sequenced on a NovaSeq 6000 sequencer (Illumina). All raw reads generated in this study were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioProject number PRJNA600334.

Table 1. Characteristics of N. gonorrhoeae isolates (n = 3) with high-level resistance to penicillin (MIC ≥ 128 μg/ml).

Strain number Penicillin MIC (μg/ml) NG-MAST
CT530 256 ST18929
CT532 128 STnovel10
CT602 256 STnovel68
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Abbreviations: MIC, minimum inhibitory concentration; NG-MAST, Neisseria gonorrhoeae multiantigen sequence typing; ST, sequence type.

Sequence assembly and bioinformatic analyses

With ONT, the base-calling of raw signals and demultiplexing were conducted using Guppy v.3.2.4. Adapter trimming was performed using Porechop v.0.2.4 (https://github.com/rrwick/Porechop). Raw reads with a short length (< 1000 bases) or low quality (mean quality score < 8) were removed with NanoFilt v.2.5.0 [25]. In the case of Illumina, the raw reads were trimmed and filtered using fastp v.0.19.5 [26]. The N50 read length was calculated using the Assembly Stats tool (https://github.com/sanger-pathogens/assembly-stats). De novo whole-genome assembly was carried out using the hybrid assembler Unicycler v.0.4.4 [27]. Genome sequencing and assembly statistics for these 3 N. gonorrhoeae genomes are summarized in S1 Table. Genome error correction, circularization, and rotation (using the dnaA gene as the starting point) were performed using Unicycler v.0.4.4. The quality of complete genome assemblies was evaluated with QUAST v.5.0.2 [28]. The genomic GC content was ascertained with a GC Content Calculator (https://www.sciencebuddies.org/science-fair-projects/references/genomics-g-c-content-calculator). The default parameter settings of all programs were used unless otherwise noted. Genome annotations were executed with the NCBI Prokaryotic Genome Annotation Pipeline v.4.11 [29].

The plasmid maps were generated and visualized using SnapGene Viewer software (http://www.snapgene.com/). Beta-lactam resistance genes and their mutations were predicted using Resistance Gene Identifier software (v.5.1.0, https://github.com/arpcard/rgi) against the Comprehensive Antibiotic Resistance Database (v.3.0.9, https://card.mcmaster.ca/) [30] and manually confirmed by alignment with their wild-type alleles from the N. gonorrhoeae strain FA1090 (GenBank accession number AE004969.1). These genes were probably related to decreased susceptibility or resistance to either penicillin or ESCs. They were ponA, penA, dacB, pbpG, dacC, mtrR, pilQ, penB, and blaTEM-1. The presence of a 57-kb gonococcal genetic island (GGI) encoding a type IV secretion system (​T4SS) and the type of tetM determinants were examined by in silico PCR using SnapGene software version 6.0.5 (from GSL Biotech; available at snapgene.com) [31, 32]. The conjugal plasmid backbones were subjected to in silico BglI digestion using SnapGene software version 6.0.5 (from GSL Biotech; available at snapgene.com) to differentiate between the American and Dutch type.

Multilocus sequence typing (MLST), NG-MAST, and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-​STAR) sequence types were assigned using the Bacterial Isolate Genome Sequence Database (BIGSdb) platform of the PubMLST Neisseria database (https://pubmlst.org/neisseria/).

Ethical considerations

The Siriraj Hospital Institutional Review Board approved this nonclinical research study before its commencement (certificates of approval Si479/2015 and Si720/2018). Informed consent was exempted by the Review Board because our retrospective and non-clinical study used bacterial isolates recovered from clinical samples taken as part of routine clinical laboratory examination and patient information was anonymized and deidentified prior to analysis.

Results

Patient data and N. gonorrhoeae isolates

Of the 117 patients with gonorrhea, 54.7% were women, and 45.3% were men (ratio, 1:1.2). The ages of 2 patients were unknown. The median age was 20 years, ranging from 15 days to 65 years. The highest prevalence of gonorrhea (20.9%) was found in patients aged 15 to 19. The samples were recovered from the urethra (46.1%), cervix (29.1%), vagina (17.9%), eyes (4.3%), and other sites (2.6%).

Prevalence and penicillin susceptibility of PPNG isolates

Of the 117 gonococcal isolates, 100 (85.5%) were PPNG isolates (39 in 2015, 33 in 2016, and 28 in 2017). The remaining isolates (17/117; 14.5%) were negative for beta-lactamase production. The ratio between the PPNG and non-PPNG isolates was approximately 5.9. The prevalence of PPNG isolates was 86.7% (39/45) in 2015, 89.2% (33/37) in 2016, and 80.0% (28/35) in 2017. The antimicrobial susceptibility testing revealed that almost all PPNG isolates (99%) were resistant to penicillin except one, which showed intermediate resistance. Most non-PPNG isolates (82.4%) had intermediate susceptibility to penicillin, whereas the remaining isolates (17.6%) were susceptible to penicillin.

Antimicrobial susceptibility testing

Most gonococcal isolates exhibited resistance to penicillin (84.6%), tetracycline (91.5%), and ciprofloxacin (96.6%), while all were susceptible to spectinomycin, cefixime, ceftriaxone, and azithromycin. None of the isolates were susceptible to tetracycline. Table 2 summarizes the antimicrobial susceptibility of the N. gonorrhoeae isolates. Regarding the MIC distributions, penicillin resistance (MIC of 64 μg/ml) was commonly found in N. gonorrhoeae isolates (23/117; 19.7%). The most common MIC values for tetracycline and ciprofloxacin were 16 μg/ml (61/117; 52.1%) and 2 μg/ml (68/117; 58.1%), respectively. In the case of spectinomycin, the proportions of N. gonorrhoeae isolates with MIC values of 16 μg/ml and 32 μg/ml were 42.7% (50/117) and 43.6% (51/117), respectively. An azithromycin MIC of 0.06 μg/ml was frequently observed in the gonococcal isolates (45/117; 38.5%). The prevalences of N. gonorrhoeae isolates susceptible to cefixime (MIC = 0.008) and ceftriaxone (MIC ≤ 0.002) were 41.9% (49/117) and 78.6% (92/117), respectively. The lowest spectinomycin MIC increased every year between 2015 and 2017. The highest azithromycin MIC (1 μg/ml) was observed in N. gonorrhoeae obtained in 2017.

Table 2. Antimicrobial susceptibility of 117 N. gonorrhoeae clinical isolates to 7 antimicrobials previously or currently used for gonorrhea treatment.

Antimicrobial No. (%) of isolates MIC (μg/ml)
Susceptible Intermediate Resistant Range MIC50 MIC90
Penicillin G 3 (2.6) 15 (12.8) 99 (84.6) 0.06–256 16 128
Tetracycline 0 10 (8.5) 107 (91.5) 0.5–64 16 32
Ciprofloxacin 3 (2.6) 1 (0.8) 113 (96.6) ≤ 0.001–16 2 8
Azithromycin 117 (100) 0 0 ≤ 0.004–1 0.06 0.12
Spectinomycin 117 (100) 0 0 2–32 16 32
Cefixime 117 (100) 0 0 ≤ 0.002–0.06 0.008 0.016
Ceftriaxone 117 (100) 0 0 ≤ 0.002–0.03 0.002 0.004
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Abbreviations: MIC, minimum inhibitory concentration; MIC50, minimum inhibitory concentration for 50% of isolates; MIC90, minimum inhibitory concentration for 90% of isolates

Characterization of azithromycin resistance determinants

As a result of antimicrobial susceptibility, N. gonorrhoeae isolate number 577 had the highest azithromycin MIC of 1 μg/ml, representing borderline resistance to azithromycin. Therefore, this isolate was selected to examine mutations in the 23S rRNA and mtrR genes as these determinants are associated with azithromycin resistance. The results showed that no mutation was detected in each 23S rRNA allele (GenBank accession numbers ON638953 to ON638956). However, an A (adenine) deletion was identified in the 13-bp inverted repeat region (5′-aAAAAGTCTTTTT-3′; the deletion is indicated by lowercase letter) between the − 10 and − 35 hexamers of the mtrR promoter region, and an H105Y substitution was found in the mtrR coding region (GenBank accession number ON638952).

Class 1 integrons, molecular epidemiology, and genetic resistance determinants

None of the N. gonorrhoeae clinical isolates carried class 1 integrons. The molecular epidemiology and genetic resistance determinants of 3 high-level penicillin-resistant strains were revealed by whole-genome sequencing (Table 3). N. gonorrhoeae strains CT530 and CT602 belonged to MLST ST8143, and the CT532 strain was assigned to MLST ST1925. Two novel and 1 known NG-MAST ST were identified: STnovel10 for CT532, STnovel68 for CT602, and ST18929 for CT530. NG-STAR analysis revealed that the N. gonorrhoeae strains CT530, CT532, and CT602 were associated with NG-STAR ST1900, ST1663, and ST1849, respectively. The 57-kb GGI was not found in any of the 3 strains.

Table 3. Genetic characteristics of Neisseria gonorrhoeae with high level resistance to penicillin (MIC ≥ 128 μg/ml).

Strain characteristics CT530 CT532 CT602
MLST ST8143 ST1925 ST8143
NG-MAST ST18929 STnovel10 STnovel68
NG-STAR ST1900 ST1663 ST1849
GGI Absent Absent Absent
ponA WT A375T A375T
penA Non-mosaic type II Non-mosaic type II Non-mosaic type II
dacB WT WT WT
pbpG WT WT WT
dacC S189A WT WT
pilQ type VI type VI type VI
porB1b G120K, A121N G120K, A121N G120R, A121D
mtrR A39T A39T, G162a A39T
mtrR promoter WT WT WT
bla TEM M182T M182T M182T
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Abbreviations: GGI, gonococcal genetic island; MLST, multilocus sequence typing; NG-MAST, Neisseria gonorrhoeae multiantigen sequence typing; NG-STAR, Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance; ST, sequence type; WT, wild type.

Regarding the determinants of penicillin resistance located on the chromosome, alterations in high-molecular-weight PBPs, including PBP1 encoded by ponA and PBP2 encoded by penA, were the main contributors to penicillin resistance in N. gonorrhoeae. Bioinformatic analyses demonstrated that the N. gonorrhoeae strains CT532 and CT602 contained the A375T substitution in ponA. In particular, there was no ponA mutation in the CT530 strain of N. gonorrhoeae. The penA alleles of all 3 strains were classified as nonmosaic penA type II due to having an insertion of aspartic acid after position 346 (D346a), F504L, A510V, and A516G alterations. The reduced extent of penicillin resistance might result from the mutations in dacB and pbpG, encoding low-molecular-weight PBPs, ie, PBP3 and PBP4, respectively; however, no mutation was found in these genes. Only the S189A mutation was found in the newly identified dacC gene of strain CT530, which encodes a low-molecular-weight PBP.

The promoter of mtrR, the gene encoding the multiple transferable resistance (Mtr) efflux pump, had no substitution in all strains. The 3 gonococcal strains showed the A39T mutation in the mtrR coding region, of which strain CT532 had an additional insertion of glycine after position 162 (G162a). Regarding the pilQ gene, which encodes the pore-forming secretin PilQ associated with type IV pilus formation, all gonococcal strains presented type VI PilQ, comprising a QAATPAKQ insertion at position 180 (180QAATPAKQ insertion), and substitutions of S341N and N648S. As to the penB gene (encoding pore-forming transmembrane porin), the 3 strains expressed PorB1b (but not PorB1a); PorB1b was previously confirmed to be associated with penicillin resistance [33]. Mutations in penB at positions 120 and 121 were detected in N. gonorrhoeae strains CT530 and CT532 (G120K and A121N) and in CT602 (G120R and A121D). Importantly, these strains harbored a beta-lactamase plasmid that encodes TEM-135 beta-lactamase, which is responsible for high-level penicillin resistance.

Sequence analysis of gonococcal plasmids

Analysis of the whole-genome sequencing data revealed that each strain of high-level penicillin-resistant N. gonorrhoeae had 3 types of plasmids: conjugative, beta-lactamase, and cryptic. The sizes of these plasmids are listed in S1 Table. As the 3 plasmid types of each N. gonorrhoeae strain were very similar, the gonococcal plasmids from N. gonorrhoeae strain CT530 were used to represent the common features of each plasmid type (Fig 1). Nonetheless, this was not appropriate for the conjugative plasmid of strain CT602 because it did not contain the tetM determinant (unlike the others; Fig 1A). The conjugative plasmids of the N. gonorrhoeae strains carried Dutch-type tetM determinants and showed a similar digestion pattern to the previously reported Dutch type plasmid (GenBank accession number GU479466) after in silico digestion with BglI [34]. The conjugative plasmids consisted of several open reading frames (ORFs) grouped into 5 modules as follows: (1) replication initiation (ssb and trfA); (2) conjugative transfer (tra); (3) mating pair formation (trb); (4) plasmid inheritance and control (kor, kle, inc, and kfr); and (5) accessory genes (Zeta/Epsilon toxin-antitoxin system, vapD, and tetM). We identified 10 tra genes and 13 trb genes in all 3 conjugative plasmids (without tetM from strain CT602 but with tetM from strains CT530 and CT532). Two genes found in the replication initiation region, ssb and trfA, encode a single-stranded DNA-binding protein and the oriV activator, respectively. The plasmid inheritance and control module comprised incC2, which encodes the ParA ATPase responsible for the partitioning system. The vapD and marR genes located in the accessory gene region encode the virulence-associated protein D or VapD toxin and a transcriptional regulator of the MarR family, respectively. This region was important due to its tetM resistance determinant that encodes a cytoplasmic ribosome protecting protein that confers resistance to tetracycline. The yegA, found in the same region of tetM, encodes a DUF882 domain-containing protein. The ngoCT_11260 gene encodes a transcription elongation factor, while ngoCT_11285 encodes a site-specific DNA methyltransferase. The remaining open reading frames encode hypothetical proteins. The tetM-containing conjugative plasmid differed from the conjugative plasmid lacking tetM by the presence of the tetM determinant and the ngoCT_11305 gene located upstream of the start site of the tetM gene.

Fig 1. Schematic representation of gonococcal plasmids.

Fig 1

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(A) Gene organization of 2 different conjugative plasmids (upper left and right); the conjugative plasmid carrying a Dutch type tetM determinant and a Dutch type backbone of N. gonorrhoeae strain CT530 and the conjugative plasmid with no tetM resistance determinant of gonococcal strain CT602. The colors depict the functional modules as follows: red, mating pair formation; purple, replication initiation; green, inheritance/control; yellow, conjugative transfer; blue, accessory genes. (B) Gene organization of the beta-lactamase plasmid (Rio/Toronto type) of N. gonorrhoeae CT530. The colors represent the following: yellow, replication initiation; blue, recombination; red, ampicillin resistance; purple, antitoxin; green, unknown function. (C) Gene organization of the cryptic plasmid of gonococcal strain CT530. The open reading frames are colored as follows: green, cryptic plasmid proteins; yellow, plasmid mobilization; purple, proteins with unknown function; blue, plasmid-replicase genes; red, toxin-antitoxin genes.

The beta-lactamase plasmids of the 3 strains were identified as the Rio/Toronto plasmid. This plasmid contained repA and repB (encoding replication initiation proteins), recombinase family protein (Tn3 resolvase), vbhA (encoding the antitoxin VbhA family protein), and the ngoCT_11335 gene (encoding a hypothetical protein). The blaTEM variant with the M182T mutation, blaTEM-135, was identified in the Rio/Toronto plasmids of the 3 strains. This gene encodes TEM-135 beta-lactamase, which could render benzylpenicillin and ampicillin inactive.

The cryptic plasmid had 9 open reading frames; 3 of these encode cryptic plasmid proteins (cppA, cppB, and cppC). The vapD and vapX encode VapD toxin and VapX antitoxin, respectively. Two open reading frames encode proteins responsible for plasmid replicase, while the mobC encodes the plasmid mobilization relaxosome protein. The ngoCT_11365 gene encoding a hypothetical protein was also detected in this cryptic plasmid.

Discussion

This work described the AMR data of clinical isolates of N. gonorrhoeae collected from 2015 to 2017 in Thailand. We also analyzed resistance determinants and plasmids of gonococcal isolates with high levels of penicillin resistance. Additionally, the presence of class 1 integrons in these clinical isolates was examined.

Penicillin was the first-line treatment for gonorrhea in Thailand until the 1980s when the rate of PPNG isolates was 71% [35]. However, a high prevalence of PPNG isolates has since been observed. In the current investigation, 85.5% of gonococcal isolates from 2015 to 2017 were PPNG isolates; these are highly resistant to penicillin. This proportion increased from the 83.6% recorded between 2005 and 2009 [36] and from the 83.8% reported from 2008 to 2014 [16]. Intermediate resistance to penicillin of most non-PPNG isolates may be attributed to chromosome-mediated resistance by point mutations. Moreover, it was previously noted that only 1 PPNG isolate presented intermediate resistance to penicillin, whereas the remaining PPNG isolates were resistant. The possible explanations for this result may be different levels of blaTEM gene expression or the Canadian plasmid carrying a unique 6 bp deletion in blaTEM-1 encoding a truncated 24 kDA TEM-1 beta-lactamase with slow activity [37].

The present work carried out agar dilution instead of the disc diffusion and E-testing used in earlier studies [16]. Agar dilution was preferred because our objective was to undertake quantitative interpretations of the MIC values of the antibiotics with as high an accuracy and reliability as possible. As expected, a high proportion of N. gonorrhoeae isolates were resistant to penicillin (84.6%), tetracycline (91.5%), and ciprofloxacin (96.6%). These findings are consistent with earlier data from Thailand [16]. The results suggested a very low possibility of reapplying the antibiotics previously used for gonorrhea treatment.

Interestingly, all gonococcal isolates were susceptible to spectinomycin, azithromycin, cefixime, and ceftriaxone. This finding was despite the highest cefixime and azithromycin MICs having increased only slightly after 2008–2014 (cefixime: from 0.023 to 0.06 μg/ml; azithromycin: from 0.25 to 1 μg/ml) [16]. The increase in MICs was partly due to the use of different methods for antimicrobial susceptibility testing (E-testing and agar dilution). Another factor that may have been involved was the implementation of combination ceftriaxone and azithromycin therapy, which exerts antimicrobial selective pressure, to treat gonorrhea in Thailand.

The MIC50 and MIC90 values of the drugs used for gonorrhea treatment (spectinomycin, azithromycin, cefixime, and ceftriaxone) were low, suggesting the high efficacy of these antibiotics against gonococcal isolates. Spectinomycin has not been available in Thailand for many years following the Thai Food and Drug Administration withdrawing its approval for use [14]. The withdrawal may explain why spectinomycin-resistant isolates were not observed in this study. At the same time, there have been no published reports of N. gonorrhoeae resistance to cefixime or ceftriaxone in Thailand. Consequently, our findings support the ongoing use of the recommended dual therapy of intramuscular ceftriaxone and oral azithromycin for gonorrhea treatment in Thailand. However, another possible cause of the absence of resistance to cefixime or ceftriaxone might be that local AMR surveillance is inadequate and not nationwide. It was of concern when N. gonorrhoeae isolates with reduced susceptibility to ceftriaxone were recently identified in Thailand by Kueakulpattana et al. [17]. In addition, decreased susceptibility or resistance to ceftriaxone and azithromycin, as a cause of treatment failures, has been sporadically described in multiple countries [6, 7].

The AMR determinants that conferred elevated MICs of azithromycin, including 23S rRNA and mtrR, were examined because azithromycin is part of dual therapy for the treatment of gonorrhea. Of the 117 gonococcal isolates, only one showed borderline resistance to azithromycin (an MIC of 1 μg/ml). Moreover, mutations in these resistance determinants have never been previously uncovered in Thai gonococcal isolates. The results showed a deletion of A within a 13-bp inverted-repeat sequence of the mtrR promoter, which can abolish the expression of mtrR to repress the expression of the mtrCDE-encoded multidrug efflux pump. This produces a higher expression of the mtrCDE operon, mainly due to the increased binding affinity of RNA polymerase to the mtrCDE promoter and a decreased susceptibility to azithromycin (to approximately a tenth) [38, 39]. In addition to an alteration in the mtrR promoter, H105Y substitution was identified in the mtrR coding region. The substitution contributes to decreased binding of MtrR to its target promoter due to the altered structure of the MtrR dimer [40]. The MIC value of azithromycin was elevated by the H105Y alteration (by approximately 2- to 4-fold) [41]. Three mutations in the peptidyl transferase loop in domain V of the 23S rRNA that influence the secondary structure of 23S rRNA (C2611T, A2058G, and A2059G) have not been identified in all 4 copies of 23S rRNA from this isolate. The C2611T mutation is associated with low- and moderate-level azithromycin resistance, whereas both A2058G and A2059G confer high-level resistance to azithromycin [22, 42].

We investigated the presence of class 1 integrons in all N. gonorrhoeae isolates. These bacteria display AMR to many classes of antibiotics, express natural competence, and exhibit a high frequency of DNA recombination [2]. Furthermore, class 1 integrons were previously detected in some bacteria in the genital area [43], indicating the high possibility of acquired integrons in N. gonorrhoeae. Surprisingly, none of our gonococcal isolates contained class 1 integrons, except the Schaalia turicensis isolate co-isolated with N. gonorrhoeae [44]. This finding supports the notion that antimicrobial resistance in N. gonorrhoeae is derived from chromosomal mutations of resistance genes or plasmids carrying resistance determinants. This finding is consistent with other reports and information from the NCBI genome database [10].

Whole-genome sequencing was performed to obtain data on antibiotic resistance determinants, genetic relationships, and plasmid composition of N. gonorrhoeae isolates with high-level resistance to penicillin as reference data for strains from Thailand. From the whole-genome sequencing analysis, N. gonorrhoeae strains CT530 and CT602 belonged to MLST ST8143. These initially emerged in Taiwan in 2012 and were monitored whether they would follow the pattern of MLST ST1901 (strain exhibiting higher MICs for both ceftriaxone and azithromycin) to become highly prevalent locally and globally [45]. There was no epidemiological link to strains from other countries for the remaining isolates. The 57-kb GGI, responsible for the AMR dissemination and increased fitness of N. gonorrhoeae, has not been found in all gonococcal strains with high-level resistance to penicillin, although it was highly prevalent in strains from other countries [46]. This genetic element was associated with the international spread of the MLST ST1901 strain that displayed decreased susceptibility to ESCs in the Western Pacific Region [46].

Concerning beta-lactam resistance, alterations in the ponA, penA, mtrR, pilQ, and penB genes combined with the presence of a beta-lactamase plasmid containing the blaTEM allele were identified. For ponA encoding PBP1, strains CT532 and CT602 harbored the A375T mutation, which was rarely found in N. gonorrhoeae isolates and appeared not to be associated with decreased susceptibility to ceftriaxone [47]. Although the association of this substitution with penicillin resistance has not yet been elucidated, the mutation position was close to the conserved active site motif of PBP1 (Ser-461 of the SXXK motif). Moreover, the amino acid change from alanine to threonine might affect the structure of the active site by forming hydrogen bonds with specific amino acid residues [48, 49]. The common mutation L421P, which increases penicillin MICs slightly by 3- to 4-fold relative to wild-type, was not found in all strains [48] and was not associated with reduced susceptibility to ceftriaxone [47]. The penA allele is classified as nonmosaic or mosaic, followed by the type of penA allele assigned to Arabic numerals according to the amino acid profiles. After Arabic numerals, a decimal number is added to indicate an existing penA allele with different DNA sequences [50]. The penA alleles of all gonococcal strains were nonmosaic penA-2 alleles containing the D346a, F504L, A510V, and A516G substitutions. The D346a mutation was found in most penicillin-resistant strains because this mutation impeded antibiotic binding or breakage of the beta-lactam ring, leading to a decrease in the penicillin acylation rate to approximately a sixth [51]. The F504L mutation reduces the rate of penicillin acylation by restricting the conformational flexibility of PBP2 [52] and has a low impact on cephalosporin MIC [53]. Next, the A510V alteration was detected in gonococcal strains with high-level resistance to penicillin and had little effect on penicillin MIC [54]. The penicillin acylation rate was previously found to be decreased to less than a half relative to the wild-type because of the A516G mutation [54]. The nonmosaic penA-2 allele was the most common type and the most allelic diversity found in N. gonorrhoeae [50]. This penA allele confers resistance to penicillin but not ESC [55]. We further investigated the mutations in the dacB gene encoding the low-molecular-weight PBP, PBP3. PBP3 plays a role in peptidoglycan modification and recycling, and previous studies have demonstrated increased susceptibility to ceftazidime (after inactivation of DacB) and ampicillin, ceftazidime, and imipenem (after the loss of both DacB and DacC [encoded by dacC]) [56]. Nonetheless, we found no mutation in this gene. Likewise, there was no mutation in the pbpG gene encoding PBP4 in any of the N. gonorrhoeae strains. The dacC gene of N. gonorrhoeae strain CT530, encoding a third low molecular mass PBP that possesses carboxypeptidase and lacks conserved active site motifs, contained the S189A substitution [55]. Although the effect of this mutation is still unknown, DacC has been found to play a role in the intrinsic level of beta-lactam resistance in P. aeruginosa by both beta-lactam hydrolysis and trapping [57]. All strains had the A39T mutation located on the DNA binding domain of MtrR, causing the lower binding of the MtrR protein to the mtrCDE promoter and thus resulting in increased resistance to hydrophobic antibiotics [47]. Only the mtrR coding region of strain CT532 contained G162a, and its role remained unidentified. The PilQ of all N. gonorrhoeae strains belonged to type VI PilQ (180QAATPAKQ insertion, S341N, and N648S), and this PilQ type had no association with penicillin resistance. A previous investigation found that the E666K mutation in the pilQ gene was involved in penicillin resistance only when presented with alterations in penA, mtrR, and penB [58]. N. gonorrhoeae fundamentally expresses 1 of 2 forms of porin: PorB1a or PorB1b. Only the substitutions at positions 120 and 121 in PorB1b contribute to the increasing MICs of beta-lactam antibiotics and tetracyclines by replacing charged amino acids, such as aspartate (D), lysine (K), and arginine (R) [59]. The mutations found in our strains were G120K, G120R, A121D, and A121N.

Regarding plasmid-mediated resistance, blaTEM-135, first identified in Thailand, encodes TEM-135 beta-lactamase and differs from blaTEM-1 by 1 mutation, M182T [36]. This mutation increases enzyme thermal stability and suppresses enzyme misfolding, proteolysis, and aggregation [60, 61]. We reported the presence of beta-lactamase plasmids (Rio/Toronto plasmids) comprising the blaTEM-135 allele in all strains with high levels of penicillin resistance. Although the driver of blaTEM-135 evolution is still inconclusive, the blaTEM-135 allele can evolve into TEM-type ESBLs that destroy ESCs (TEM-20 [G238S], TEM-106 [E104K], or TEM-126 [D179E]) by acquiring 1 additional mutation at those specific positions.

In summary, the high-level resistance to penicillin in N. gonorrhoeae strains resulted from alterations in penA (D346a, F504L, A510V, and A516G), mtrR (A39T), and penB resistance genes (amino acid substitutions at positions 120 and 121) as well as the presence of Rio/Toronto plasmids carrying the blaTEM-135 allele. It is noteworthy that the A375T substitution in the ponA gene was identified in our 2 N. gonorrhoeae strains with high-level resistance to penicillin. Consequently, further investigation of this mutation related to penicillin resistance is needed.

We provide a genomic analysis of all plasmids in N. gonorrhoeae strains from Thailand. The results from plasmid analysis showed that all strains carried conjugative plasmids with the Dutch type tetM determinant and Dutch type plasmid backbones, beta-lactamase (Rio/Toronto plasmids), and cryptic plasmids, except for strain CT602, which carried conjugative plasmids without the tetM determinant. The gene composition of all types of plasmids in this study was indistinguishable from previous reports [62]. The plasmid backbones of conjugative plasmids could be discriminated into Dutch type or American type due to dissimilar restriction endonuclease patterns. All 3 strains carried the beta-lactamase plasmids harboring the blaTEM-135 allele with an amino acid substitution from methionine to threonine at position 182 (M182T), which could develop into an ESBL if it acquires 1 additional mutation. These plasmids were of the Rio/Toronto type. It is the derivative of the prototypical Asia plasmid (7.4 kb) and (as observed in our strains) confers high-level resistance to penicillin. Beta-lactamase plasmids were commonly found along with conjugative plasmids due to the capability of conjugative plasmids to comobilize beta-lactamase plasmids. Most gonococci contain the cryptic plasmid, and the absence of this plasmid has been reported to be associated with a novel chromosomal type IV secretion system found in N. gonorrhoeae [62].

This study has some limitations. First, the samples were only taken from one center, the routine microbiology laboratory at Siriraj Hospital, Bangkok. This results in a low number of gonococcal isolates each year and limited information on N. gonorrhoeae for Thailand. Additionally, since demographic, behavioral, and clinical data (including previous antibiotic exposure) of the patients were largely unavailable, it was not possible to identify the risk factors in patients that may have been associated with gonococcal infections or to correlate patient characteristics with specific patterns of resistance in N. gonorrhoeae.

Conclusions

The majority of N. gonorrhoeae isolates in Thailand from 2015 to 2017 were resistant to previously used antibiotics for gonorrhea treatment, including penicillin, tetracycline, and ciprofloxacin. No N. gonorrhoeae isolates were resistant to spectinomycin, azithromycin, cefixime, and ceftriaxone, warranting the continued use of ceftriaxone plus azithromycin as the first-line treatment for gonorrhea in Thailand. Nonetheless, borderline resistance to azithromycin (MIC of 1 μg/ml) was observed in 1 gonococcal isolate, with mutations in the mtrR promoter (A deletion) and coding regions (H105Y). The PPNG isolates were highly prevalent (85.5%), and almost all PPNG isolates were resistant to penicillin. No class 1 integrons were identified in our gonococcal isolates. Genetic analysis of N. gonorrhoeae with high-level resistance to penicillin revealed that the mutations in penA (D346a, F504L, A510V, and A516G), mtrR (A39T), and penB determinants (G120K, G120R, A121N, and A121D) and the presence of Rio/Toronto plasmids with the blaTEM-135 allele, contributed to a high level of penicillin resistance. The A375T mutation in ponA was identified as a putative mutation for penicillin resistance. Three types of plasmids were identified in N. gonorrhoeae isolates (conjugative, beta-lactamase, and cryptic plasmids), and they had the same gene composition as reported elsewhere [62]. Our results indicate that sustained and expanded AMR surveillance coupled with the characterization of AMR resistance determinants are critical. Doing so will enable the early detection of emergent isolates resistant to ceftriaxone or azithromycin, inform treatment guidelines, and enhance the understanding of AMR evolution.

Supporting information

S1 Table. Genome sequencing and assembly statistics for 3 Neisseria gonorrhoeae strains with high-level resistance to penicillin.

(DOCX)

Click here for additional data file. (16.6KB, docx)
S2 Table. Specimen source and antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Thailand, 2015–2017.

(XLSX)

Click here for additional data file. (19.1KB, xlsx)

Acknowledgments

We are grateful to Dr. Piriyaporn Chongtrakool for providing a steers replicator to perform the agar dilution testing. We also thank the microbiology laboratory staff, Department of Microbiology at the Siriraj Hospital, Bangkok, Thailand, for collecting and providing clinical isolates for this study. We are also indebted to Mr. David Park for English-language editing.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was financially supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (grant number PHD/0025/2557 awarded to N.N.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Ayesha Sabah Rahman

24 May 2022

PONE-D-22-11066Whole-genome sequence analysis of high-level penicillin-resistant strains and antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from ThailandPLOS ONE

Dear Dr. Chanwit Tribuddharat,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

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Ayesha Sabah Rahman, PhD

Academic Editor

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript is well written and seem scientifically sound to me. I have listed below a number of comments and suggestions that I think should be addressed before publication in PLoS One.

Major Issues

Line 338. Sequence analysis of gonococcal plasmids - can these plasmids be assigned to an compatibility group (I think they are normally IncP-1) and / or a relaxase group e.g. using MOBscan?

Line 365. Figure 1 is so low resolution as to be unreadable - I am not sure if this is due to compression during production of the proof pdf, or because the authors supplied a low resolution image.

Lines 402-403. "The possible explanations for this result..." - I assume his has been double or triple checked? If not then it should be; if it has been then a measurement error seems unlikely?

Lines 540-541. "The plasmid backbones of conjugative plasmids could not be discriminated..." this should be reported in the Results section - but I'm not clear what this means? Surely with the whole plasmid genome sequence available a virtual restriction digestion could be carried out, or if sequences are available for prototypic Dutch / American plasmids then some sequence comparisons could be made to allow assignment?

Minor Issues

Lines 32-33. "Nanopore and Illumina sequencing characterized beta-lactam resistance determinants and plasmids..." please clarify this was WGS, not only plasmids and resistance genes.

Lines 43-44. "...carried a conjugative plasmid or the conjugative plasmid with the Dutch type tetM determinant" is confusing - please rewrite for clarity.

Line 48. "one strain"?

Line 76. "The penicillin resistance..." - delete "The".

Lines 78-79. "horizontal gene transfer of a beta lactamase plasmid" i.e. there is more than 1 possible plasmid ?

Line 80. "The chromosomal mutations..."

Line 88. "The beta-lactamase plasmid contains" - is there only a single "clone" / type known of this plasmid? If not, this should be plural "plasmids"?

Line 103. "carries up to 3 types of plasmid"?

Line 297. "However, an A (adenine) deletion was identified..." a figure or some text showing this 13 bp repeat sequence with mutation might be useful.

Line 331. "a QAATPAKQ motif"?

Line 346. Specify "The conjugative plasmids".

Line 380. "encoding a hypothetical protein"

Line 391. "resistance determinants and plasmids of..."

Line 407. Delete 2nd "was" - "Agar dilution was preferred because...". Also - it is unclear to me whether this is a CLSI recommendation?

Line 494-495. "The penicillin acylation rate was decreased to...". Please clarify - this was not shown here? "was previously found to be decreased..."?

Line 535. This sentence is unclear and should be re-written - you have not provided the first genomic analysis of all plasmids in N. gonorrhoeae strains from Thailand?

Reviewer #2: the manuscript titled; Whole-genome sequence analysis of high-level penicillin-resistant strains and

2 antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from Thailand is a sound piece of scientific work with valuable input,

Please consider the comment given in the attached document to further improve the manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: Reviewer comments PLOS 1.docx

Click here for additional data file. (13.1KB, docx)
PLoS One. 2022 Jul 29;17(7):e0271657. doi: 10.1371/journal.pone.0271657.r002

Author response to Decision Letter 0

19 Jun 2022

Journal Requirements

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf.

Answer: The manuscript has been revised according to the PLOS ONE style templates to meet PLOS ONE's style requirements.

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

Answer: Additional details regarding informed consent and statement of anonymized patient data have been added in ethical consideration section.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Answer: The minimal data set underlying the results has been added in our manuscript (provided as accession numbers) and submitted as Supporting Information files. This information has been added and described within our revised cover letter. For our Data Availability statement, all relevant data are within the paper and its Supporting Information files.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ.

Answer: an ORCID iD of the corresponding author has been updated.

5. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Answer: Our reference list has been reviewed and updated. Changes to the reference list has been shown in the rebuttal letter.

Reviewer #1

Major Issues

1. Line 338. Sequence analysis of gonococcal plasmids - can these plasmids be assigned to an compatibility group (I think they are normally IncP-1) and / or a relaxase group e.g. using MOBscan?

Answer: Some of gonococcal plasmids can be assigned to incompatibility groups based on phylogenetic analysis of plasmid backbone, including origin of replication, replicase and partitioning genes. Though incompatibility tests should be carried out to give a definitive answer. As a preliminary result, phylogenetic analysis of the backbone sequences (TraI, TraG, TrbE and TrfA proteins) of the gonococcal conjugative plasmids showed that they belong to a novel IncP1 subfamily. In spite of reviewing the literature and in silico detection based on PCR-based replicon typing, incompatibility groups of gonococcal beta-lactamase (Rio/Toronto type) and cryptic plasmids could not be identified. It is noteworthy that the gonococcal Asia-type plasmid belongs to incompatibility group W and carries a silent IncFII determinant, in contrast to the Africa-type plasmid, belongs to the IncFII group. Nonetheless, there are no established plasmid incompatibility groups among the gonococcal plasmids. As for plasmid classification using relaxases, phylogenetic trees are constructed from the family alignments to allow the classification of conjugative transfer systems in six MOB families: MOBF, MOBH, MOBQ, MOBC, MOBP and MOBV. For this respect, functional analysis and annotation of all gonococcal plasmids are required to identify the relaxase genes. This notion is supported by MOBscan result as it cannot classify any of our gonococcal plasmids into any of the nine MOB families.

2. Line 365. Figure 1 is so low resolution as to be unreadable - I am not sure if this is due to compression during production of the proof pdf, or because the authors supplied a low resolution image.

Answer: Figure 1 was validated with the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool to ensure that it meets PLOS requirements. Therefore, the low-resolution issue may occur during the compression process of the file for reviewers.

3. Lines 402-403. "The possible explanations for this result..." - I assume his has been double or triple checked? If not then it should be; if it has been then a measurement error seems unlikely?

Answer: Since the agar dilution method has been done in duplicate to ensure the measured MICs, an MIC measurement error should be minimized, but not totally excluded. In addition, the different MICs among these isolates could be from different levels of blaTEM gene expression or a truncated 24 kDA TEM-1 enzyme. The sentence has been edited.

4. Lines 540-541. "The plasmid backbones of conjugative plasmids could not be discriminated..." this should be reported in the Results section - but I'm not clear what this means? Surely with the whole plasmid genome sequence available a virtual restriction digestion could be carried out, or if sequences are available for prototypic Dutch / American plasmids then some sequence comparisons could be made to allow assignment?

Answer: The sentence has been removed. We performed in silico BglI digestion with the gonococcal conjugative plasmids containing Dutch type tetM determinants and found that they showed a similar digestion pattern to the previously reported Dutch type plasmid. Consequently, using in silico BglI digestion the plasmid backbones of conjugative plasmids could be discriminated into American or Dutch type. All relevant details in the Materials and methods, Results, and Discussion were added and updated.

Minor Issues

5. Lines 32-33. "Nanopore and Illumina sequencing characterized beta-lactam resistance determinants and plasmids..." please clarify this was WGS, not only plasmids and resistance genes.

Answer: The sentence has been edited.

6. Lines 43-44. "...carried a conjugative plasmid or the conjugative plasmid with the Dutch type tetM determinant" is confusing - please rewrite for clarity.

Answer: The sentence has been rewritten to "All isolates with high-level penicillin resistance carried the conjugative plasmids with or without the Dutch type tetM determinant, the beta-lactamase plasmid (Rio/Toronto), and the cryptic plasmid."

7. Line 48. "one strain"?

Answer: It has been changed to “1 strain” following the American Medical Association Manual of Style.

8. Line 76. "The penicillin resistance..." - delete "The".

Answer: “The” has been deleted.

9. Lines 78-79. "horizontal gene transfer of a beta lactamase plasmid" i.e. there is more than 1 possible plasmid ?

Answer: It has been edited to “horizontal gene transfer of the beta-lactamase plasmids”.

10. Line 80. "The chromosomal mutations..."

Answer: It has been added.

11. Line 88. "The beta-lactamase plasmid contains" - is there only a single "clone" / type known of this plasmid? If not, this should be plural "plasmids"?

Answer: It is not a single plasmid clone. The sentence has been corrected.

12. Line 103. "carries up to 3 types of plasmid"?

Answer: It has been edited.

13. Line 297. "However, an A (adenine) deletion was identified..." a figure or some text showing this 13 bp repeat sequence with mutation might be useful.

Answer: The 13-bp inverted repeat sequence with an adenine deletion position in the mtrR promoter region has been added.

14. Line 331. "a QAATPAKQ motif"?

Answer: It has been edited to “a QAATPAKQ insertion” because this is an insertion mutation of QAATPAKQ motif.

15. Line 346. Specify "The conjugative plasmids".

Answer: It has been specified.

16. Line 380. "encoding a hypothetical protein"

Answer: It has been corrected.

17. Line 391. "resistance determinants and plasmids of..."

Answer: It has been corrected.

18. Line 407. Delete 2nd "was" - "Agar dilution was preferred because...". Also - it is unclear to me whether this is a CLSI recommendation?

Answer: It has been deleted. The disk diffusion and agar dilution methods were recommended by the CLSI for testing antimicrobial susceptibility in N. gonorrhoeae isolates. However, agar dilution susceptibility testing is the “gold standard” for susceptibility testing of N. gonorrhoeae. The suitable method depends on the time, availability of resources, and research aims.

19. Line 494-495. "The penicillin acylation rate was decreased to...". Please clarify - this was not shown here? "was previously found to be decreased..."?

Answer: This sentence referred to the previous report cited in reference (54).

20. Line 535. This sentence is unclear and should be re-written - you have not provided the first genomic analysis of all plasmids in N. gonorrhoeae strains from Thailand?

Answer: The given sentence has been rewritten by excluding overstatement.

Reviewer #2

Abstract/ Introduction

1. Adding statement to justify the importance of study regionally and globally;

As this study is from Thailand (the study region), giving a general impact of gonococcal infection and the effect of AMR in the study region would elaborate on the relevance and importance of the study locally and globally.

Answer: The statement regarding the importance of study regionally and globally has been added in the introduction section. Continued antimicrobial surveillance (including molecular studies) as in this study is necessary to detect patterns of resistance to ensure treatment efficacy against gonococcal infection at regional and national level. With regards to this surveillance, it can help to provide additional details on the emergence and dissemination of resistant gonococcal strains, so that international countries can make control and prevent these strains. This is due to the fact that most of ceftriaxone-resistant isolates have emerged in Asia and subsequently spread globally. Additionally, one of the gonococcal strains with ceftriaxone resistance combined with high-level resistance to azithromycin was linked to Thailand. Quality-assured local antimicrobial resistance surveillance data could also partially help to improve the WHO global gonorrhoeae treatment guidelines.

2. Justification of sample size/patient recruitment

Answer: The sample size was consulted with a statistician of our institution and calculated based on the prevalence of cefixime- or ceftriaxone-resistant N. gonorrhoeae isolates using a sample size application (called “n4Studies”). The first aim of this study was to investigate the presence of those isolates using antimicrobial susceptibility testing. Since cefixime- or ceftriaxone-resistant N. gonorrhoeae isolates have not been reported in Thailand and barely found in other countries, a statistician suggested that the more sample you collect, the more chance you will get these isolates. Therefore, 117 gonococcal isolates we had in the retrospective collection were used for the study. There is no patient recruitment.

3. Justification of only 03 strains analysis

Answer: To uncover penicillin resistance determinants and plasmid composition, we have selected 3 N. gonorrhoeae strains from a total of 18 strains based on their MIC to penicillin (MIC ≥ 128 μg/ml) and different NG-MAST STs. The number of gonococcal strains is limited due to time constraints and shrinking budgets. Furthermore, our purpose is to provide a brief overview and discussion of the results.

4. Abstract line 32; mtrR were performed. Need to define mtrR

Answer: Additional information has been added.

5. Line 38: notifying the significance of the genes/genetic characteristics will highlight the importance of the study further

Answer: The significance of genes and their mutations associated with antimicrobial resistance in N. gonorrhoeae has been highlighted in the introduction as well as the discussion section.

6. 46, 47 The gonococcal population in Thailand showed high susceptibility to all antimicrobials 47 recommended for gonorrhoea treatment.

But the line 37: All isolates were susceptible to spectinomycin, can you clarify the comment in lines 46,47

Answer: The sentence in lines 46 and 47 is ambiguous, indeed it actually means that the gonococcal population in Thailand showed high susceptibility to antimicrobials currently recommended for gonorrhea treatment, ceftriaxone and azithromycin. However, this sentence has been edited.

7. 48: expanded and enhanced surveillance of antimicrobial susceptibility 49 and study of genetic resistance determinants are essential to improve treatment guidelines.

Can you clarify why this is needed by considering the actual prevalence of gonococcal infection prevalence in Thailand?

Answer: Gonorrhea is one of the most commonly reported STD in Thailand. Prevalence of gonorrhea was estimated to be 13.1 per 100,000 population in 2015 (as available). In general, gonococcal infections acquired in or from Asia represent most verified ceftriaxone treatment failures, and several ceftriaxone-resistant strains have emerged in Asia and subsequently spread globally. In Thailand, 0.5% of isolates showed resistance or decreased susceptibility to ceftriaxone. Taking these aspects into account, expanded and enhanced surveillance of antimicrobial susceptibility in N. gonorrhoeae is imperative to inform the Thailand national treatment guideline. Nonetheless, limited geographical representativeness of the isolates (mostly from Bangkok) and the lack of periodic antimicrobial susceptibility surveillance are a hassle.

Introduction

8. Line 74: What is the significance of gonococcal infection prevalence in Thailand?

Answer: The significance is in the lines 90-93 about emerging ESBL NG producers.

9. Line 90; (M182T) should be referred to as strain?

Answer: M182T is referred to an amino acid substitution. This information has been added.

10. Line 122: should bring to earlier and also compare with global statistics to show why this study is important to Thailand

Answer: This paragraph has been moved to earlier. The global cases of ceftriaxone-resistant gonococcal strains in other countries are reported and referred in the lines 65-71, comparing to Thailand where these strains have never been reported. Since ceftriaxone-resistant strains remain rare, a statement underlining the presence of these strains should be better used to elaborate. The importance of this study to Thailand is given in the lines 145-148. Additional details have been added in lines 67 and 125-126.

11. Line 155: do the participants considered have received the antibiotic treatment before the study?

Answer The study was retrospective and no patient involvement. The clinical data on previous antibiotic exposure of patients are difficult to retrieve and largely unavailable; therefore, it has not been considered for this study.

12. What is the statistical method used for Patient data and Prevalence and penicillin susceptibility of PPNG isolate

Answer: No statistical methods were used for analyzing patient data and prevalence and penicillin susceptibility of PPNG isolates.

13. Line 255: Do the study participants give their consent if so mention this in this section.

Answer: No informed consent was required due to the retrospective nature of the study. In addition, this non-clinical study used bacterial isolates recovered from clinical samples taken as part of routine clinical laboratory examination and patient information was anonymized and deidentified prior to analysis. The ethical reviewing committee has approved the study as non-human research. This statement has been added in the ethical consideration section.

14. Line 181: interpretative criteria of the guidelines; can you clarify further with regards to MIC

Answer: The obtained MIC values were interpreted based on the interpretative criteria of the CLSI 2020 guidelines as follows: penicillin, S≤0.06, I=0.12-1, R≥2; tetracycline, S≤0.25, I=0.5-1, R≥2; ciprofloxacin, S≤0.06, I=0.12-0.5, R≥1; spectinomycin, S≤32, I=64, R≥128; cefixime, S≤0.25; ceftriaxone, S≤0.25. “CLSI” has been added to specify guidelines.

15. Line 183; What is the coverage of the N gonorrhoeae genome? Are the AMR determinants considered located in short or long read sequences? Justify the usage of both sequencing methodologies.

Answer: The coverage of each N. gonorrhoeae is the number of bases that cover the genome. It describes how frequently, on average, based from read coverage of the genome sequence (Table x1). The AMR genes of N. gonorrhoeae is covered by both short- (illumina) and long-reads (Nanopore) as shown in Table x2 and Figure x1. As example, we plotted read coverage of each AMR genes of N. gonorrhoeae strain CT530 in Figure x1 using IGV software. The methodology of de novo assembly was written in the method section. The AMR genes were based on the literature review and predicted from each assemble genome using Resistance Gene Identifier software (v.5.1.0, https://github.com/arpcard/rgi) against the Comprehensive Antibiotic Resistance Database (v.3.0.9, https://card.mcmaster.ca/). Whole-genome sequencing with both Illumina and Nanopore platforms was performed to generate complete genome sequences of N. gonorrhoeae. As a result, we can obtain the full-length of all antimicrobial resistance genes and the entire plasmid sequences for analysis.

Table x1. The read coverage of the N. gonorrhoeae genome

STRAIN GENOMESIZE ILLUMINA_TOTAL_BASES ILLUMINA_COV NANOPORE_TOTAL_BASES NANOPORE_COV

CT530 2220590 1546958433 697 488748095 220

CT532 2216438 1469088082 663 472676167 213

CT602 2211092 1747893851 791 1118067306 506

Table x2. The read coverage of AMR genes

CONTIG GENE CT530 CT532 CT602

Nanopore Illumina Nanopore Illumina Nanopore Illumina

PLASMID blaTEM-1-WT 24826 16869 33518 24732 52891 30099

CHROMOSOME dacB (PBP3)-WT 80 624 72 570 246 653

CHROMOSOME dacC-WT 89 562 58 479 248 647

CHROMOSOME mtrR-WT 83 537 73 480 222 546

CHROMOSOME pbpG (PBP4)-WT 84 565 59 498 244 600

CHROMOSOME penA (PBP2)-WT 89 632 65 560 216 658

CHROMOSOME pilQ-WT 93 589 64 536 212 647

CHROMOSOME ponA (PBP1)-WT 101 620 65 580 237 681

CHROMOSOME porB1b-WT 113 686 75 654 273 755

Figure x1. The example of AMR genes in N. gonorrhoeae strain CT530

16. Line 465 what is the significance of the MLST ST8143, clarify

Answer: In Taiwan, the MLST ST8143 isolates were abruptly emerged after 2012. These isolates may follow the pattern of ST1901 and become highly prevalent locally and globally; therefore, they should be monitored. Of note, the circulating ST1901 strains generally exhibited higher MICs for both ceftriaxone and azithromycin. Moreover, the MLST ST8143 isolates were predominantly identified in the United States and less found in Ireland. The significance of the MLST ST8143 has been added.

17. Line 528: is there a positive correlation between the MIC and the corresponding genetic determinants/ it is not very clear

Answer: There is no experiment to show a positive correlation between the MIC and the penicillin resistance determinants in this study. Nonetheless, we made an attempt to correlate the high penicillin MIC value (≥ 128 μg/ml) of 3 representative gonococcal strains with well-known penicillin resistance determinants (penA, mtrR, penB and blaTEM-135). All specified mutations in these resistance determinants stated in the sentence have been previously studied and shown to increase penicillin MICs.

18. Line 535: how to justify the statement; We provide the first genomic analysis of all plasmids in N. gonorrhoeae strains from Thailand. Clarify it further

Answer: This statement is overstated in terms of novelty. The sentence has been edited.

19. Line 555: it is worth mention the previous antibiotic treatment data is also not available

Is the risk factors referring to the patients?

Answer: The lack of previous antibiotic treatment data of patients has been added. The risk factors refer to the patients. Additional details have been added.

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Decision Letter 1

Ayesha Sabah Rahman

6 Jul 2022

Whole-genome sequence analysis of high-level penicillin-resistant strains and antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from Thailand

PONE-D-22-11066R1

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Associated Data

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    Supplementary Materials

    S1 Table. Genome sequencing and assembly statistics for 3 Neisseria gonorrhoeae strains with high-level resistance to penicillin.

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    S2 Table. Specimen source and antimicrobial susceptibility of Neisseria gonorrhoeae isolates from Thailand, 2015–2017.

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