Background

The sexually transmitted infection (STI) gonorrhea caused by the bacterium Neisseria gonorrhoeae remains a major global public health concern because of its capacity to evolve high levels of resistance to antibiotics available for treatment [1, 2]. The superbug has developed plasmid-mediated and/or chromosomally mediated antimicrobial resistance (AMR) that has compromised the management of gonorrhea worldwide [3]. The AMR mechanisms are usually present in the gonococcal cell and/or a combination of genes with mutations within specific genes to cause resistance to antibiotics [4,5,6]. The World Health Organization (WHO) has declared N. gonorrhoeae as a priority pathogen because of its resistance to third-generation cephalosporins (3GS) and fluoroquinolones [7].

Whole genome sequencing (WGS) technology has allowed the tracking of transmission and prediction of AMR to control gonococcal infections [8,9,10]. Multi-locus sequence typing (MLST), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) are different typing tools that have been used to study the molecular epidemiology of N. gonorrhoeae in terms of genetic lineages and clonal relationships to control the spread of drug-resistant genotypes [11,12,13,14,15]. MLST is based on the detection of sequence variation using seven conserved housekeeping genes, putative ABC transporter (abcZ), adenylate kinase (adk), shikimate dehydrogenase (aroE), furamase hydrase (fumC), glucose-6-phosphate dehydrogenase (gdh), pyruvate dehydrogenase subunit (pdhC), and phosphoglucomutase (pgm) [12]. The NG-MAST analyses are based on the variable internal fragments of highly polymorphic porin B (porB) and transferrin binding protein B (tbpB) [16]. The NG-STAR is based on AMR determinants (penA, mtrR, porB1b, ponA, gyrA, parC, and 23rRNA) [17]. However, the cost of WGS remains high and is not available in many parts of Africa [13, 18].

In 2020, the World Health Organization (WHO) estimated 82.4 million incident global cases of gonorrhea among adults 15–49 years of age [2]. The highest incidence rate of gonorrhea was found in sub-Africa with an increase of 0.2% in women and 1.1% in men every year [19, 20]. The development of effective vaccines and novel therapeutics would mitigate the emergence and spread of untreatable gonorrhea [21]. Gonorrhea can be concomitant with HIV and enhances its transmission [22,23,24,25,26].

The treatment of STIs is according to syndromic management guidelines which have contributed highly to the AMR due to empirical treatment in sub-Sahara Africa [27,28,29,30]. The Zambian standard treatment guidelines recommended the use of a single dose of ciprofloxacin in the treatment of gonorrhea (Ciprofloxacin 500 mg PO stat plus doxycycline 100 bd PO X 7/7) [31]. The WGS data that provides opportunities to understand the population structure of N. gonorrhoeae for prevention and control of gonorrhea was sparse despite AMR being an emerging phenomenon in Zambia [32].

This study aimed to determine the genetic diversity of N. gonorrhoeae isolated from patients attending STI clinics in urban hospitals in Lusaka, Zambia.

Methods

Study Design and Population

A cross-sectional study on 38 Neisseria gonorrhoeae isolated from 122 patients with gonorrhea attending STI clinics in urban hospitals in Lusaka, Zambia. The urethral and endocervical specimens were collected from patients who presented with a discharge from September 2019 to August 2020. The gonococcal isolates were submitted to the University Teaching Hospital (UTH) molecular laboratory for antimicrobial susceptibility testing (AST) and DNA extraction, and to Kilimanjaro Clinical Research Institute (KCRI) at the biotechnology laboratory (KCRI-BL) for molecular testing. The KCRI-BL is GCLP accredited, ISBN 978-1-904610-00-7 operated by Qualogy.

Antimicrobial susceptibility testing

The minimum inhibitory concentrations (MICs; µg/mL) of the ciprofloxacin, ceftriaxone, spectinomycin, azithromycin, penicillin, and tetracycline were determined by the E-test (bioMerieux, Marcy-I’Etoile, France), on GC-chocolate with 1% Vitox supplement (Beckton Dickison, France) following the manufacturer’s instructions. The interpretation of MIC dilutions in susceptible (S), intermediate (I) and resistance (R) categories was according to Clinical and Laboratory Standard Institute (CLSI) criteria [33]. The plates were inoculated by dipping a sterile swab into a bacterial cell suspension adjusted to 0.5 McFarland standards using a turbidometer (Oxoid Integrated Technologies Ltd, England). The standardized inoculum was then streaked across the surface of the GC-chocolate agar. The plates were dried at ambient temperature for 5 min before applying the E-test strips and incubated at 36oC ± 1oC in 5% CO2 for 24 h. The SIR categories for antimicrobial agents in µg/mL were as follows: Ciprofloxacin (CIP) S; ≤0.06, I; 0.12–0.5, R; ≥1, ceftriaxone (CTX) S; ≤0.25, R; >0.25, spectinomycin (SPEC) S; ≤32, I; 64, R ≥ 128, cefixime (CFX) S; ≤0.25, R; >0.25, azithromycin (AZT) S; ≤1, R; >1, penicillin (PEN) S; ≤0.06, I; 0.12-1, R; ≥2, and tetracycline (TET) S; ≤0.25, I; 0.12-1, R; ≥2. Neisseria gonorrhoeae American Type Culture Collection (ATCC) 49,226 was used as a reference strain and was within the acceptable quality control ranges.

Extraction of genomic DNA

DNA was isolated using the NucliSens easyMaG Nucleic Extraction platform (BioMerieux, Marcy-I’Etoile, France), according to the manufacturer’s instructions. A loopful of Neisseria gonorrhoeae from pure cultures grown on chocolate agar (Mast Diagnostics, Merseyside, UK) were transferred into a microcentrifuge tube containing 400µL of 1x TE buffer (10mM Tris-HCl [pH 8.0], 0.1mM EDTA [pH 8.0]) for the bacterial suspension. The bacterial preparation was transferred to the sample strip well of the extractor with the elution of 50µL. The DNA preparation was then preserved at -20oC before further analysis.

DNA quantification and sequencing

The cgDNA was quantified using a Qubit V4.0 fluorometer (Invitrogen by Thermo Fisher Scientific). The volume of 2µL of DNA was quantified before sequencing. The whole genome sequencing (WGS) was performed using Illumina DNA library preparation protocol, document 1,000,000,025,416 v09 (Illumina Inc., San Diego, CA, USA). Input cgDNA of 100ng was used for library preparation. The amplified DNA library was cleaned using double-sided beads and purified in resuspension buffer (RSB). The paired-ends 150 bp indexed reads were generated using the mid-output protocol on the Illumina Nextseq550 platform (Illumina Inc., San Diego, CA, USA).

Bioinformatics analyses

The raw sequence data was checked using FastQC 0.11.9 and screened for contamination using FastQScreen 0.15.1 against the Knead-Data human reference with decoy, and NCBI UniVec Core [34,35,36]. Untrimmed reads were processed by the KCRI-CGE Bacterial Analysis Pipeline (BAP) 3.6.5 using default settings [37, 38]. The BAP workflow was comprised of genome assembly with SKESA 2.4.0 and computation of assembly metrics with uf-stats 1.3.1 [39, 40]. Identification of N. gonorrhoeae species was performed with KmerFinder 3.0.2, and MLST typing with CGE MLST 2.0.9 and KCST 1.2.6 [41,42,43,44,45,46]. The AMR detection with ResFinder 4.2.3 and plasmid identification with PlasmidFinder 2.1.6 and pMLST 2.0.3 [47, 48] while the core genome MLST assignment was performed with cgMLSTFinder 1.1.5 [43, 45]. The assemblies were assessed for genome completeness and bacterial contamination with CheckM 1.2.2 and GUNC 1.0.5 + post1 [49, 50]. The Reference was made of 160 genomes for Neisseria gonorrhoeae with assembly level “Complete Genome” or “Chromosome” downloaded from NCBI RefSeq on 17 Oct 2022 and were annotated with their MLST using KCST 1.2.6 [14]. Pairwise average nucleotide identities (ANI) were computed between all study genomes and all reference genomes with FastANI 1.33 [51]. A phylogenetic tree was estimated from genome assemblies using SANS serif in ‘strict’ mode using default settings [52].

Results

A total of 38 isolates of N. gonorrhoeae were successfully sequenced and various sequence types (ST) were identified. The most frequent STs with 18.4% (7/38) of isolates each were ST7363, ST1921 and ST1582, followed by ST1583 (13%), novel ST17026 (7.9%), ST1588 (7.9%), ST1596 (5.3%) and ST11181 (5.3%). The ST11750 and ST11241 (2.6%) each had one representative (Fig. 1).

Fig. 1
figure 1

Frequency and percentage of sequence types of N. gonorrhoeae

Full size image

The majority of isolates contained both blaTEM-1B and tetM (55%, 21/38), while 18.4% (7/38) contained blaTEM-1B only, 15.8% (6/38) contained tetM only, and one isolate contained blaTEM-1B, ermT, and tetL (Fig. 2). The Beta lactamase plasmid TEM-1 was detected in 84.2% (32/38) and gonococcal genetic island (GGI) was detected in all isolates.

Fig. 2
figure 2

Frequency and percentage of AMR determinants of N. gonorrhoeae

Full size image

The most prevalent combination of mutations of the isolates 23.7% (9/38) was gyrA (S91F), parC (E91G), ponA (L421) and rpsJ (V57M) followed by 18.4% (7/38) in gyrA (S91F), ponA (L421P), rpsJ (V57M), and 18.4% (7/38) in gyrA (D95G, S91F), ponA (L421P), and rpsJ (V57M). The combination of mutations 13.2% (5/38) gyrA (D95G, S91F) and rpsJ (V57M), and 13.2% (5/38) in the combination of gyrA (D95G, S91F), parC (E91F), ponA (L421P) and rpsJ (V57M) were detected. All isolates showed mutations in gyrA and rpsJ with S91F and V57M substitutions respectively (Fig. 3).

Fig. 3
figure 3

Frequency and percentage of AMR mutations from 38 N. gonorrhoeae isolates

Full size image

The isolates with MLST ST7363, ST1921, ST1582, ST1588, ST1596, ST11181, and ST11750 were associated with ciprofloxacin, penicillin and tetracycline resistance with a combination of blaTEM-1B and tetM AMR genetic determinants. Phenotypic resistance to tetracycline was detected in isolates carrying tetM and rpsJ genes while 2.6% of the isolates each exhibited AMR phenotype to cefixime and azithromycin (Table 1). The G45A deletion in mtrR promoter was identified in 10.6% (4/38) of the isolates. All the isolates had non-mosaic penA alleles which were associated with susceptibility to ceftriaxone. The identical F504L type II non-mosaic penA allele: 2.002 penicillin MIC 0.5 µg/mL was detected in 66% (25/38), and Type XIX non-mosaic penA allele: 19.001 penicillin MIC 0.5 µg/mL was detected in 34% (13/38) of the isolates.

Table 1 Phenotypic and genotypic characterization of isolates in MLST clusters (n = 38)
Full size table

The multiple sequence alignment CCphylotree was generated using the reference strain WHO O with GenBank accession number NZ_LT592146/GCF_900087625 with MLST sequence type ST1902 because the majority of the isolates were closest to that reference strain. The multiple sequence alignments included 160 reference genomes for N. gonorrhoeae at assembly levels “Complete Genome” and “Chromosome”, retrieved on 17 Oct 2022. The isolates clustered amongst themselves rather than around the reference strain (Fig. 4). The clades showed inter-clade distances of 5000 SNPs.

Fig. 4
figure 4

MLST phylogenetic tree of 37 Neisseria gonorrhoeae isolates with WHO O reference strain with GenBank accession number NZ_LT592146.1 in Lusaka, Zambia. Symbol key: NOV = Novel sequence types. The scale bar represents the estimated evolutionary divergence of the isolates

Full size image

Discussion

The AMR of N. gonorrhoeae has been on the increase and is considered a public health challenge in various regions around the world [7]. Nowadays, WGS has been used widely in the typing and monitoring of resistant strains of N. gonorrhoeae [53]. Data presented in this study showed genetically diverse N. gonorrhoeae having 10 different MLSTs, which includes novel ST17026 identified in 8% (3/38) of the isolates. The three isolates with novel ST shared a single MLST profile with a novel combination of abcZ:109 adk:39 aroE:67 fumC:771 gdh:148 pdhC:71 pgm:65 [54]. The substantial number of the novelty of the isolates corresponds with findings observed in South Africa [55]. The ST7363, ST1921, and ST1582 representing 55% (21/38) were the most circulating sequence types in Lusaka, Zambia, and highly resistant to ciprofloxacin, penicillin, and tetracycline [33]. The combinations of amino substitutions in the gyrA (S91F, D95G) and parC (E91G) were associated with resistance to ciprofloxacin. The study findings are in agreement with studies in Tanzania and South Africa where 70% of the isolates with MLST ST7363 and ST1901 were resistant to ciprofloxacin, penicillin, and tetracycline [59, 60]. Penicillinase-producing N. gonorrhoeae (PPNG) strains 84.2% (32/38) were detected in all the STs, and 2.6% (1/38) of the isolates had reduced susceptibility to cefixime with MIC 0.75 µg/mL (Additional file 1: Table S1). The PPNG isolates are highly prevalent and the global concern is that the blaTEM−1 gene encoding TEM−1 β-lactamase requires few specific single nucleotide polymorphisms (SNPs) to evolve into a gene encoding an extended-spectrum β-lactamase (ESBL) which could degrade all cephalosporins including ceftriaxone [61, 62]. All isolates harbored the GGI which is a type IV secretion system (T4SS) implicated in AMR to multiple antimicrobials [63]. Mutations within the mtrR gene are usually found amongst multidrug-resistant isolates of N. gonorrhoeae and are associated with the outflow of antimicrobials from the bacterial cell [64]. Furthermore, 2.6% of the isolates exhibited resistance to azithromycin, and all the isolates were susceptible to ceftriaxone [33]. The rise in the number of reports on treatment failures with 3GS demands urgent development of new antimicrobials for the treatment of gonorrhea and increased AMR surveillance remains vital to the prevention and control of gonorrhea worldwide [65–67].

Conclusion

The genomic analysis of the study showed a remarkable genetic diversity of N. gonorrhoeae with blaTEM−1, tetM, ponA, gyrA, and parC genes associated with high resistance to penicillin, tetracycline and ciprofloxacin demanding for revision of the Zambian standard treatment guidelines. The detection of N. gonorrhoeae resistant to azithromycin demands improved antimicrobial stewardship to prevent an epidemic of untreatable gonorrhea in Zambia.

Limitation of the study

The isolates studied were only from hospitals in Lusaka and might not be representative of other settings in Zambia.