Seven Years of Culture Collection of Neisseria gonorrhoeae: Antimicrobial Resistance and Molecular Epidemiology

The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobials, in particular to ceftriaxone monotherapy or ceftriaxone plus azithromycin, represents a global public health concern. This study aimed to analyze the trend of antimicrobial resistance in a 7-year isolate collection retrospective analysis in Italy. Molecular typing on a subsample of gonococci was also included. A total of 1,810 culture-positive gonorrhea cases, collected from 2013 to 2019, were investigated by antimicrobial susceptibility, using gradient diffusion method, and by the N. gonorrhoeae multiantigen sequence typing (NG-MAST). The majority of infections occurred among men with urogenital infections and 57.9% of male patients were men who have sex with men. Overall, the cefixime resistance remained stable during the time. An increase of azithromycin resistance was observed until 2018 (26.5%) with a slight decrease in the last year. In 2019, gonococci showing azithromycin minimum inhibitory concentration above the EUCAST epidemiological cutoff value (ECOFF) accounted for 9.9%. Ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae (PPNG) percentages increased reaching 79.1% and 18.7% in 2019, respectively. The most common sequence types identified were 5,441, 1,407, 6,360, and 5,624. The predominant genogroup (G) was the 1,407; moreover, a new genogroup G13070 was also detected. A variation in the antimicrobial resistance rates and high genetic variability were observed in this study. The main phenotypic and genotypic characteristics of N. gonorrhoeae isolates were described to monitor the spread of drug-resistant gonorrhea.


Introduction
A ntimicrobial resistance is the main issue of Neisseria gonorrhoeae and represents a global public health emergency. 1,2 In 2018, 100,673 cases of gonorrhea were reported by 28 European Union (EU)/European Economic Area (EEA) with the overall notification of 26.4 cases per 100,000 population. 3 In Italy, 905 confirmed cases were described in 2018, with a rate of 1.5 per 100,000 population. 3 As described, 3 gonorrhea cases are likely underestimated and underreported for the majority of EU/EEA countries and, N. gonorrhoeae isolates resistant to antimicrobials used previously for treatment (sulfonamides, penicillin, tetracycline, fluoroquinolones) and/or currently in use (third-generation extended-spectrum cephalosporins-ESCs and broad-spectrum macrolides) have been widely spread all over the world. [4][5][6] In 2019, the European Gonococcal Antimicrobial Surveillance Program (Euro-GASP) 7,8 reported a decrease in gonococci resistant to cefixime compared with 2018 (0.9% vs. 1.4%). 8,9 Ceftriaxone resistance is very rare in Europe and, in 2019, three isolates were described in Norway, Portugal, and Belgium, 8 instead, gonococci showing azithromycin minimum inhibitory concentration (MIC) above the EUCAST epidemiological cutoff value (ECOFF; MIC >1 mg/L) increased from 7.6% (in 2018) to 10.1% (in 2019) and 15 high-level resistant isolates (HLR, ‡256 mg/L) were reported. 8 Similarly, in 2019, the ciprofloxacin resistance significantly increased (57.3%) compared with 2018 (50.4%) 8,9 and the percentage of Penicillin-Producing Neisseria gonorrhoeae (PPNG) was 14.9% in 2016, in Europe. 10 In Italy, the percentages of isolates resistant to cefixime and azithromycin were 17.1% and 23.7%, respectively, in 2009 and decreased to 3.9% and 3% in 2012 11 ; no ceftriaxone-resistant isolates were found. 11 Furthermore, in 2012, 64% of gonococci were resistant to ciprofloxacin and 7% were PPNG. 12 Many countries report declining in vitro susceptibility of azithromycin and ceftriaxone plus azithromycin treatment failures were described, which is a concern because azithromycin and ceftriaxone are the current recommended dual treatment for gonorrhea. [13][14][15] Moreover, the Centers for Disease Control and Prevention (CDC) 16 and the British Association for Sexual Health and HIV (BASHH) 17 revised the guidelines due to the spread of azithromycin resistance and the emergence of ceftriaxone resistance, among gonococci, worldwide, 14,15,18 recommending ceftriaxone as monotherapy and the use of azithromycin as a second-line treatment to reduce the selective pressure on gonococcal isolates. 16 Due to this alarming situation, both the World Health Organization (WHO) and the European Center for Disease Prevention and Control (ECDC) 2,7-9 have developed action plans to control the spread of antimicrobial resistance in N. gonorrhoeae focusing on programs to collect clinical and microbiological data with a special attention to antimicrobial susceptibility pattern.
For this purpose, the surveillance of N. gonorrhoeae antimicrobial susceptibility permits to detect emerging and increasing antimicrobial resistance, with particular regard to those suggested for gonorrhea therapy.
Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) has been used globally to type gonococcal isolates. 19,20 As previously described in a pilot study of 2018, the G1407 clone spread in Europe 19 and globally with a prevalence of 23.3% in the period 2009-2010. 19,20 This clone is characterized by cefixime and ciprofloxacin resistance 19 and reduced susceptibility or resistance to azithromycin. 19 In the 2013 European study, 21 G1407 was confirmed as the predominant genogroup, however, a decrease in the percentage of ST1407 (7.6% in 2013 vs. 15.6% in 2009-2010) was observed.
The aims of this study are to describe the proportion and trends of N. gonorrhoeae resistance to previous and current antimicrobial treatment options, together with the genotypic analysis by NG-MAST. A retrospective evaluation, from 2013 to 2019, on antimicrobial susceptibility and molecular epidemiology of gonococci isolated in Italy is reported in this study.

Neisseria gonorrhoeae isolates and patient data
In this study, from January 2013 to December 2019, 1,810 culture-positive gonorrhea cases were investigated, of which 1,525 (84.2%) viable isolates were available. Primary isolation, identification, and collection of gonococci were completed following standard microbiological procedures by the 18 collaborating laboratories from universities and sexually transmitted infection and dermatology/venereology clinics (11 in the North, 5 in the Center, and 2 in the South of Italy) collaborating with Istituto Superiore di Sanità (ISS). During the study period, 11 collaborating laboratories, out of 18, provided isolates annually; whereas, 4 not for entire period and 3 joined the network since 2018.
Briefly, gonococci, after growth at 37°C in a 5% CO 2 atmosphere on Thayer-Martin agar plates, were stored at -80°C in brain heart infusion medium (Oxoid, Ltd.) with 20% glycerol. Cultivated isolates were sent bimonthly to ISS.
Unlinked pseudoanonymous data of patients were received and recorded by ISS using Epi Info software (version 3.5.4, 2012).

Patient characteristics
Taking into account the 7 years, 1,786 patients with one infection episode at a single anatomical site were included. Two episodes of infection were reported for 7 patients and 86 CARANNANTE ET AL. lected in two or three infected anatomical sites for a total of 1,810 culture-positive gonorrhea cases (Supplementary Table S1). The infections were confirmed by culture, microscopy, and/or nucleic acid amplification tests (NAATs). Culture was the most common diagnostic test accounting for 30.6% of confirmed cases. NAAT was used to diagnose 1.6% of cases and microscopy 3.3%. Moreover, it was reported as 21.9% for the use of culture plus microscopy, 17.5% for culture, microscopy plus NAAT, 13.1% for culture plus NAAT (13.1%), and 12.0% for microscopy plus NAAT. Among 1,786 patients, 95.8% were men (median age 34.5 years) and the 57.9% were men who had sex with men (MSM). Women accounted for 4.2% (median age 35.3 years).
The samples used to diagnose N. gonorrhoeae were cervical or urethral discharge; cervical, vaginal, anorectal, urethral, or pharyngeal swabs; blood; urine; seminal fluid; and peritoneal liquid. As shown in Supplementary Table S1, 85.1% were genital infections, 11.5% anorectal, 2.5% pharyngeal, and 0.8% others, including peritonitis as PID complication in a woman. 22 Finally, the majority of patients reported Italy as a possible country of infection (95.6%).
After the introduction of the azithromycin ECOFF value, the MIC values were interpreted by referring to both the 2018 and 2021 EUCAST clinical breakpoint criteria (version 8.1, 2018 and version 11, 2021). 23,24 For testing purposes, the azithromycin ECOFF is 1 mg/L, 23 and isolates with MIC values >1 mg/L were considered resistant to azithromycin. For gentamicin, the breakpoints were not available. According to the Euro-GASP Reporting Protocol, 7 for the isolates with cefixime MIC value ‡0.25 mg/L and azithromycin MIC value ‡256 mg/L, the MIC was repeated and the identification was confirmed. The World Health Organization (WHO) N. gonorrhoeae G, K, M, O, and P reference strains were used as controls. 25

NG-MAST and phylogenetic analysis
Gonococcal DNA was extracted using the QiAmp Mini Kit (Qiagen, Hilden, Germany) from an overnight culture, following the manufacturer's procedure.
NG-MAST was performed on 743 DNAs from viable isolates selected by year (48.7%). All the cefixime and azithromycin-resistant gonococci were included in the analysis. The porB and tbpB alleles were amplified as previously described 26 and sequence types (STs) were assigned using the NG-MAST website, following the interpretative procedures. 26 Closely related STs were defined using the published definition, 19,21 as well as, the genogroup (G) definition. 19,21 Multiple sequence alignments were performed using CromasPro version 2.6.6 and the Clustal Omega website.
For each ST, by NG-MAST, excluding the singletons that did not belong to any genogroup, porB and tbpB sequences were concatenated and imported into the alignment tool. A phylogenetic tree was constructed using MEGA6 software following the maximum likelihood method (Kimura 2parameter model, gamma distributed).

Comparative genomic analysis
Whole-genome sequencing (WGS) was performed using the Illumina HiSeq platform, as previously described. 27 The assemblies were uploaded in the pubMLST database running the BIGSdb genomic platform. 28 The genetic relationship between a subsample (n = 55) of Italian gonococcal genomes, randomly selected, and a panel of N. gonorrhoeae genomes (n = 521), available from other European countries, were evaluated. Genomes of gonococcal isolates from European countries were selected according to the year of isolation, source of isolation, clinical manifestations, and age of patients available on the PubMLST Neisseria database (accessed 12/05/22; See Supplementary Table S2 for the isolate details).
Genome comparison was performed through a gene-bygene approach using the core genome MLST (cgMLST) scheme, referring to a set of 1,668 loci of the gonococcal genome (N. gonorrhoeae cgMLST V.1.0) available on PubMLST.org/neisseria. Incomplete loci were automatically removed from the distance matrix calculation for the neighbor-net graphs. A core genome threshold of 95% were considered for comparison. The resulting distance matrix was visualized as a Neighbor-net in SplitTree4 (version 4.17.1).

Statistical analysis
p-value was calculated by Chi-square for linear trend (Extended Mantel-Haenszel; EpiCalc 2000 statistical calculator) of antimicrobial resistance rate and genogroups. A p-value of 0.05 was considered statistically significant. One-way ANOVA statistical analysis was performed by years of data collection to investigate the effect of the year on the MIC values' distribution. The mean was found statistically significant at p < 0.0001. Statistical analysis was performed using GraphPad Prism software version 6.0.

Antimicrobial susceptibility
The percentage of resistant N. gonorrhoeae by antimicrobial and year on 1,525 viable isolates is shown in Fig. 1.
The proportion of gonococci resistant to cefixime remained quite stable: 2.3% in 2013 versus 2.7% in 2019, ( p = 0.6454). The lowest cefixime resistance percentage was recorded in 2016 (1.1%) and no resistant gonococci were identified in 2015. During the period, none of the 33 isolates resistant to cefixime showed HLR to azithromycin and a MIC value close to breakpoint of 0.125 mg/L for ceftriaxone.
Referring to 2018 EUCAST guidelines, the azithromycin resistance increased from 4.6% in 2013 to 26.5% in 2018, ( p < 0.0001) with a slight decrease in 2019 (24.3%). Considering the most recent EUCAST breakpoints published in 2021, the percentage of gonococci showing azithromycin MIC value above the ECOFF of 1 mg/L increased from 2.3% in 2013 to 12.3% in 2018. In 2019, about 10% of the isolates showed a MIC above the ECOFF value for azithromycin. Isolates with MIC values of 0.75 or 1 mg/L were considered susceptible to azithromycin.
Ten isolates showed a multidrug-resistant (MDR) pattern for azithromycin, cefixime, and ciprofloxacin. Six gonococci were resistant to azithromycin, ciprofloxacin, and resulted PPNG.
No resistance to ceftriaxone (MIC >0.125 mg/L) or spectinomycin (MIC >64 mg/L) was reported. For ceftriaxone, spectinomycin, and gentamicin the mean MIC values were found significantly different during the 7-year study period ( p < 0.0001).

Neisseria gonorrhoeae multiantigen sequence typing
On a subsample of 743 gonococci, 288 STs, of which 182 comprise a single isolate, were identified. Onehundred thirty-seven STs had not been previously described: 76 due to a new porB allele, 12 for a new tbpB allele, 3 for both of these alleles, and 46 for a new combination of them.
A total of 390 gonococci showed closely related STs and clustered together in 20 different genogroups (G) (Fig. 2B); the STs included in the main genogroups are shown in Table 1.
All the 33 gonococci resistant to cefixime (isolated from 2013 to 2019) were typed resulting in 22 different STs, of which 13 belonged to G1407 with the prevalent type ST1407 (n = 6).
Fourteen gonococci, out of 19 with ceftriaxone MIC values close to breakpoint of 0.125 mg/L, were distributed into 11 different STs, of which 5 belonged to G1407.
The seven azithromycin HLR gonococci (MIC ‡256 mg/L) had diverse epidemiological, phenotypic, and molecular characteristics showing low linkages with each other, except for the three isolates with ST3935 ( Table 2).
The maximum likelihood phylogenetic analysis grouped the STs into three main clades (A, B, and C), which were divided into subclades, except for the clade C. Figure 3 shows the genetic relationship among porB and tbpB sequences of 144 STs. All the NG-MAST types found in the analysis were included, except the singletons unless they belonged to a genogroup.
Of note, sequences of clade B2 belonged to isolates showing a similar antimicrobial resistance profile of those belonging to G1407. In B2, clustered sequences obtained from gonococci belonging to G13070 were found for the first time in 2015, and from gonococci resistant to ciprofloxacin (n = 12; 100%), cefixime (n = 3; 25%), and azithromycin (n = 2; 16.6%).

Genomic comparison between Italian and European gonococci
A subsample of 55 genomes were examined by cgMLST for a high-resolution comparison to investigate the genetic relatedness together with a representative panel of genomes from gonococci collected from other European countries (n = 521, Supplementary Fig. S2).
The resulting NeighborNet network, reconstructed on the estimated allelic distances, revealed a high genetic variability with a star-like structure, where Italian isolates were intermixed with European gonococci and grouped by genogroup ( Supplementary Fig. S2). Of all the European genomes, those from Portugal and Spain were more similar to those collected in Italy (Supplementary Fig. S2).

Discussion
Antimicrobial resistance in N. gonorrhoeae remains a global public health concern, considering that in 2020, the World Health Organization (WHO) estimated an annual incidence of 82 million gonorrhea cases among adults. 29 To estimate the proportion of resistant N. gonorrhoeae isolates and the molecular variability, 1,525 viable gonococci collected in Italy, from 2013 to 2019, were evaluated.
In agreement with previous reports and in EU/EEA countries, 8,9 most of the gonococcal isolates were collected from the urogenital tract (85.1%) of Italian males (95.8%), the MSM represents the 57.9% and the HIV-positive patients the 14.4%.
As reported in the EU/EEA countries, 9 the cefixime resistance remained quite stable with a proportion around 2% from 2014 to 2018, reaching the 2.7% in 2019. On the contrary, an increase in azithromycin resistance was de-

CARANNANTE ET AL.
Although the European and the International treatment guidelines 13,16 were updated in 2012, a certain percentage of gonococci resistant to cefixime (2.7%) and azithromycin (9.9%) were identified in Italy in 2019. As reported previously in the country, 11 despite the combined treatment being in use, the cefixime as monotherapy was prescribed when the guidelines did no longer support its use (after 2013), except when an intramuscular injection was not possible or refused by the patient. 13,16 Moreover, coinfections with Chlamydia Chlamydia trachomatis were likely treated, considering the European treatment guidelines, 13 with azithromycin as monotherapy.
Unlike other European countries, 8 no ceftriaxoneresistant isolate was found and the majority of gonococci showed a MIC value of 0.002 mg/L or lower. Nevertheless, during the period of the analysis, 19 gonococci with ceftriaxone MIC values close to the breakpoint of 0.125 mg/L were identified, of which 1 (in 2014) was with MIC value of 0.094 mg/L, and the remaining with MIC value of 0.064 mg/L. The data might suggest a still efficacy of both ceftriaxone and azithromycin to treat gonorrhea.
In Italy, an increase in ciprofloxacin resistance and of PPNG was observed reaching 79.1% and 18.7%, respectively, in 2019. The spread of ciprofloxacin-resistant isolates might likely be the result of its use to treat other infectious diseases. 30 Finally, less than 1% of gonococci were MDR. An isolate with high-level resistance to spectinomycin was reported in China, where spectinomycin is used as firstline monotherapy as an alternative to ceftriaxone. 31 In our country, as well as in Germany and the United Kingdom, 32,33 no spectinomycin resistance was identified likely due to the limited availability of this antimicrobial, and also capable to select resistant isolates. 5,34 Gentamicin is still under evaluation for the gonorrhea treatment. A randomized controlled trial reported a noninferiority in the efficacy of gentamicin compared with ceftriaxone in the clearance of N. gonorrhoeae in all the anatomic and/or possible other anatomical infected sites, including the urogenital tract. 35 A breakpoint for gentamicin is not yet established and the gentamicin MIC values, con-sidered in this study, were similar to those reported by other countries. 18,36 The identification of 288 STs of the gonococci considered in the study, including 182 singleton STs, underlined a high genetic diversity of the gonococci as also previously described (2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012). 12 As also reported, 19,21 ST1407, ST6360, ST5624, ST2400, ST2992, and ST4995 were widespread in EU/EEA countries, whereas, ST5441 was almost predominant in Italy and previously identified in Spain. 34 ST13070, also described in Spain 34 as to be associated with gonococci showing decreased susceptibility or resistance to cefixime and resistance to ciprofloxacin and azithromycin, appeared for the first time in Italy in 2015 reaching a peak in 2017. 37 According to other reports, 19,21 and as previously described in our country, 12 G1407, comprising gonococci resistant to cefixime, ciprofloxacin, and azithromycin, 12,19,21 remains the predominant genogroup, despite a slowly, but constant decrease over the years (22.3% in 2013 vs. 3.5% in 2019). In fact, a decrease in the percentage of ST1407, one of the main ST of this genogroup (30.5% in 2013 vs. 5.5% in 2018) was observed also in Italy. 21 G1407 was identified among gonococci with ceftriaxone MIC values close to the breakpoint of 0.125 mg/L. In addition, G2400 was the predominant genogroup among azithromycin-resistant gonococci, with the ST6360 and ST10128 as the most frequent STs. The seven HLR azithromycin isolates showed five different STs of which three of them were ST3935.
Comparison between genomes obtained from gonococci collected in Italy and those from European gonococcal isolates confirmed the high level of genetic diversity of N. gonorrhoeae isolates. However, despite this, the majority of circulating gonococci up to 2019 belonged to the distinct genogroups, which are disseminate and spread internationally.
Some limitations have to be mentioned, first of all, the absence of N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) and whole-genome sequencing (WGS) performed only on a subsample of gonococci Overall, the results confirm some important characteristics of the isolates, as previously described, 11,12 which, however, have to be continuously monitored for circulating gonococci: (i) a stable resistance against cefixime, (ii) a wide-spread resistant isolates to ciprofloxacin and certain proportion of PPNG, (iii) an increase of azithromycin resistance but with a slight decrease in 2019 (the last year of this analysis) and, finally, the lack of ceftriaxone-resistant isolates among the collection analyzed.
The molecular investigation supported the high genetic variability statement for gonococci and the identification of ST13070 and its genogroup, as well as the clear consolidation of the international clone G1407.
Gonococcal AMR surveillance is crucial to monitor antimicrobial susceptibility and to highlight the main circulating clones useful to update, if needed, the treatment guidelines and to reinforce preventive actions. and drafted the article; S.F. performed the phylogenetic analysis; I.D.C., V.G., M.C., G.P., C.V., A.M.B., E.S., M.B., A.M., M.R., M.A.D.F., C.L.B., P.I., M.A.L., E.R., F.P., I.C.C., and G.S. collected the isolates and patient data; L.A. contributed to the molecular characterization and data analysis; S.B. performed statistical analysis; A.C. and F.L. performed whole-genome sequencing; P.S. conceived the study, provided expertise, and contributed toward the draft and review of the article. All authors participated in the drafting and revision of this article and gave their final approval of this version.

Ethics Statement
Gonococcal isolates were collected, processed, and stored as part of routine clinical care by the collaborating laboratories participating in the network. All the personal information is kept confidential and data analysis was performed in aggregated pseudonymous modalities and considering the EU's 2016 General Data Protection Regulation GDPR.

Disclosure Statement
No competing financial interests exist.

Supplementary Material
Supplementary  Figure S2