High tetracycline and azithromycin resistance of Neisseria gonorrhoeae impacting treatment and doxyPEP use - antimicrobial susceptibility patterns of isolates from a prospective cohort of men who have sex with men in Wroclaw, Lower Silesia, Poland 2022–2024 – final report

Abstract

Objectives

The aim was to ascertain Neisseria gonorrhoeae (NG) antimicrobial resistance patterns among men who have sex with men (MSM), evaluate their impact on treatment and prophylaxis, identify risk factors for infection, and assess whether expanding the sample size beyond the preliminary cohort significantly altered the primary outcomes (1).

Methods

This trial was conducted at the outpatient All Saints’ HIV clinic in Wroclaw, Poland, between 2022 and 2024. Rectal, urethral, and oropharyngeal swabs were collected from 200 MSM who were wither living with HIV (PLWH) or using pre-exposure prophylaxis (PrEP). Included participants reported high-risk sexual contacts, symptoms, or prior positive results. NG isolates were evaluated for susceptibility to ceftriaxone, cefixime, azithromycin, ciprofloxacin, tetracycline, and benzylpenicillin.

Results

Of the 200 MSM enrolled, 33.5% were PLWH and 66.5% were PrEP users. This extended analysis identified 23 additional isolates alongside the initial 26, yielding 43 viable isolates for susceptibility testing. PrEP users were slightly younger than PLWH (median 34 vs. 36 years) and reported more oral contacts, more sexual partners outside of Poland, and more partners of foreign nationality within Poland. Doxycycline post-exposure prophylaxis (doxyPEP) was more frequently utilized by PLWH (30.3% vs. 14.18%). NG culture positivity rates were similar between PLWH (21.7%) and PrEP users (23.3%). In multivariate logistic regression, NG infection was significantly associated with higher number of oral contacts (OR = 4.05; 95% CI: 1.15–14.27; p = 0.029). While all isolates retained full susceptibility to ceftriaxone and cefixime, resistance to azithromycin was 30.2% (13/43) and tetracycline 48.8% (21/43), demonstrating stability between the preliminary and final analyses.

Conclusions

Ceftriaxone and cefixime remain highly effective first-line agents against NG in Poland, whereas high resistance rates dictate that azithromycin should be relegated to alternative therapy status in national guidelines. Furthermore, the high prevalence of tetracycline resistance may compromise the efficacy of doxyPEP. Finally, the initial cohort of 100 MSM was statistically sufficient to accurately ascertain core antimicrobial resistance patterns.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12879-026-14073-0.

What is already known on this topic

In recent years, gonorrhoea incidence rates among MSM have continued to increase, substantially exceeding those observed in the general population. Neisseria gonorrhoea (NG) has developed resistance to nearly all antimicrobial classes used for treatment and is classified as a high-priority pathogen by the World Health Organisation. Despite this global threat, there is currently minimal to no structured monitoring of NG antimicrobial resistance (AMR) patterns among key populations in Poland and several other European countries.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12879-026-14073-0.

What this study adds

This study demonstrates a complete absence of resistance to ceftriaxone and cefixime, contrasted with alarmingly high rates of resistance to both azithromycin and tetracycline among local NG isolates. To our knowledge, this is the first prospective report on NG resistance patterns among MSM in Poland, providing crucial contemporary data on AMR and its immediate implications for treatment guidelines and biomedical prophylaxis.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12879-026-14073-0.

How this study might affect research, practice or policy

Bacterial culture and susceptibility testing must remain an integral component of routine diagnostic workflows to monitor evolving resistance patterns. High susceptibility (low minimum inhibitory concentrations [MICs]) supports the continued use of highly effective ceftriaxone monotherapy. Conversely, national guidelines recommending ceftriaxone plus azithromycin combination therapy should be revised, relegating azithromycin to an alternative treatment status.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12879-026-14073-0.

Introduction

The World Health Organisation (WHO) has designated Neisseria gonorrhoeae (NG) as a high-priority pathogen urgently requiring the research and development of new therapeutic agents, driven by escalating resistance to legacy antimicrobials [1]. Gonorrhoea remains a prevalent sexually transmitted infection (STI) that disproportionately affects key populations, including men who have sex with men, sex workers, adolescents, young adults, and transgender women [2]. Because extragenital NG infections – specifically oropharyngeal and rectal – are predominantly asymptomatic in men [3–5], timely diagnosis represents a significant clinical challenge [6]. Paradoxically, while routine screening of asymptomatic individuals and subsequent targeted treatment within dense transmission networks is standard practice across various clinical guidelines, it may serve as an evolutionary driver for antimicrobial resistance, an effect further compounded by rising background antibiotic consumption in the general population [6, 7].

We previously reported a preliminary analysis of 26 NG isolates cultured from a cohort of 100 MSM in the Lower Silesia region between 2022 and 2023 [8]. Expanding upon this surveillance is critical, given that gonorrhoea incidence among MSM continuously outpaces that of the general population, and the pathogen has sequentially developed resistance to nearly all historical first-line treatments [1, 2, 9–11]. Despite this pressing public health threat, contemporary longitudinal data regarding NG resistance patterns in Poland remain sparse; existing literature is largely restricted to outdated datasets or constrained by small sample sizes [12–15].

To address this critical gap, the present study aims to characterize the definite phenotypic susceptibility profiles of NG isolates within a prospective cohort of MSM in Wroclaw, while evaluating the behavioural risk factors associated with infection. Furthermore, we sought to evaluate whether doubling our initial sample size would significantly alter or refine our previously reported primary outcomes, thereby establishing a robust baseline for local treatment guidelines and biomedical prophylaxis strategies.

Methods

To expand upon our preliminary analysis and enhance statistical power, the cohort was extended to include an additional 100 MSM either living with HIV or using PrEP. Because robust historical data regarding NG prevalence and representative sample sizes within the Polish MSM population are lacking, doubling the initial cohort size was essential to provide deeper insights into the affected population. Furthermore, this expansion served to establish a baseline for sample size requirements in future multicentre surveillance trials planned across other regions in Poland.

Participants fulfilling the inclusion criteria were recruited consecutively at All Saints’ Clinic in Wroclaw, Poland – an outpatient HIV/PrEP/STI facility – between October 2022 and May 2024. Inclusion criteria were age > 18 years and self-reported high-risk sexual behaviour, defined as having more than two sexual partners in the last 3 months or having sexual contact with a partner with confirmed NG infection. Both symptomatic and asymptomatic individuals (the latter presenting either with a positive PCR result or following a contact with a confirmed NG-positive partner) were included. Symptoms suggestive of NG infection included:

• Urogenital symptoms: dysuria, urethral discharge, or testicular pain/swelling.

• Anorectal symptoms: rectal discharge, anal pruritus, soreness, bleeding, or painful defecation.

• Other symptoms: painful lymphadenopathy and pharyngeal soreness or dryness.

At baseline, patients completed a structured, study-specific questionnaire capturing sociodemographic characteristics, sexual behaviour (number of partners in the last month, types of intercourse [oral/anal], and condom use), engagement in Chemsex, substance use (alcohol, tobacco, recreational drugs), PrEP use, history of sexually transmitted infections (including syphilis, gonorrhoea, chlamydia, and hepatitis C virus), and the use of doxyPEP. The English translation of the questionnaire is available as supplemental material.

Oropharyngeal, urethral, and rectal swabs were collected using Amies transport medium with charcoal. Specimen processing and bacterial cultures were conducted at the Department of Pharmaceutical Microbiology and Parasitology, Wroclaw Medical University, Poland. Swabs were inoculated onto Modified Thayer Martin, GC, and chocolate agar media and incubated under a 5% CO₂ atmosphere within 24 h of collection. Phenotypic identification of N. gonorrhoeae was performed using the API NH system (bioMerieux). Confirmed isolates were cryopreserved at -80 °C for subsequent testing.

Antimicrobial susceptibility was evaluated for six antimicrobials: ceftriaxone, cefixime, azithromycin, ciprofloxacin, tetracycline, and benzylpenicillin. Minimum inhibitory concentrations (MICs; mg/L) were determined via the gradient diffusion method (Etest) on Chocolate II and GC agar bases. Phenotypic resistance profiles were interpreted in accordance with the clinical breakpoints established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; version 14.0).

Statistical analyses were performed using Epi Info (version 7.2.3.1) and Statistica (version 13.3). For all studied groups, the number of cases (N), the mean (X), median (M), range (min-max), lower and upper quartile (25q-75q) and standard deviation (SD) of the parameters were calculated. Median (interquartile range [IQR]) was used for description of non-normally distributed data. Qualitative variables were presented as absolute values and percentages (%). The normality assumption was assessed by carrying out the Shapiro-Wilk test for the groups of data. Levene’s test was carried out to assess the homogeneity of variances assumption. For qualitative parameters, the frequency of the traits or characteristics in groups was analysed using the χ²_df test with the appropriate number of degrees of freedom df (df=(m-1)x(n-1), where m – number of rows, n – number of columns). The verification of the hypothesis of equality of means groups of heterogeneous variance was performed by the non-parametric Mann-Whitney U test. P ≤ 0.05 was considered statistically significant. Variables that were statistically significant or showed a trend toward significance in univariate analyses of associations with culture positivity were included in the multivariable logistic regression model. Multivariate analysis was conducted using logistic regression via the quasi-Newton estimation method.

Results

Demographics and study population

The primary 100 individuals from the interim analysis [8] was combined with an additional 100 high-risk MSM, yielding a final sample size of 200 participants. This expanded cohort comprised: 67 (33.5%) PLWH and 133 (66.5%) HIV-negative PrEP users (using co-formulated oral emtricitabine/tenofovir [FTC/TDF]).

The vast majority of participants (83%) lived in metropolitan areas with populations over 500,000, with an equal distribution between study arms (p = 0.909).

Age and sexual behaviour profiles

PrEP users were slightly younger than PLWH (median age 34 vs. 36 years respectively; p = 0.0104), reported a significantly higher number of sexual partners in the last 3 months (median of 5 vs. 4.5 respectively; p = 0.0136) and a greater number of oral sexual contacts during the last month (median of 5 vs. 3 respectively; p = 0.0222). Conversely, no significant differences were observed between the two arms regarding the median number of anal sexual contacts in the last month (a median of 3 in both groups) or condom use during oral and anal contacts (a median condom use of 0% in both groups). A small subset of bisexual men in both arms reported additional sexual contacts with women, with no significant variance between the groups (4.5% [n = 6] of PrEP users vs. 9.1% [n = 6] among PLWH; p = 0.196).

Geographic partner distribution, STIs, and prophylaxis

While participants in both arms had sexual contacts predominantly within Poland or with Polish partners, PrEP users accounted for a significantly higher proportion of individuals reporting sexual contacts outside of Poland or with foreign nationals within Poland (76.62% vs. 23.38% among PLWH; p = 0.0220).

Among participants reporting a history of STIs (syphilis, gonorrhoea, and chlamydiosis), 65.7% were PrEP users and 34.3% were PLWH (p = 0.505). Similarly, of those engaging in Chemsex, 63.4% were PrEP users compared to 36.1 who were PLWH (p = 0.426); however, neither of these trends achieved statistical significance.

In contrast, doxyPEP use was significantly more common among PLWH, who accounted for 51.3% of users compared to 48.7% among PrEP users (p = 0.0194). Notably, a uniform deficit in comprehensive knowledge regarding doxyPEP was observed across both groups and reported by 14.2% of PrEP users and 15.2% of PLWH.

Full baseline characteristic of the study cohort are detailed in Table 1. Crucially, the demographic, behavioural, and clinical trends remained highly consistent, demonstrating no substantial deviations between the primary interim analysis (n = 100) and this final expanded analysis (n = 200).

Table 1.

Group characteristics

	PrEP users (n = 134)			PLWH (n = 66)			p
	25Q	Median	75Q	25Q	Median	75Q
Age	28	34	39	32	36	42	0.0104
Number of partners during the last 3 months [n]	3	5	10	2	4,5	8	0.0136
Number of oral contacts during the last month [n]	2	5	10	2	3	6	0.0222
Number of anal contacts during the last month [n]	2	3	7	1	3	5	0.0749
Oral contacts with condoms [%]	0	0	0	0	0	3	0.757
Anal contacts with condoms [%]	0	0	30	0	0	30	0.875

Microbiological and clinical data

Clinical symptoms suggestive of an STI were reported by 101 participants, comprising 29 (43.3%) PLWH and 72 (54.1%) PrEP users; this difference was not statistically significant (p = 0.147).

Bacterial cultures confirmed Neisseria gonorrhoeae infection in 44 patients, yielding an overall positivity rate of 22% (44/200). Among these, 42 individuals presented with single-site infections and two with multi-site infection, resulting in a total recovery of 46 viable isolates.

Positivity rates did not differ significantly between symptomatic and asymptomatic participants, regardless of the anatomical site (p = 0.570). This represents a notable shift from the preliminary analysis (n = 100), where positive cultures were significantly clustered among symptomatic individuals (p = 0.004). This divergence should be primarily attributed to a structural shift in the overall study population composition, as the total proportion of symptomatic patients declined from 58% to 38% in the final expanded cohort. Four urethral (18.2% of site-specific isolates), four oropharyngeal (33.3% of site-specific isolates) and three rectal (30% of site-specific isolates) isolates were cultured from asymptomatic patients, displaying a high prevalence across extragenital sites.

Anatomical distribution and group comparison

The anatomical site distribution for the 46 isolates included 25 urethral, 12 oropharyngeal and 9 rectal isolates.

Culture positivity rates were highly comparable between the two groups, standing at 20.9% (14/67) among PLWH and 24.1% (32/133) among PrEP users (p = 0.801; see Table 2). Notably, both instances of dual-site infections occurred exclusively within the PLWH cohort, manifesting as concurrent rectal/urethral and oropharyngeal/urethral positive cultures.

Table 2.

Positive Neisseria gonorrhoeae cultures – site specific

	urethral site	oropharyngeal site	rectal site
PrEP users	16	11	5
PLWH	9	1	4
Total	25	12	9

p = 0.258

Consistent with the demographic trends, transitioning from the preliminary interim cohort (n = 100) to the final combined cohort (n = 200) revealed no significant deviations or shifts in the primary clinical or microbiological outcomes.

Univariate and multivariate risk factor analysis

In univariate analysis, culture-positive patients reported a significantly higher number of oral sexual contacts compared to culture-negative patients (p = 0.0112), whereas the total number of sexual partners did not differ significantly between the two groups (p = 0.276). Variables demonstrating statistical significance or a strong trend toward significance in the univariable screening were subsequently entered into the multivariate logistic regression model (Table 3).

Table 3.

Final multivariate logistic regression model for factors associated with positive culture results

Variable	OR	95% CI	p-value
≥ 2 oral contacts (vs. ≤ 1)	4.05	1.15–14.27	0.029
≥ 30% condom use during anal sex	0.30	0.11–0.82	0.018
Sexual contacts only with male	0.22	0.06–0.79	0.020

Model statistics: χ²(3) = 16.3, p = 0.0010

Abbreviations: OR – odds ratio; CI – confidence interval

In the final multivariate model, the following independent association with NG infection were identified:

• Oral contacts: A higher number of oral exposures remained independently associated with increased odds of infection (OR = 4.05; 95% CI: 1.15–14.27; p = 0.029). This risk was specifically observed among participants reporting ≥ 2 oral contacts compared with those reporting ≤ 1 contact.

• Condom utilization: Reporting at least 30% condom use during anal intercourse was significantly protective, and associated with a 70% reduction in the odds of NG diagnosis (OR 0.3; 95% CI 0.11–0.82; p = 0.018).

• Partner gender dynamics: Engaging in sexual contacts exclusively with men was also associated with lower odds of infection (OR = 0.22; 95% CI: 0.06–0.79; p = 0.020). However, this specific finding must be interpreted with caution due to the small sample size of bisexual men enrolled in the study (n = 12).

Antimicrobial susceptibility profiles

Antimicrobial susceptibility profiles of the recovered NG isolates are summarizes in Table 4; Fig. 1. Of the 46 isolated strains, three exhibited stunned growth during susceptibility testing and were excluded from the analysis, yielding a final analytical sample of 43 viable isolates. Phenotypic resistance patterns demonstrated high stability, with no statistically significant shifts between the preliminary interim analysis and this final expanded dataset.

Table 4.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates

	PENICILIN		CEFIXIME		CEFTRIAXONE		CIPROFLOXACIN		TETRACYCLINE		AZITHROMYCIN
	Number of isolates	%	Number of isolates	%	Number of isolates	%	Number of isolates	%	Number of isolates	%	Number of isolates	%
S – susceptible	10	23.3	43	100.0	43	100.0	10	23.3	22	51.2	30	69.8
I - intermediate	24	55.8		0.0		0.0	3	7.0		0.0		0.0
R – resistant	9	20.9		0.0		0.0	30	69.8	21	48.8	13	30.2
total	43	100.0	43	100.0	43	100.0	43	100.0	43	100.0	43	100.0

Fig. 1.

Antibiotic resistance patterns among NG isolates [%]

Third-generation cephalosporins

Full phenotypic susceptibility was maintained for both first-line extended spectrum cephalosporins:

• Cetriaxone: All 43 isolates (100%) were fully susceptible. Minimum inhibitory concentrations (MICs) were ≤ 0.016 mg/L in 95.3% (n = 41) of isolates and equal to 0.02 mg/L in the remaining 4.7% (n = 2).

• Cefixime: Full susceptibility was likewise observed across all 43 isolates (100%). MICs were ≤ 0.016 mg/L in 79.1% (n = 34) of isolates, ranged between 0.032 and 0.047 mg/L in 18.6% (n = 8), and reached 0.064 mg/L in a single isolate (2.3%).

Other antimicrobial classes

Resistance to older antimicrobial classes and macrolides was highly prevalent across the cohort:

• Ciprofloxacin: Fluoroquinolone resistance was the most pervasive phenotypic trait, identified in 69.8% (30/43) of the tested isolates.

• Tetracycline: Resistance was confirmed in 48.8% (21/43) if isolates.

• Azithromycin: Resistance was identified in 30.2% (13/43) of isolates.

• Benzylpenicillin: Phenotypic resistance was observed in 20.9% (9/43) of isolates, while an additional 55.8% (24/43) exhibited intermediate susceptibility.

The specific MIC distributions (mg/L) for ceftriaxone, cefixime, tetracycline, and azithromycin are visually detailed in Figs. 2, 3, 4 and 5.

Fig. 2.

MICs for ceftriaxone among NG isolates [n] (S ≤ 0.125, R > 0.125)

Fig. 3.

MICs for cefixime among NG isolates [n] (S ≤ 0.125, R > 0.125)

Fig. 4.

MICs for tetracycline among NG isolates [n] (S ≤ 0.5, R > 0.5)

Fig. 5.

MICs for azithromycin among NG isolates [n] (S ≤ 1.0, R > 1.0)

Discussion

The study provides the first contemporary, prospective cohort data evaluating the antimicrobial resistance profiles of Neisseria gonorrhoea specifically among MSM in Poland. By consecutively enrolling participants, we minimized selection bias and achieved a highly representative regional sample. Furthermore, relying on phenotypic culture diagnostics prioritized isolates from actively infectious cases.

Longitudinal AMR monitoring remains sparse in Central and Eastern Europe. Sentinel surveillance is significantly underutilized in Poland; for context, the 2022 European Centre for Disease Prevention and Control (ECDC) report included a mere 15 NG isolates for the entire country, all drawn from the general population [13]. In contrast, our single-centre study recovered 46 isolates over a comparable timeframe, highlighting the vital role of targeted, clinic-based surveillance. While the MICs for ceftriaxone in our cohort mirrored broader European trends reported by the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP), elevated cefixime MICs (specifically the highest observed value of 0.064 mg/L) were notably less prevalent in our sample than across Europe (2.3% vs. 10%, respectively) [13].

Our findings indicate a stark escalation in resistance compared to historical benchmarks. A recent genomic study by Golparian et al., which included 23 Polish isolates collected across 2013, 2018, and 2020, demonstrated an absence of cephalosporin resistance alongside 50% resistance rate to ciprofloxacin and a 35% resistance rate to azithromycin [12]. Notably, their 2020 data suggested that azithromycin resistance in Poland occurred two- to ten-fold more frequently than in neighbouring nations. However, interpretation was limited by small sample sizes and potential sampling bias [12].

The high prevalence of azithromycin resistance observed in our cohort (30.2%) may be driven by two primary factors: antibiotic overprescribing (widespread, empirical use of macrolides for respiratory tract infection within the Polish healthcare system) and horizontal gene transfer. Commensal, non-pathogenic oropharyngeal Neisseria species frequently harbour macrolide resistance determinants, which can be readily transferred to NG during localized co-infection or exposure, as documented by Gaspari et al. [10, 16].

Historically, Euro-GASP networks reported much lower macrolide resistance in Poland, such as a 5.4% prevalence out of 56 isolates in 2015 [17]. Even older data from 201 to 2012 by Młynarczyk-Bonikowska et al. identified azithromycin resistance at just 10%, with tetracycline resistance hovering between 38% and 47% [15]. Taken together, these temporal data underscore stable, creeping resistance in Poland – a trajectory that mirrors broader European epidemiological shifts.

Clinical guideline implications – shifting to monotherapy

Major international bodies, including European AIDS Clinical Society (EACS) [18], the British Association for Sexual Health and HIV (BASHH) [19] as well as the US Centres for Disease Control and Prevention (CDC) [20], now strongly advocate for ceftriaxone monotherapy as the standard of care for uncomplicated gonorrhoea. Because oral cefixime exhibits lower efficacy against oropharyngeal infections, ceftriaxone should be used whenever clinically feasible. Conversely, International Union against Sexually Transmitted Infections (IUSTI) guidelines [21] require rapid revision, as dual-therapy frameworks utilizing azithromycin are increasingly compromised by regional AMR trends. Clinicians must account for the prolonged washout period of azithromycin. Sub-MIC concentrations persist for up to 2 weeks post-treatment, creating an evolutionary window that drives resistance mutations if a patient gets reinfected during this time. For ceftriaxone monotherapy, a 7-day post-treatment abstinence period is sufficient.

Interestingly, the 2023 Euro-GASP report showed that while most European clinics have transitioned to ceftriaxone monotherapy (with fewer than 20% utilizing combined therapy) [22], the reported azithromycin resistance rate for Poland paradoxically dropped to 16.7% across 30 isolates. These sharp, year-over-year fluctuation in national data strongly imply localized sampling artifacts rather than genuine epidemiological shifts, further emphasizing the need for a standardized, robust national gonococcal surveillance framework.

Diagnostic performance and screening dynamics

The observed NG culture positivity rate of 22% exceeds the 17.7% reported in a structurally similar Spanish cohort by Serra-Pladevall et al. [23]. This high diagnostic yield is notable given our longer sample transport times (up to 24 h vs. 4 h in the Spanish study). This can be largely attributed to our use of Amies charcoal transport media, which has been shown by Boiko et al. to preserve isolate viability exceptionally well during prolonged transport [24].

Conversely, the high proportion of symptomatic patients who ultimately tested negative highlights a common clinical challenge. As noted by Wong et al., up to 50.2% of anorectal, 19.6% of scrotal, and 7.3% of urethral symptomatic presentations result in unnecessary empirical treatment due to the overlapping clinical presentation of other pathogens [25]. In their study, factors inversely corelating with Neisseria gonorrhoeae or Chlamydia trachomatis diagnosis included specific localized symptoms, an STI diagnosis in the prior year, known contact with an STI-positive partner, and living with HIV – leading the authors to advocate for routine point-of-care molecular diagnostics [25].

Crucially, the equivalent culture positivity rates found between symptomatic and asymptomatic individuals in our study reinforce the understanding that asymptomatic individuals can sometimes be a silent reservoir within dense transmission networks. Future targeted trials are required to fully ascertain the power thresholds necessary to refine screening intervals as our study was not powered to confirm which of these patients were infectious and which were not.

Behavioural nuances and prophylaxis access disparities

DoxyPEP was used twice as often by PLWH than PrEP users (30% vs. 14%), despite identical levels of baseline declared knowledge regarding this intervention. Because doxyPEP was an emerging biomedical tool during our study period, we hypothesize that PLWH benefited from more established, stable, and trusting patient-doctor relationships within specialized HIV clinics, facilitating earlier adoption.

Historically, MSM living with HIV have been at the vanguard of adopting novel biomedical prevention strategies. However, structural barriers must be dismantled for PrEP users, who in our cohort were younger, reported higher numbers of sexual partners, engaged in more frequent oral contacts, and had more geographically diverse sexual networks. Younger age is frequently intersected by financial, social, and educational exclusion, magnifying acquisition risks – particularly when compounded by engagement in Chemsex [9, 26]. Given that an increased frequency of oral contacts independently drives NG acquisition, younger PrEP users represent a critical target demographic requiring equitable, barrier-free access to evidence-based prophylaxis.

Doubling our sample size from 100 to 200 participants did not significantly alter the observed phenotypic AMR patters, suggesting that in highly concentrated, high-prevalence transmission networks, smaller sentinel samples can accurately capture core resistance profiles. However, populations with lower background prevalence will inherently require larger sample sizes to maintain epidemiological representativeness. Ultimately, expanding our cohort proved invaluable as it provided the statistical power necessary to construct a highly reliable, stable multivariable regression model.

Limitations

Diagnostic modality constraints: Financial constraints precluded the universal use of NAAT/PCR testing for all participants and sites. Because phenotypic culture has lower sensitivity than molecular assays, some low-burden or fastidious infections may have been missed, despite the use of optimized charcoal transport media.

Empirical treatment masking: Symptomatic patients received immediate empirical therapy (typically a single dose of 1 g intramuscular ceftriaxone or 800 mg oral cefixime, paired with a 7-day course of oral doxycycline) prior to culture confirmation. While all patients symptoms resolved, this clinical approach means that false-negative cultures or infections caused by other STIs (such as C. trachomatis or Mycoplasma genitalium) could not be fully differentiated.

Generalizability: We specifically enrolled a high-risk, urban MSM population characterized by dense transmission networks. Consequently, these specific behavioural risk factors and high AMR rates cannot be directly generalized to the broader, heterosexual population In Poland.

Conclusions

Ceftriaxone and cefixime retain full, uncompromised phenotypic activity against N. gonorrhoeae in the Lower Silesia region, supporting their role as the first-line therapeutic agents.

Dual therapy combining ceftriaxone with azithromycin should be discontinued, and azithromycin should be relegated strictly to alternative-therapy status in national Polish guidelines.

Phenotypic bacterial culture and susceptibility testing must remain a fundamental component of routine diagnostic workflows to prospectively monitor evolving AMR trends.

The high baseline prevalence of tetracycline resistance (49%) underscores an urgent need for continuous monitoring of local strains, as it may possibly comprise the long-term clinical efficacy of doxyPEP.

Given the concentrated nature of the local epidemic, 100 high-risk MSM cohort appears epidemiologically sufficient to reliably determine core regional antimicrobial resistance patterns.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Bartosz Szetela, Martyna Biała – idea for the study, methodology, sampling, data curation and analysis, manuscript preparation, Beata Mączyńska, Daria Zaręba, Danuta Rurańska-Smutnicka, Paulina Szuba – cultures, microbiological methodology, data analysis, manuscript preparation.

Funding

This study was funded by Wroclaw Medical University, Poland, grant number SUBZ.C170.23.093 (Martyna Biala), SUBZ.D230.26.016 (Beata Maczynska) and by the Polish AIDS Scientific Society grant (Bartosz Szetela).

Data availability

Data available from the authors at request: Bartosz Szetela at bartoszetela@gmail.com and Martyna Biala at biala.martyna@interia.pl.

Declarations

Ethics approval and consent to participate

The study was approved by the Wroclaw Medical University Ethics Committee in accordance with the Declaration of Helsinki – KB-375/22 and KB-944/2022. Informed consent was obtained from patients involved in the study.

Consent for publication

All authors confirm they have read and accepted the manuscript and consent to the publication.

Abbreviations

Explained in text if used.

Competing interests

The authors declare no competing interests.