Ceftriaxone resistance in Neisseria gonorrhoeae is an escalating global health concern, threatening the efficacy of empiric treatment. In the Asia-Pacific region, a rapid surge in ceftriaxone resistance has been observed, primarily associated with the penA-60.001 allele. This study, conducted in Hanoi, Vietnam between 2023 and 2024, analyzed 352 N. gonorrhoeae isolates and found a 27% prevalence of ceftriaxone resistance. Whole-genome sequencing identified that resistance was predominantly driven by the mosaic penA-237.001 allele, with a smaller contribution from penA-60.001. The high prevalence of resistance and the emergence of new alleles underscore the urgent threat to ceftriaxone as an empiric treatment option.

BACKGROUND:commensal Neisseria species are part of the oropharyngeal microbiome and play an important role in nitrate reduction and protecting against colonization by pathogenic bacteria. They do, however, also serve as a reservoir of antimicrobial resistance. Little is known about the prevalence of these species in the general population, how this varies by age and how antimicrobial susceptibility varies between species. METHODS:we assessed the prevalence and antimicrobial susceptibility of commensal Neisseria species in the parents (n = 38) and children (n = 50) of 35 families in Belgium. RESULTS:various commensal Neisseria (n = 5) could be isolated from the participants. Most abundant were N. subflava and N. mucosa. Neisseria subflava was detected in 77 of 88 (87.5%) individuals and N. mucosa in 64 of 88 (72.7%). Neisseria mucosa was more prevalent in children [41/50 (82%)] than parents [23/38 (60.5%); P < .05], while N. bacilliformis was more prevalent in parents [7/36 (19.4%)] than children [2/50 (4%); P < .05]. Neisseria bacilliformis had high ceftriaxone minimum inhibitory concentrations (MICs; median MIC 0.5 mg/l; IQR 0.38-0.75). The ceftriaxone MICs of all Neisseria isolates were higher in the parents than in the children. This could be explained by a higher prevalence of N. bacilliformis in the parents. INTERPRETATION:the N. bacilliformis isolates had uniformly high ceftriaxone MICs which warrant further investigation.

Introduction. Listerine is a bactericidal mouthwash widely used to prevent oral health problems such as dental plaque and gingivitis. However, whether it promotes or undermines a healthy oral microbiome is unclear. Hypothesis/Gap Statement. We hypothesized that the daily use of Listerine Cool Mint would have a significant impact on the oropharyngeal microbiome. Aim. We aimed to assess if daily usage of Listerine Cool Mint influenced the composition of the pharyngeal microbiome. Methodology. The current microbiome substudy is part of the Preventing Resistance in Gonorrhoea trial. This was a doubleblind single- centre, crossover, randomized controlled trial of antibacterial versus placebo mouthwash to reduce the incidence of gonorrhoea/chlamydia/syphilis in men who have sex with men (MSM) taking HIV pre- exposure prophylaxis (PrEP). Fiftynine MSM taking HIV PrEP were enrolled. In this crossover trial, participants received 3 months of daily Listerine followed by 3 months of placebo mouthwash or vice versa. Oropharyngeal swabs were taken at baseline and after 3 months use of each mouthwash. DNA was extracted for shotgun metagenomic sequencing (Illumina Inc.). Non- host reads were taxonomically classified with MiniKraken and Bracken. The alpha and beta diversity indices were compared between baseline and after each mouthwash use. Differentially abundant bacterial taxa were identified using ANOVA- like differential expression analysis. Results. Streptococcus was the most abundant genus in most samples (n n = 103, 61.7 %) with a median relative abundance of 31.5% (IQR 20.6-44.8), followed by Prevotella [13.5% (IQR 4.8-22.6)] and Veillonella [10.0% (IQR 4.0-16.8)]. Compared to baseline, the composition of the oral microbiome at the genus level (beta diversity) was significantly different after 3 months of Listerine (P P = 0.006, pseudo- F = 2.29) or placebo (P P = 0.003, pseudo- F = 2.49, permutational multivariate analysis of variance) use. Fusobacterium nucleatum and Streptococcus anginosus were significantly more abundant after Listerine use compared to baseline. Conclusion. Listerine use was associated with an increased abundance of common oral opportunistic bacteria previously reported to be enriched in periodontal diseases, oesophageal and colorectal cancer, and systemic diseases. These findings suggest that the regular use of Listerine mouthwash should be carefully considered.

One of the most promising new treatments for gonorrhoea currently in phase 3 clinical trials is zoliflodacin. Studies have found very little resistance to zoliflodacin in currently circulating N. gonorrhoeae strains, and in-vitro experiments demonstrated that it is difficult to induce resistance. However, zoliflodacin resistance may emerge in commensal Neisseria spp., which could then be transferred to N. gonorrhoeae via transformation. In this study, we investigated this commensal-resistance-pathway hypothesis for zoliflodacin. To induce zoliflodacin resistance, ten wild-type susceptible isolates belonging to 5 Neisseria species were serially passaged for up to 48 h on gonococcal agar plates containing increasing zoliflodacin concentrations. Within 7 to 10 days, all strains except N. lactamica , exhibited MICs of ≥ 4 µg/mL, resulting in MIC increase ranging from 8- to 64-fold. The last passaged strains and their baseline were sequenced. We detected mutations previously reported to cause zoliflodacin resistance in GyrB (D429N and S467N), novel mutations in the quinolone resistance determining region (QRDR) (M464R and T472P) and mutations outside the QRDR at amino acid positions 28 and 29 associated with low level resistance (MIC 2 µg/mL). Genomic DNA from the laboratory evolved zoliflodacin-resistant strains was transformed into the respective baseline wild-type strain, resulting in MICs of ≥ 8 µg/mL in most cases. WGS of transformants with decreased zoliflodacin susceptibility revealed presence of the same zoliflodacin resistance determinants as observed in the donor strains. Two inter-species transformation experiments were conducted to investigate whether zoliflodacin resistance determinants of commensal Neisseria spp. could be acquired by N. gonorrhoeae . N. gonorrhoeae strain WHO P was exposed to (i) pooled genomic DNA from the two resistant N. mucosa strains and (ii) a gyrB amplicon of the resistant N. subflava strain 45/1_8. Transformants of both experiments exhibited an MIC of 2 µg/mL and whole genome analysis revealed uptake of the mutations detected in the donor strains. This is the first in-vitro study to report that zoliflodacin resistance can be induced in commensal Neisseria spp. and subsequently transformed into N. gonorrhoeae.

BACKGROUND:In antibiotic naïve populations, there is a strong association between the use of an antimicrobial and resistance to this antimicrobial. Less evidence is available as to whether this relationship is weakened in populations highly exposed to antimicrobials. Individuals taking HIV preexposure prophylaxis (PrEP) have a high intake of antimicrobials. We previously found that there was no difference in the prevalence of pheno- and genotypic antimicrobial resistance between two groups of PrEP clients who had, and had not, taken antimicrobials in the prior 6 months. Both groups did, however, have a higher prevalence of resistance than a sample of the general population.METHODS:In the current study, we used zero-inflated negative binomial regression models to evaluate if there was an individual level association between the consumption of antimicrobials and 1. the minimum inhibitory susceptibilities of oral Neisseria subflava and 2. the abundance of antimicrobial resistance genes in the oropharynges of these individuals.RESULTS:We found no evidence of an association between the consumption of antimicrobials and the minimum inhibitory susceptibilities of oral Neisseria subflava or the abundance of antimicrobial resistance genes in these individuals.CONCLUSIONS:We conclude that in high-antimicrobial-consumption populations, the association between antimicrobial consumption and resistance may be attenuated. This conclusion would not apply to lower-consumption populations.

Introduction. Commensal Neisseria spp. are highly prevalent in the oropharynx as part of the healthy microbiome. N. meningitidis can colonise the oropharynx too from where it can cause invasive meningococcal disease. To identify N. meningitidis, clinical microbiology laboratories often rely on Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS). Hypothesis/Gap statement. N. meningitidis may be misidentified by MALDI-TOF MS. Aim. To conduct genomic surveillance of oropharyngeal Neisseria spp. in order to: (i) verify MALDI-TOF MS species identification, and (ii) characterize commensal Neisseria spp. genomes. Methodology. We analysed whole genome sequence (WGS) data from 119 Neisseria spp. isolates from a surveillance programme for oropharyngeal Neisseria spp. in Belgium. Different species identification methods were compared: (i) MALDI-TOF MS, (ii) Ribosomal Multilocus Sequence Typing (rMLST) and (iii) rplF gene species identification. WGS data were used to further characterize Neisseria species found with supplementary analyses of Neisseria cinerea genomes. Results. Based on genomic species identification, isolates from the oropharyngeal Neisseria surveilence study were composed of the following species: N. meningitidis (n=23), N. subflava (n=61), N. mucosa (n=15), N. oralis (n=8), N. cinerea (n=5), N. elongata (n=3), N. lactamica (n=2), N. bacilliformis (n=1) and N. polysaccharea (n=1). Of these 119 isolates, four isolates identified as N. meningitidis (n=3) and N. subflava (n=1) by MALDI-TOF MS, were determined to be N. polysaccharea (n=1), N. cinerea (n=2) and N. mucosa (n=1) by rMLST. Phylogenetic analyses revealed that N. cinerea isolates from the general population (n=3, cluster one) were distinct from those obtained from men who have sex with men (MSM, n=2, cluster two). The latter contained genomes misidentified as N. meningitidis using MALDI-TOF MS. These two N. cinerea clusters persisted after the inclusion of published N. cinerea WGS (n=42). Both N. cinerea clusters were further defined through pangenome and Average Nucleotide Identity (ANI) analyses. Conclusion. This study provides insights into the importance of genomic genus-wide Neisseria surveillance studies to improve the characterization and identification of the Neisseria genus.

We hypothesized that the residual concentrations of fluoroquinolones allowed in food (acceptable daily intake-ADIs) could select for ciprofloxacin resistance in our resident microbiota. We developed models of chronic Escherichia coli and Klebsiella pneumoniae infection in Galleria mellonella larvae and exposed them to ADI doses of ciprofloxacin via single dosing and daily dosing regimens. The emergence of ciprofloxacin resistance was assessed via isolation of the target bacteria in selective agar plates. Exposure to as low as one-tenth of the ADI dose of the single and daily dosing regimens of ciprofloxacin resulted in the selection of ciprofloxacin resistance in K. pneumoniae but not E. coli. This resistance was associated with cross-resistance to doxycycline and ceftriaxone. Whole genome sequencing revealed inactivating mutations in the transcription repressors, ramR and rrf2, as well as mutations in gyrA and gyrB. We found that ciprofloxacin doses 10-fold lower than those classified as acceptable for daily intake could induce resistance to ciprofloxacin in K. pneumoniae. These results suggest that it would be prudent to include the induction of antimicrobial resistance as a significant criterion for determining ADIs and the associated maximum residue limits in food.IMPORTANCEThis study found that the concentrations of ciprofloxacin/enrofloxacin allowed in food can induce de novo ciprofloxacin resistance in Klebsiella pneumoniae. This suggests that it would be prudent to reconsider the criteria used to determine "safe" upper concentration limits in food.

Background: Phenotypic studies have found high levels of antimicrobial resistance to cephalosporins, macrolides and fluoroquinolones in commensal Neisseria species in the oropharynx of men who have sex with men (MSM) using HIV pre-exposure prophylaxis (PrEP). These species include Neisseria subflava and Neisseria mucosa. This may represent a risk to pathogens like Neisseria gonorrhoeae which tend to take up antibiotic resistance genes (ARGs) from other bacteria. We aimed to explore to what extent the orophar-yngeal resistome of MSM using PrEP differed from the general population. Methods: We collected oropharyngeal swabs from 32 individuals of the general population and from 64 MSM using PrEP. Thirty-two MSM had consumed antibiotics in the previous six months, whereas none of the other participants had. Samples underwent shotgun metagenomic sequencing. Sequencing reads were mapped against MEGARes 2.0 to estimate ARG abundance. ARG abundance was compared between groups by zero-inflated negative binomial regression. Findings: ARG abundance was significantly lower in the general population than in MSM (ratio 0.41, 95% CI 0.26-0.65). More specifically, this was the case for fluoroquinolones (0.33, 95% CI 0.15-0.69), macrolides (0.37, 95% CI 0.25-0.56), tetracyclines (0.41, 95% CI 0.25-0.69), and multidrug efflux pumps (0.11, 95% CI 0.03-0.33), but not for beta-lactams (1.38, 95% CI 0.73-2.61). There were no significant differences in ARG abundance between MSM who had used antibiotics and those that had not. Interpretation: The resistome of MSM using PrEP is enriched with ARGs, independent of recent antibiotic use. Stewardship campaigns should aim to reduce antibiotic consumption in populations at high risk for STIs. (c) 2023 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Background:In antibiotic naïve populations, there is a strong association between the use of an antimicrobial and resistance to this antimicrobial. Less evidence is available as to whether this relationship is weakened in populations highly exposed to antimicrobials. Individuals taking HIV PrEP have a high intake of antimicrobials. We previously found that there was no difference in the prevalence of pheno- and genotypic antimicrobial resistance between two groups of PrEP clients who had, and had not, taken antimicrobials in the prior 6-months. Both groups did, however, have a higher prevalence of resistance than a sample of the general population. Methods: In the current study, we used zero-inflated negative binomial regression models to evaluate if there was an individual level association between the consumption of antimicrobials and 1. the minimum inhibitory susceptibilities of oral Neisseria subflava and 2. the abundance of antimicrobial resistance genes in the oropharynges of these individuals. Results:We found no evidence of an association between the consumption of antimicrobials and the minimum inhibitory susceptibilities of oral Neisseria subflava or the abundance of antimicrobial resistance genes in these individuals. Conclusions:We conclude that in high antimicrobial consumption populations, the association between antimicrobial consumption and resistance may be attenuated.

Background No randomized controlled trial (RCT) has compared the impact on the resistome of ceftriaxone (CRO) plus azithromycin (AZM) vs CRO for the treatment of Neisseria gonorrhoea (NG).Methods This was an open-label, single-center, RCT comparing the effect on the resistome of CRO plus AZM vs CRO for the treatment of NG. Men who have sex with men (MSM) with genital, anorectal, or pharyngeal NG infection were randomized into the CRO/AZM and CRO arms. Oral rinse and anorectal samples were taken for culture and resistome profiling at 2 visits (baseline and day 14). The primary outcome was the ratio of mean macrolide resistance determinants in anorectal samples from day 14 between arms.Results Twenty individuals were randomized into the CRO/AZM arm and 22 into the CRO arm. We found no significant difference in the mean macrolide resistance determinants in the day 14 anorectal samples between arms (ratio, 1.05; 95% CI, 0.55-1.83; P = .102). The prevalence of baseline macrolide resistance was high (CRO/AZM arm = 95.00%; CRO arm = 90.91%).Conclusions We could not demonstrate a significant effect of dual CRO/AZM therapy on the resistome compared with CRO alone, likely due to a high baseline resistance to AZM. Interventions to prevent the emergence of antimicrobial resistance in MSM are needed.

In addition to antimicrobial resistance, bacteria contain other mechanisms to survive antibiotic exposure such as tolerance, defined as the ability to slow metabolism by the extension of the lag phase without altering antimicrobial susceptibility. In a number of bacterial species, tolerance has been associated with treatment failure and infection chronicity and is found to precede and facilitate antimicrobial resistance. It is unknown if tolerance can be induced in Neisseria gonorrhoeae. In this study, we determined if tolerance to ceftriaxone (CRO) can be induced in N. gonorrhoeae and detected in clinical isolates. To induce tolerance, WHO P N. gonorrhoeae reference strain samples were grown under daily 3 h intermittent CRO exposure (10× the MIC), partitioned by overnight growth in GC broth. This cyclic exposure was performed for 7 consecutive days in sextuplicate, with two control cultures to which GC medium without antibiotics was added. To detect tolerance and assess CRO susceptibility, modified Tolerance Disc (TD) and Epsilometer tests were performed on isolates after each CRO exposure cycle. Additionally, this experiment was carried out on 18 clinical N. gonorrhoeae isolates. Tolerance was first detected after two CRO exposure cycles in five out of six samples. The phenotype differed per cycle with no clear pattern. No tolerance was found in control samples but was detected in 10 out of 18 clinical isolates. The present study is the first to demonstrate the induction of tolerance to CRO in N. gonorrhoeae through antibiotic exposure. In addition, tolerance to CRO was found in clinical samples.

Background: Concentrations of fluoroquinolones up to 200-fold lower than the minimal inhibitory concentration (MIC) have been shown to be able to select for antimicrobial resistance in E. coli and Salmonella spp. (the minimum selection concentration—MSC). We hypothesized that the low concentrations of quinolones found in meat may play a role in the genesis of quinolone resistance in Neisseria gonorrhoeae. We aimed to (i) establish the ciprofloxacin MSC for N. gonorrhoeae and (ii) assess if, at the ecological level, the prevalence of gonococcal ciprofloxacin resistance is associated with the concentration of quinolones used in food animal production, which is an important determinant of long-term low-dose exposure to ciprofloxacin in humans. Methods: (i) To assess if subinhibitory ciprofloxacin concentrations could select for de novo generated resistant mutants, a susceptible WHO-P N. gonorrhoeae isolate was serially passaged at 1, 1:10, 1:100 and 1:1000 of the ciprofloxacin MIC of WHO-P (0.004 mg/L) on GC agar plates. (ii) Spearman’s correlation was used to assess the association between the prevalence of ciprofloxacin resistance in N. gonorrhoeae and quinolone use for animals and quinolone consumption by humans. Results: Ciprofloxacin concentrations as low as 0.004 µg/L (1/1000 of the MIC of WHO-P) were able to select for ciprofloxacin resistance. The prevalence of ciprofloxacin resistance in N. gonorrhoeae was positively associated with quinolone use for food animals (ρ = 0.47; p = 0.004; N = 34). Conclusion: Further individual level research is required to assess if low doses of ciprofloxacin from ingested foodstuffs are able to select for ciprofloxacin resistance in bacteria colonizing humans and other species.

Commensal Neisseria provide a reservoir of resistance genes that can be transferred to the pathogens Neisseria gonorrhoeae and N. meningitidis in the human oropharynx. Surveillance programs are thus needed to monitor resistance in oropharyngeal commensal Neisseria, but currently the isolation and antimicrobial susceptibility testing of these commensals is laborious, complex and expensive. In addition, the posterior oropharyngeal/tonsillar swab, which is commonly used to sample oropharyngeal Neisseria, is poorly tolerated by many individuals. We evaluated an alternative non-invasive method to isolate oropharyngeal commensal Neisseria and to detect decreased susceptibility to azithromycin using selective media (LBVT.SNR) with and without azithromycin (2 µg/mL). In this pilot study, we compared paired posterior oropharyngeal/tonsillar swabs and oral rinse-and-gargle samples from 10 participants and demonstrated that a similar Neisseria species diversity and number of colonies were isolated from both sample types. Moreover, the proportion of Neisseria colonies that had a decreased susceptibility to azithromycin was similar in the rinse samples compared to the swabs. This pilot study has produced encouraging data that a simple protocol of oral rinse-and-gargle and culture on plates selective for commensal Neisseria with and without a target antimicrobial can be used as a surveillance tool to monitor antimicrobial susceptibility in commensal oropharyngeal Neisseria. Larger studies are required to validate these findings.

Antibiotic-sparing treatments are required to prevent the further emergence of antimicrobial resistance in Neisseria gonorrhoeae. Commensal Neisseria species have previously been found to inhibit the growth of pathogenic Neisseria species. For example, a previous study found that three out of five historical isolates of Neisseria mucosa could inhibit the growth of N. gonorrhoeae. In this study, we used agar overlay assays to assess if 24 circulating and historical isolates of Neisseria mucosa could inhibit the growth of 28 circulating and historical isolates of N. gonorrhoeae. Although pitting around each colony of N. mucosa created an optical illusion of decreased growth of N. gonorrhoeae, we found no evidence of inhibition (n = 24). In contrast, positive controls of Streptococcus pneumoniae and Escherichia coli demonstrated a strong inhibitory effect against the growth of N. gonorrhoeae.

Resistance acquisition via natural transformation is a common process in the Neisseria genus. Transformation has played an important role in the emergence of resistance to many antimicrobials in Neisseria gonorrhoeae and Neisseria meningitidis. In a previous study, we found that currently circulating isolates of Neisseria subflava had acquired an msr(D) gene that has been found to result in macrolide resistance in other bacteria but never found in Neisseria species before. To determine if this resistance mechanism is transferable among Neisseria species, we assessed if we could transform the msr(D) gene into other commensal and pathogenic Neisseria under low dose azithromycin pressure. Intraspecies recombination in commensal N. subflava was confirmed with PCR and resulted in high-level macrolide resistance. Whole-genome sequencing of these transformed strains identified the complete uptake of the msr(D) integration fragment. Sequence analysis showed that a large fragment of DNA (5 and 12 kb) was transferred through a single horizontal gene transfer event. Furthermore, uptake of the msr(D) gene had no apparent fitness cost. Interspecies transformation of msr(D) from N. subflava to N. gonorrhoeae was, however, not successful.

Background: The effect of sequential exposure to different antibiotics is an underexplored topic. Azithromycin can be detected in humans for up to 28 days post-ingestion and may prime bacterial responses to subsequently ingested antibiotics. Methods: In this in vitro study, we assessed if preexposure to azithromycin could accelerate the acquisition of resistance to ciprofloxacin in Neisseria gonorrhoeae reference strain, WHO–F. In a morbidostat, we set two conditions in 3 vials each: mono-exposure (preexposure to Gonococcal Broth followed by exposure to ciprofloxacin) and dual sequential exposure (preexposure to azithromycin followed by exposure to ciprofloxacin).The growth of the cultures was measured by a software (MATLAB). The program decided if gonococcal broth or antibiotics were added to the vials in order to keep the evolution of the cultures. Samples were taken twice a week until the end of the experiment i.e. until resistance was achieved or cellular death. Additionally, six replicates of WHO–F WT and WHO–F with rplV mutation, caused by azithromycin, were exposed to increasing concentrations of ciprofloxacin in plates to assess if there were differences in the rate of resistance emergence. Results: We found that after 12 hours of pre-exposure to azithromycin, N. gonorrhoeae's resilience to ciprofloxacin exposure increased. Pre-exposure to azithromycin did not, however, accelerate the speed to acquisition of ciprofloxacin resistance. Conclusions: We found that azithromycin does not accelerate the emergence of ciprofloxacin resistance, but there were differences in the molecular pathways to the acquisition of ciprofloxacin resistance: the strains preexpossed to azithromycin followed a different route (GyrA: S91F pathway) than the ones without antibiotic preexposure (GyrA:D95N pathway). However, the number of isolates is too small to draw such strong conclusions.

Emerging resistance to ceftriaxone and azithromycin has led to renewed interest in using ciprofloxacin to treat Neisseria gonorrhoeae. This could lead to the rapid emergence and spread of ciprofloxacin resistance. Previous studies investigating the emergence of fluoroquinolone resistance have been limited to a single strain of N. gonorrhoeae. It is unknown if different genetic backgrounds affect the evolution of fluoroquinolone resistance in N. gonorrhoeae, as has been shown in other bacterial species. This study evaluated the molecular pathways leading to ciprofloxacin resistance in two reference strains of N.gonorrhoeae-WHO-F and WHO-P. Three clones of each of the two strains of N.gonorrhoeae were evolved in the presence of ciprofloxacin, and isolates from different time points were whole-genome sequenced. We found evidence of strain-specific differences in the emergence of ciprofloxacin resistance. Two out of three clones from WHO-P followed the canonical pathway to resistance proceeding via substitutions in GyrA-S91F, GyrA-D95N and ParC. None of the three WHO-F clones followed this pathway. In addition, mutations in gyrB, uvrA and rne frequently occurred in WHO-F clones, whereas mutations in yhgF, porB and potA occurred in WHO-P.

Objectives Gonococcal infections with a higher bacterial load may pose a higher risk of transmission. We assessed the association between gonococcal bacterial load and coinfection with Mycoplasma genitalium. Methods From September 2015 until May 2018, 200 men and transgender women who have sex with men participated in an HIV pre-exposure prophylaxis (PrEP) demonstration trial in Antwerp, Belgium. They underwent 3-monthly 3-site (anus, urine, and pharynx) molecular testing for N. gonorrhoeae and C. trachomatis and M. genitalium, irrespective of symptoms. Gonococcal bacterial load was determined on remnant DNA extracts using an in-house quantitative PCR. Results were expressed as log(10) transformed copies/mL and analyzed with a linear regression model. Results Gonococcal bacterial load could be determined for 82 (80.4%) of 102 anal, 17 (73.9%) of 23 urine, and 64 (90.1%) of 71 pharyngeal samples. M. genitalium was detected in five of these anal, two urine, and two pharyngeal samples and C. trachomatis was detected in 16 anal, one urine, and two pharyngeal samples. Gonococcal bacterial load was significantly higher in the presence of M. genitalium (difference 0.92 log copies/mL, 95% CI 0.16-1.67). Conclusions Gonococcal bacterial load was higher with M. genitalium coinfection. M. genitalium may thus be a cofactor in gonococcal transmission.

Non-pathogenic Neisseria are a reservoir of antimicrobial resistance genes for pathogenic Neisseria meningitidis and Neisseria gonorrhoeae . Men who have sex with men (MSM) are at risk of co-colonization with resistant non-pathogenic and pathogenic Neisseria . We assessed if the antimicrobial susceptibility of non-pathogenic Neisseria among MSM differs from a general population and if antimicrobial exposure impacts susceptibility. We recruited 96 participants at our center in Belgium: 32 employees, 32 MSM who did not use antibiotics in the previous 6 months, and 32 MSM who did. Oropharyngeal Neisseria were cultured and identified with MALDI-TOF–MS. Minimum inhibitory concentrations for azithromycin, ceftriaxone and ciprofloxacin were determined using E-tests ® and compared between groups with non-parametric tests. Non-pathogenic Neisseria from employees as well as MSM were remarkably resistant. Those from MSM were significantly less susceptible than employees to azithromycin and ciprofloxacin ( p < 0.0001, p < 0.001), but not ceftriaxone ( p = 0.3). Susceptibility did not differ significantly according to recent antimicrobial exposure in MSM. Surveilling antimicrobial susceptibility of non-pathogenic Neisseria may be a sensitive way to assess impact of antimicrobial exposure in a population. The high levels of antimicrobial resistance in this survey indicate that novel resistance determinants may be readily available for future transfer from non-pathogenic to pathogenic Neisseria .

Purpose: STI clinic clients frequently receive antibiotics to treat sexually transmitted infections (STIs). This may select for antibiotic resistance among their commensal microbiota. We hypothesized that non-pathogenic oropharyngeal neisseriae of men who have sex with men (MSM) are less susceptible to antibiotics than those of the general population. Methods & Materials: In 2019-2020 we enrolled 96 participants at our STI clinic in Antwerp, Belgium: one group of 32 clinic employees representing the “general population” (group I) and two groups of 32 MSM (group II and III). Group I and II did not use antibiotics in the previous 6 months, whereas group III did. EswabsTM were taken from their oropharynx, plated onto modified Thayer-Martin and blood agar and incubated in 5% carbon dioxide at 36.5°C for 24 hours. Neisseria-like colonies were identified to the species level with MALDI-TOF. Minimum inhibitory concentrations (MICs) of azithromycin, ciprofloxacin and ceftriaxone were determined by E-tests®. Species diversity and MICs were compared between groups using non-parametric tests. Results: Participants’ median age was 35 (IQR 35 – 47.5). We isolated 172 non-pathogenic Neisseria colonies, representing seven different species. Data did not differ significantly between MSM of group II and III, so their data were combined. Prevalence of non-pathogenic neisseriae was 100.0% among employees and 51.6% among MSM (p<0.0001). MICs of azithromycin and ciprofloxacin were significantly higher among MSM compared with employees (azithromycin: 8.0 mg/L [IQR 3.5–268.0 mg/L] versus 3.0 mg/L, [IQR 2.0–3.6 mg/L] p<0.001; ciprofloxacin: 0.250 mg/L [IQR 0.032–0.381 mg/L] versus 0.024 mg/L [IQR 0.016–0.101 mg/L], p<0.001). MIC of ceftriaxone did not differ significantly between MSM (0.056 [IQR 0.032–0.086 mg/L]) and employees (0.043 mg/L [IQR 0.028–0.056 mg/L], p=0.25). Conclusion: MICs of oropharyngeal neisseriae from members of the general Belgian population were remarkably high, and those from STI clinic clients were even higher. This probably results from repeated antibiotic exposure at the population level and warns us that novel resistance determinants may be readily available in these neisseriae for transfer to pathogenic neisseriae. This finding urges us to critically reconsider side-effects of current population-based interventions such as screening and treatment for asymptomatic STIs.