Assessing novel partner antimicrobials to protect ceftriaxone against gonococcal resistance: An in vitro evaluation.

BACKGROUND: The emergence of ceftriaxone-resistant Neisseria gonorrhoeae poses a significant threat to existing treatment regimens. Our study aimed to assess the efficacy of antimicrobials that could be combined with ceftriaxone to reduce the probability of ceftriaxone resistance emerging and spreading in N. gonorrhoeae. METHODS AND RESULTS: Broth microdilution was used to determine the minimal inhibitory concentrations (MICs) for a panel of ceftriaxone-resistant (WHO X, Y, Z) and ceftriaxone-susceptible (WHO L, N, P) N. gonorrhoeae WHO reference strains for the following antimicrobials: ceftriaxone, doxycycline, azithromycin, zoliflodacin, fosfomycin, pristinamycin, ramoplanin, gentamicin and NAI-107. The MICs for zoliflodacin and pristinamycin for all strains were lower than or equal to the available breakpoints. A checkerboard assay was used to determine the drug-drug combination effect, which showed either an indifferent or an additive effect for all the combinations tested with ceftriaxone. CONCLUSIONS: The low MICs of zoliflodacin and pristinamycin for the three ceftriaxone-resistant strains suggest that these antimicrobials could be used as partner drugs with ceftriaxone to reduce the probability of ceftriaxone resistance spreading in areas with a high prevalence of ceftriaxone resistance.

Doxycycline post-exposure prophylaxis among men who have sex with men and transgender women in Belgium: awareness, use and antimicrobial resistance concerns in a cross-sectional online survey.

OBJECTIVES: We aimed to assess the awareness, willingness to use and use of doxycycline post-exposure prophylaxis (doxyPEP) among men who have sex with men (MSM) and transgender women (TGW) in Belgium. Additionally, we aimed to identify factors associated with doxyPEP use and concerns regarding antimicrobial resistance (AMR). METHODS: Cross-sectional online survey among MSM and TGW in Belgium in April 2024. Participants were recruited through sexual networking applications and social media of community-based organisations. Numerical variables were compared with Wilcoxon rank-sum test and categorical variables with χ2 or Fisher's exact tests. Factors associated with doxyPEP use were assessed using logistic regression. Willingness to use doxyPEP and concerns about side effects/AMR were assessed before and after presenting a brief paragraph on the potential effects of doxyPEP on AMR. RESULTS: 875 individuals initiated the survey. Almost all identified as men (860/875, 98.3%) with a median age of 40 years (IQR 32-48), 40.4% (n=352/875) had heard of doxyPEP and 9.4% (n=82/875) had used it, among whom the majority used it within the previous 6 months (70/81, 86.4%). In multivariable logistic regression, doxyPEP use was associated with reporting ≥1 sexually transmitted infection (STI) in the previous 12 months, engagement in chemsex, HIV status and pre-exposure prophylaxis use, and education level.About 80% of the participants initially reported being willing to use doxyPEP, and about 50% reported being concerned about side effects. After reading about the potential effects of doxyPEP on AMR, willingness to use decreased to 60% and concerns of side effects/AMR increased to around 70%. CONCLUSIONS: Approximately 1 in 10 MSM in Belgium reported using doxyPEP. A recent history of STIs and STI risk factors were positively associated with doxyPEP use. Importantly, concerns about AMR and side effect influenced willingness to use doxyPEP. If doxyPEP is introduced, informing patients about doxyPEP benefits and risks is crucial to enable informed decision-making.

Genomic oropharyngeal Neisseria surveillance detects MALDI-TOF MS species misidentifications and reveals a novel Neisseria cinerea clade.

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.

Antimicrobial susceptibility of commensal Neisseria spp. in parents and their children in Belgium: a cross-sectional survey.

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.

The Prevalence of Antibiotic Tolerance in Neisseria gonorrhoeae Varies by Anatomical Site.

BACKGROUND: Tolerance enables bacteria to survive intermittent antibiotic exposure without an increase in antimicrobial susceptibility. In this study, we investigated the presence of tolerance to three antimicrobials, ceftriaxone, azithromycin and ciprofloxacin, in clinical isolates and the WHO (World Health Organization) reference panel of Neisseria gonorrhoeae. METHODS: We used the modified tolerance disk (TD test) to assess for tolerance to ceftriaxone, azithromycin and ciprofloxacin in 14 WHO reference strains and 62 N. gonorrhoeae clinical isolates-evenly divided between anorectal and urogenital infections. The isolates underwent a three-step incubation process wherein the isolates were exposed to an antibiotic disk for 20 h of incubation (Step I), followed by the replacement of the antibiotic disk with a nutrient disk for overnight incubation (Step II) and additional overnight incubation with extra nutrients (Step III). RESULTS: A total of 4 of the 62 clinical anorectal isolates and none of the urogenital isolates exhibited tolerance to azithromycin (p = 0.033). Tolerance to ceftriaxone and ciprofloxacin was observed in eight and four isolates, respectively, with no difference between infection sites. Tolerance was also detected in 8 (K, M, N, O, P, U, V, W) out of the 14 WHO reference strains, with varying patterns of tolerance to ceftriaxone (n = 8), ciprofloxacin (n = 2) and azithromycin (n = 1). CONCLUSIONS: This study identified ceftriaxone, azithromycin and ciprofloxacin tolerance in clinical and WHO reference N. gonorrhoeae isolates. Azithromycin tolerance was more common in anorectal than urogenital infections.

Understanding adaptive responses in PrEP service delivery in Belgian HIV clinics: a multiple case study using an implementation science framework.

INTRODUCTION: In Belgium, oral HIV pre-exposure prophylaxis (PrEP) is primarily provided in specialized clinical settings. Optimal implementation of PrEP services can help to substantially reduce HIV transmission. However, insights into implementation processes, and their complex interactions with local context, are limited. This study examined factors that influence providers' adaptive responses in the implementation of PrEP services in Belgian HIV clinics. METHODS: We conducted a qualitative multiple case study on PrEP care implementation in eight HIV clinics. Thirty-six semi-structured interviews were conducted between January 2021 and May 2022 with a purposive sample of PrEP care providers (e.g. physicians, nurses, psychologists), supplemented by 50 hours of observations of healthcare settings and clinical interactions. Field notes from observations and verbatim interview transcripts were thematically analysed guided by a refined iteration of extended Normalisation Process Theory. RESULTS: Implementing PrEP care in a centralized service delivery system required considerable adaptive capacity of providers to balance the increasing workload with an adequate response to PrEP users' individual care needs. As a result, clinic structures were re-organized to allow for more efficient PrEP care processes, compatible with other clinic-level priorities. Providers adapted clinical and policy norms on PrEP care (e.g. related to PrEP prescribing practices and which providers can deliver PrEP services), to flexibly tailor care to individual clients' situations. Interprofessional relationships were reconfigured in line with organizational and clinical adaptations; these included task-shifting from physicians to nurses, leading them to become increasingly trained and specialized in PrEP care. As nurse involvement grew, they adopted a crucial role in responding to PrEP users' non-medical needs (e.g. providing psychosocial support). Moreover, clinicians' growing collaboration with sexologists and psychologists, and interactions with PrEP users' family physician, became crucial in addressing complex psychosocial needs of PrEP clients, while also alleviating the burden of care on busy HIV clinics. CONCLUSIONS: Our study in Belgian HIV clinics reveals that the implementation of PrEP care presents a complex-multifaceted-undertaking that requires substantial adaptive work to ensure seamless integration within existing health services. To optimize integration in different settings, policies and guidelines governing PrEP care implementation should allow for sufficient flexibility and tailoring according to respective local health systems.

Ciprofloxacin Concentrations 100-Fold Lower than the MIC Can Select for Ciprofloxacin Resistance in Neisseria subflava: An In Vitro Study.

Neisseria gonorrhoeae can acquire antimicrobial resistance (AMR) through horizontal gene transfer (HGT) from other Neisseria spp. such as commensals like Neisseria subflava. Low doses of antimicrobials in food could select for AMR in N. subflava, which could then be transferred to N. gonorrhoeae. In this study, we aimed to determine the lowest concentration of ciprofloxacin that can induce ciprofloxacin resistance (minimum selection concentration-MSC) in a N. subflava isolate (ID-Co000790/2, a clinical isolate collected from a previous community study conducted at ITM). In this study, Neisseria subflava was serially passaged on gonococcal (GC) medium agar plates containing ciprofloxacin concentrations ranging from 1:100 to 1:10,000 below its ciprofloxacin MIC (0.006 µg/mL) for 6 days. After 6 days of serial passaging at ciprofloxacin concentrations of 1/100th of the MIC, 24 colonies emerged on the plate containing 0.06 µg/mL ciprofloxacin, which corresponds to the EUCAST breakpoint for N. gonorrhoeae. Their ciprofloxacin MICs were between 0.19 to 0.25 µg/mL, and whole genome sequencing revealed a missense mutation T91I in the gyrA gene, which has previously been found to cause reduced susceptibility to fluoroquinolones. The N. subflava MSCde novo was determined to be 0.06 ng/mL (0.00006 µg/mL), which is 100×-fold lower than the ciprofloxacin MIC. The implications of this finding are that the low concentrations of fluoroquinolones found in certain environmental samples, such as soil, river water, and even the food we eat, may be able to select for ciprofloxacin resistance in N. subflava.

Potential determinants of the decline in mpox cases in Belgium: A behavioral, epidemiological and seroprevalence study.

OBJECTIVES: The 2022 mpox epidemic reached a peak in Belgium and the rest of Europe in July 2022, after which it unexpectedly subsided. This study investigates epidemiological, behavioral, and immunological factors behind the waning of the epidemic in Belgium. METHODS: We investigated temporal evolutions in the characteristics and behavior of mpox patients using national surveillance data and data from a prospective registry of mpox patients in the Institute of Tropical Medicine (Antwerp). We studied behavioral changes in the population at risk using a survey among HIV-preexposure prophylaxis (PrEP) users. We determined the seroprevalence of anti-orthopoxvirus antibodies among HIV-PrEP users across four-time points in 2022. RESULTS: Mpox patients diagnosed at the end of the epidemic had less sexual risk behavior compared to those diagnosed earlier: they engaged less in sex at mass events, had fewer sexual partners, and were less likely to belong to the sexual network's central group. Among HIV-PrEP users there were no notable changes in sexual behavior. Anti-orthopoxvirus seroprevalence did not notably increase before the start of national vaccination campaigns. CONCLUSION: The observed changes in group immunity and behavior in the population at greater risk of exposure to mpox seem unable to explain the waning of the mpox epidemic. A change in the profile of mpox patients might have contributed to the decline in cases.

Effect of erythromycin residuals in food on the development of resistance in Streptococcus pneumoniae: an in vivo study in Galleria mellonella.

Background: The use of antimicrobials to treat food animals may result in antimicrobial residues in foodstuffs of animal origin. The European Medicines Association (EMA) and World Health Organization (WHO) define safe antimicrobial concentrations in food based on acceptable daily intakes (ADIs). It is unknown if ADI doses of antimicrobials in food could influence the antimicrobial susceptibility of human-associated bacteria. Objectives: This aim of this study was to evaluate if the consumption of ADI doses of erythromycin could select for erythromycin resistance in a Galleria mellonella model of Streptococcus pneumoniae infection. Methods: A chronic model of S. pneumoniae infection in G. mellonella larvae was used for the experiment. Inoculation of larvae with S. pneumoniae was followed by injections of erythromycin ADI doses (0.0875 and 0.012 µg/ml according to EMA and WHO, respectively). Isolation of S. pneumoniae colonies was then performed on selective agar plates. Minimum inhibitory concentrations (MICs) of resistant colonies were measured, and whole genome sequencing (WGS) was performed followed by variant calling to determine the genetic modifications. Results: Exposure to single doses of both EMA and WHO ADI doses of erythromycin resulted in the emergence of erythromycin resistance in S. pneumoniae. Emergent resistance to erythromycin was associated with a mutation in rplA, which codes for the L1 ribosomal protein and has been linked to macrolide resistance in previous studies. Conclusion: In our in vivo model, even single doses of erythromycin that are classified as acceptable by the WHO and EMA induced significant increases in erythromycin MICs in S. pneumoniae. These results suggest the need to include the induction of antimicrobial resistance (AMR) as a significant criterion for determining ADIs.

Could the effect of antimicrobials on antimicrobial resistance be saturated at high-antimicrobial consumption? A comparison of the MORDOR and ResistAZM studies.

OBJECTIVES: Antimicrobial resistance poses a considerable threat in high-antimicrobial-consumption populations, such as men who have sex with men (MSM) taking HIV pre-exposure prophylaxis. While the ResistAZM trial found no increase in macrolide resistance genes in MSM with gonorrhea after azithromycin treatment, the MORDOR trial observed an increase in these genes after mass azithromycin distribution. We hypothesized that this could be due to saturation of the resistome. To test this hypothesis, we compared the abundance of macrolide resistance determinants in anorectal samples between the baselines of the two trials. METHODS: Shotgun metagenome reads from the anorectal baseline samples from the ResistAZM (n = 42) and MORDOR (n = 30) trials were analyzed using AMRPlusPlus. Nonhost reads were mapped to the MEGARes database to detect antibiotic resistance genes (ARG). Antimicrobial resistance (AMR) was normalized using cumulative sum scaling, and ARG abundance was estimated. RESULTS: Macrolide, lincosamides, and streptogramins determinants were approximately 10-fold more abundant in the ResistAZM than the MORDOR samples (P ≤ 0.001). CONCLUSION: The findings are compatible with our hypothesis. Thus, in populations with high-antimicrobial use, the relationship between antimicrobial consumption and AMR may be diminished due to saturation. These findings are vital for future studies investigating the resistogencity of novel interventions, such as doxycycline post-exposure prophylaxis, in populations with high preceding consumption of antimicrobials.

Belgian 2024 guidance on the use of pre-exposure prophylaxis.

OBJECTIVES: We aimed to develop a guidance on the use of pre-exposure prophylaxis (PrEP) for HIV tailored to the Belgian context. METHODS: Different aspects of PrEP care were judged by an expert group of nine Belgian clinicians, seeking consensus for areas of controversies. RESULTS: PrEP should be considered in HIV negative patients at high risk of acquiring HIV. Currently, only oral tenofovir/emtricitabine is available in Belgium for PrEP, which can be used daily, or also event-driven in cisgender men and trans women who are not taking exogenous estradiol-based hormones. Personal counselling directed at medication adherence and sexual health should have a central role in PrEP care. At the initial assessment clinicians should give attention to symptoms of an acute HIV infection, the patients' immunization status and renal function. A regular follow-up must be set up to diagnose HIV seroconversion, treat sexually transmitted infections, and manage side effects of PrEP. CONCLUSION: The Belgian guidance on the use of PrEP provides a point of reference for standard PrEP care in Belgium and will be periodically updated.

Could traces of fluoroquinolones in food induce ciprofloxacin resistance in Escherichia coli and Klebsiella pneumoniae? An in vivo study in Galleria mellonella with important implications for maximum residue limits in food.

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.

Oral streptococcal susceptibility to azithromycin may be associated with doxycycline use: A post-hoc analysis with implications for doxycycline post exposure prophylaxis.

OBJECTIVE: Tetracycline and macrolide resistance are frequently linked in streptococci and other species. We aimed to assess the association between doxycycline use and azithromycin MICs in oral streptococci. METHODS: Linear regression was used to assess the association between doxycycline use in the prior year and the median MIC per participant of oral streptococcal colonies isolated at the baseline visit of the ResistAZM study. The analysis controlled for receipt of other antimicrobials as well as time since antimicrobial consumption. RESULTS: Fifty-six individual colonies confirmed to be streptococci were isolated from 19 individuals at baseline. The azithromycin MICs of these isolates varied considerably between 0.25 mg/L and >256 mg/L (median 28 mg/L; IQR 1-192 mg/L). The consumption of doxycycline in the preceding 12 months was positively associated with median streptococcal azithromycin MIC (coef. 151.6 [95% CI 10.6-292.7]; p = .037). CONCLUSION: This post-hoc analysis found that doxycycline use was associated with streptococcal azithromycin susceptibility. Numerous limitations of the study design mean that this study is best considered hypothesis generating. Prospective studies are required to assess if the use of doxycycline could select for macrolide resistance in oral streptococci.

45 years of tetracycline post exposure prophylaxis for STIs and the risk of tetracycline resistance: a systematic review and meta-analysis.

There is considerable interest in the use of doxycycline post exposure prophylaxis (PEP) to reduce the incidence of bacterial sexually transmitted infections (STIs). An important concern is that this could select for tetracycline resistance in these STIs and other species. We searched PubMed and Google Scholar, (1948-2023) for randomized controlled trials comparing tetracycline PEP with non-tetracycline controls. The primary outcome was antimicrobial resistance (AMR) to tetracyclines in all bacterial species with available data. Our search yielded 140 studies, of which three met the inclusion criteria. Tetracycline PEP was associated with an increasedprevalence of tetracycline resistance in Neisseria gonorrhoeae, but this effect was not statistically significant (Pooled OR 2.3, 95% CI 0.9-3.4). PEP had a marked effect on the N. gonorrhoeae tetracycline MIC distribution in the one study where this was assessed. Prophylactic efficacy was 100% at low MICs and 0% at high MICs. In the one study where this was assessed, PEP resulted in a significant increase in tetracycline resistance in commensal Neisseria species compared to the control group (OR 2.9, 95% CI 1.5-5.5) but no significant effect on the prevalence of tetracycline resistance in Staphylococcus aureus. The available evidence suggests that PEP with tetracyclines could be associated with selecting tetracycline resistance in N. gonorrhoeae and commensal Neisseria species.

Four recent insights suggest the need for more refined methods to assess the resistogenicity of doxycycline post exposure prophylaxis.

Two recently published randomized trials of doxycycline post exposure prophylaxis (PEP) have concluded that this intervention is highly effective at reducing the incidence of bacterial sexually transmitted infections (STIs) and has little or no risk of promoting the spread of antimicrobial resistance (AMR). In this perspective piece, we review four types of evidence that suggest that the risk of promoting AMR has been inadequately assessed in these studies. 1) The studies have all used proportion resistant as the outcome measure. This is a less sensitive measure of resistogenicity than MIC distribution. 2) These RCTs have not considered population-level pathways of AMR selection. 3) In populations with very high antimicrobial consumption such as PrEP cohorts, the relationship between antimicrobial consumption and resistance may be saturated. 4) Genetic linkage of AMR means that increased tetracycline use may select for AMR to not only tetracyclines but also other antimicrobials in STIs and other bacterial species. We recommend novel study designs to more adequately assess the AMR-inducing risk of doxycycline PEP.

Effect of mass treatment on the long-term prevalence of gonorrhoea, chlamydia and syphilis-a systematic review.

BACKGROUND: Selective mass treatment of STIs may lead to a durable reduction in the prevalence of STIs or a temporary reduction associated with an increased probability of antimicrobial resistance emerging. METHODS: We searched PubMed and Google Scholar for studies evaluating the impact of mass STI treatment on the long-term prevalence of chlamydia, gonorrhoea, syphilis and chancroid. The primary outcomes were the long term (≥3 months post the intervention) impact of the intervention on prevalence/incidence of the STI and on antimicrobial resistance. RESULTS: Our search yielded 269 studies, of which 4 met the inclusion criteria. With the exception of the Carletonville study, where this was not assessed, three of the four studies found that intensive STI treatment was associated with a reduced prevalence of the targeted STI during or immediately after the intervention. In all four studies, there was no evidence that the intense treatment had a long-term effect on prevalence. In the only study where this was assessed, the intensive use of penicillin to reduce gonococcal prevalence was associated with the emergence of reduced susceptibility to penicillin in N. gonorrhoeae. CONCLUSION: The available evidence suggests that mass treatment of chlamydia, gonorrhoea and syphilis in high prevalence populations is only associated with a temporary reduction in the prevalence of these infections and may select for antimicrobial resistance.