Protective effect of microbisporicin (NAI-107) against vancomycin resistant Enterococcus faecium infection in a Galleria mellonella model.

Increasing antimicrobial resistance in Enterococcus faecium necessitates the search for novel treatment agents, such as bacteriocins. In this study, we conducted an in vivo assessment of five bacteriocins, namely Lacticin Z, Lacticin Q, Garvicin KS (ABC), Aureocin A53 and Microbisporicin (NAI-107), against vanB-resistant Enterococcus faecium using a Galleria mellonella model. Our in vitro experiments demonstrated the efficacy of all five bacteriocins against vanB-resistant E. faecium with only NAI-107 demonstrating in vivo efficacy. Notably, NAI-107 exhibited efficacy across a range of tested doses, with the highest efficacy observed at a concentration of 16 µg/mL. Mortality rates in the group treated with 16 µg/mL NAI-107 were lower than those observed in the linezolid-treated group. These findings strongly suggest that NAI-107 holds promise as a potential alternative therapeutic agent for treating infections caused by resistant E. faecium and warrants further investigation.

Gonococcal resistance to zoliflodacin could emerge via transformation from commensal Neisseria species. An in-vitro transformation study.

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.

Chronic high level parasitemia in HIV-infected individuals with or without visceral leishmaniasis in an endemic area in North-West Ethiopia: potential superspreaders?

BACKGROUND: HIV patients with recurrent visceral leishmaniasis (VL) could potentially drive Leishmania transmission in areas with anthroponotic transmission such as East-Africa, but studies are lacking. Leishmania parasitemia has been used as proxy for infectiousness. METHODS: This study is nested within the PreLeish prospective cohort study, following a total of 490 HIV infected individuals free of VL at enrollment for upto 24-37 months in North-West Ethiopia. Blood Leishmania PCR was done systematically. This case series reports on ten HIV-coinfected individuals with chronic VL (≥3 VL episodes during follow-up) for upto 37 months, and three individuals with asymptomatic Leishmania infection for upto 24 months. RESULTS: All ten chronic VL cases were male, on antiretroviral treatment, with 0-11 relapses before enrollment. Median baseline CD4 counts were 82 cells/µL. They displayed three to six VL treatment episodes over a period upto 37 months. Leishmania blood PCR levels were strongly positive for almost the entire follow-up time (median Ct value 26 (IQR 23-30), including during periods between VL treatment. Additionally, we describe three HIV-infected individuals with asymptomatic Leishmania infection and without VL history, with equally strong Leishmania parasitemia over a period of upto 24 months without developing VL. All were on antiretroviral treatment at enrollment, with baseline CD4 counts ranging from 78 to 350 cells/µL. CONCLUSION: These are the first data on chronic parasitemia in HIV-infected individuals from L donovani endemic areas. HIV patients with asymptomatic and symptomatic Leishmania infection could potentially be highly infectious and constitute Leishmania superspreaders. Xenodiagnosis studies are required to confirm infectiousness.

Ramoplanin as a novel therapy for Neisseria gonorrhoeae infection: an in vitro and in vivo study in Galleria mellonella.

Neisseria gonorrhoeae is a bacterial pathogen that causes gonorrhoea, a sexually transmitted infection. Increasing antimicrobial resistance in N. gonorrhoeae is providing motivation to develop new treatment options. In this study, we investigated the effectiveness of the antibiotic ramoplanin as a treatment for N. gonorrhoeae infection. We tested the effectiveness of ramoplanin in vitro against 14 World Health Organization (WHO) reference strains of N. gonorrhoeae and found that it was active against all 14 strains tested. Furthermore, in a Galleria mellonella infection model of N. gonorrhoeae WHO P, we demonstrated that ramoplanin was active in vivo without any evidence of toxicity. This suggests that ramoplanin might be a new promising antibiotic treatment for gonorrhoea.

Genome Mining Uncovers NRPS and PKS Clusters in Rothia dentocariosa with Inhibitory Activity against Neisseria Species.

The growing global threat of antimicrobial resistance is reaching a crisis point as common bacterial infections, including those caused by pathogenic Neisseria species, are becoming increasingly untreatable. This is compelling the scientific community to search for new antimicrobial agents, taking advantage of computational mining and using whole genome sequences to discover natural products from the human microbiome with antibiotic effects. In this study, we investigated the crude extract from a Rothia dentocariosa strain with demonstrated antimicrobial activity against pathogenic Neisseria spp. by spot-on-lawn assay. The genomic DNA of the R. dentocariosa strain was sequenced, and bioinformatic evaluation was performed using antiSMASH and PRISM to search for biosynthetic gene clusters (BGCs). The crude extract with potential antimicrobial activity was run on Tricine-SDS-PAGE, and the putative peptides were characterised using liquid chromatography-tandem mass spectrometry (LC-MS). The crude extract inhibited the growth of the pathogenic Neisseria spp. Six BGCs were identified corresponding to non-ribosomal peptide synthases (NRPSs), polyketide synthases (PKSs), and ribosomally synthesised and post-translationally modified peptides. Three peptides were also identified corresponding to Actinorhodin polyketide putative beta-ketoacyl synthase 1. These findings serve as a useful reference to facilitate the research and development of NRPS and PKS as antimicrobial products against multidrug-resistant N. gonorrhoeae.

Microbisporicin (NAI-107) protects Galleria mellonella from infection with Neisseria gonorrhoeae.

IMPORTANCE: We screened 66 bacteriocins to see if they exhibited anti-gonococcal activity. We found 12 bacteriocins with anti-gonococcal effects, and 4 bacteriocins showed higher anti-gonococcal activity. Three bacteriocins, lacticin Z, lacticin Q, and Garvicin KS (ABC), showed in vitro anti-gonococcal activity but no in vivo inhibitory effects against the Neisseria gonorrhoeae (WHO-P) isolate. On the other hand, NAI-107 showed in vivo anti-gonococcal activity. The findings suggest that NAI-107 is a promising alternative to treat gonorrhea infections.

Doxycycline PEP can induce doxycycline resistance in Klebsiella pneumoniae in a Galleria mellonella model of PEP.

Background: Four randomized controlled trials have now established that doxycycline post exposure (sex) prophylaxis (PEP) can reduce the incidence of chlamydia and syphilis in men who have sex with men. These studies have concluded that the risk of selecting for antimicrobial resistance is low. We evaluated this risk in vitro and in vivo using a Galleria mellonella infection model. Methods: We evaluated how long it took for doxycycline resistance to emerge during passage on doxycycline containing agar plates in 4 species - Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae and Neisseria subflava. We then assessed if K. pneumoniae could acquire resistance to doxycycline (and cross resistance to other antimicrobials) during intermittent exposure to doxycycline in a Galleria mellonella model of doxycycline PEP. Results: In our passage experiments, we found that resistance first emerged in K. pneumoniae. By day 7 the K. pneumoniae MIC had increased from 2 mg/L to a median of 96 mg/L (IQR 64-96). Under various simulations of doxycycline PEP in the G. mellonella model, the doxycycline MIC of K. pneumoniae increased from 2 mg/L to 48 mg/L (IQR 48-84). Ceftriaxone and ciprofloxacin MICs increased over ten-fold. Whole genome sequencing revealed acquired mutations in ramR which regulates the expression of the AcrAB-TolC efflux pump. Conclusion: Doxycycline PEP can select for doxycycline, ceftriaxone and ciprofloxacin resistance in K. pneumoniae in a G. mellonella model. The emergent ramR mutations were similar to those seen in circulating strains of K. pneumoniae. These findings suggest that we need to assess the effect of doxycycline PEP on resistance induction on a broader range of bacterial species than has hitherto been the case.

Enolase Is Implicated in the Emergence of Gonococcal Tolerance to Ceftriaxone.

Antibiotic tolerance is associated with antibiotic treatment failure, and molecular mechanisms underlying tolerance are poorly understood. We recently succeeded in inducing tolerance to ceftriaxone (CRO) in an N. gonorrhoeae reference isolate. In a prior in vitro study, six biological replicates of WHO P strains were exposed to CRO (10× the MIC) followed by overnight growth, and tolerance was assessed using a modified Tolerance Disc (T.D.) test. In the current study, we characterized the mutation profile of these CRO-tolerant phenotypes. The whole genome was sequenced from isolates from different replicates and time points. We identified mutations in four genes that may contribute to ceftriaxone tolerance in N. gonorrhoeae, including a mutation in the enolase (eno) gene that arose independently in three lineages.

Detection of asymptomatic Leishmania infection in blood donors at two blood banks in Ethiopia.

Visceral leishmaniasis (VL) is a disease caused by Leishmania parasites. While predominantly transmitted by sandflies, cases of VL transmitted through blood transfusion have been reported, particularly in immunocompromised recipients. Although Leishmania parasites have been found in blood donors in some VL endemic areas, this has never been studied in East-Africa, where HIV prevalence is relatively high. We established the prevalence of asymptomatic Leishmania infection and associated socio-demographic factors among blood donors presenting at two blood bank sites (Metema and Gondar) in northwest Ethiopia between June and December 2020. Metema is located in a VL-endemic area; Gondar has historically been considered VL non-endemic but as an outbreak of VL has occurred around Gondar, it was defined as previously VL non-endemic. Blood samples were tested by the rK39 rapid diagnostic test (RDT), rK39 ELISA, direct agglutination test (DAT) and qPCR targeting kinetoplast DNA (kDNA). Asymptomatic infection was defined as positive by any of these tests in a healthy person. A total of 426 voluntary blood donors were included. The median age was 22 years (IQR, 19-28 years); 59% were male and 81% resided in urban areas. Only one participant had a history of VL and three had a family history of VL. Asymptomatic infection was detected in 15.0% (n = 32/213) in Metema and 4.2% (n = 9/213) in Gondar. The rK39 ELISA was positive in 5.4% (n = 23/426), the rK39 RDT in 2.6% (11/426), PCR in 2.6% (11/420) and DAT in 0.5% (2/426). There were six individuals with two positive tests: one positive on rK39 RDT and PCR and five positive on rK39 RDT and ELISA. The prevalence of asymptomatic infection was higher in Metema (VL-endemic) and males but was not associated with age, a history of VL amongst family members or living in a rural area. Antibodies against Leishmania and parasite DNA was detected in a substantial number of blood donors. Future research should be directed at better defining the risk to recipients, including parasite viability studies and longitudinal studies amongst recipients.

The oropharynx of men using HIV pre-exposure prophylaxis is enriched with antibiotic resistance genes: A cross-sectional observational metagenomic study.

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 oropharyngeal 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.

The Discovery of Oropharyngeal Microbiota with Inhibitory Activity against Pathogenic Neisseria gonorrhoeae and Neisseria meningitidis: An In Vitro Study of Clinical Isolates.

With increasing incidence of pathogenic Neisseria infections coupled with emerging resistance to antimicrobials, alternative approaches to limit the spread are sought. We investigated the inhibitory effect of oropharyngeal microbiota on the growth of N. gonorrhoeae and N. meningitidis and the impact of the essential oil-based mouthwash Listerine Cool Mint® (Listerine). Oropharyngeal swabs from 64 men who have sex with men (n = 118) from a previous study (PReGo study) were analysed (ClinicalTrials.gov, NCT03881007). These included 64 baseline and 54 samples following three months of daily use of Listerine. Inhibition was confirmed by agar overlay assay, and inhibitory bacteria isolated using replica plating and identified using MALDI-TOF. The number of inhibitory isolates were compared before and after Listerine use. Thirty-one pharyngeal samples (26%) showed inhibitory activity against N. gonorrhoeae and/or N. meningitidis, and 62 inhibitory isolates were characterised. Fourteen species belonging to the genera Streptococci and Rothia were identified. More inhibitory isolates were observed following Listerine use compared to baseline, although this effect was not statistically significant (p = 0.073). This study isolated and identified inhibitory bacteria against pathogenic Neisseria spp. and established that daily Listerine use did not decrease their prevalence. These findings could provide a new approach for the prevention and treatment of pharyngeal Neisseria infections.

Tolerance to Ceftriaxone in Neisseria gonorrhoeae: Rapid Induction in WHO P Reference Strain and Detection in Clinical Isolates.

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.

Ciprofloxacin Concentrations 1/1000th the MIC Can Select for Antimicrobial Resistance in N. gonorrhoeae-Important Implications for Maximum Residue Limits in Food.

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.

Successful Intra- but Not Inter-species Recombination of msr(D) in Neisseria subflava.

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.

Alternative Pathways to Ciprofloxacin Resistance in Neisseria gonorrhoeae: An In Vitro Study of the WHO-P and WHO-F Reference Strains

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.

Screening of Anorectal and Oropharyngeal Samples Fails to Detect Bacteriophages Infecting Neisseria gonorrhoeae.

There are real concerns that Neisseria gonorrhoeae may become untreatable in the near future due to the rapid emergence of antimicrobial resistance. Alternative therapies are thus urgently required. Bacteriophages active against N. gonorrhoeae could play an important role as an antibiotic-sparing therapy. To the best of our knowledge, no bacteriophages active against N. gonorrhoeae have ever been found. The aim of this study was to screen for bacteriophages able to lyse N. gonorrhoeae in oropharyngeal and anorectal swabs of 74 men who have sex with men attending a sexual health clinic in Antwerp, Belgium. We screened 210 swabs but were unable to identify an anti-gonococcal bacteriophage. This is the first report of a pilot screening that systematically searched for anti-gonococcal phages directly from clinical swabs. Further studies may consider screening for phages at other anatomical sites (e.g., stool samples, urine) or in environmental settings (e.g., toilet sewage water of sex clubs or sexually transmitted infection clinics) where N. gonorrhoeae can be found.

A Novel Method to Assess Antimicrobial Susceptibility in Commensal Oropharyngeal Neisseria-A Pilot Study.

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.

Antimicrobial susceptibility of commensal Neisseria in a general population and men who have sex with men in Belgium.

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.

Pre-exposure to azithromycin enhances gonococcal resilience to subsequent ciprofloxacin exposure: an in vitro study.

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.