Diagnosing Viral Infections Through T-Cell Receptor Sequencing of Activated CD8+ T Cells.

T-cell-based diagnostic tools identify pathogen exposure but lack differentiation between recent and historical exposures in acute infectious diseases. Here, T-cell receptor (TCR) RNA sequencing was performed on HLA-DR+/CD38+CD8+ T-cell subsets of hospitalized coronavirus disease 2019 (COVID-19) patients (n = 30) and healthy controls (n = 30; 10 of whom had previously been exposed to severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]). CDR3α and CDR3ß TCR regions were clustered separately before epitope specificity annotation using a database of SARS-CoV-2-associated CDR3α and CDR3ß sequences corresponding to >1000 SARS-CoV-2 epitopes. The depth of the SARS-CoV-2-associated CDR3α/ß sequences differentiated COVID-19 patients from the healthy controls with a receiver operating characteristic area under the curve of 0.84 ± 0.10. Hence, annotating TCR sequences of activated CD8+ T cells can be used to diagnose an acute viral infection and discriminate it from historical exposure. In essence, this work presents a new paradigm for applying the T-cell repertoire to accomplish TCR-based diagnostics.

Breakthrough Mpox Outbreak Investigation, the Delicate Balance Between Host Immune Response and Viral Immune Escape.

BACKGROUND: Limited data are available on Mpox breakthrough infections. PURPOSE: The purpose of this study is to investigate a Mpox breakthrough outbreak in 3 vaccinated individuals. METHODS: Study participants provided informed consent. Serology testing was performed in one involved individual (ID-1) using an in-house assay detecting anti-orthopoxvirus IgG. Whole genome sequencing (WGS) was carried out and compared with the reference sequence ON563414.3 ( https://www.ncbi.nlm.nih.gov/nuccore/ON563414.3/ ). RESULTS: Three individuals vaccinated with modified vaccinia Ankara-Bavaria Nordic contracted Mpox following one sexual intercourse event. One of them (ID-1) had received only one vaccine dose, while the other two were fully vaccinated. ID-1 presented to the sexual health clinic of the Universitair Ziekenhuis Brussel with proctitis related to Mpox. Despite one vaccination, serology testing Three months post vaccine showed absence of Mpox virus (MPXV) specific antibodies in ID-1. In contrast, 2 weeks after the sexual intercourse, seroconversion occurred. Whole genome sequencing of the isolated MPXV showed, compared with the reference sequence, a total of seven single nucleotide variants with four of them indicating protein amino-acid changes. CONCLUSION: Incomplete MPXV vaccination as well as MPXV variants might result in breakthrough infections. Preventive measures, such as MPVX vaccination, could maintain immunity in individuals with higher risk of MPXV infection, and might lower disease severity.

Detection of mpox virus in ambient air in a sexual health clinic.

Although transmitted mainly through direct (sexual) contact, mpox virus (MPXV) can be detected in ambient air. We explored the use of air sampling for diagnosis or (genomic) surveillance of mpox in a sexual health clinic. For six out of six patients who were infected with MPXV, all four of our ambient air PCR tests were positive. For 14 uninfected patients, PCR was positive in three ambient air samples, albeit with higher cycle threshold (Ct) values. Genomic sequencing of samples from two positive patients showed matching sequences between air and clinical samples.

Global epidemiology of antimicrobial resistance in commensal Neisseria species: A systematic review.

BACKGROUND: Commensal Neisseria species (spp). represent an important reservoir of antimicrobial resistance genes for pathogenic Neisseria spp. In this systematic review, we aimed to assess the antimicrobial susceptibility of commensal Neisseria spp. and how this has evolved over time. We also aimed to assess if commensal Neisseria spp. showed intrinsic resistance to four antimicrobials - penicillin, azithromycin, ceftriaxone and ciprofloxacin. METHODS: Pubmed and Google Scholar were searched following the PRISMA guidelines. Articles reporting MICs of commensal Neisseria spp. were included according to inclusion/exclusion criteria, and the quality of the articles was assessed using a pre-designed tool. Individual and summary measures of penicillin, azithromycin, ceftriaxone and ciprofloxacin MICs were collected. Additional data was sought to perform a comparison between the MICs of pathogenic and commensal Neisseria spp. RESULTS: A total of 15 studies met our criteria.We found no evidence of intrinsic AMR in commensal Neisseria spp. We did find evidence of an increasing trend in MICs of commensal Neisseria spp. over time for all antimicrobials assessed. These findings were similar in various countries. Eight additional studies were included to compare pathogenic and commensal Neisseria spp. CONCLUSION: The MICs of commensal Neisseria spp. appear to be increasing in multiple countries. Surveillance of MICs in commensals could be used as an early warning system for antimicrobial resistance emergence in pathogens. Our findings underline the need for antibiotic stewardship interventions, particularly in populations with high antimicrobial consumption.

Severe mpox (formerly monkeypox) disease in five patients after recent vaccination with MVA-BN vaccine, Belgium, July to October 2022.

Vaccination is important in containing the 2022 mpox (formerly monkeypox) epidemic. We describe five Belgian patients with localised severe symptoms of proctitis and penile oedema, occurring between 4 and 35 days after post-exposure preventive vaccination or after one- or two-dose off-label pre-exposure preventive vaccination with MVA-BN vaccine. Genome sequencing did not reveal evidence for immune escape variants. Healthcare workers and those at risk should be aware of possible infections occurring shortly after vaccination and the need for other preventive measures.

Lack of Association between Antimicrobial Consumption and Antimicrobial Resistance in a HIV Preexposure Prophylaxis Population: A Cross-Sectional Study.

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.

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.

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.

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. N. subflava was detected in 77 of 88 (87.5%) individuals and N. mucosa in 64 of 88 (72.7%). N. mucosa was more prevalent in children (41/50 [82%]) than parents (23/38 [60.5%]; P < 0.05), while N. bacilliformis was more prevalent in parents (7/36 [19.4%]) than children (2/50 [4%]; P < 0.05). N. 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.

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.

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.

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.

Effect on the Resistome of Dual vs Monotherapy for the Treatment of Neisseria gonorrhoeae: Results From a Randomized Controlled Trial (ResistAZM Trial).

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.

Leveraging T-cell receptor - epitope recognition models to disentangle unique and cross-reactive T-cell response to SARS-CoV-2 during COVID-19 progression/resolution.

Despite the general agreement on the significance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain. Understanding this aspect could provide insights for adjusting vaccines and maintaining robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to SARS-CoV-2 epitopes unique to the virus (SC2-unique) or shared with other coronaviruses (CoV-common), we trained a large number of T-cell receptor (TCR) - epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. These models were then applied to longitudinal CD8+ TCR repertoires from critical and non-critical COVID-19 patients. In spite of comparable initial CoV-common TCR repertoire depth and CD8+ T-cell depletion, the temporal dynamics of SC2-unique TCRs differed depending on the disease severity. Specifically, while non-critical patients demonstrated a large and diverse SC2-unique TCR repertoire by the second week of the disease, critical patients did not. Furthermore, only non-critical patients exhibited redundancy in the CD8+ T-cell response to both groups of epitopes, SC2-unique and CoV-common. These findings indicate a valuable contribution of the SC2-unique CD8+ TCR repertoires. Therefore, a combination of specific and cross-reactive CD8+ T-cell responses may offer a stronger clinical advantage. Besides tracking the specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire, our analytical framework can be expanded to more epitopes and assist in the assessment and monitoring of CD8+ T-cell response to other infections.

Salmonella Typhi whole genome sequencing in Rwanda shows a diverse historical population with recent introduction of haplotype H58.

Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, presenting high rates of morbidity and mortality in low- and middle-income countries. The H58 haplotype shows high levels of antimicrobial resistance (AMR) and is the dominant S. Typhi haplotype in endemic areas of Asia and East sub-Saharan Africa. The situation in Rwanda is currently unknown and therefore to reveal the genetic diversity and AMR of S. Typhi in Rwanda, 25 historical (1984-1985) and 26 recent (2010-2018) isolates from Rwanda were analysed using whole genome sequencing (WGS). WGS was locally implemented using Illumina MiniSeq and web-based analysis tools, thereafter complemented with bioinformatic approaches for more in-depth analyses. Whereas historical S. Typhi isolates were found to be fully susceptible to antimicrobials and show a diversity of genotypes, i.e 2.2.2, 2.5, 3.3.1 and 4.1; the recent isolates showed high AMR rates and were predominantly associated with genotype 4.3.1.2 (H58, 22/26; 84,6%), possibly resulting from a single introduction in Rwanda from South Asia before 2010. We identified practical challenges for the use of WGS in endemic regions, including a high cost for shipment of molecular reagents and lack of high-end computational infrastructure for the analyses, but also identified WGS to be feasible in the studied setting and giving opportunity for synergy with other programs.

A global genomic analysis of Salmonella Concord reveals lineages with high antimicrobial resistance in Ethiopia.

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance.

A genomic appraisal of invasive Salmonella Typhimurium and associated antibiotic resistance in sub-Saharan Africa.

Invasive non-typhoidal Salmonella (iNTS) disease manifesting as bloodstream infection with high mortality is responsible for a huge public health burden in sub-Saharan Africa. Salmonella enterica serovar Typhimurium (S. Typhimurium) is the main cause of iNTS disease in Africa. By analysing whole genome sequence data from 1303 S. Typhimurium isolates originating from 19 African countries and isolated between 1979 and 2017, here we show a thorough scaled appraisal of the population structure of iNTS disease caused by S. Typhimurium across many of Africa's most impacted countries. At least six invasive S. Typhimurium clades have already emerged, with ST313 lineage 2 or ST313-L2 driving the current pandemic. ST313-L2 likely emerged in the Democratic Republic of Congo around 1980 and further spread in the mid 1990s. We observed plasmid-borne as well as chromosomally encoded fluoroquinolone resistance underlying emergences of extensive-drug and pan-drug resistance. Our work provides an overview of the evolution of invasive S. Typhimurium disease, and can be exploited to target control measures.

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.

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.

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.