In Vitro Investigation of Therapy-Induced Senescence and Senescence Escape in Breast Cancer Cells Using Novel Flow Cytometry-Based Methods.

Although cellular senescence was originally defined as an irreversible form of cell cycle arrest, in therapy-induced senescence models, the emergence of proliferative senescence-escaped cancer cells has been reported by several groups, challenging the definition of senescence. Indeed, senescence-escaped cancer cells may contribute to resistance to cancer treatment. Here, to study senescence escape and isolate senescence-escaped cells, we developed novel flow cytometry-based methods using the proliferation marker Ki-67 and CellTrace CFSE live-staining. We investigated the role of a novel senescence marker (DPP4/CD26) and a senolytic drug (azithromycin) on the senescence-escaping ability of MCF-7 and MDA-MB-231 breast cancer cells. Our results show that the expression of DPP4/CD26 is significantly increased in both senescent MCF-7 and MDA-MB-231 cells. While not essential for senescence induction, DPP4/CD26 contributed to promoting senescence escape in MCF-7 cells but not in MDA-MB-231 cells. Our results also confirmed the potential senolytic effect of azithromycin in senescent cancer cells. Importantly, the combination of azithromycin and a DPP4 inhibitor (sitagliptin) demonstrated a synergistic effect in senescent MCF-7 cells and reduced the number of senescence-escaped cells. Although further research is needed, our results and novel methods could contribute to the investigation of the mechanisms of senescence escape and the identification of potential therapeutic targets. Indeed, DPP4/CD26 could be a promising marker and a novel target to potentially decrease senescence escape in cancer.

In Vivo Immune-Modulatory Activity of Lefamulin in an Influenza Virus A (H1N1) Infection Model in Mice.

Lefamulin is a first-in-class systemic pleuromutilin antimicrobial and potent inhibitor of bacterial translation, and the most recent novel antimicrobial approved for the treatment of community-acquired pneumonia (CAP). It exhibits potent antibacterial activity against the most prevalent bacterial pathogens that cause typical and atypical pneumonia and other infectious diseases. Early studies indicate additional anti-inflammatory activity. In this study, we further investigated the immune-modulatory activity of lefamulin in the influenza A/H1N1 acute respiratory distress syndrome (ARDS) model in BALB/c mice. Comparators included azithromycin, an anti-inflammatory antimicrobial, and the antiviral oseltamivir. Lefamulin significantly decreased the total immune cell infiltration, specifically the neutrophils, inflammatory monocytes, CD4+ and CD8+ T-cells, NK cells, and B-cells into the lung by Day 6 at both doses tested compared to the untreated vehicle control group (placebo), whereas azithromycin and oseltamivir did not significantly affect the total immune cell counts at the tested dosing regimens. Bronchioalveolar lavage fluid concentrations of pro-inflammatory cytokines and chemokines including TNF-α, IL-6, IL-12p70, IL-17A, IFN-γ, and GM-CSF were significantly reduced, and MCP-1 concentrations were lowered (not significantly) by lefamulin at the clinically relevant 'low' dose on Day 3 when the viral load peaked. Similar effects were also observed for oseltamivir and azithromycin. Lefamulin also decreased the viral load (TCID50) by half a log10 by Day 6 and showed positive effects on the gross lung pathology and survival. Oseltamivir and lefamulin were efficacious in the suppression of the development of influenza-induced bronchi-interstitial pneumonia, whereas azithromycin did not show reduced pathology at the tested treatment regimen. The observed anti-inflammatory and immune-modulatory activity of lefamulin at the tested treatment regimens highlights a promising secondary pharmacological property of lefamulin. While these results require confirmation in a clinical trial, they indicate that lefamulin may provide an immune-modulatory activity beyond its proven potent antibacterial activity. This additional activity may benefit CAP patients and potentially prevent acute lung injury (ALI) and ARDS.

Molecular characterization of Treponema pallidum isolates from Brazil.

Syphilis remains a public health concern in Brazil, and the data on the characterization and resistance of Treponema pallidum in Brazil is limited. The present study aimed to detect Treponema DNA in the lesions and blood samples obtained from individuals diagnosed with syphilis. The Brazilian isolates were submitted to the Enhanced Centers for Disease Control and Prevention (ECDC) scheme and also analyzed for resistance gene. Treponemal DNA from 18 lesions and 18 blood specimens were submitted for amplification using Polymerase Chain Reaction (PCR) and Polymerase Chain Reaction in Real Time (RT-PCR). Eight samples from lesions and eight from blood were positive in the RT-PCR analysis. Eight lesions and three blood samples were positive using PCR. Two samples exhibited azithromycin resistance. The Brazilian isolate types 14d/g, 14 d/c, 15d/c, and 15d/e were identified using the ECDC scheme. The three subtypes 14d/c, 15d/c, and 15d/e have been identified in Brazil for the first time.

Using host-mimicking conditions and a murine cutaneous abscess model to identify synergistic antibiotic combinations effective against Pseudomonas aeruginosa.

Antibiotic drug combination therapy is critical for the successful treatment of infections caused by multidrug resistant pathogens. We investigated the efficacy of ß-lactam and ß-lactam/ß-lactamase inhibitor combinations with other antibiotics, against the hypervirulent, ceftazidime/avibactam resistant Pseudomonas aeruginosa Liverpool epidemic strain (LES) B58. Although minimum inhibitory concentrations in vitro differed by up to eighty-fold between standard and host-mimicking media, combinatorial effects only marginally changed between conditions for some combinations. Effective combinations in vitro were further tested in a chronic, high-density murine infection model. Colistin and azithromycin demonstrated combinatorial effects with ceftazidime and ceftazidime/avibactam both in vitro and in vivo. Conversely, while tobramycin and tigecycline exhibited strong synergy in vitro, this effect was not observed in vivo. Our approach of using host-mimicking conditions and a sophisticated animal model to evaluate drug synergy against bacterial pathogens represents a promising approach. This methodology may offer insights into the prediction of combination therapy outcomes and the identification of potential treatment failures.

Designing of a new transdermal antibiotic delivery polymeric membrane modified by functionalized SBA-15 mesoporous filler.

A new drug delivery system using an asymmetric polyethersulfone (PES) membrane modified by SBA-15 and glutamine-modified SBA-15 (SBA-Q) was prepared in this study by the aim of azithromycin delivery enhancement in both in vitro and ex vivo experiments. The research focused on optimizing membrane performance by adjusting critical parameters including drug concentration, membrane thickness, modifier percentage, polymer percentage, and pore maker percentage. To characterize the fabricated membranes, various techniques were employed, including scanning electron microscopy, water contact angle, and tensile strength assessments. Following optimization, membrane composition of 17% PES, 2% polyvinylpyrrolidone, 1% SBA-15, and 0.5% SBA-Q emerged as the most effective. The optimized membranes demonstrated a substantial increase in drug release (906 mg/L) compared to the unmodified membrane (440 mg/L). The unique membrane structure, with a dense top layer facilitating sustained drug release and a porous sub-layer acting as a drug reservoir, contributed to this improvement. Biocompatibility assessments, antibacterial activity analysis, blood compatibility tests, and post-diffusion tissue integrity evaluations confirmed the promising biocompatibility of the optimized membranes. Moreover, long-term performance evaluations involving ten repeated usages underscored the reusability of the optimized membrane, highlighting its potential for sustained and reliable drug delivery applications.

Detection of OXA-181 Carbapenemase in Shigella flexneri.

We report the detection of OXA-181 carbapenemase in an azithromycin-resistant Shigella spp. bacteria in an immunocompromised patient. The emergence of OXA-181 in Shigella spp. bacteria raises concerns about the global dissemination of carbapenem resistance in Enterobacterales and its implications for the treatment of infections caused by Shigella bacteria.

Antimicrobial-resistant Neisseria gonorrhoeae in Europe in 2020 compared with in 2013 and 2018: a retrospective genomic surveillance study.

BACKGROUND: Regular quality-assured whole-genome sequencing linked to antimicrobial resistance (AMR) and patient metadata is imperative to elucidate the shifting gonorrhoea epidemiology, both nationally and internationally. We aimed to examine the gonococcal population in the European Economic Area (EEA) in 2020, elucidate emerging and disappearing gonococcal lineages associated with AMR and patient metadata, compare with 2013 and 2018 whole-genome sequencing data, and explain changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: In this retrospective genomic surveillance study, we analysed consecutive gonococcal isolates that were collected in EEA countries through the European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) in 2020, and made comparisons with Euro-GASP data from 2013 and 2018. All isolates had linked AMR data (based on minimum inhibitory concentration determination) and patient metadata. We performed whole-genome sequencing and molecular typing and AMR determinants were derived from quality-checked whole-genome sequencing data. Links between genomic lineages, AMR, and patient metadata were examined. FINDINGS: 1932 gonococcal isolates collected in 2020 in 21 EEA countries were included. The majority (81·2%, 147 of 181 isolates) of azithromycin resistance (present in 9·4%, 181 of 1932) was explained by the continued expansion of the Neisseria gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) clonal complexes (CCs) 63, 168, and 213 (with mtrD/mtrR promoter mosaic 2) and the novel NG-STAR CC1031 (semi-mosaic mtrD variant 13), associated with men who have sex with men and anorectal or oropharyngeal infections. The declining cefixime resistance (0·5%, nine of 1932) and negligible ceftriaxone resistance (0·1%, one of 1932) was largely because of the progressive disappearance of NG-STAR CC90 (with mosaic penA allele), which was predominant in 2013. No known resistance determinants for novel antimicrobials (zoliflodacin, gepotidacin, and lefamulin) were found. INTERPRETATION: Azithromycin-resistant clones, mainly with mtrD mosaic or semi-mosaic variants, appear to be stabilising at a relatively high level in the EEA. This mostly low-level azithromycin resistance might threaten the recommended ceftriaxone-azithromycin therapy, but the negligible ceftriaxone resistance is encouraging. The decreased genomic population diversity and increased clonality could be explained in part by the COVID-19 pandemic resulting in lower importation of novel strains into Europe. FUNDING: European Centre for Disease Prevention and Control and Örebro University Hospital.

Azithromycin and moxifloxacin resistance determinants in Mycoplasma genitalium in Lleida, Spain.

OBJECTIVE: Mycoplasma genitalium (MG) is a microorganism related to sexually transmitted infections. Antibiotic resistance of MG leads to an increase in treatment failure rates and the persistence of the infection. The aim of this study was to describe the most frequent mutations associated with azithromycin and moxifloxacin resistance in our geographical area. METHODS: A prospective study from May 2019 to May 2023 was performed. MG-positive samples were collected. Real-time PCRs (AllplexTM MG-AziR Assay and AllplexTM MG-MoxiR Assay, Seegene) were performed in MG positive samples to detect mutations in 23S rRNA V domain and parC gene. RESULTS: A 37.1% of samples presented resistance determinants to azithromycin and the most common mutation detected was A2059G (57.9%). Resistance to moxifloxacin was studied in 72 azithromycin-resistant samples and 36.1% showed mutations, being G248T the most prevalent (73.1%). CONCLUSIONS: The resistance to different lines of treat ment suggests the need for a targeted therapy and the performing of a test of cure afterwards.

Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies.

Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future.

Keratin Formed Bioadhesive Ophthalmic Gel for the Bacterial Conjunctivitis Treatment.

Keratin has the potential to function as the gel matrix in an ophthalmic formulation for the encapsulation of the macrolide antibiotic azithromycin. The quality of this formulation was thoroughly evaluated through various analyses, such as in vitro release assessment, rheological examination, intraocular retention studies in rabbits, assessment of bacteriostatic efficacy, and safety evaluations. It is worth mentioning that the gel demonstrated shear thinning properties and exhibited characteristics of an elastic solid, thereby confirming its structural stability. The gel demonstrated a notable affinity for mucosal surfaces in comparison to traditional azithromycin aqueous solutions. In vitro release testing revealed that drug release transpired via diffusion mechanisms, following a first-order kinetic release pattern. Additionally, the formulated gel exhibited remarkable antibacterial efficacy against Staphylococcus aureus and Pseudomonas aeruginosa in bacteriostatic evaluations. Lastly, safety assessments confirmed that the gel eye drops induced minimal irritation and displayed no apparent cytotoxicity, indicating their good safety and biocompatibility for ocular application. Thus, these findings indicated that the prepared azithromycin gel eye drops complied with the requisite standards for ophthalmic preparations.

Minimum inhibitory concentrations of Neisseria gonorrhoeae strains in clients of the Amsterdam sexual health clinic with a Dutch versus an international sexual network.

OBJECTIVES: International travel combined with sex may contribute to dissemination of antimicrobial-resistant (AMR) Neisseria gonorrhoeae (Ng). To assess the role of travel in Ng strain susceptibility, we compared minimum inhibitory concentrations (MICs) for five antibiotics (ie, azithromycin, ceftriaxone, cefotaxime, cefixime and ciprofloxacin) in strains from clients with an exclusively Dutch sexual network and clients with an additional international sexual network. METHODS: From 2013 to 2019, we recorded recent residence of sexual partners of clients (and of their partners) with Ng at the Center for Sexual Health of Amsterdam. We categorised clients as having: (1) exclusively sexual partners residing in the Netherlands ('Dutch only') or (2) at least one partner residing outside the Netherlands. We categorised the country of residence of sexual partners by World Bank/EuroVoc regions. We analysed the difference of log-transformed MIC of Ng strains between categories using linear or hurdle regression for each antibiotic. RESULTS: We included 3367 gay and bisexual men who had sex with men (GBMSM), 516 women and 525 men who exclusively had sex with women (MSW) with Ng. Compared with GBMSM with a 'Dutch only' network, GBMSM with: (1) a Western European network had higher MICs for ceftriaxone (ß=0.19, 95% CI=0.08 to 0.29), cefotaxime (ß=0.19, 95% CI=0.08 to 0.31) and cefixime (ß=0.06, 95% CI=0.001 to 0.11); (2) a Southern European network had a higher MIC for cefixime (ß=0.10, 95% CI=0.02 to 0.17); and (3) a sub-Saharan African network had a lower MIC for ciprofloxacin (ß=-1.79, 95% CI=-2.84 to -0.74). In women and MSW, higher MICs were found for ceftriaxone in clients with a Latin American and Caribbean network (ß=0.26, 95% CI=0.02 to 0.51). CONCLUSIONS: For three cephalosporin antibiotics, we found Ng strains with slightly higher MICs in clients with partner(s) from Europe or Latin America and the Caribbean. International travel might contribute to the spread of Ng with lower susceptibility. More understanding of the emergence of AMR Ng is needed.

Multidrug-resistant conjugative plasmid carrying mphA confers increased antimicrobial resistance in Shigella.

Shigellosis remains a common gastrointestinal disease mostly in children < 5 years of age in developing countries. Azithromycin (AZM), a macrolide, is currently the first-line treatment for shigellosis in Bangladesh; ciprofloxacin (CIP) and ceftriaxone (CRO) are also used frequently. We aimed to evaluate the current epidemiology of antimicrobial resistance (AMR) and mechanism(s) of increasing macrolide resistance in Shigella in Bangladesh. A total of 2407 clinical isolates of Shigella from 2009 to 2016 were studied. Over the study period, Shigella sonnei was gradually increasing and become predominant (55%) over Shigella flexneri (36%) by 2016. We used CLSI-guided epidemiological cut-off value (ECV) for AZM in Shigella to set resistance breakpoints (zone-diameter ≤ 15 mm for S. flexneri and ≤ 11 mm for S. sonnei). Between 2009 and 2016, AZM resistance increased from 22% to approximately 60%, CIP resistance increased by 40%, and CRO resistance increased from zero to 15%. The mphA gene was the key macrolide resistance factor in Shigella; a 63MDa conjugative middle-range plasmid was harboring AZM and CRO resistance factors. Our findings show that, especially after 2014, there has been a rapid increase in resistance to the three most effective antibiotics. The rapid spread of macrolide (AZM) resistance genes among Shigella are driven by horizontal gene transfer rather than direct lineage.

Fluorescent reporters give new insights into antibiotics-induced nonsense and frameshift mistranslation.

We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.

Injectable Hydrogel To Deliver Bone Mesenchymal Stem Cells Preloaded with Azithromycin To Promote Spinal Cord Repair.

Spinal cord injury is a disease that causes severe damage to the central nervous system. Currently, there is no cure for spinal cord injury. Azithromycin is commonly used as an antibiotic, but it can also exert anti-inflammatory effects by down-regulating M1-type macrophage genes and up-regulating M2-type macrophage genes, which may make it effective for treating spinal cord injury. Bone mesenchymal stem cells possess tissue regenerative capabilities that may help promote the repair of the injured spinal cord. In this study, our objective was to explore the potential of promoting repair in the injured spinal cord by delivering bone mesenchymal stem cells that had internalized nanoparticles preloaded with azithromycin. To achieve this objective, we formulated azithromycin into nanoparticles along with a trans-activating transcriptional activator, which should enhance nanoparticle uptake by bone mesenchymal stem cells. These stem cells were then incorporated into an injectable hydrogel. The therapeutic effects of this formulation were analyzed in vitro using a mouse microglial cell line and a human neuroblastoma cell line, as well as in vivo using a rat model of spinal cord injury. The results showed that the formulation exhibited anti-inflammatory and neuroprotective effects in vitro as well as therapeutic effects in vivo. These results highlight the potential of a hydrogel containing bone mesenchymal stem cells preloaded with azithromycin and trans-activating transcriptional activator to mitigate spinal cord injury and promote tissue repair.

Recent dynamics in Neisseria gonorrhoeae genomic epidemiology in Brazil: antimicrobial resistance and genomic lineages in 2017-20 compared to 2015-16.

OBJECTIVES: Regular quality-assured WGS with antimicrobial resistance (AMR) and epidemiological data of patients is imperative to elucidate the shifting gonorrhoea epidemiology, nationally and internationally. We describe the dynamics of the gonococcal population in 11 cities in Brazil between 2017 and 2020 and elucidate emerging and disappearing gonococcal lineages associated with AMR, compare to Brazilian WGS and AMR data from 2015 to 2016, and explain recent changes in gonococcal AMR and gonorrhoea epidemiology. METHODS: WGS was performed using Illumina NextSeq 550 and genomes of 623 gonococcal isolates were used for downstream analysis. Molecular typing and AMR determinants were obtained and links between genomic lineages and AMR (determined by agar dilution/Etest) examined. RESULTS: Azithromycin resistance (15.6%, 97/623) had substantially increased and was mainly explained by clonal expansions of strains with 23S rRNA C2611T (mostly NG-STAR CC124) and mtr mosaics (mostly NG-STAR CC63, MLST ST9363). Resistance to ceftriaxone and cefixime remained at the same levels as in 2015-16, i.e. at 0% and 0.2% (1/623), respectively. Regarding novel gonorrhoea treatments, no known zoliflodacin-resistance gyrB mutations or gepotidacin-resistance gyrA mutations were found. Genomic lineages and sublineages showed a phylogenomic shift from sublineage A5 to sublineages A1-A4, while isolates within lineage B remained diverse in Brazil. CONCLUSIONS: Azithromycin resistance, mainly caused by 23S rRNA C2611T and mtrD mosaics/semi-mosaics, had substantially increased in Brazil. This mostly low-level azithromycin resistance may threaten the recommended ceftriaxone-azithromycin therapy, but the lack of ceftriaxone resistance is encouraging. Enhanced gonococcal AMR surveillance, including WGS, is imperative in Brazil and other Latin American and Caribbean countries.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

Detection of antimicrobial resistance in <5 h in Neisseria gonorrhoeae isolates using flow cytometry-proof of concept for seven clinically relevant antimicrobials.

INTRODUCTION: Antimicrobial resistance in Neisseria gonorrhoeae compromises gonorrhoea treatment and rapid antimicrobial susceptibility testing (AST) would be valuable. We have developed a rapid and accurate flow cytometry method (FCM) for AST of gonococci. METHODS: The 2016 WHO gonococcal reference strains, and WHO Q, R and S (n = 17) were tested against seven clinically relevant antibiotics (ceftriaxone, cefixime, azithromycin, spectinomycin, ciprofloxacin, tetracycline and gentamicin). After 4.5 h incubation of inoculated broth, the fluorescent dye Syto™ 9 was added, followed by FCM analysis. After gating, the relative remaining population of gonococci, compared with unexposed growth control samples, was plotted against antimicrobial concentration, followed by non-linear curve regression analysis. Furthermore, the response at one single concentration/tested antibiotic was evaluated with the intention to use as a screening test for detection of resistant gonococci. RESULTS: A dose-dependent response was seen in susceptible isolates for all tested antimicrobials. There was a clear separation between susceptible/WT and resistant/non-WT isolates for ceftriaxone, cefixime, spectinomycin, ciprofloxacin and tetracycline. In contrast, for azithromycin, only high-level-resistant isolates were distinguished, while resistant isolates with MICs of 4 mg/L were indistinguishable from WT (MIC ≤ 1 mg/L) isolates. For gentamicin, all tested 17 isolates were WT and FCM analysis resulted in uniform dose-response curves. Using a single antibiotic concentration and a 50% remaining cell population cut-off, the overall sensitivity and specificity for resistance detection were 93% and 99%, respectively. CONCLUSIONS: By providing results in <5 h for gonococcal isolates, FCM-based AST can become a rapid screening method for antimicrobial resistance or antimicrobial susceptibility in gonococci.

Azithromycin-loaded liposomes and niosomes for the treatment of skin infections: Influence of excipients and preparative methods on the functional properties.

The aim of this study was to develop azithromycin (AZT)-loaded liposomes (LP) and niosomes (NS) useful for the treatment of bacterial skin infections and acne. LP based on phosphatidylcholine from egg yolk (EPC) or from soybean lecithin (SPC), and NS composed of sorbitan monopalmitate (Span 40) or sorbitan monostearate (Span 60) were prepared through the thin film hydration (TFH) and the ethanol injection (EI) methods. The formulations were subsequently characterized for their physico-chemical and functional properties. Vesicles prepared through TFH showed higher average sizes than the corresponding formulations obtained by EI. All the vesicles presented adequate encapsulation efficiency and a negative ζ potential, which assured good stability during the storage period (except for LP-SPC). Formulations prepared with TFH showed a more prolonged AZT release than those prepared through EI, due to their lower surface area and multilamellar structure, as confirmed by atomic force microscopy nanomechanical characterization. Finally, among all the formulations, NS-Span 40-TFH and LP-EPC-TFH allowed the highest drug accumulation in the skin, retained the antimicrobial activity and did not alter fibroblast metabolism and viability. Overall, they could ensure to minimize the dosing and the administration frequency, thus representing promising candidates for the treatment of bacterial skin infections and acne.

Transmission of azithromycin-resistant gene, erm(T), of Gram-positive bacteria origin to Klebsiella pneumoniae.

The erm(T) gene encodes the 23 s rRNA methyltransferase and confers erythromycin resistance in Gram-positive bacteria, while has rarely been identified in Gram-negative bacteria. In this study, we identified a small IncQ1 plasmid of 6135 bp harboring the erm(T) gene in a clinical K. pneumoniae strain and confirmed the role of the erm(T) gene in mediating azithromycin resistance. This plasmid was found to be generated by incorporating the erm(T) gene from mobile elements into an IncQ1 plasmid. Our data indicated the spread of the erm(T) gene from Gram-positive bacteria to Gram-negative bacteria and the clonal spread of the ST11-KL47 type K. pneumoniae strains carrying this plasmid. Generation of this kind of multi-host plasmid will promote the dissemination of the erm(T) gene among Gram-negative bacteria and result in failures of azithromycin in treating bacterial infections.

Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates among individuals at high risk for sexually transmitted diseases in Zurich, Switzerland.

BACKGROUND: While ceftriaxone resistance remains scarce in Switzerland, global Neisseria gonorrhoeae (NG) antimicrobial resistance poses an urgent threat. This study describes clinical characteristics in MSM (men who have sex with men) diagnosed with NG infection and analyses NG resistance by phenotypic and genotypic means. METHODS: Data of MSM enrolled in three clinical cohorts with a positive polymerase chain reaction test (PCR) for NG were analysed between January 2019 and December 2021 and linked with antibiotic susceptibility testing. Bacterial isolates were subjected to whole genome sequencing (WGS). RESULTS: Of 142 participants, 141 (99%) were MSM and 118 (84%) living with HIV. Participants were treated with ceftriaxone (N = 79), azithromycin (N = 2), or a combination of both (N = 61). No clinical or microbiological failures were observed. From 182 positive PCR samples taken, 23 were available for detailed analysis. Based on minimal inhibitory concentrations (MICs), all isolates were susceptible to ceftriaxone, gentamicin, cefixime, cefpodoxime, ertapenem, zoliflodacin, and spectinomycin. Resistance to azithromycin, tetracyclines and ciprofloxacin was observed in 10 (43%), 23 (100%) and 11 (48%) of the cases, respectively. Analysis of WGS data revealed combinations of resistance determinants that matched with the corresponding phenotypic resistance pattern of each isolate. CONCLUSION: Among the MSM diagnosed with NG mainly acquired in Switzerland, ceftriaxone MICs were low for a subset of bacterial isolates studied and no treatment failures were observed. For azithromycin, high occurrences of in vitro resistance were found. Gentamicin, cefixime, cefpodoxime, ertapenem, spectinomycin, and zoliflodacin displayed excellent in vitro activity against the 23 isolates underscoring their potential as alternative agents to ceftriaxone.