OBJECTIVES:Fusidic acid resistance in Staphylococcus aureus is a growing clinical concern, but its temporal dynamics and mechanism evolution at a global scale are poorly characterised. METHODS:We combined 2512 fusidic acid-resistant S. aureus isolates from Pathogenwatch (1964-2021) with 189 resistant isolates identified from 5051 NCBI GenBank genomes deposited January 2022-April 2026, yielding 2701 isolates across 72 countries (2549 with collection years, 1964-2025). AMR determinants were identified using AMRFinderPlus v4.2.7 and sequence types using mlst v2.23.0. Temporal mechanism shifts were tested by chi-squared across six eras. RESULTS:fusC (52.0%), fusA (35.1%), and fusB (12.8%) were the primary mechanisms. A highly significant temporal shift was observed (chi-squared = 283.7, P = 4.4 × 10⁻⁵⁵): fusB rose from 15% (pre-2000) to 61.5% (2021-2025), overtaking both fusC and fusA. The earliest chromosomal events were fusA P404L in ST250 (1964) and P404Q in ST247 (1965). 93.9% of isolates were MDR; fusA independently predicted quinolone co-resistance (adjusted OR = 12.47). PVL positivity was 13.3%, driven by clonal background. ST121 (EEFIC) showed late emergence (92% in 2011-2025). CONCLUSION:Fusidic acid resistance is shifting from chromosomal fusA towards plasmid-mediated fusB, supporting integrated molecular surveillance to guide therapy and stewardship.

PURPOSE:This study characterizes 109 MRSA isolates collected at a major hospital network in Antwerp, Belgium, focusing on their classification as community-acquired (CA-MRSA) or hospital-acquired (HA-MRSA), antimicrobial susceptibility profiles, genotypic resistance determinants, virulence factors, and sequence type (ST) distribution. METHODS:This study was conducted retrospectively. MRSA isolates were collected from patients presenting to facilities within the "Ziekenhuis aan de Stroom (ZAS)" hospital network. The strains were prepared for whole genome sequencing with the Nextera XT sample preparation kit (Illumina), followed by sequencing on the Illumina MiSeq platform. RESULTS:Of the 109 sequenced isolates, 83 (76,1%) were classified as CA-MRSA and 26 (23,9%) as HA-MRSA. Seventy-three (67,0%) isolates originated from clinical samples and 36 (33,0%) from screening samples. MLST revealed substantial genetic diversity. CA-MRSA clinical isolates were most frequently associated with ST5, ST6, ST152, and ST30, whereas HA-MRSA clinical isolates were dominated by ST8. Panton-Valentine leukocidin (PVL) genes were identified in 29 of the 109 isolates (26,6%). Antimicrobial susceptibility differed between CA-MRSA and HA-MRSA and was influenced by sequence type. CONCLUSION:In this study, the majority of isolates were classified as CA-MRSA based on a pragmatic epidemiological classification. Multiple sequence types (STs) are involved and different STs appear to be associated with varying degrees of clinical severity and virulence genes. Antimicrobial susceptibility profiles are influenced by the circulating ST, which may in turn affect empirical treatment strategies. These findings underscore the need for nationwide surveillance systems for CA-MRSA.

BackgroundAntibiotic tolerance slows bacterial killing during drug exposure without changing minimum inhibitory concentration (MIC) and is often missed by MIC-based testing in Neisseria gonorrhoeae.ObjectivesTo quantify ceftriaxone tolerance using complementary laboratory assays and to integrate these readouts with genomic typing to interpret recurrent urethritis within one patient series.MethodsFour clinical isolates obtained between 2022 and 2025 underwent tolerance disc testing. From the most recent isolate (25355), tolerant and non-tolerant variants were derived for functional study, including growth curves (log10 CFU/mL), minimum duration needed to kill 99% of cells (MDK99) across ceftriaxone concentrations, and a Galleria mellonella infection model. Whole-genome sequencing with in silico typing compared strain type between episodes.ResultsTolerance disc testing was positive for isolates 22073 and 25355. Across the growth trajectory, tolerant variants showed consistently lower bacterial counts than non-tolerant variants. Tolerant variants tended to exhibit longer MDK99 values, and in Galleria they declined more slowly under ceftriaxone exposure. Sequencing revealed distinct sequence types across episodes, supporting reinfection rather than within-host persistence.ConclusionsAssays that capture killing kinetics detected ceftriaxone tolerance that was not captured by MIC-based testing. Genomic analysis distinguished reinfection from persistence. This integrated workflow may improve the evaluation of suspected treatment failure in gonorrhoea.

Intracranial aneurysms can rupture and cause subarachnoid haemorrhage, yet their pathophysiology remains incompletely understood. Bacterial involvement has been proposed, although evidence remains inconsistent. We investigated the presence of bacterial genomic material in aneurysm vessel walls and characterised the oral and rectal microbiome using high-resolution 16 S rRNA amplicon sequencing with DADA2-based amplicon sequence variant (ASV) analysis and PICRUSt2-inferred functional profiling. In this cross-sectional study, 34 neurosurgical patients (16 with intracranial aneurysms, 18 neurosurgical controls) were included. No bacterial genomic material was detected in aneurysm walls or surrounding intracranial tissues. Microbiome profiling of oral (n = 29) and rectal (n = 27) mucosal swabs identified 9,132 ASVs across all samples. Alpha diversity (Shannon entropy, Chao1 richness, Pielou’s evenness, observed ASVs, and Faith’s phylogenetic diversity) did not differ between groups at either site. Beta diversity community composition was similar across Bray–Curtis dissimilarity and weighted and unweighted UniFrac distances (all PERMANOVA p ≥ 0.27, R²<0.05). No genus reached FDR significance after Benjamini–Hochberg correction (178 oral and 201 rectal genera tested). Machine learning classifiers showed no discriminatory ability (Random Forest and XGBoost AUC 0.53–0.60). PICRUSt2-inferred functional profiling of MetaCyc pathways, EC numbers, and KEGG Orthology gene families likewise revealed no FDR-significant differences at either site; nominally elevated pathways in intracranial aneurysm patients included galactitol degradation and NAD biosynthesis II, consistent with trends in Bifidobacterium abundance, but none survived multiple-testing correction. A preliminary OTU-based analysis suggested oral microbiome differences, but these were not reproduced with ASV-level resolution. These findings do not support a distinct mucosal microbiome or functional signature in intracranial aneurysm patients and highlight the importance of high-resolution analytical methods and adequate study power for microbiome research.

Klebsiella pneumoniae sequence type 48 (Kp-ST48) is a globally distributed clone linked to antimicrobial resistance (AMR) yet lacks a comprehensive genomic analysis. Here, we investigated the persistence, transmission dynamics and global context of ST48 in a large tertiary hospital in Berlin, Germany. Between 2014 and 2022, 48 surveillance and 15 putative outbreak Kp-ST48 isolates were isolated in a tertiary care, multi-site hospital in Berlin, Germany. Genomic diversity was analysed by short- and long-read sequencing. Additionally, we included 223 publicly available Kp-ST48 genomes from five continents over 40 years (1982-2022) in the phylodynamic analysis. We identified two genetically distinct clades (A and B) within the global Kp-ST48 population. The global spread of Kp-ST48 was driven by clade B, which included all the genomes from the Berlin hospital. Two hospital-specific lineages (1 and 2) were identified with distinct population dynamics. Lineage 2 was transient and linked to a putative outbreak in 2019. Meanwhile, lineage 1 was first detected in 2014 and persisted for over 8 years until 2022, with multiple putative patient-to-patient and indirect transmission events identified. Carbapenem resistance determinants (ompK35/36 mutations, bla KPC, bla NDM, bla OXA-48, bla VIM) were present in 57% (n=163/286) of genomes, and up to three bla CTX-M-15 copies were found integrated into chromosomes. Although Kp-ST48 generally did not contain a high number of virulence genes, 19 genomes showed potential for AMR-hypervirulence convergence. This study reveals the endemic persistence with outbreak potentials of Kp-ST48 in a hospital over 8 years, characterized by high genome plasticity. Our results highlight the global distribution of this clone, which warrants continuous surveillance.

This study aims to characterize the phenotypic behavior and in vivo persistence of a ceftriaxone-tolerant Neisseria gonorrhoeae clinical isolate from a single patient and evaluate the potential role of tolerance in treatment failure. A previously identified ceftriaxone-tolerant vaginal isolate was compared with isogenic and clinical non-tolerant strains. Bacterial growth was assessed in vitro, and tolerance was quantified using the minimum duration required to kill 99% of the population (MDK99), and persistence was evaluated in an in vivo Galleria mellonella infection model. Whole-genome sequencing (WGS) and transcriptomic (RNA-sequencing [RNA-seq]) profiling were performed to identify tolerance-associated genetic and transcriptional signatures. The tolerant strain exhibited prolonged MDK99 values across ceftriaxone concentrations, persisting for up to 24 hours under drug exposure. It also showed delayed early-phase growth, suggesting a fitness cost. In vivo, the tolerant strain remained viable up to 8 hours after treatment, whereas non-tolerant strains were cleared. WGS revealed identical gene content across all isolates, but non-synonymous mutations in pilE_3, a type IV pilin gene, were exclusively present in tolerant strains. RNA-seq analysis showed upregulation of pilin-associated genes and downregulation of zinc-independent ribosomal paralogs (rpmE2 and ykgO), suggesting a combined mechanism of surface remodeling and translational suppression associated with the tolerant phenotype. Ceftriaxone tolerance enables prolonged survival of N. gonorrhoeae despite apparent susceptibility by standard MIC-based testing. This phenotype may contribute to treatment failure, recurrent infection, and ongoing transmission, indicating the need for revised diagnostic and therapeutic strategies.IMPORTANCECeftriaxone remains the last reliable option for gonorrhea therapy, yet recurrent infections can occur despite isolates being classified as susceptible by MIC testing. One possible explanation is antibiotic tolerance, a phenotype that allows survival during drug exposure without changes in MIC. Although tolerance has been described in other pathogens, its role in gonococcal infection has remained poorly defined. In this study, we provide the first detailed characterization of a ceftriaxone-tolerant Neisseria gonorrhoeae clinical isolate associated with repeated treatment failure. By combining in vitro killing assays, an in vivo Galleria mellonella infection model, whole-genome sequencing, and transcriptomic profiling, we demonstrate that tolerance enables prolonged survival under ceftriaxone and is linked to pilin gene variation and ribosomal remodeling. These findings illustrate how a clinically observed phenomenon can be mechanistically dissected and emphasize tolerance as a hidden factor contributing to gonococcal persistence and potential treatment failure.

Cefiderocol-resistant bacteria pose a growing concern in both clinical and environmental settings. This study investigates cefiderocol-resistant bacteria in wastewater from six German tertiary care hospitals. A total of 36 samples were analysed using a culture-dependent approach involving cefiderocol pre-enrichment, yielding 97 cefiderocol-resistant isolates—primarily Enterobacter roggenkampii, Klebsiella oxytoca, Serratia marcescens, and Citrobacter farmeri. Most isolates exhibited high minimum inhibitory concentrations against cefiderocol and resistance to multiple antibiotics. Resistance rates were lower for meropenem-vaborbactam (10.3%) and imipenem-relebactam (33.0%), while all isolates remained susceptible to aztreonam-avibactam. Whole-genome sequencing of 79 isolates revealed a diverse resistome, with 78.5% (62/79) carrying carbapenemase genes. Some isolates harbored up to six distinct β-lactamase genes, including combinations of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and one or multiple carbapenemases—such as as blaOXA−48 co-occurring with blaNDM−1 or blaVIM−1. In addition, biocide and heavy metal resistance genes were prevalent, highlighting bacterial adaptation to harsh environments. Plasmid profiling showed significant interspecies variation, with C. farmeri and K. oxytoca displaying the highest plasmid loads. Across all isolates, 38 unique plasmid incompatibility types were detected, 18 of which were species-specific. These findings highlight the multidrug-resistant nature of wastewater-derived pathogens and the importance of monitoring resistance dissemination in healthcare environments.

Background : Nakaseomyces glabratus (Candida gabrata) poses a significant clinical challenge due to common drug resistance. We report a case of a complicated urinary tract infection (UTI) progressing to prostatitis and urosepsis, with the emergence of a hyper-multidrug-resistant isolate with low stress tolerance, slow growth and a short life span. This study elucidates the genetic mechanisms and phenotypic characteristics underlying antifungal hyper-resistance with strong fitness trade-offs, and explores potential alternative therapies for resistant UTIs. Methods : Whole-genome sequencing was performed to identify resistance-associated mutations and gene knock-out strains were generated to assess the relative impact of putative loss-of-function (LoF) mutations on antifungal resistance, fitness and membrane sterol composition. Drug susceptibility testing of the antibiotic nitroxoline and related compounds was conducted to evaluate it as a therapeutic alternative and study the mechanism of action. Findings : Loss-of-function mutations in ERG3 and ERG11 were identified and linked to the accumulation of 4,14-dimethylzymosterol and lanosterol instead of ergosterol. Engineered ERG3Δ+ERG11Δ strains recapitulated the clinical isolate's hyper-multidrug resistance and associated fitness deficits. While ERG3Δ strains showed no resistance but enhanced thermotolerance, ERG11Δ and ERG3Δ+ERG11Δ strains exhibited multidrug resistance with severe fitness trade-offs. Interestingly, ERG3Δ+ERG11Δ strains showed mild resistance to flucytosine, but an additional FUR1 mutation in the clinical isolate most probably underlies hyper-resistance to flucytosine. The UTI antibiotic nitroxoline demonstrated high antifungal activity against all strains, and the LoF of ERG3 and/or ERG11 induced collateral sensitivity to this drug. Testing of related compounds suggest a mode of action beyond iron chelation. Interpretation : This case demonstrates that hyper-resistant strains of N. glabratus can emerge despite significant fitness costs and persist under prolonged antifungal therapy in specific clinical settings. These findings underscore the importance of vigilant antifungal resistance monitoring and highlight nitroxoline as a promising alternative treatment for complicated fungal UTIs. These results challenge the notion that strains with fitness deficits are clinically irrelevant and emphasize the need for novel therapeutic strategies including repurposed agents. ### Competing Interest Statement KL received consultancy fees from Mundipharma, speaker fees from Pfizer, Gilead, Mundipharma and FUJIFILM Wako chemicals Europe GmbH, a service fee from TECOmedical, a fee for Advisory Board participation from Pfizer and travel support from Pfizer, Gilead and AstraZeneca. All other authors declare no competing interests.

Colistin is one of the last-line antibiotics against multi-drug-resistant (MDR) gram-negative pathogens, such as Klebsiella pneumoniae. Using long-read sequencing, we observed remarkable genome-wide transposition events in MDR K. pneumoniae exposed to colistin in patients receiving treatment for respiratory infections or as part of selective decolonization strategies and further confirmed these on in vitro selection experiments. These data add yet another dimension to the role of antibiotics in mediating specific processes in bacteria beyond antibiotic resistance.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT02208154.

BACKGROUND:Pseudomonas aeruginosa and Enterobacter cloacae, both members of the ESKAPE group, are multidrug-resistant pathogens that pose significant challenges in clinical care. The high GC-content (∼67 %) of the P. aeruginosa genome complicates DNA extraction and long-read sequencing, with downstream effects on genome assembly and analyses. We compared three commercial extraction kits for isolating high-quality DNA suitable for Oxford Nanopore sequencing from clinical isolates of P. aeruginosa and E. cloacae. METHODS:Genomic DNA was extracted from P. aeruginosa (n = 63; 3 × 21) and E. cloacae (n = 96; 3 × 32) clinical isolates using the MagAttract HMW DNA Kit (Qiagen), the DNeasy UltraClean Microbial Kit (Qiagen), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (ThermoFisher). DNA quantity/quality was assessed by spectrophotometry (NanoDrop, Thermo Fisher Scientific), fluorometry (Qubit, ThermoFisher), and capillary electrophoresis (TapeStation 2200, Agilent). Libraries were prepared and sequenced using Oxford Nanopore Technologies platforms. Assemblies were generated with Unicycler (v.0.5.1) and Flye (v.2.9.6); quality was assessed with QUAST (v.5.3). Genome completeness was evaluated by CheckM (v.1.1.6). Antimicrobial resistance determinants were identified with AMRFinderPlus (v.4.1.19). RESULTS:DNeasy yielded up to 4.7× higher DNA and ∼ 50 % higher sequencing output than MagAttract, while MagAttract produced higher DNA integrity and more contiguous assemblies. The choice of assembly had a greater impact on the detection of AMR determinants than the extraction method alone. Across workflows, Flye outperformed Unicycler, increasing detection by 2-14 percentage points. The best-performing combination (DNeasy + Flye) achieved 95.2 % AMR determinants, compared to 67.8 % for MagMAX + Unicycler, with the most difference (37.5 %) in efflux pump genes. CONCLUSIONS:This systematic comparison highlights trade-offs between DNA yield, integrity, and downstream assembly performance, demonstrating that assembler choice critically impacts the detection of AMR determinants. These findings provide practical guidance for optimizing long-read-based sequencing workflows to support AMR surveillance and genomic epidemiology.

Serratia marcescens is an opportunistic pathogen with a propensity to cause nosocomial outbreaks, particularly in neonatal intensive care units (NICUs). We present a sustained outbreak spanning over 18 months (1 January 2022-29 August 2023) in a NICU in Antwerp, Belgium, affecting 61 neonates, identified through samples taken for diagnostic purposes and by rectal screening. Ten neonates were infected: five with lower respiratory tract infection, four with conjunctivitis and one fatal case with sepsis. In a logistic regression analysis, nursing in an incubator was significantly associated with acquisition of S. marcescens (odds ratio (OR): 2.99; 95% confidence interval (CI): 1.14-8.25; p < 0.05). Whole genome sequencing-based multilocus sequence typing (wgMLST) and core genome single nucleotide polymorphism (cgSNP) analysis of isolates from clinical (n = 4), screening (n = 52) and environmental samples (n = 8), identified eight clusters and five singletons not associated with the clusters. Although outbreak measures were successful in containing further spread within the ward during sudden surges when > 4 cases per week were identified (peak events), several peaks with different clonal clusters occurred. The emergence of similar outbreaks in Belgian hospitals underscores the need of continuous surveillance and NICU-specific infection prevention and control (IPC) measures.

Multidrug-resistant Acinetobacter baumannii is a major concern in healthcare institutions worldwide. Several reports described the dissemination of A. baumannii high-risk clones that are responsible for a high number of difficult-to-treat infections. In our study, 19 multidrug-resistant A. baumannii strains from Budapest, Hungary, were investigated based on whole-genome sequencing (WGS). The obtained results were analysed together with data from 433 strains of A. baumannii from the Pathogenwatch database. WGS analysis of 19 A. baumannii strains detected that 12 belonged to ST2 and seven belonged to ST636. Among ST2 strains, 11 out of 12 carried either blaOXA-23 or blaOXA-58 genes; however, all strains of ST636 uniformly carried blaOXA-72 gene. All strains of ST2 and ST636 carried blaOXA-66 and blaADC-25 genes. Based on core genome multilocus sequence typing (cgMLST), 10 strains of ST2 belonged to cgMLST906, one strain to cgMLST458, and one strain to cgMLST1320; by contrast, all strains of ST636 belonged to cgMLST1178. Certain virulence determinants were present in all strains of both ST2 and ST636, namely, Ata, Bap, BfmRS, T2SS and PNAG. Interestingly, OmpA was present in all strains of ST2, but it was absent in all strains of ST636. Comparative analysis of 19 strains of this study and the collection of 433 isolates from Pathogenwatch database, proved a diverse clonal distribution of high-risk A. baumannii clones in Europe. The major clone in Europe is ST2, which is present all over the continent. However, ST636 has been mainly reported in Eastern Europe. Interestingly, cgMLSTs of ST2 correspond to the production of different beta-lactamases, namely, OXA-82 in cgMLST116, OXA-72 in cgMLST506, and cgMLST556, PER-1 in cgMLST456 and cgMLST1041. Our study demonstrates that the ST2 high-risk clone of A. baumannii is the most widespread in Europe; however, based on cgMLST analysis, a detailed detection of beta-lactamase production can be determined.

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

The impact of community carriage on the influx of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) into hospitals remains understudied. In this prospective 2-year single-centre study, we investigate the community ESBL-E influx and trace the colonisation, nosocomial acquisition, transmission, and infection dynamics of ESBL-producing Escherichia coli (ESBL-Ec) in non-ICU wards at a tertiary care hospital. This study reports primary and post hoc outcomes of the clinical trial NCT01208519 in which hospitalised patients were screened for rectal carriage of ESBL-E. ESBL-Ec isolates from ≈50

Klebsiella pneumoniae (Kp) associated with hospital acquired infections are extensively-drug resistant (XDR), making treatment problematic. Understanding the genetic epidemiology of XDR -Kp can determine their potential to be hypervirulent (hv) through the presence of siderophores. We characterized genomes of 18 colistin-resistant XDR-Kp isolated from 14 patients with complicated urinary tract infection in an Indian healthcare facility. 18 organisms comprised STs: ST14 (9/18), ST147 (5/18), ST231 (2/18), ST2096 (1/18), and ST25 (1/18). Many patients in one ward were infected with the same ST, indicating a common infection source. Some patients had recurrent infections with multiple STs that were circulating in that ward, providing evidence for hospital transmission. Beta lactamase genes (blaCTX-M-1, blaSHV, and blaampH) were present in all isolates. blaNDM-1 was present in isolates 15/18, blaOXA-1 was present in isolates 16/18, blaTEM-1D was present in 13/18 isolates, and blaOXA-48 was present in isolates 14,19 and 30. Disruption of mgrB with various IS elements was responsible for colistin resistance in 6 isolates. The most common K type among these isolates was K2 (10/18). One XDR convergent hv-Kp ST2096 was associated with prolonged hospitalisation (iuc+ybt+blaOXA-1+blaOXA-48).Convergent XDR-hv-Kp detected has outbreak potential, warranting effective antimicrobial stewardship and infection control.

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

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.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

BACKGROUND:Colistin serves as the last line of defense against multidrug resistant Gram-negative bacterial infections in both human and veterinary medicine. This study aimed to investigate the occurrence and spread of colistin-resistant Enterobacterales (ColR-E) using a One Health approach in Belgium and in the Netherlands.METHODS:In a transnational research project, a total of 998 hospitalized patients, 1430 long-term care facility (LTCF) residents, 947 children attending day care centres, 1597 pigs and 1691 broilers were sampled for the presence of ColR-E in 2017 and 2018, followed by a second round twelve months later for hospitalized patients and animals. Colistin treatment incidence in livestock farms was used to determine the association between colistin use and resistance. Selective cultures and colistin minimum inhibitory concentrations (MIC) were employed to identify ColR-E. A combination of short-read and long-read sequencing was utilized to investigate the molecular characteristics of 562 colistin-resistant isolates. Core genome multi-locus sequence typing (cgMLST) was applied to examine potential transmission events.RESULTS:The presence of ColR-E was observed in all One Health sectors. In Dutch hospitalized patients, ColR-E proportions (11.3 and 11.8% in both measurements) were higher than in Belgian patients (4.4 and 7.9% in both measurements), while the occurrence of ColR-E in Belgian LTCF residents (10.2%) and children in day care centres (17.6%) was higher than in their Dutch counterparts (5.6% and 12.8%, respectively). Colistin use in pig farms was associated with the occurrence of colistin resistance. The percentage of pigs carrying ColR-E was 21.8 and 23.3% in Belgium and 14.6% and 8.9% in the Netherlands during both measurements. The proportion of broilers carrying ColR-E in the Netherlands (5.3 and 1.5%) was higher compared to Belgium (1.5 and 0.7%) in both measurements. mcr-harboring E. coli were detected in 17.4% (31/178) of the screened pigs from 7 Belgian pig farms. Concurrently, four human-related Enterobacter spp. isolates harbored mcr-9.1 and mcr-10 genes. The majority of colistin-resistant isolates (419/473, 88.6% E. coli; 126/166, 75.9% Klebsiella spp.; 50/75, 66.7% Enterobacter spp.) were susceptible to the critically important antibiotics (extended-spectrum cephalosporins, fluoroquinolones, carbapenems and aminoglycosides). Chromosomal colistin resistance mutations have been identified in globally prevalent high-risk clonal lineages, including E. coli ST131 (n = 17) and ST1193 (n = 4). Clonally related isolates were detected in different patients, healthy individuals and livestock animals of the same site suggesting local transmission. Clonal clustering of E. coli ST10 and K. pneumoniae ST45 was identified in different sites from both countries suggesting that these clones have the potential to spread colistin resistance through the human population or were acquired by exposure to a common (food) source. In pig farms, the continuous circulation of related isolates was observed over time. Inter-host transmission between humans and livestock animals was not detected.CONCLUSIONS:The findings of this study contribute to a broader understanding of ColR-E prevalence and the possible pathways of transmission, offering insights valuable to both academic research and public health policy development.

Objectives Escherichia coli can cause infections in the urinary tract and in normally sterile body sites leading to invasive E. coli disease (IED), including bacteraemia and sepsis, with older populations at increased risk. We aimed to estimate the theoretical coverage rate by the ExPEC4V and 9V vaccine candidates. In addition, we aimed at better understanding the diversity of E. coli isolates, including their genetic and phenotypic antimicrobial resistance (AMR), sequence types (STs), O-serotypes and the bacterial population structure.Methods Blood and urine culture E. coli isolates (n = 304) were collected from hospitalized patients >= 60 years (n = 238) with IED during a multicentric, observational study across three continents. All isolates were tested for antimicrobial susceptibility, O-serotyped, whole-genome sequenced and bioinformatically analysed.Results A large diversity of STs and of O-serotypes were identified across all centres, with O25b-ST131, O6-ST73 and O1-ST95 being the most prevalent types. A total of 45.4% and 64.7% of all isolates were found to have an O-serotype covered by the ExPEC4V and ExPEC9V vaccine candidates, respectively. The overall frequency of MDR was 37.4% and ST131 was predominant among MDR isolates. Low in-patient genetic variability was observed in cases where multiple isolates were collected from the same patient.Conclusions Our results highlight the predominance of MDR O25b-ST131 E. coli isolates across diverse geographic areas. These findings provide further baseline data on the theoretical coverage of novel vaccines targeting E. coli associated with IED in older adults and their associated AMR levels.