JAC-Antimicrobial Resistance

Background Using a population-based cohort from the Calgary Health Zone (CHZ), Canada, we integrated longitudinal antimicrobial susceptibility and prescribing data with the whole genome sequences of five major pathogens. We aimed to assess how antimicrobial resistance (AMR) responds to prescribing changes and determine which bacterial strains shape these dynamics. Methods We analysed antibiotic prescribing rates, clinical and genomic data from 7,271 Staphylococcus aureus, 1,609 Enterococcus faecalis, 801 Enterococcus faecium, 11,363 Escherichia coli, and 2,319 Klebsiella pneumoniae isolates, associated with bacteraemia episodes in the CHZ between 2006-2022. Genomic clusters (referred to as strains) were identified using StrainGST and assigned to known sequence types (STs) or clonal complexes (CCs). Strain-level incidence, stratified by community-onset (isolates collected [&le;]48h after admission) and hospital-onset (>48h after admission), AMR phenotypes, and prescribing rates were modelled using negative-binomial and binomial regression. Temporal trends were quantified using average annual percentage change (AAPC). Findings Between 2010-2022, fluoroquinolone prescribing declined in both community (AAPC=-6.8% [95% CI -8.1, -5.4]; p<0.0001) and hospital settings (AAPC=-5.1% [-6.5, -3.7]; p<0.0001). This was accompanied by a significant reduction in fluoroquinolone resistance among Gram-positive species. Specifically, S aureus bacteraemia resistant to clinically important antibiotics, cloxacillin, ciprofloxacin, erythromycin, and clindamycin, declined from 2006 to 2022, mostly in hospital-onset cases (AAPC=-16.0%, [-19.3%, -12.7%], p<0.0001). In E coli, ceftriaxone and ciprofloxacin resistance were clustered in ST131 and the emerging ST1193; the latter increased steadily, particularly in community-onset cases (AAPC=17.7%, [0.0%, 30.0%], p<0.0001). CTX-M-27-producing E coli ST131 strains increased (AAPC=23.8%, [17.4%, 30.5%], p<0.0001) between 20082022, while CTX-M-14-producing E coli ST131 declined (AAPC=-15.9%, [-21.3%, -10.2%], p<0.0001) between 2013-2022. These trends were paralleled by an increase in community cephalosporin prescribing (AAPC=7.3%, [4.2%, 10.5%], p<0.0001) between 2010-2022. For K pneumoniae, hypervirulent ST23 was most common (N=88) with an increasing trend in incidence (AAPC=3.0%, [-2.8%, 9.2%]) between 2006-2019. Conclusions The contrasting resistance trends between Gram-positive and Gram-negative species underscore the complexity of AMR control efforts. Effective strategies will require stewardship efforts targeting multiple drug classes, genomic surveillance for emerging resistant strains, and interventions extending beyond hospital settings.

Public genomic repositories can support antimicrobial resistance (AMR) surveillance, but unequal sampling can bias interpretation. We characterised AMR determinants, multicountry genomic cluster overlap and surveillance gaps across Africa using an NCBI Pathogen Detection snapshot retrieved on 24 August 2026 for 55 African Union member states. Records were validated and deduplicated by BioSample, and complete AMRFinderPlus calls were summarised across five United Nations M49 subregions and eight overlapping regional economic communities (RECs). Country-pair cluster overlap was assessed using the Jaccard index, while project-based and composition-standardised sensitivity analyses evaluated repository bias. The dataset contained 86,829 unique BioSamples from 51 states; South Africa, Malawi and Kenya contributed 55.8%. Complete extended-spectrum {beta}-lactamase calls were detected in 21,513 isolates and carbapenemase calls in 4,642. blaCTX-M-15 dominated the ESBL profile, while NDM and OXA types predominated. Seventy clusters contained carbapenemase-positive isolates from at least two countries. A shared REC covered all participating countries in 38 clusters, while 32 crossed REC boundaries. Normalised country-pair overlap was low, with a maximum Jaccard index of 9.5%. Project balancing reduced the Northern African carbapenemase estimate from 32.3% to 17.9% and the Eastern African ESBL estimate from 36.9% to 12.5%. Public repositories identify determinants and clusters for investigation but do not estimate prevalence or transmission. AMR surveillance should combine national confirmation, regional institution-led investigation where countries share an REC, and continent-wide coordination through Africa CDC for cross-REC signals, supported by representative One Health sampling, standardised metadata and sustained African sequencing capacity.

Objective As antimicrobial resistance (AMR) continues to threaten global public health, bacteriophage therapy products (BTPs) offer a promising alternative to conventional antimicrobials. However, translation into routine clinical practice requires best practice standards for manufacturing and quality control to ensure the consistent safety, quality, and reliability of personalised BTPs produced for individual patients or small cohorts. Design A modified Delphi methodology was used to develop consensus statements, engaging experts from Australia's National Bacteriophage Therapy Regulatory Working Group across the fields of clinical microbiology, phage biology, good manufacturing practice (GMP), regulatory science, and government. The process comprised three iterative phases: (1) structured statement development, (2) an anonymous REDCap survey, and (3) a hybrid consensus meeting. The strength of evidence and recommendations was assessed using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) framework. Results Consensus was reached on 35 statements to provide best practice manufacture and quality control guidance for BTPs. These statements address requirements for phage identification and characterisation; define the point at which GMP-aligned processes commence for ubiquitous phages; outline quality control expectations for phage active pharmaceutical ingredient (pAPI) production and maintenance of BTP and host cell repositories. Additional guidance covers quality management systems, including documentation, traceability, and governance. Conclusion These consensus statements provide comprehensive best practice recommendations for the manufacture and quality control of BTPs in Australia. By promoting consistent, safe, and quality-assured approaches to personalised BTPs, they aim to facilitate clinical implementation while remaining aligned with existing international pharmacopoeial standards and regulatory frameworks.

Enteric fever is endemic to many low- and middle-income countries (LMICs), particularly those in sub-Saharan Africa, South and South-East Asia. The causative agents are typhoidal serovars of Salmonella enterica, including Typhi (S. Typhi) and Paratyphi A (SPA). There are no vaccines currently licensed for SPA, leaving antimicrobials as the only therapeutic option. Multi-drug resistance (MDR) S. Typhi is increasingly prevalent, but to date has not been detected in SPA. In Australia, cases of SPA are notifiable. Here we report on the genomic epidemiology of 208 cases of SPA in returned travellers to Australia, and their close contacts, from 2018 to 2025. A total of 15 unique genotypes were detected, and these were correlated with geographical regions of reported travel. There was a low incidence of antimicrobial resistance with only a single isolate carrying acquired resistance genes. Mutations in quinolone resistance determining regions were common across the genotypes, detected in 95.7% of isolates. A single isolate in a traveller returning from India was resistant to several first line antibiotics including: ampicillin, amoxicillin plus clavulanic acid, ceftriaxone, azithromycin and ciprofloxacin. The isolate carried a plasmid encoding an extended spectrum beta-lactamase (blaCTX-M-231), two macrolide resistance genes (mphA and ermB) and a quinolone resistance gene (qnrS1). Elements of the pangenome were explored, with stable maintenance of small plasmids encoding hypothetical proteins detected in four genotypes. Copy number variation in genes encoding surface antigen biosynthesis genes were detected in six genotypes. These biosynthesis genes are targets for one of the two SPA vaccines in development, and the potential variation in surface antigens could have implications for vaccine efficacy. Linking epidemiological data with genomic studies of SPA provides an opportunity to improve understanding of the emergence, spread and risk of drug-resistant SPA infections, and to better inform empirical treatment guidelines in returned travellers.

Carbapenem-resistant Escherichia coli (CR-E. coli) is an emerging One Health threat, but recent shifts in predominant lineages and genomic links between clinical and food reservoirs in Hong Kong remain poorly defined. We analyzed 271 CR-E. coli isolates from four hospitals (2022-2026) and 585 isolates recovered from 4,917 retail food samples (2022-2025). Isolates underwent antimicrobial susceptibility testing, whole-genome sequencing, multilocus sequence typing, resistance-gene and plasmid profiling, core-genome SNP phylogenetics, and comparative genomics. Food isolates were mainly from raw pork (268/585, 45.8%) and raw chicken (231/585, 39.5%). blaNDM-5 was detected in 527/585 (90.1%) food and 241/271 (88.9%) clinical isolates. ST69 was the most frequent defined sequence type in both collections, representing 44/585 (7.5%) food and 36/271 (13.3%) clinical isolates, in contrast to the heterogeneous lineages and carbapenemases previously reported in Hong Kong. Applying a predefined [&le;]50-pairwise-SNP threshold for close genomic relatedness, core-genome phylogeny of 80 ST69 isolates identified two major mixed-source clusters collectively comprising 28 clinical and 27 food isolates. Clustered isolates showed similar antimicrobial resistance profiles, carried blaNDM-5 and blaTEM-1, and were associated with IncI1 MLST | ST136 plasmids. Comparative analyses showed >99.85% average nucleotide identity and broad conservation of the blaNDM-5-associated plasmid backbone across sources. These findings indicate the emergence of blaNDM-5-carrying ST69 as a prominent CR-E. coli lineage in Hong Kong and demonstrate close genomic relatedness between selected clinical and retail food isolates. Although transmission direction have not been inferred yet, the findings support integrated One Health surveillance and source-tracing across clinical, food, animal, and environmental sectors.

Background Rising antimicrobial resistance (AMR) in the African region contributes to high morbidity and mortality. Continuous national AMR surveillance is critical in understanding the spread of AMR and informing policies on containment. We present results of national AMR surveillance in Kenya Methods Passive surveillance was prospectively conducted in 20 sites in Kenya between 2021 and 2025. Sites included national and sub-national level tertiary public and private hospital laboratories. Non-duplicate isolates of WHO priority Gram-negative and Gram-positive pathogens were included in this analysis. Bacterial isolates were identified using either conventional identification methods, Analytical Profile Index or automated systems while antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method or automated systems and interpreted using the Clinical and Laboratory Standards Institute guidelines. The primary outcomes were the proportions of various priority bacteria isolated and the proportions resistant to commonly used antibiotics. Results Between 2021 and 2025, there were 15,124 priority pathogens isolated with 7,592 (50.2%) from urine, 5,430 (35.9%) from blood (35.9%), and 1,784 (11.8%) from respiratory specimens. Escherichia coli and Klebsiella pneumoniae accounted for 76.3% of the priority pathogens. Resistance to 3rd generation cephalosporins was 63.2% for Escherichia coli and 79.1% for Klebsiella pneumoniae for the period 2021 to 2025 while carbapenem-resistance was 30.4% for Klebsiella pneumoniae and 7.2% for Escherichia coli. Resistance to carbapenems by Klebsiella pneumoniae increased from 17.9% in 2021 to 35.9% in 2025 while Methicillin resistance in Staphylococcus aureus increased from 36.5% in 2021 to 56.4% in 2025. Conclusion Resistance to critical antibiotics is a significant problem in Kenya, with alarming rates of Methicillin Resistant Staphylococcus aureus and carbapenem resistant Klebsiella pneumoniae. Ugent and sustained infection prevention and control measures and appropriate antimicrobial stewardship activities should be instituted across all health facilities in the country. There is need for improved access to antibiotics with activity against these resistant pathogens.

Antimicrobial resistance generates a vast, rapidly growing literature, yet no resource offers a comprehensive, evidence-linked repository of AMR findings at scale. We present ResLit, an automated pipeline and public database that mines the AMR literature for resistance genes, mutations, organisms, and mechanisms. From 2 million candidate PubMed records, BioMistral-7B screened abstracts to 356,000 relevant papers; multi-tier retrieval yielded 117,000 full texts, from which Qwen3-30B performed two-step extraction. ResLit contains 3,120 genes and 13,593 mutations, cross-linked to CARD, ResFinder, and NCBI Reference Gene Catalog across four evidence tiers. It further supports community-driven curation of automated outputs and reference databases. Freely available at www.reslit.info.

The global dissemination of antimicrobial resistance is increasingly driven by bacterial clones combining antimicrobial resistance with enhanced virulence and environmental adaptability. Escherichia coli sequence type 131 (ST131) has historically been regarded as a major disseminator of the extended-spectrum {beta}-lactamase (ESBL) blaCTX-M-15. However, the emergence of E. coli ST1193 carrying blaCTX-M-15 may represent an ongoing shift in the epidemiology of this resistance determinant. Here, we investigated the prevalence, genomic characteristics, virulence and antimicrobial resistance potential of ST1193 in comparison with ST131. A total of 1,136 E. coli isolates were recovered from touristic and non-touristic environments, hospital-associated samples, and aircraft toilets in Guadeloupe. Isolates were whole-genome sequenced and analysed for antimicrobial resistance and virulence determinants. Additionally, publicly available genomic data comprising 1,215 blaCTX-M-15-positive ST131 and ST1193 isolates were analysed to assess temporal and geographical trends. ST1193 was significantly associated with aircraft-associated samples and exhibited a higher antimicrobial resistance gene burden than ST131, while maintaining a comparable virulence factor content. Analysis of publicly available genomes revealed similar temporal emergence patterns for blaCTX-M-15-positive ST1193 and ST131, with ST1193 showing a more recent distribution and a higher number of deposited isolates in recent years, consistent with a potential ongoing clonal replacement. Comparative genomic analysis identified numerous virulence and adaptation-associated genes shared between both sequence types, while ST1193 additionally carried distinct determinants, including components of the transmissible locus of stress tolerance. Furthermore, quinolone resistance-associated mutations were strongly linked to blaCTX-M-15 carriage, particularly among ST1193 isolates. Together, these findings identify E. coli ST1193 as an emerging high-risk clone with substantial potential for blaCTX-M-15 dissemination. Its association with aircraft-associated samples further highlights the potential role of air travel in long-distance transmission and underscores the need to reconsider current surveillance strategies focused predominantly on ST131.

IntroductionIn sub-Saharan Africa, and more specifically in Cameroon, antimicrobial resistance (AMR) represents a major public health threat. This is underlined by the increasing appearance of multidrug-resistant bacteria. Extended-spectrum {beta}-lactamase producing Escherichia coli (ESBL-Ec), a critical priority bacterium, is increasingly implicated in life-threatening infections in hospital and community settings in Cameroon. Data on the genetic composition of ciprofloxacin-resistant Escherichia coli are limited in Cameroon. This study aimed to investigate the prevalence, genetic diversity, resistance mechanisms in multidrug-resistant Escherichia coli organisms isolated from clinical samples in two hospitals in Yaounde, Cameroon. MethodA cross-sectional study was conducted from February to June 2025 in two healthcare facilities in Yaounde, Cameroon. All clinical samples from in- and out-patients were analysed. After culturing, identification was performed using API20E as per the manufacturers instructions and ESBL production was screened in CHROMagarTM ESBL. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method. Polymerase chain reaction (PCR) was used to detect ESBL and plasmid mediated quinolone resistance (PMQR)genes, as well as mutations in quinolone resistance-determining region (QRDR) (gyrA/parC) Horizontal. plasmid transfer was also investigated. Finally, phylogroup analysis was assessed. ResultThe prevalence of MDR E. coli was 50.7% (n=33/65), all of which (100%) were ESBL producers and 91% were ciprofloxacin-resistant. Highest resistance rates were observed for cefotaxime (100%), ceftriaxone (100%), and ciprofloxacin (91%). The most frequent ESBL genes were blaTEM (36.3%; n=12/33). Among PMQR genes, qnrB was detected in 16.6% (n=5/30) of isolates. Only the ESBL genes were carried by plasmids; the most prevalent plasmid-borne gene was blaTEM (40%), followed by blaCTX-M (26.7%). Mutations within the topoisomerase QRDR (parC gene) were identified in 36.6% (n=11/30) of ciprofloxacin-resistant strains. Phylogroup analysis revealed a predominance of phylogroup A, followed by group B. ConclusionThis study reveals a high prevalence of multidrug-resistance, ESBL (blaTEM dominant) and fluoroquinolone resistance in E. coli in Yaounde, with plasmid dissemination of ESBL genes and chromosomal stabilization of PMQR determinants. The predominance of commensal phylogroups in clinical samples underlines the role of the community reservoir. It is urgent to reinforce " real-time One Health" genomic surveillance in Cameroon.

BackgroundThe epidemiology of Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa is rapidly evolving in China. While blaKPC-2 remains the predominant KPC variant in P. aeruginosa, blaKPC-3 has rarely been documented in this pathogen. This study investigated the molecular epidemiology, resistance and virulence characteristics, and plasmid features of blaKPC-3-producing CRPA isolates collected from a tertiary hospital in eastern China. MethodsA total of 65 non-duplicate CRPA isolates collected in 2023 were subjected to whole-genome sequencing. Antimicrobial susceptibility testing, phylogenetic analysis, plasmid characterization, conjugation experiments, and virulence assays were performed. ResultsAmong the 65 CRPA isolates, 37 (56.9%) carried blaKPC-3. These blaKPC-3-positive isolates belonged to four sequence types (STs), including ST1076 (62.2%), ST463 (21.6%), ST646 (10.8%), and ST3393 (5.4%). To our knowledge, this is the first report of blaKPC-3 in P. aeruginosa ST463, ST646 and ST3393. All isolates exhibited extensive drug resistance, and 51.8% were resistant to ceftazidime-avibactam. Phylogenetic analysis indicated that blaKPC-3 dissemination was driven by both clonal expansion and horizontal transmission. Comparative genomic analysis identified three kinds of blaKPC-3 -carrying plasmid. A transferable IncP-2 megaplasmid was widely distributed among ST1076, ST646, and ST3393 isolates, whereas non-transferable IncP-10 plasmids were primarily restricted to ST463. The genetic environments and plasmid backbones of blaKPC-3 were highly conserved and closely related to those of blaKPC-2 and its variants, suggesting evolution from pre-existing blaKPC-2-associated plasmids. Virulence analysis demonstrated marked heterogeneity across lineages. ST463 isolates co-harbored exoU and exoS, exhibited enhanced biofilm formation and pyocyanin production, and caused significantly higher mortality in the G. mellonella infection model, indicating a hypervirulent phenotype. ConclusionsThe blaKPC-3 is becoming an increasingly important determinant of carbapenem resistance in P. aeruginosa in China. The IncP-2 megaplasmid and IncP-10 plasmid derived blaKPC-3 spread across multiple lineages. Continuous genomic surveillance and enhanced infection control measures are urgently needed to prevent its further prevalence in clinical settings.

Antimicrobial resistance is a public health challenge, driving the need for rapid, cost-effective diagnostic support tools. Artificial intelligence (AI) may enable prediction of susceptibility to untested antibiotics from known susceptibility results, but prospective clinical validation is required before routine use. We evaluated an AI-based decision support method, trained on invasive isolates from the European Surveillance System (TESSy), for prediction of antibiotic susceptibility in clinical Escherichia coli urine isolates. The evaluation included 99 E. coli isolates from urine samples with diversity in age, sex, and antibiotic susceptibility. Predictions were evaluated for 14 antibiotics using patient metadata and susceptibility results for 4-8 antibiotics as input. Prediction uncertainty was handled using conformal prediction, allowing abstention when confidence was insufficient. EUCAST disk diffusion test results were used as reference and genomic sequence data was used to explore mechanisms of the AI performance. Without conformal prediction, 84% of predictions were correct when susceptibility results of six antibiotics were used to predict susceptibility to eight additional antibiotics. Across all predictions generated using susceptibility results for six antibiotics as input, the major and very major error rates were 19% and 12%, respectively. Prediction errors varied between antibiotics and were associated with certain phenotypic and genotypic resistance patterns. Conformal prediction reduced errors but increased abstentions; at confidence levels of 90%, 95%, and 97.5%, the model abstained in 9.6%, 14%, and 22% of instances. The method showed promising performance, but its clinical use remains limited and may require diagnostic data beyond susceptibility test results and demographic variables.

Hospital wastewater (WW) and wastewater biofilms (WWB) are increasingly recognized as important reservoirs of carbapenemase-producing Enterobacterales (CPE), yet their long-term ecological dynamics and relationship with contemporaneous clinical isolates remain poorly understood. Here, we performed longitudinal CPE surveillance of WW and WWB over a 17-month period, combining culture-based screening and comparative whole-genome sequencing of environmental isolates with CPE isolates recovered from patients hospitalized in the same hospital building. A total of 42 environmental and 21 clinical CPE isolates were characterized. Environmental CPE populations underwent a marked ecological shift, with blaOXA-48 -producing Citrobacter spp. progressively replaced by blaVIM-4-producing Serratia nevei. In contrast, clinical isolates remained taxonomically diverse throughout the study period, with a range of betalactamases including blaOXA-48, blaVIM-4, and blaNDM, with no comparable temporal replacement. Comparative genomic analyses revealed a strong association between resistance genes and mobile genetic elements (MGEs), with MGE dynamics largely following those of their hosts. blaOXA-48 was predominantly associated with highly conserved IncL/M plasmid backbones shared across environmental and clinical compartments, whereas blaVIM-4 was consistently embedded within conserved class 1 integron-associated genetic contexts on IncHI2A-rep1088 plasmids. In contrast, blaNDM displayed heterogeneous genomic organizations involving multiple plasmid backgrounds and frequent chromosomal integration. Together, our findings show that bacterial hosts and carbapenemase-carrying genetic elements follow distinct ecological trajectories within hospital WW ecosystems. Integrating longitudinal environmental surveillance with comparative genomics provides new insights into the persistence of clinically important carbapenemases across interconnected environmental and clinical reservoirs.

Background Carbapenem-resistant bacteria are a major global public health threat, classified as critical priority pathogens by the WHO. In Indonesia, despite a national antimicrobial resistance control programme established by the Ministry of Health in 2015, resistance rates continue to rise, including increasing carbapenem resistance among clinically important bacteria. Strengthening approaches to directly interrupt transmission is essential, yet transmission pathways remain poorly understood with limited research and policy guidance within the Indonesian context. Methods and analysis The INTERCEPT study is a UK-Indonesia multidisciplinary collaboration aiming to identify transmission routes of carbapenem-resistant bacteria across healthcare and community settings, and the mechanisms of resistance gene transfer between bacteria and mobile genetic elementss. We will conduct genomic surveillance of hospital inpatients, healthcare workers, hospital environments, and surrounding communities, including wastewater systems, combined with genomic analyses and mathematical transmission modelling. A cohort of patients with bloodstream infections will be recruited to evaluate resistant bacteria, treatment practices, and clinical outcomes. Qualitative research will explore behavioural and system-level factors influencing transmission and intervention implementation. Findings will inform stakeholder workshops to co-design context-specific interventions, with pilot intervention over 9 months with pre- and post-intervention assessment to guide scalable strategies to reduce AMR transmission. Discussion The INTERCEPT study addresses carbapenem resistance in Indonesia using an integrated approach combining microbiological surveillance, genomics, modelling, and qualitative methods. Strengths include cross-sectoral analysis (patients, workers, environment) and participatory intervention design. Limitations include geographic scope restricted to Central Java, Indonesia.

Reduced cephalosporin resistance in Enterococcus faecium has traditionally been reported in laboratory mutants and, more recently, in a single clinical ampicillin-susceptible (AmpS) isolate. Herein, we investigated whether this phenotype is widespread by analysing 95 clinical enterococcal isolates (78 AmpS and 17 ampicillin resistant [AmpR]) collected from three hospitals in Spain and Portugal (2009-2025). Low ceftriaxone MICs ([&le;]4 mg/L) were detected in 19/51 (37.3%) AmpS E. faecium and 7/27 (25.9%) E. lactis but in none of the AmpR isolates. Low ceftriaxone MICs were associated with older patient age in both species and with prior ampicillin therapy in E. faecium, but not with other clinical or epidemiological variables. Ceftaroline MICs were consistently low among AmpS isolates, while ceftriaxone and cefotaxime showed greater variability. Low-MIC isolates were distributed across multiple clonal lineages and hospitals and did not share a distinctive resistance or virulence gene profile. PBP5 phylogeny and variation at the psr-pbp5 region separated AmpS from AmpR E. faecium but did not explain variability in ceftriaxone MICs. Five AmpS isolates with reduced ceftriaxone MICs carried chromosomal deletions that included the psr-pbp5 region and genes with diverse cellular functions. Variation in other candidate resistance genes (pbpA, ponA, pbpF, croRS, stpA/stk and murAA) did not consistently explain the MIC differences. These results reveal unexpected heterogeneity in intrinsic cephalosporin resistance in clinical E. faecium and E. lactis and suggest that additional genetic or regulatory mechanisms underlie reduced susceptibility.

Background Nontuberculous mycobacteria (NTM) are an increasingly common group of pathogens that remain challenging to diagnose and treat effectively. The lack of standardization of NTM management, from identification to antibiotic resistance prediction, results in imperfect correlations between treatment and outcomes. This study characterizes the genetic heterogeneity of a previously uncharacterized NTM during a 29-month bacteremia with acquired drug resistance. Results In contrast to the initial diagnostic result identifying M. nebraskense, a rare NTM causing disease in humans, whole genome sequencing (WGS) identified Mycobacterium sp. SMC-2, a species with only one publicly available genome. High-resolution analysis of variants revealed 444 unique SNPs and 26 indels in 12 longitudinal isolates, with the highest number of low-frequency mutations between 3-5% frequency. Seven candidate drug-resistance mutations across five evolutionary trajectories showed frequency shifts that correlated with changes in minimum inhibitory concentrations to the corresponding antibiotics. These included a 23S rRNA clarithromycin-resistance SNP detected at 7% frequency when phenotypic resistance emerged, suggesting that low-frequency variants drive subpopulation evolution. Acquisition of drug resistance during therapy was associated with several low-frequency mutations in genes associated with resistance to antibiotics, including clarithromycin and quinolones, in other NTM species. Conclusion This study highlights the importance of low-frequency variants as drivers of intra-patient bacterial population diversity, allowing subpopulations to adapt to antibiotic pressure and ultimately contributing to treatment failure. Additionally, it underscores their potential implications for the development of molecular diagnostic tests for NTM resistance prediction.

BackgroundDespite the importance of oropharyngeal gonorrhoea in transmission, suboptimal antimicrobial responses and propensity for horizontal transfer of antimicrobial resistance at this site, it remains understudied. An oropharyngeal N. gonorrhoeae controlled human infection model (CHIM) represents a promising tool to study infection and undertake translational research. MethodsA panel of five contemporary N. gonorrhoeae isolates were subject to detailed characterisation to assess antimicrobial susceptibility, in vitro infectivity, cytotoxicity and serum sensitivity to inform challenge agent selection. A method for challenge agent manufacture, including release testing, was developed and validated. FindingsAll candidate isolates were able to infect the surface of pharyngeal and cervical cells in vitro. One isolate displayed an invasive phenotype, induced higher inflammatory cytokine production and displayed elevated serum resistance and was excluded. The remaining four isolates were minimally inflammatory, did not induce cytotoxicity and were susceptible to serum killing. Three of the four isolates grew in a defined liquid medium. Together these results led to the selection of a contemporary N. gonorrhoeae isolate suitable for use in CHIM. A challenge agent manufacture workflow was established and shown to reliably and reproducibly generate doses suitable for direct inoculation in an oropharyngeal CHIM. ConclusionPhenotypic characterization of candidate N. gonorrhoeae challenge agents led to the successful identification of a contemporary isolate suitable for implementation in a novel oropharyngeal gonorrhoea CHIM. We demonstrate the feasibility of a challenge inoculum manufacturing process that aligns with international best practice guidelines.

The global spread of antimicrobial-resistant (AMR) bacteria represents one of the greatest threats to modern medicine, making the development of novel therapeutic strategies increasingly urgent. Phage therapy, which exploits bacteriophages (phages, viruses that specifically infect and kill bacteria) has regained attention as a therapeutic approach for multidrug-resistant infections. One critical determinant of treatment outcome is the capacity of phages to sustain bacterial growth suppression; however, no metric has previously existed to directly quantify the duration of effective lytic activity. Here, we propose the Sustainability Index (SusI), a novel metric that quantifies both the duration and extent of phage-mediated bacterial growth suppression, which is restricted to the primary lysis period from lysis initiation to resistance emergence. Evaluation of individual phages and two-phage cocktails against both laboratory and clinical isolates of Escherichia coli demonstrated that SusI provides information independent of the Virulence Index, which primarily reflects bactericidal activity during the initial phase of infection, and serves as a complementary metric to the Suppression Index, which may incorporate behavior beyond primary lysis. Cocktails composed of phages targeting different receptors specificities consistently exhibited higher SusI values, consistent with the notion that multifaceted selective pressure delays resistance emergence. Furthermore, in a mouse model of systemic infection established by intraperitoneal administration, cocktails with higher SusI values demonstrated superior therapeutic efficacy. These results confirm SusI as a practical metric for rational phage cocktail design. As phage therapy advances toward clinical implementation, standardized quantitative metrics such as SusI are expected to facilitate evidence-based selection of therapeutic phages across diverse pathogens and infection conditions. ImportanceThe global spread of antimicrobial-resistant bacteria is making bacterial infections increasingly difficult to treat. Phage therapy, which uses bacteriophages (viruses that specifically infect bacteria), has re-emerged as a therapeutic alternative; however, reliable methods to determine in advance which phages will be therapeutically effective remain limited. Current evaluation metrics are well-suited for quantifying how rapidly phages kill bacteria but were not designed to directly measure how long lytic activity is sustained before resistant bacteria emerge. Here, we developed the Sustainability Index (SusI), a novel metric that specifically quantifies the duration of effective bacterial growth suppression. Evaluation of multiple phages and their combinations (cocktails) against both laboratory and clinical bacterial isolates demonstrated that SusI can distinguish phage combinations that existing metrics fail to differentiate. Moreover, in a mouse model of lethal bacterial infection, higher SusI values correlated with improved therapeutic outcomes. SusI has potential as a practical tool for selecting phages with greater likelihood of therapeutic success.

OXA-48-like carbapenemases have been historically rare, however steady increases both locally and globally have warranted further investigation into their spread. Here we present the largest genomic analysis of blaOXA-181-producing bacteria in Australia to date, focusing on a single jurisdiction over seven years (2017 -- 2024). The initial investigation was prompted by an outbreak in 2017, where enhanced genomic surveillance in a single hospital identified 85 outbreak isolates related to an imported Escherichia coli ST38, carrying blaOXA-181 on an IncX3/colKP3 plasmid (previously reported as pOXA181). After four months of intensive infection control, the initial outbreak strain was eliminated. To confirm the outbreak plasmid was also contained, we collected all blaOXA-181-positive isolates from the same jurisdiction over subsequent years and sequenced with both Illumina and Oxford Nanopore Technologies to investigate clonal and mobile genetic element mediated spread. While continued surveillance post-2017 did not identify the same E. coli strain following the outbreak, pOXA181 plasmids were identified in >70% of surveillance isolates, with minimal genetic changes, which initially suggested local plasmid-mediated spread. Additional comparison to a global collection of pOXA181 plasmids found that epidemiologically unrelated pOXA181 plasmids were near identical, with no rearrangements and low, or no, single nucleotide polymorphisms. This suggests the mutation rate of pOXA-181 is incompatible with recent genomic transmission inference. This study highlights the current genomic epidemiology and drivers of blaOXA-181 and further demonstrates the necessity for detailed understanding of plasmid evolutionary rates to inform genomic surveillance.

ObjectiveCarbapenem-resistant Klebsiella pneumoniae harboring blaNDM poses a serious threat to public health; however, blaNDM-15 remains poorly characterized outside the dominant epidemic lineages. MethodsWe characterized K. pneumoniae strain ETFK6090, isolated from a perianal surveillance swab of an 11-month-old immunocompromised child in a paediatric intensive care unit. Investigations included antimicrobial susceptibility testing, broth conjugation, S1 nuclease PFGE with Southern blotting, complete genome sequencing, and comparative genomic analysis against 465 curated blaNDM-positive K. pneumoniae genomes from 37 countries. ResultsETFK6090 belonged to ST580 and exhibited resistance to carbapenems, ceftazidime-avibactam, broad-spectrum cephalosporins, fluoroquinolones, gentamicin, chloramphenicol and trimethoprim-sulfamethoxazole; amikacin and fosfomycin retained low MICs. The complete genome comprised one chromosome and five plasmids, blaNDM-15 was localized on a 46,161-bp IncX3 plasmid, confirmed by Southern blotting. Conjugation into Escherichia coli EC600 transferred carbapenem and cephalosporin resistance, confirming in vitro mobility. The blaNDM-15 genetic environment retained a conserved blaNDM module, with IS-mediated rearrangements at the downstream boundary. In the global comparison, blaNDM-1 and blaNDM-5 predominated, the ST580-blaNDM-15 combination was exceedingly rare, and ETFK6090 constituted a distinct branch apart from major epidemic lineages. ConclusionsA transferable IncX3-blaNDM-15 plasmid can emerge in an uncommon ST580 background, underscoring the necessity to extend genomic surveillance of carbapenem-resistant K. pneumoniae beyond dominant epidemic clones, particularly in high-risk paediatric and intensive-care settings.

Objectives: To investigate whether ethambutol resistance in Mycobacterium avium complex is associated with the emergence of macrolide resistance. Methods: Patients who developed macrolide resistance during guideline-based treatment were included, and longitudinal analyses of minimum inhibitory concentrations and mutations in embB or the upstream region of embA were performed. Clinical, microbiological, and radiological characteristics were compared according to the mutation status of embB or embA upstream region, prior to the emergence of macrolide resistance. We further evaluated the impact of embB mutation on the development of macrolide resistance using in vitro time-kill assays. Results: Sixteen patients developed macrolide resistance during guideline-based treatment. None of these patients had an ethambutol minimum inhibitory concentration >=16 ug/mL or embB or embA upstream mutations at treatment initiation; however, 8/16 patients (50.0%) had an ethambutol minimum inhibitory concentration >=16 ug/mL at the time of macrolide resistance detection, and 7/16 (43.8%) had developed embB or embA upstream mutations prior to the emergence of macrolide resistance. Cavitary lesions were present in 1/7 (14.3%) patients with embB or embA upstream mutations. In strains with embB mutations, the minimum inhibitory concentration of ethambutol increased by 1-2 dilutions relative to that of pretreatment isolates, with a corresponding increase in the concentration required to suppress macrolide resistance. Conclusions: Ethambutol resistance may contribute to the development of macrolide resistance in patients with M. avium complex pulmonary disease, particularly in those without cavitary lesions.