Antimicrobial resistance (AMR) is a serious and growing threat in Mexico. To support cross-sector dialogue and policy development around AMR, the Science and Technology Network, the Department for Business and Trade and the Microbiology Society convened a one-day workshop at the British Embassy in Mexico City. This commentary synthesises the key themes from the workshop discussions and presents the desired outcomes, challenges and policy interventions identified by participants.

Introduction. Clostridioides difficile is an opportunistic enteric pathogen posing significant challenges in human and animal healthcare due to the recurrent nature of infections and associated financial burden. The molecular chaperone protein, DnaK (Hsp70), encoded by dnaK, plays a central role in the heat stress response and protein homeostasis. Disruption of dnaK in C. difficile 630Δerm has previously been associated with increased biofilm formation and multiple phenotypic alterations.Gap Statement. Although disruption of dnaK has been shown to alter biofilm formation and multiple phenotypes in C. difficile, the global transcriptional mechanisms linking DnaK to biofilm development and stress adaptation remain poorly understood.Aim. This study aimed to characterize the global transcriptional response associated with dnaK disruption during planktonic and biofilm growth to identify pathways linking the DnaK-mediated stress response with biofilm formation.Methods. This study investigated the global transcriptional changes associated with dnaK disruption during planktonic and colony biofilm culture. Colony biofilms of C. difficile strains 630, 630Δerm and 630Δerm:dnaK were cultured on mixed nitrocellulose ester semi-permeable membranes and examined structurally using low-vacuum scanning electron microscopy. Total RNA was extracted from both planktonic cultures and 24 h colony biofilms, followed by Illumina RNA sequencing. Differential gene expression analysis was performed using DESeq (P adj<0.05). Gene set enrichment analysis (Kyoto Encyclopedia of Genes and Genomes [KEGG] and Gene Ontology) was conducted to identify significantly enriched metabolic pathways and biological processes.Results. Disruption of dnaK resulted in global transcriptomic remodelling, particularly during biofilm culture, where 72.4% of genes were significantly differentially expressed relative to planktonic culture. A core number of pathways were involved in biofilm formation in all strains, including increased sporulation with reduced translation, amino acid biosynthesis and flagellar assembly. The dnaK mutant exhibited a unique pattern of enrichment of pathways associated with cell division, peptidoglycan synthesis, carbon metabolism and amino acid biosynthesis, alongside a coordinated reduction of motility and chemotaxis pathways.Conclusion. Disruption of the dnaK gene appears to link the stress response to biofilm formation through a number of pathways associated with biofilm development. Understanding this link may identify systems relevant to persistent and recurrent infections.

Introduction. While rhinovirus was thought to cause mild infections such as the common cold, severe infections such as community-acquired pneumonia and asthma exacerbations can also occur. Children with chronic medical conditions have been shown to have increased risks of severe infections.Hypothesis/Gap statement. There is a difference in the risks of serious in-hospital outcomes among adults hospitalized for rhinovirus or influenza virus infection. Multiple morbidities in adult patients are associated with severe infections among patients hospitalized for rhinovirus infections.Aim. To compare the risks of serious in-hospital outcomes among adults hospitalized for rhinovirus or influenza virus infection and assess the association between multiple morbidities as represented by Charlson comorbidity index (CCI) in predicting risks of severe infections among patients hospitalized for rhinovirus infections.Methodology. We conducted a territory-wide retrospective study on adults hospitalized for rhinovirus or influenza virus infection between 1 January 2016 and 30 June 2023 in Hong Kong. The in-patient mortality, severe respiratory failure (SRF), secondary bacterial pneumonia and acute kidney injury (AKI) were compared. The association between multi-morbidities as reflected by CCI and severe in-hospital outcomes among adult patients hospitalized for rhinovirus infections was also assessed.Results. A total of 41,206 and 2,222 patients were hospitalized for influenza and rhinovirus infections, respectively. Patients with rhinovirus infection showed a significantly higher risk of SRF, secondary bacterial pneumonia and AKI compared to those with seasonal influenza (P<0.001 for all). Higher CCI (as both continuous variables, by grading as well as cut-off identified by receiver operating characteristic curve analysis) was associated with increased risks of severe in-hospital outcomes.Conclusion. Adults hospitalized for rhinovirus infection were associated with a significantly increased risk of adverse respiratory and kidney outcomes compared with those with seasonal influenza. More medical comorbidities, as reflected by CCI, were shown to be associated with an increased risk of severe in-hospital outcomes.

Introduction. The ability of Cryptococcus neoformans to adapt to host temperature is an important feature of its pathogenic potential and requires extensive cellular remodelling.Hypothesis/Gap Statement. Although C. neoformans undergoes substantial molecular changes at host temperature, the extent to which transcriptomic and proteomic responses are concordant during this adaptation remains incompletely understood.Aim. We investigated the molecular and metabolic responses of the C. neoformans H99 strain grown at 30 and 37 °C using an integrated transcriptomic, proteomic and functional approach.Methodology. RNA sequencing and quantitative proteomics were performed after 24 h of growth at 30 and 37 °C. Differentially expressed transcripts and proteins were identified, and matched transcript-protein features were analysed for directional concordance and quantitative correlation. Cellular metabolic activity was assessed using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay.Results. RNA-seq analysis identified 344 upregulated and 428 downregulated transcripts at 37 °C. Upregulated transcripts were enriched in functions related to ribosome biogenesis, translation, protein folding and mitochondrial-associated processes, whereas downregulated transcripts were associated with carbohydrate metabolism and other metabolic functions. Proteomic analysis revealed a more selective response, with increased abundance of proteins associated with oxidoreductase activity, sterol and isoprenoid biosynthesis, glutamine metabolism and metal-binding functions. Integration of the transcriptomic and proteomic datasets identified 305 matched features, of which 162 (53.1%) showed directional concordance. However, no significant quantitative correlation was observed between transcript and protein fold changes (Spearman ρ = -0.005, P=0.934; Pearson r=0.003, P=0.959). Cells grown at 37 °C also showed significantly increased XTT reduction, indicating higher cellular metabolic activity under the conditions tested.Conclusion. Adaptation to host temperature involves broad transcriptional remodelling accompanied by selective proteomic changes and increased cellular metabolic activity. These findings provide a molecular framework for further investigating the mechanisms underlying thermoadaptation and their contribution to the pathogenic potential of C. neoformans.

Blastocystis is an anaerobic stramenopile that colonizes the large intestine of humans and many animals worldwide. Small-subunit rRNA-based typing reveals deep genetic diversity: at least 44 genetic variants, namely, subtypes (STs), are recognized, with ST1-ST4 accounting for most human carriage. Its medical relevance remains contested. Blastocystis carriage is frequently asymptomatic, and while associations with non-specific gastrointestinal symptoms (e.g. diarrhoea, abdominal pain and bloating), irritable bowel syndrome and extraintestinal manifestations such as urticaria have been proposed. In parallel, multiple microbiome studies, including a recent population-scale analysis, link Blastocystis carriage to higher bacterial diversity, healthier diets and favourable cardiometabolic profiles, suggesting that in many settings, it may be a marker of a resilient gut ecosystem rather than a primary pathogen. Transmission is primarily faecal-oral, most likely via environmentally resilient cysts, with potential for zoonotic and waterborne spread. Diagnosis using quantitative PCR is the most sensitive method; microscopy has limited sensitivity and can be confounded by morphological plasticity, though its diagnostic accuracy varies considerably according to the experience of the microscopist. Treatment is typically based on excluding alternative causes rather than confirmed causality; while metronidazole is commonly used, clinical outcomes are highly variable due to potential co-infections, inconsistent links between parasite eradication and symptom relief and reports of reduced drug susceptibility.

Introduction. Antimicrobials are essential for maintaining animal health; however, their use exerts selective pressure that promotes antimicrobial resistance. Therefore, monitoring antimicrobial use (AMU) is critical for managing the emergence and selection of resistance and various metrics have been developed in veterinary medicine to monitor these usage patterns.Gap statement. Although metrics such as sales volume are widely used, they do not account for differences in antimicrobial potency. Currently, no defined daily dose metric specific to food-producing animals in Korea has been established to accurately assess domestic usage patterns.Aim. In this study, we aimed to establish defined daily doses for Republic of Korea (DDDkr) for antimicrobial agents approved for use in cattle, pigs and poultry and to compare these values with the defined daily doses for animals (DDDvet) established by the European Medicines Agency (EMA).Methodology. A total of 931 veterinary antimicrobial products approved and marketed with recorded sales volumes for food-producing animals in the Republic of Korea as of 2023 were analysed. DDDkr were defined following EMA principles, based on species, administration route and antimicrobial agents. To quantitatively compare the derived DDDkr values with existing DDDvet standards, the quotient of daily defined doses (QDDD) was calculated.Results. We established 200 DDDkr values: 80 for cattle, 80 for pigs and 40 for poultry. Existing DDDvet values covered 57.5% of Korean veterinary antimicrobials, as certain formulations (sprays, intramammary and intrauterine products) lacked comparable EMA values. Median QDDD values were 0.87 for cattle, 0.79 for pigs and 0.75 for poultry, reflecting significant differences between DDDkr and DDDvet for cattle and pigs (P<0.05), whereas no significant difference was observed in poultry. Comparative analysis showed that most QDDD values were less than 1.Conclusion. This study is the first to establish a DDDkr for food-producing animals in Korea. The results indicate that national dosages are generally lower than those defined in European standards, highlighting the need for country-specific metrics such as DDDkr to ensure more accurate domestic AMU monitoring.

Introduction. Enteropathogenic Escherichia coli (EPEC) is a diarrhoeagenic pathotype of E. coli, historically associated with gastrointestinal (GI) disease in infants and young children.Hypothesis/Gap statement. Although EPEC was the subject of extensive microbiological and epidemiological investigations throughout the twentieth century, in recent years, surveillance of diarrhoeagenic E. coli (DEC) has focused on Shiga toxin-producing E. coli. Consequently, the true clinical and community burden of EPEC is unknown.Aim. The aim of the study was to review the microbiological typing data and demographic data linked to EPEC isolates and cases diagnosed from 2011 to 2024 in England and to assess the risk to public health.Methodology. Faecal samples that tested positive by PCR for DEC at local microbiology diagnostic laboratories were referred to the UK Health Security Agency (UKHSA) for confirmation and culture. Isolates identified as EPEC were sequenced on the Illumina HiSeq and NextSeq platforms. Sequence type, serotype and antimicrobial resistance (AMR) profile were derived from the genome sequence. Age, sex and travel histories were linked to the typing data.Results. A total of 3,091 EPEC isolates from cases resident in England were notified to the UKHSA between January 2011 and October 2024, with 96% classified as atypical EPEC (aEPEC). The number of referred isolates increased more than eightfold, from 78 in 2011 to 654 in 2024, likely due to the widespread implementation of PCR for the detection of GI pathogens at the local hospital level. There was a slightly higher proportion of females (51.7%), and the highest proportion of cases occurred in the 0-4 age group (30.1%). Reported symptoms included diarrhoea (86.57%) and/or bloody diarrhoea (24.25%) and abdominal pain (9.05%), and 33 cases were diagnosed clinically with haemolytic uraemic syndrome. The population structure was diverse with 447 different serotypes identified, and 24.5% of isolates were resistant to three or more antibiotic classes.Conclusion. A high proportion of cases were infants or young children. Strains causing travellers' diarrhoea exhibited multidrug resistance and extensive drug resistance. Further research on potential animal reservoirs, likely food vehicles, exposure risks and pathogenic potential of EPEC, is warranted. To facilitate future investigations, we recommend that EPEC should be part of the diagnostic algorithm in the UK and that surveillance strategies should include analysis of travel data and monitoring AMR to inform the global perspective.

Introduction. Influenza A is the major cause of hospitalization due to acute respiratory virus infections (ARIs) worldwide, while other respiratory viruses are thought to play lesser roles. However, viral co-infection, host demographics and environmental factors, including air quality, may influence clinical presentation and hospital admissions. This study characterizes their clinical associations during the last influenza A outbreak in Qatar before the emergence of severe acute respiratory syndrome coronavirus 2.Gap statement. The relative contributions of host demographics, viral co-infection and environmental factors to ARI hospital admissions remain incompletely understood.Aim. To assess the association of host demographics, viral co-infection and air quality on influenza A admissions for pneumonia, bronchiolitis and reactive airway exacerbations.Methodology. Between 1 November 2019 and 28 March 2020, 1,801 patients admitted with ARIs were classified using ICD-10 codes into bronchiolitis, reactive airway exacerbations and pneumonia. Respiratory samples were analysed by real-time PCR (RT-PCR) for influenza A and other common respiratory viruses, with non-influenza A grouped collectively as other respiratory viruses (ORVs). Air quality metrics at admission were ozone, sulphur dioxide, nitrogen dioxide, carbon monoxide, nitrogen oxide and fine particulate matter. Associations between influenza A, air quality, viral co-infection, age, gender and nationality were assessed.Results. Distinct waves of influenza A and respiratory syncytial virus (RSV) were observed, while human rhinovirus (HRV) transmission persisted at a high level, with remaining viruses at low levels. Influenza A was significantly associated with pneumonia, while ORVs were the main causes of bronchiolitis (RSV) and reactive airway exacerbations (HRV); viral co-infections were uncommon. Bronchiolitis predominantly affected infants <12 months, whereas reactive airway exacerbations were seen across wider age bands. No consistent independent associations between air quality metrics and ARIs were observed after multivariable adjustment during the 5-month study period. Combining all the respiratory viruses showed a strong correlation with patterns of hospital admissions (r=0.88, 95% confidence interval: 0.72, 0.95).Conclusion. Age and viral type were the most significant determinants of admission. While the associations of influenza A, HRV and RSV with pneumonia, acute exacerbations and bronchiolitis, respectively, were confirmed, all viruses had similar associations, and the strongest correlation was observed when all viruses were treated as a single wave.

Introduction. Third-generation cephalosporin-resistant Klebsiella pneumoniae (3GC-R-KP) is a significant global health concern. The widespread production of β-lactamases and virulence factors contributes to multidrug resistance and complicates treatment.Hypothesis/Gap Statement. The potential co-evolution of resistance and virulence in 3GC-R-KP remains unclear, raising the risk of emerging 'highly resistant-hypervirulent' clones.Aim. To analyse the resistance spectrum, virulence characteristics and biofilm formation ability of 3GC-R-KP and to clarify its enzyme production types and carriage of virulence factors.Methodology. The antimicrobial susceptibility of each strain, the hypermucoviscous (HM) phenotype (determined by the string test) and the biofilm-forming capacity (assessed using the crystal violet staining method) were examined. Three β-lactamase phenotypes were detected using the double disc synergy test, the disc approximation test and the modified carbapenem inactivation method, respectively. Simultaneously, multiplex PCR technology was employed to screen for drug resistance genes and virulence genes, and statistical analysis was performed on the detection results.Results. Most isolates were multidrug-resistant strains with biofilm-forming ability, and the main resistance phenotypes were extended-spectrum β-lactamase (ESBL) and carbapenemase production. Genotypically, bla SHV (95.8%; note: this represents only the carriage rate of the gene, and the wild-type does not possess ESBL activity) and bla KPC (76%) were the most common. The virulence genes entB (99%) and mrkD (96.9%) were detected at extremely high rates, indicating that drug-resistant strains generally possess high virulence potential, and there is a coexistence of resistance and virulence genes.Conclusion. 3GC-R-KP exhibits a high rate of multidrug resistance and widespread carriage of hypervirulence genes. Traditional hypervirulent K. pneumoniae (hvKP) is evolving into 'dual-risk' strains that possess both hypervirulence and multidrug resistance by acquiring resistance genes, thereby posing an increased clinical threat. The HM phenotype alone is insufficient to define hvKP, and accurate typing requires the detection of virulence genes. The co-occurrence of resistance genes and virulence factors suggests possible coevolution between them.

Introduction. BK polyomavirus (BKPyV) reactivation is a significant health risk among renal transplant recipients that can lead to nephropathy and allograft loss.Hypothesis/Gap statement. While the microbiota is increasingly recognized as an important determinant of viral infection and pathogenesis, as well as itself undergoing compositional changes in response to infection, the urinary microbiome has yet to be investigated in the context of BK polyomavirus reactivation.Aim. This study aimed to investigate associations between the urinary microbiome and BKPyV-DNAemia in renal transplant patients.Methodology. Shotgun metagenomics of the urinary microbiome was conducted for 22 renal transplant recipients, 11 of whom had BKPyV-DNAemia. Sequence data were analysed using two complementary approaches to identify common microbiome associations with BKPyV-DNAemia: (1) Kaiju - a DNA-to-Protein method that captures bacteria, archaea, fungi, microeukaryotes and DNA viruses and (2) MetaPhlAn4 - a DNA-to-Marker method using a reference database of specific marker genes of prokaryotes.Results. We found increased observed diversity of bacterial taxa in control patients compared to those with BKPyV-DNAemia for data analysed with MetaPhlAn4 (P=0.037) but not Kaiju (P>0.05), which followed a similar trend. Significant differences in microbial beta diversity between the control and BKPyV-DNAemia patient group were identified for the Kaiju dataset (P=0.027) but not for MetaPhlAn4 (P>0.05), with viral reads likely driving these differences in the Kaiju dataset. Both Kaiju and MetaPhlAn4 identified Proteobacteria, Firmicutes and Actinobacteria as bacterial phyla with greatest relative abundance across samples. Screening bacterial species data generated from Kaiju and MetaPhlAn4 against a database of 243 human pathogens identified 8 pathogenic species recovered from both datasets that were present in the urinary microbiome of renal transplant patients.Conclusion. The observed evidence for differences in microbiome diversity and composition associated with BKPyV-DNAemia may play an important role in its pathology and guide the development of diagnostic biomarkers. Our findings warrant further investigation across larger patient cohorts that are more evenly balanced for gender.

Prosthetic joint infections (PJIs) remain among the most devastating complications of arthroplasty, imposing substantial clinical, economic and patient burdens. Although culture-based diagnostics underpin current clinical practice, PJIs are biofilm-driven infections shaped by taxonomic diversity, spatial organization, host responses and surface interactions, meaning conventional approaches provide only a partial and often decontextualized view of the infection process. We examine how convergent methodologies can transform PJI research by integrating approaches that have traditionally been studied in isolation, including sequencing, transcriptomics, metabolomics, advanced imaging and culture-based characterization. We discuss how whole-genome sequencing, shotgun metagenomics, transcriptomic and metabolomic approaches resolve pathogen identity, functional activity and adaptive persistence and how cross-scale imaging and spatial biology techniques reveal where microbes colonize, interact and survive across implant surfaces. We highlight emerging opportunities to unify these datasets into coherent frameworks that capture both the molecular and physical dimensions of PJIs. Integrating these complementary approaches will enable a multi-layered understanding of PJIs that link composition, function and spatial organization. Ultimately, this provides a foundation for predictive diagnostics, precision antimicrobial strategies and improved implant design and supports a shift towards more effective, mechanism-informed management of implant-associated infection.

Tetracyclines are bacteriostatic antimicrobials used in the treatment of both bacterial and protozoan pathogens. They abolish protein synthesis by binding to the 16S rRNA, blocking aminoacyl-tRNA access and preventing polypeptide elongation. Tetracyclines are polyketide antimicrobials, with members of this drug family (doxycycline, minocycline and tigecycline) on the World Health Organization (WHO) list of essential medicines. Bacterial resistance to this antimicrobial family is widespread and arises via multiple mechanisms. The most common of these resistance mechanisms are tetracycline efflux, ribosomal protection proteins and the newly emerging tetracycline destructases (TDases). The rise of resistance has limited the clinical use of first- and second-generation tetracyclines; however, third-generation tetracyclines are now reserved as antimicrobials of last resort for complicated bacterial infections. However, genetically mobile TDases have now been identified in opportunistic pathogens, with this mechanism of resistance capable of inactivating third-generation tetracyclines. This presents the question, are third-generation tetracyclines doomed to fail in the same manner as first-generation tetracyclines?

Background. Histological examination and rabbit intratesticular inoculation of clinical specimens are traditionally used to diagnose syphilis and characterize the aetiological agent, Treponema pallidum. Usually, these methods provide only qualitative data of the bacterium. For quantitative analysis, dark-field microscopy and real-time PCR are commonly used but both methods have important limitations. We investigated intradermal inoculations in rabbits as a sensitive and quantitative alternative test method.Methods. We prepared 10-fold serial dilutions of a treponemal stock ranging from 4×10[3] to 4 treponemes ml[-1]. Each dilution series was injected intradermally in two rabbits, ten sites per rabbit. Similarly, each dilution was inoculated intratesticularly into two rabbits, four dilutions in a total of eight rabbits. We scored and measured the size of lesions at the injection sites in rabbits inoculated intradermally. We monitored intratesticularly infected rabbits for orchitis. Sera from all animals were tested for seroconversion. We also compared the analytical sensitivity of animal bioassays to that of treponemal PCR.Results. The infectivity titre of T. pallidum was the same whether measured by intradermal or intratesticular inoculations, but the former used 75% fewer animals. Bacterial concentration at 50% endpoint was lower, 4 treponemes ml[-1], with intratesticular route compared to 25 treponemes ml[-1] using intradermal injections. Nested PCR demonstrated an analytical sensitivity of 40 treponemes ml[-1], comparable to that obtained with animal bioassays. We also showed a correlation between lesion size and treponemal counts that, under standardized conditions, might be exploited to estimate the number of treponemes in a sample.Conclusions. Our studies showed that all methods tested detected T. pallidum with similar sensitivities. However, the intradermal route might be better suited to investigate clinical specimens as it offered desirable features such as treponemal quantification, minimal number of animals required and detection of live infectious bacteria, all in a single test.