Ashlee M. Earl

The gastrointestinal (GI) tract serves as a reservoir of Klebsiella pneumoniae (Kp), facilitating transmission of invasive strains and dissemination of antimicrobial resistance genes (ARG). Studies of drug-resistant strains in hospitalized patients, during care, and post-discharge, have shown that Kp may persist for months to years in the GI tract. In contrast, there are limited longitudinal data characterizing the duration of Kp GI carriage in healthy populations. In this study, we evaluated a previously described population from an urban neighborhood of Dhaka, Bangladesh, using metagenomic analyses of Kp-enriched fecal cultures to characterize strain-level and ARG carriage dynamics over a 2-month period, and we compared this with Kp carriage data from a contemporaneously enrolled hospitalized cohort from Dhaka Medical College Hospital. Our results show high rates of Kp carriage in both groups, along with frequent co-carriage of multiple drug-resistant strains. Overall, Kp strains were genetically diverse, and carriage of individual strains was typically short-lived, with extensive turnover between time points. We also observed age-associated increases in IgG antibody responses to common Kp surface antigens, which we postulate reflect the effect of repeated, frequent exposures to Kp in these populations. Together, these findings provide insights into the carriage dynamics of Kp in a highly endemic setting. IMPORTANCE: Klebsiella pneumoniae is a leading cause of antibiotic-resistant healthcare-associated infections, with the World Health Organization listing carbapenem-resistant strains as a priority pathogen. Gastrointestinal (GI) carriage is recognized as a risk factor for invasive disease in hospitalized patients, with the infecting strain often originating from the patient’s own gut microbiota. Less is known about non-hospitalized populations, where GI carriage is common but asymptomatic. By defining strain-level dynamics and characterizing the antimicrobial resistance genes (ARGs) circulating in a highly endemic community, this study provides a valuable counterpoint to previous research focused on hospital settings and contributes to our understanding of the diversity and dynamics of circulating strains in healthy populations where the burden of Klebsiella pneumoniae is high.

Enterococci appear to have originated in the guts of early terrestrializing arthropods and invertebrates over 425 million years ago—hosts that are now highly diverse and widespread in nature today. Yet most knowledge of the genus comes from human infection-associated lineages with genomes swollen by the recent accretion of foreign DNA conveyed by mobile elements. Because invertebrates dominate terrestrial animal diversity and biomass, they would be predicted to constitute a major but little-explored reservoir of enterococcal diversity. We therefore systematically examined Enterococcus association and species diversification in invertebrate hosts of the comparatively natural, isolated, but well-characterized environment of the Azorean island of Terceira. Over 100 invertebrate specimens were examined for associated enterococci, which were taxonomically classified by whole-genome sequencing. Supporting the existence of a large pool of uncharacterized enterococci and Enterococcus -adapted genes, 40% (eight of 20) of the Enterococcus species identified were either undescribed, including four candidate new species described here, or very recently discovered. In contrast, control isolates from vertebrates were exclusively of known species typical of sampling elsewhere, discounting geographic isolation as a main driver of the novelty observed. Further, because of the abundance of E. casseliflavus and E. flavescens in this collection, we obtained the resolution necessary to quantify the divergence and decipher the drivers of speciation in the controversial division between these naturally vancomycin-resistant species. These findings provide robust support for the existence of a large pool of new species and unexplored adaptive traits in invertebrate-associated enterococci—diverse environmental survival traits optimized for expression in an enterococcal background, and well positioned for transmission into human-associated enterococcal strains. IMPORTANCE: Enterococci are auxotrophic gut-associated bacteria that co-evolved with their terrestrial hosts over many eons. In the last 75 years—the “antibiotic era” —E. faecalis and E. faecium gained genes for antibiotic resistance and enhanced virulence, emerging as leading causes of multidrug-resistant infection. Little is known about the source of those genes or the pathway by which they entered human-associated strains. A recent global survey suggested a potentially large repository of uncharacterized genetic diversity in the enterococci of invertebrates. We directly tested this prospect by examining enterococci of invertebrate hosts in a largely natural and pastoral environment. Our findings provide clear evidence that invertebrates naturally harbor vast unexplored enterococcal diversity. Moreover, associations are likely driven by intrinsic host selection factors rather than geographic isolation. This expands our knowledge of Enterococcus biodiversity, including the identification of four novel species, identifying a vast reservoir of enterococcal genes available to species that colonize and infect humans.

Summary: 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 ≤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]; pS 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%], pE 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%, [9·0%, 30·0%], pE coli ST131 strains increased (AAPC=23·8%, [17·4%, 30·5%], pE coli ST131 declined (AAPC=−15·9%, [−21·3%, −10·2%], pK pneumoniae , hypervirulent ST23 was most common (N=88) with an increasing trend in incidence (AAPC=2·3%, [−4·4%, 9·4%]) 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. Funding: This study was funded by a Large Scale Applied Research Project competition award (2017) from Genome Canada, administered via Genome Alberta. IAL is supported by an Alberta Innovates Translational Health Chair and a UCalgary Research Excellence Chair, with additional support from the Canada Foundation for Innovation (CFI-JELF 34986), the Natural Sciences and Engineering Research Council (DG 04547). Microbial isolates were prepared for analysis at the Alberta Centre for Advanced Diagnostics, which is supported by PrairiesCan (000022734). This work was also supported by federal funds from the US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110818 to the Broad Institute. JTS is additionally supported by the NIH/NIAID F32AI179151. Research in context: Evidence before this study: We searched PubMed for articles published in English up to Dec 23, 2025, using (antibiotic[tiab] OR antimicrobial[tiab]) AND (use[tiab] OR prescribing[tiab] OR consumption[tiab]) AND (resistance[tiab] OR “multidrug resistance“[tiab]) AND (relationship[tiab] OR association[tiab] OR correlation[tiab] OR effect[tiab] OR impact[tiab] OR influence[tiab] OR regression[tiab]) AND (genomic OR genetic OR “”molecular epidemiology) AND (“Staphylococcus aureus” OR “Escherichia coli” OR “Klebsiella pneumoniae” OR “Enterococcus faecalis” OR “Enterococcus faecium”) NOT animal. This search yielded 739 articles. Numerous studies have characterised the molecular epidemiology of these species over the past two decades: For Staphylococcus aureus , extensive genomic surveillance has revealed that a limited number of sequence types have dominated global infections over the past two to three decades. Pandemic hospital-associated methicillin resistant S aureus (MRSA) strains such as CC5, CC22, CC80, and CC30 were widely established in the 1990s and early 2000s, often carrying large multidrug-resistant staphylococcal cassette chromosome mec (SCCmec) elements. Since then, community-associated MRSA strains, including ST8 (CC8, USA300), ST59, and ST80, have emerged and expanded, frequently harbouring SCCmec IV or V. Livestock-associated MRSA ST398 has also become widespread, particularly in Europe and North America. While vancomycin resistance in S aureus remains rare, reduced susceptibility has been reported sporadically, especially in ST5 and ST8. Overall, the distribution of S aureus sequence types continues to shift regionally, with the spread of community-associated strains into hospitals. These patterns largely reflect data from high-income countries whereas strain distribution tends to be more heterogeneous in low-and middle-income countries. In Enterococcus faecium , genomic analyses have revealed a split population structure: clade A1 (clonal complex 17) comprises the vast majority of clinical, multidrug-resistant isolates, while clade B includes largely commensal, less drug-resistant strains circulating in the community. Globally, most clinical isolates now belong to clade A1 (CC17), which is characterised by near-universal ampicillin resistance, frequent vancomycin resistance, and additional resistance to aminoglycosides and fluoroquinolones. Since its emergence, CC17 E faecium has disseminated globally, with distinct subclones predominating in specific regions and time periods. In Europe and North America, ST17 and ST117 have become the predominant sequence types, commonly associated with vancomycin resistance and implicated in bloodstream infections. Although vancomycin resistance rates remain high in the USA and low in Canada, they have been relatively stable in recent years. Genomic epidemiology has shown that Enterococcus faecalis is more genetically diverse than E faecium , with a highly recombinogenic genome and no clear separation into distinct clades. Early vancomycin-resistant E faecalis reports in the 1990s were sporadic and genetically diverse, but by the 2000s and 2010s, expansions of hospital-associated strains such as ST6, ST87, and ST179 became more prominent, particularly in Europe and North America, frequently producing beta-lactamase and exhibiting resistance to vancomycin, and high-level gentamicin. For Klebsiella pneumoniae and Escherichia coli, studies have focused on the surveillance of extended-spectrum-beta-lactamase (ESBL) producing clones. The most common epidemic clones associated with multidrug resistance in E coli were ST131, ST1193, and ST69. The majority of ESBL-producing strains are attributed to CTX-M alleles with CTX-M-15 and CTX-M-14 being the predominant genotypes. Since 2000, the emergence of CTX-M-27, a single-nucleotide variant of CTX-M-14, has been increasingly reported. ESBL rates among hospital-associated E coli infections showed increasing trends in North America, Europe, and in many countries in Southeast and East Asia. ESBL rates varied considerably for K pneumoniae across different countries (15%–60%) with high rates primarily detected in Asia and in Southern and Eastern Europe. K pneumoniae bacteraemia was characterized by diverse strains with only a few associated with clonal outbreaks and hypervirulence, such as ST20, ST23, ST258, ST1. Only one study has comprehensively integrated clinical, genomic, and antibiotic prescribing data. Pöntinen and colleagues (2024) examined E coli bloodstream infections, showing that non-penicillin β-lactam use influenced the success of widespread multidrug-resistant, extended-spectrum beta-lactamase (ESBL)-producing E coli clones. However, no previous study has combined longitudinal clinical, genomic, and antibiotic use data across multiple major pathogens. Added value of this study: To our knowledge, this study is the first to integrate longitudinal clinical, genomic, and antibiotic prescribing data for five key bacterial pathogens for an entire region over almost two decades. This combined dataset provides a unique opportunity to analyse the impact of changing antibiotic use on antimicrobial resistance trends across multiple organisms, as well as explore how pathogen-specific strains adapt to these changes over time. Implications of all the available evidence: This study underscores the critical importance of integrated, pathogen-specific surveillance programs for guiding antimicrobial stewardship. The observed association between declining fluoroquinolone prescribing and reduced resistance among Gram-positive pathogens illustrates the potential impact of targeted prescribing interventions. At the same time, the heterogeneous responses across bacterial species to this change in practice highlight that stewardship strategies must be tailored, rather than one-size-fits-all. Continuous genomic surveillance is essential for anticipating resistance shifts and enabling timely detection and response to emerging threats, particularly from high-risk clones with the capacity for rapid dissemination. Finally, reducing antimicrobial resistance will require an integrated approach with hospital-based interventions along with coordinated efforts in the community to limit transmission and overall antibiotic exposure.

UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite used to build the peptidoglycan cell wall that protects bacteria against lysis caused by high turgor pressure. More than thirty years ago GlmU, which contains both acetyltransferase and uridyltransferase activities, was discovered to make UDP-GlcNAc in E. coli, and it is now widely considered essential for UDP-GlcNAc synthesis across bacteria. Here we report that the clinically important pathogen Staphylococcus aureus relies on a previously uncharacterized uridyltransferase called NagU to synthesize most of its UDP-GlcNAc for cell wall synthesis. We also report that NagU binds to and negatively regulates DacA, the diadenylate cyclase that makes the essential second messenger cyclic di-AMP, which regulates turgor pressure. We propose a model in which increasing NagU expression simultaneously increases flux into peptidoglycan and increases turgor pressure to enable cell wall expansion. Finally, an evolutionary analysis shows that many bacteria beyond Staphylococcaceae encode NagU homologues but lack GlmU either entirely or in part, suggesting that alternative UDP-GlcNAc biosynthesis pathways are not rare.

Antimicrobial resistance (AMR) is a health threat disproportionately affecting nursing home (NH) residents. Surveillance and infection control in NHs are restricted to nares or perirectal cultures, overlooking skin colonization and multidrug-resistant organisms (MDROs) not recovered by selective media. Here, within the PROTECT trial NCT03118232, we show, that NH residents’ skin serves as a reservoir of transmissible MDROs. We analyzed 207 groin and axilla swabs from 38 residents across 15 California NHs using metagenomics, culturing, and genome sequencing. Culture detected MDROs in 10 of 38 residents (26.3%), including 4 (10.5%) with ESBL-producing Escherichia coli sequence type (ST)131/ST648 and 7 (18.4%) with methicillin-resistant Staphylococcus aureus . Skin microbiome analysis by metagenome-assembled genomes identified broader MDRO colonization, including 27 (71.1%) with E. coli ST93, 14 (36.8%) with Staphylococcus epidermidis ST2, 16 (42.1%) with Proteus mirabilis , 7 (18.4%) with Providencia stuartii , 7 (18.4%) with Enterococcus faecalis , and 5 (13.2%) with Pseudomonas aeruginosa . Colonization persisted after bathing. Clonal E. coli ST93 was shared by 27 residents across 9 facilities, and 5 resident pairs carried clonally related strains of ≥2 MDRO species, suggesting polymicrobial transmission. We confirmed skin as a reservoir of MDROs, utilizing metagenomics to detect colonization and transmission pathways, supporting AMR surveillance in long-term care.

This response addresses the comments raised by Dr. Epain and colleagues in their Letter to the Editor titled ‘On using clustering statistics for assessing plasmid binning tools accuracy’ in response to our paper ‘Circling in on plasmids: benchmarking plasmid detection and reconstruction tools for short-read data from diverse species’. In their letter, the authors caution against using homogeneity and completeness measures to evaluate the accuracy of plasmid binning tools due to issues related to the length and content of contigs. They also refer to PlasEval, a plasmid binning evaluation tool that they recently developed. In response, we evaluated the impact of contig size and content on the results of our study, and also repeated the benchmarking of plasmid reconstruction tools using PlasEval. Though the absolute values of metrics differed across tests, we observed nearly identical rankings among plasmid reconstruction tools as originally reported in our study, with gplas2 outperforming all other tools. Nevertheless, approaches specifically designed for plasmid data may be better suited than clustering metrics to evaluate plasmid reconstruction.

There are profound sex differences in the prevalence and outcomes of urinary tract infections (UTI). While females comprise the majority of infections, males exhibit higher morbidity and mortality with upper-tract UTI. Correspondingly, preclinical modeling has demonstrated that male and androgen-exposed female mice are highly susceptible to severe high-titer pyelonephritis, a phenotype observed in < 20% of females. Here we subject kidneys from female, male, and androgen-exposed female C3H/HeN mice with pyelonephritis and PBS-exposed control mice to single-nucleus RNA sequencing, creating (to our knowledge) the first whole-kidney single-nucleus transcriptomic dataset reflecting an infected state, comprising 248,483 nuclei. We differentiate healthy cell populations from those affected during UTI and show sex-discrepant responses that extend to kidney cell types beyond those directly interacting with bacteria. Female responses to UTI comprise a more limited range of cell types exhibiting significant upregulation of genes within KEGG pathways and pro-inflammatory transcription factor regulons. Meanwhile, males evidence predisposition to injury pathways even with control (saline) inoculation and responded to UTI with less intensity but across more cell types than females. In total, these data illuminate sex-discrepant transcriptional responses and outcomes in renal infection and enable detailed dissection of these responses at the cellular and molecular level.

In response to the comparatively sudden application of industrial scale levels of antibiotics to an ecosystem where naturally produced antibiotics are scarce, namely the ecologies within and around agricultural settings, animal-associated microbes have had to rapidly adapt, mostly relying on mobile genetic elements (MGEs) taken up due to loss of CRISPR functionality. Due to selection for resistance and other adaptive traits carried by dynamic and rapidly recombining MGEs, plasmids and transposons have rapidly accumulated in this human-proximal environment. Because of the occurrence of Enterococcus faecalis in a wide range of hosts up and down the food chain, and the fact that this species represents the greatest generalist of the genus, we comprehensively examined the mobilome of multidrug-resistant E. faecalis (ST330, ST591, ST710, and ST711) from healthy piglets raised on dispersed Brazilian farms, using long-read sequencing, analysis of plasmid pangenomes, and conjugation assays with these strains serving as donors. Genomes ranged from ∼2.8–3.1 Mb, with diverse MGEs constituting ∼7–15% of those genomes. Large modular antimicrobial resistance-encoding gene blocks (∼40 Kb) were observed to be integrated into a ∼67 Kb chromosomal segment of the pathogenicity island AF454824. Prophages contributed up to 70% of the chromosomal mobile element content, integrating into both CRISPR-deficient genomes and those with intact type II-A CRISPR1 arrays, which were enriched with Caudoviricetes phage-targeting spacers across all strains. Plasmid content showed pronounced mosaicism driven by diverse insertion sequences, transposons, and related mobile elements, many directly implicated in AMR gene cluster acquisitions. RepA_N, Inc18, and Rep3 plasmids, mostly conjugative, also carried various persistence-related traits, suggesting they may actively enhance agricultural fitness rather than passively accumulate due to loss of CRISPR protection.

Bacterial infections are a major cause of morbidity and mortality among children under five in low- and middle-income countries (LMICs). Children in LMICs are exposed to and colonized by a range of pathogenic bacteria, yet patterns of bacterial exchange between humans are not well known, in part because culturing and sequencing single bacterial isolates is labor-intensive. Here, we apply a machine learning strain tracking approach to metagenomic data from 511 stool samples from children and mothers across urban and rural Kenyan communities to characterize bacterial dissemination and assess if community-wide water chlorination disrupts transmission. We identified distinct strain-sharing dynamics across species; potentially pathogenic taxa (e.g., Escherichia , Enterococcus , Campylobacter ) exhibited distance-dependent dissemination driven by young children, while commensal taxa (e.g., Bifidobacterium , Bacteroides ) showed patterns consistent with dietary exposure. Drinking water chlorination reduced community-level strain-sharing in rural communities. Our study provides the first strain-level insights into multi-species bacterial transmission dynamics in LMIC communities, identifying distinct dissemination pathways for facultative versus mostly anaerobic bacteria. Moreover, our findings highlight the utility of metagenomic strain tracking to uncover how community spread can be disrupted.