Abstract Conjugative plasmids drive bacterial evolution and niche adaptation, yet how their active acquisition reshapes host transcription remains poorly understood. Most studies focus on stable plasmid carriage, overlooking the dynamic transcriptional changes during conjugation itself. Here, we investigate the active conjugation of RP4, a prototypical broad-host range plasmid, to E. coli , and K. pneumoniae recipients. We observe that an immediate, host- and surface factor-dependent transcriptional response occurs. This includes activation of non-SOS stress pathways, motility, exopolysaccharide production, anaerobic respiration, and metabolic adaptation. These responses do not inhibit conjugation, suggesting they serve to maintain host homeostasis, particularly of envelope encoded functions. Capsule expression prevents these responses and inhibits RP4 activation by physically blocking donor-recipient contact. Unexpectedly, RP4 can bypass the capsular barrier through opportunistic transfer into a sub-population of phenotypically thin-capsulated cells. These findings show how RP4, the hosts species, and surface architecture shape the active conjugation transcriptional landscape, which may have implications for plasmid dissemination and bacterial evolution.

The colonic epithelium is organized in functional regions and interacts with an abundant microbial community. In Cell, Rispal et al. discover that regionalization depends on microbes, with proximal identity regulated by microbial nicotinic acid-induced PPARα activation in the epithelium. Changes in tissue identity reshape zones of tissue injury.

Clostridium perfringens is a Gram-positive anaerobe responsible for a wide spectrum of diseases in humans and animals, driven by an expanding arsenal of toxins. Notably, many of these virulence factors are encoded on mobile genetic elements, particularly conjugative plasmids, positioning mobile DNA at the centre of C. perfringens pathogenicity. Over the past decade, the pace of discovery has accelerated, with at least 11 new putative toxin-encoding genes identified, revealing an increasingly complex and dynamic toxin landscape. Here, we review advances in our understanding of toxin diversity and plasmid biology in C. perfringens, highlighting the remarkable plasticity of its mobile genome. We discuss how plasmid-encoded virulence, horizontal gene transfer and the emergence of novel plasmid families are reshaping current models of pathogenesis and evolution. We also consider key gaps in knowledge, including plasmid function, mobility and ecological context, and outline priorities for future genomic and functional studies. Together, these advances position C. perfringens as a compelling model to understand how mobile genetic elements drive virulence, adaptation and disease in bacterial pathogens.

BACKGROUND:Recent European Society of Clinical Microbiology and Infection and Australasian Society of Infectious Diseases Clostridioides difficile infection (CDI) management guidelines endorse the addition of intravenous tigecycline in patients who are progressing to fulminant infection. However, there are very limited data regarding clinical outcomes with this approach. AIMS:We aimed to evaluate mortality and adverse outcomes after tigecycline combination therapy was commenced at the time of fulminant CDI. METHODS:This retrospective single-site observational study included patients between January 2018 and December 2022 treated with adjunctive intravenous tigecycline for fulminant CDI in addition to oral vancomycin and intravenous metronidazole. The primary outcome was all-cause mortality at 30 days. Secondary outcomes included clinical cure, partial response, in-hospital CDI-attributable and 90-day all-cause mortality, colectomy and adverse events. RESULTS:Eighteen patient episodes occurred, with median age 67.5 years, requiring intensive care admission and inotropic support in 66.7% and 50% of episodes respectively. All-cause mortality at 30 days was 22.2%. Clinical cure occurred at 14 days in 16.7% of cases and partial response requiring further oral therapy in 55.6%. There was one colectomy and two serious adverse events, including a hyperkalaemic cardiac arrest in a haemodialysis patient and one significant intra-abdominal bleeding episode requiring surgery. CONCLUSIONS:Tigecycline combination therapy resulted in improved all-cause 30-day mortality in high-risk CDI patients versus historical data, without the use of faecal microbiota transplantation. Poor outcomes were seen in three patients with pre-existing renal failure, and we question the efficacy and safety of tigecycline in this group. This is a useful salvage strategy, but monitoring for toxicity is essential.

Soil-transmitted helminths are one of the most common infections globally, yet how to promote effective gut-associated humoral responses is not well understood. We identify the histone methyltransferase MLL1 as a key target to promote IgA-driven responses. Mll1 was increased in germinal center B cells in gut-associated lymphoid tissues, and Mll1-deficiency led to changes in the histone modification H3K4me3 on key B cell and immune-regulatory genes. Correspondingly, MLL1-deficient B cells had defective germinal centers and IgG1 in response to the helminth Trichuris muris. Yet Mll1f/fCd23cre/+ mice expelled worms more rapidly compared to control mice. Accelerated worm clearance correlated with elevated immunoglobulin A (IgA)+ plasma cells, as well as both serum and fecal IgA. RNA-sequencing identified CCR9 as a key MLL1-regulated molecule. As such, Mll1f/fCd23cre/+ mice infected with T. muris had increased IgA+CCR9+ PC localized in the large intestine. Regulation of IgA by MLL1 was confirmed beyond T. muris infection. In vitro cultures confirmed Mll1-deficiency increased IgA+ plasma cells in a B cell-intrinsic manner, and IgA production was also increased in Mll1f/fCd23cre/+ mice infected with the bacterium Citrobacter rodentium. This study reveals MLL1 as a key target to promote IgA responses to gut-associated infections.

Abstract Spore-forming bacteria produce two distinct cell types: vegetative cells and resilient spores. While antibiotic resistance is typically associated with vegetative cells, spores play a critical role in disseminating resistance genes due to their durability and transmissibility. We previously demonstrated that cephamycin antibiotics target the conserved spore-specific protein SpoVD, significantly reducing spore formation in pathogens including Clostridioides difficile . Here, we show that when C. difficile acquires CdmecA , a homologue of Staphylococcus aureus mecA , one of the most globally burdensome resistance genes, the anti-sporulation effect of cephamycins is bypassed. Cd MecA functionally replaces Cd SpoVD, restoring sporulation and producing phenotypically distinct spores. We further show that mecA is prevalent across C. difficile strains and other pathogenic, gut, and environmental spore-formers. Since SpoVD is conserved, MecA may broadly co-opt sporulation; we confirm this in Clostridium perfringens . This work reveals an unusual resistance mechanism with unexpected physiological consequences, reshaping our understanding of antibiotic resistance within the context of sporulation and microbial adaptation.

BACKGROUND:Damage to the enteric nervous system in murine Clostridioides difficile infection (CDI) has recently been postulated as an explanation for the paradoxical dysmotility, including ileus, observed in a disease generally characterized by diarrheal onset. However, the clinical spectrum and mortality implications of acute gastrointestinal dysmotility remain under-explored in human CDI. METHODS:We conducted the first retrospective cohort study of hospitalized adults (≥18 years) with CDI at a single tertiary health service in Australia between January 2018 and December 2021, defining clinical and radiologic features of gastrointestinal dysmotility at diagnosis, during admission, and in relation to the timing of diarrheal onset. Atypical presentations were defined as 1 or more CDI-associated symptoms persisting for >24 hours before diarrheal onset. Outcomes included time to diagnostic testing and directed therapy, 30- and 90-day mortality. Multivariable logistic and Cox regression models were used assessing independent risk factors associated with death. RESULTS:Among 467 CDI episodes, dysmotility occurred in 32% at diagnosis and persisted radiologically for up to 6 weeks. Atypical presentations (91/467 [19.5%] episodes) were associated with significantly delayed treatment (median, IQR 4 [1-9] versus 2 [0-8] days, P < .001) and increased 90-day all-cause mortality compared with typical presentations (24.2% versus 9.6%, HR 2.62 [1.51-4.57], P = .001). Small-bowel involvement was identified in 44/270 (16.3%) baseline imaging episodes. CONCLUSIONS:Both large- and small-bowel dysmotility occur frequently in hospitalized adults with CDI, contributing to diagnostic delays and increased mortality among patients with atypical presentations. These findings support extension of global CDI guidance addressing diagnostic pathways and empiric therapy.

Conjugative plasmids can drive the global spread of antimicrobial resistance (AMR) in Enterobacterales. Hypervirulent Klebsiella pneumoniae (hvKp) increasingly acquire AMR plasmids, raising concern about convergent hypervirulent drug-resistant clones. Yet little is known about plasmid transmission dynamics in hvKp. Using an antibiotic-perturbed murine gut model with hvKp and human commensal E. coli, we discover that broad-host range IncP plasmids belonging to different phylogenetic branches (clade I (PTU-P1) and clade II (PTU-P2)) transfer differentially in the gut, mirroring the higher prevalence of PTU-P2 plasmids in human-associated samples. Statistical modelling and experimental results show that secondary transfer by transconjugants sustains gut transmission without continuous donor input. Furthermore, the hvKp capsule exerts a modest effect on transfer in vivo compared to in vitro aerobic conditions. Under anaerobic conditions, hvKp capsule mucoviscosity is markedly reduced, and PTU-P2 plasmids conjugate more efficiently than PTU-P1 counterparts. Our findings reveal that the hypermucoviscous capsule may not substantially impede gene exchange in the gut where microenvironments shape plasmid transfer dynamics, highlighting the pitfalls of extrapolating in vitro data to relevant ecological niches. Our work also emphasizes the high-risk nature of gut-adapted PTU-P2 plasmids and the ease with which hvKp can acquire them, underscoring the need for continued surveillance.

In Australia, most mentoring programs are organization-based, rather than discipline-based. To address the need to support our early career microbiologists, we leveraged the professional membership of the Australian Society for Microbiology (ASM) to develop and evaluate the performance of a mentoring program. The ASM Mentoring Program (ASMMP) was a national 6-month program in which 33 mentor-mentee pairs were matched and met monthly from June to December 2023. Over 80% of respondents agreed ASMMP was a beneficial, rewarding experience and would recommend the program to a colleague. Mentees reported a sense of improved career planning, networking skills, and a better working relationship with their line manager/supervisor. Most mentors reported a sense of fulfilment from being able to support and guide their mentee. Overall, ASMMP was a manageable and cost-effective strategy for the ASM to support the professional development of early career members while addressing an unmet mentoring need.

Large-scale metagenomic and data-mining efforts have revealed an expansive diversity of bacteriophages (phages) within the human gut1-3. However, functional understanding of phage-host interactions within this complex environment is limited, largely due to a lack of cultured isolates available for experimental validation. Here we characterize 134 inducible prophages originating from 252 human gut bacterial isolates using 10 different induction conditions to expand the experimentally validated temperate phage-host pairs originating from the human gut. Importantly, only 18% of computationally predicted prophages could be induced in pure cultures. Moreover, we construct a 78-member synthetic microbiome that, when co-cultured in the presence of human colonic cells (Caco2), led to the induction of 35% phage species. Using cultured isolates, we demonstrate that human host-associated cellular products may act as induction agents, providing a possible link between gastrointestinal cell lysis and temperate phage populations4,5. We provide key insights into prophage diversity and genetics, including a genetic pathway for domestication, finding that polylysogeny was common and resulted in coordinated prophage induction, and that differential induction can be influenced by divergent prophage integration sites. More broadly, our study highlights the importance of culture-based techniques, alongside experimental validation, genomics and computational prediction, to understand the biology and function of temperate phages in the human gut microbiome. These culture-based approaches will enable applications across synthetic biology, biotechnology and microbiome fields.

Conjugative plasmids drive bacterial evolution and niche adaptation, yet how their active acquisition reshapes host transcription remains poorly understood. Most studies focus on stable plasmid carriage, overlooking the dynamic transcriptional changes during conjugation itself. Here, we show that active RP4 conjugation triggers an immediate, host- and surface factor-dependent transcriptional response. This includes activation of non-SOS stress pathways, motility, exopolysaccharide production, anaerobic respiration, and metabolic adaptation. These responses do not inhibit conjugation, suggesting they serve to maintain host homeostasis. Capsule expression blocks these responses by preventing conjugation, and also inhibits RP4 activation by physically blocking donor-recipient contact. Unexpectedly, RP4 overcomes this barrier by exploiting single-cell variation in recipient capsule thickness, successfully conjugating with thin-capsulated recipients. These findings reveal a striking interplay between plasmid, host, and surface architecture in shaping the conjugation transcriptional landscape, with broad implications for plasmid dissemination and bacterial evolution. ### Competing Interest Statement The authors have declared no competing interest. Australian Research Council, https://ror.org/05mmh0f86, DP250103521, FL210100258 National Medical Research Council, OFIRG20NOV-0045

OBJECTIVE:The IMP-4 carbapenemase is an endemic cause of carbapenem resistance in the Asia-Pacific region. Our aim was to determine the dissemination mechanism of the blaIMP-4 gene. METHODS:Twelve representative Australian IMP-4 clinical isolates from The Alfred Hospital (Victoria, Australia) were characterised using antimicrobial susceptibility testing, with their genome and plasmid assemblies analysed. The conjugation efficiencies of different plasmids were investigated using filter mating with four recipient strains across two species. RESULTS:Selected IMP-4 isolates included six species and four genera (Enterobacter, Klebsiella, Serratia, and Acinetobacter), whereby isolates of the same species belonged to the same sequence type and were closely related. Four IMP-4 plasmid types were noted: IncHI2A types 1 and 2 (Klebsiella spp. and Enterobacter hormaechei, respectively), IncC (Serratia marcescens and Klebsiella pneumoniae), and a novel type in Acinetobacter pittii. Sequence homology was observed across all plasmids at the blaIMP-4 location, termed Region I, with IS26 on IncHI2A, and IS5075 and Tn3 resistance gene cassettes present on IncC plasmids. Genomic rearrangements mediated by IS26 or Tn3 and IS5075 were identified in Region I of plasmids from the same Inc type. The plasmids of each Inc type were capable of conjugative transfer with varying efficiency. IncH12A plasmids and K. pneumoniae IncC displayed higher transfer efficiencies than other plasmids examined in this study when using the recipient E. coli strain J53 (with conjugation efficiencies of 1.17×10-2 to 5.02×10-5, P < 0.001). CONCLUSIONS:Clonal spread, Inc type, homologous region, and insertion sequences are important mobility factors in the dissemination and evolution of blaIMP-4 plasmids.

Disruption of hydrogen (H2) cycling in the gut is linked to gastrointestinal disorders, infections and cancers. However, the mechanisms and microorganisms controlling H2 production in the gut remain unresolved. Here we show that gut H2 production is primarily driven by the microbial group B [FeFe]-hydrogenase. Metagenomics and metatranscriptomics of stool and tissue biopsy samples show that hydrogenase-encoding genes are widely present and transcribed in gut bacteria. Assessment of 19 taxonomically diverse gut isolates revealed that the group B [FeFe]-hydrogenases produce large amounts of H2 gas and support fermentative growth of Bacteroidetes and Firmicutes. Further biochemical and spectroscopic characterization of purified enzymes show that they are catalytically active, bind a di-iron active site and reoxidize ferredoxin derived from the pyruvate:ferredoxin oxidoreductase reaction. Group B hydrogenase-encoding genes are significantly depleted in favour of other fermentative hydrogenases in patients with Crohn's disease. Finally, metabolically flexible respiratory bacteria may be the dominant hydrogenotrophs in the gut, rather than acetogens, methanogens and sulfate reducers. These results uncover the enzymes and microorganisms controlling H2 cycling in the healthy human gut.

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404. Identification of an N-acetylmuramoyl-l-alanine amidase in the ribotype 027 C. difficile strain M7404, involved in the active secretion of the large clostridial glucosylating toxins, TcdA and TcdB.

The rapid and accurate identification of pathogenic bacteria is crucial for combating the growing threat of antibiotic resistance, nosocomial infections, and food safety concerns. This study presents a novel and comprehensive comparison of two vibrational spectroscopic techniques - attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and a low-cost miniature near-infrared (NIR) spectrometer - for distinguishing Gram-positive and Gram-negative bacterial samples grown using the same stock media solution. This is the first report of NIR spectroscopy being applied to differentiate Gram-positive and Gram-negative bacteria, as well as the first direct comparison of ATR-FTIR and NIR for the combined multimodal analysis of clinical bacterial isolates. Using a data set of five Gram-positive and seven Gram-negative species and recording spectra in triplicate, the study employed advanced data fusion and multivariate analysis techniques to classify the spectra and facilitate NIR band assignment. 2D correlation analysis revealed strong positive correlations between key spectral markers identified in both modalities. Partial least-squares- and support vector machine discriminant analysis models were validated using a methodology based on 100 repeated random sampling of calibration and test sets. Models demonstrated that both the standalone ATR-FTIR and the combined ATR-FTIR/NIR approach achieved exceptional classification accuracy (>98%) in differentiating the two bacterial groups. Differences observed in the spectra were attributed to the distinct cell wall compositions of Gram-Positive and Gram-negative bacteria. Notably, the low-cost NIR technique also showed promising performance, with classification accuracy values above 90%. The findings highlight the potential of these rapid, noninvasive, and cost-effective vibrational spectroscopic techniques, particularly the NIR method, for point-of-care applications in clinical microbiology and food safety monitoring. The combination of ATR-FTIR and NIR data further enhances the robustness and reliability of bacterial identification, paving the way for broader adoption of these advanced analytical tools in various healthcare and food safety settings.

Molecular hydrogen (H2) is among the most central, but least understood, metabolites in the human gastrointestinal tract (gut). H2 gas is produced in large quantities during bacterial fermentation and consumed as an energy source by bacteria and archaea. Disruption of H2 cycling is linked to gastrointestinal disorders, infections, and cancers, with H2 used as an indicator of gut dysfunction through breath tests. Despite this, the microorganisms, pathways, and enzymes mediating H2 production remain unresolved. Here we show that a previously uncharacterised enzyme, the group B [FeFe]-hydrogenase, drives most fermentative H2 production in the human gut. Analysis of stool, biopsy, and isolate (meta)genomes and (meta)transcriptomes show this hydrogenase is encoded by most gut bacteria and is highly expressed. Through analysis of 19 taxonomically diverse gut isolates, the group B [FeFe]-hydrogenase produces large amounts of H2 gas and supports fermentative growth of both Bacteroidetes and Firmicutes. Bacteroides particularly dominate H2 production. Biochemical and spectroscopic characterisation shows purified group B [FeFe]-hydrogenases are catalytically active and bind a di-iron active site. These hydrogenases are highly enriched in the guts of healthy individuals, but significantly depleted in favour of other fermentative hydrogenases in Crohn's disease. Furthermore, we show that metabolically flexible respiratory bacteria are the most abundant H2 oxidizers in the gut, not sulfate reducers, methanogens, and acetogens as previously thought. This combination of enzymatic, cellular, and ecosystem-level analysis provides the first detailed understanding of H2 cycling in the human gut and reveals new links between microbiota function and gastrointestinal health. ### Competing Interest Statement The authors have declared no competing interest.

Pathogenic and antimicrobial-resistant (AMR) microorganisms are continually transmitted between human, animal, and environmental reservoirs, contributing to the high burden of infectious disease and driving the growing global AMR crisis. The sheer diversity of pathogens, AMR mechanisms, and transmission pathways connecting these reservoirs create the need for comprehensive cross-sectoral surveillance to effectively monitor risks. Current approaches are often siloed by discipline and sector, focusing independently on parts of the whole. Here we advocate that integrated surveillance approaches, developed through transdisciplinary cross-sector collaboration, are key to addressing the dual crises of infectious diseases and AMR. We first review the areas of need, challenges, and benefits of cross-sectoral surveillance, then summarise and evaluate the major detection methods already available to achieve this (culture, quantitative PCR, and metagenomic sequencing). Finally, we outline how cross-sectoral surveillance initiatives can be fostered at multiple scales of action, and present key considerations for implementation and the development of effective systems to manage and integrate this information for the benefit of multiple sectors. While methods and technologies are increasingly available and affordable for comprehensive pathogen and AMR surveillance across different reservoirs, it is imperative that systems are strengthened to effectively manage and integrate this information.

Apicomplexan infections, such as giardiasis and cryptosporidiosis, negatively impact a considerable proportion of human and commercial livestock populations. Despite this, the molecular mechanisms of disease, particularly the effect on the body beyond the gastrointestinal tract, are still poorly understood. To highlight host–parasite–microbiome biochemical interactions, we utilised integrated metabolomics-16S rRNA genomics and metabolomics–proteomics approaches in a C57BL/6J mouse model of giardiasis and compared these to Cryptosporidium and uropathogenic Escherichia coli (UPEC) infections. Comprehensive samples (faeces, blood, liver, and luminal contents from duodenum, jejunum, ileum, caecum and colon) were collected 10 days post infection and subjected to proteome and metabolome analysis by liquid and gas chromatography–mass spectrometry, respectively. Microbial populations in faeces and luminal washes were examined using 16S rRNA metagenomics. Proteome–metabolome analyses indicated that 12 and 16 key pathways were significantly altered in the gut and liver, respectively, during giardiasis with respect to other infections. Energy pathways including glycolysis and supporting pathways of glyoxylate and dicarboxylate metabolism, and the redox pathway of glutathione metabolism, were upregulated in small intestinal luminal contents and the liver during giardiasis. Metabolomics-16S rRNA genetics integration indicated that populations of three bacterial families—Autopobiaceae (Up), Desulfovibrionaceae (Up), and Akkermanasiaceae (Down)—were most significantly affected across the gut during giardiasis, causing upregulated glycolysis and short-chained fatty acid (SCFA) metabolism. In particular, the perturbed Akkermanasiaceae population seemed to cause oxidative stress responses along the gut–liver axis. Overall, the systems biology approach applied in this study highlighted that the effects of host–parasite–microbiome biochemical interactions extended beyond the gut ecosystem to the gut–liver axis. These findings form the first steps in a comprehensive comparison to ascertain the major molecular and biochemical contributors of host–parasite interactions and contribute towards the development of biomarker discovery and precision health solutions for apicomplexan infections.

With the aim of discovering small molecule inhibitors of the sporulation process in Clostridioides difficile, we prepared a series of C-7 α-(4-substituted-1H-1,2,3-triazol-1-yl)acetamide analogues of cefotetan, a known inhibitor of the C. difficile sporulation-specific protein target CdSpoVD. These analogues were evaluated using both in vitro binding assays with CdSpoVD and antisporulation assays against C. difficile. Further design concepts were aided utilizing the predicted docking scores (DS) using both AlphaFold (AF) models, and a crystal structure of the CdSpoVD protein (PDB 7RCZ). Despite being 1 order of magnitude more potent as a sporulation inhibitor than cefotetan, in vivo studies on compound 6a in a murine-model of C. difficile infection demonstrated comparable spore shedding capabilities as cefotetan. Importantly, compound 6a had no concerning broad spectrum antibacterial activities, toxicity, or hemolytic activity and thus has potential for further drug development.