Antimicrobial resistance (AMR) is a global public health concern, and wastewater-impacted aquatic environments are recognized as reservoirs and dissemination pathways for antimicrobial-resistant bacteria (ARB) and genes (ARGs). However, harmonized environmental AMR surveillance frameworks that integrate culture-based ARB enumeration with molecular ARG monitoring across wastewater and receiving surface waters remain limited. This pilot study, conducted in Ireland as part of a European harmonized monitoring initiative, assessed ARB and ARG abundances in wastewaters and surface waters. Wastewater treatment plant influent (n=3), effluent (n=3) and surface waters upstream and downstream of the discharge point (n=3 each) were sampled on three occasions in late 2024. Culture-based methods enumerated total and extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, while quantitative real-time PCR quantified 16S rRNA and five AMR-associated genes (intI1, ermB, aadA1, bla CTX-M-1 and vanA). Total E. coli concentrations were highest in influent (~105-106 c.f.u. dl-1), decreased in effluent (~103-105 c.f.u. dl-1) and lowest in surface waters (~10¹-10² c.f.u. dl-1). ESBL-producing E. coli were consistently detected in influent (~104 c.f.u. dl-1) and effluent (~101-103 c.f.u. dl-1) but were not recovered from seawater. ARG abundances were highest in influent, reaching up to ~1011 copies dl-1 for intl1, remained elevated in effluent (up to ~1010 copies dl-1) and were ~2-3 orders of magnitude lower in surface waters relative to effluent. Downstream seawater exhibited higher ARG levels than upstream freshwater despite low culturable E. coli. Peak ARG concentrations in effluent and surface waters were observed following a period of heavy rainfall; however, the limited number of sampling events precluded assessment of any statistical association. These findings highlight the impact of wastewater discharges on environmental AMR dissemination and suggest that faecal indicator-based monitoring may underestimate emerging risks, supporting integration of AMR indicators into EU water quality frameworks.
Bacteria live in dense communities where competition influences the composition and, therefore, the function of these communities. Beyond competing for resources, bacteria engage in antagonism by deploying a range of molecular weapon systems to inhibit and kill other bacteria. Investing in antagonism is expected to incur a fitness trade-off, but the nature of this trade-off at the level of molecular physiology remains underexplained. Applying recent advances about the physiological constraints faced by bacterial cells may help us better understand existing studies and design new investigations into interbacterial antagonism. Bacterial cells face two important constraints: a finite amount of protein and a maximum translation speed for ribosomes. As a result, the only way for a cell to grow faster is to allocate more of its finite proteome to synthesizing ribosomes. A cell choosing to attack competitors must therefore allocate some of its limited proteome budget to antagonistic proteins instead of other functions. Conversely, being attacked and resisting the effects of such attacks also require an investment of proteomic resources. The extent to which proteome allocation constraints influence bacterial physiology is not fully understood; consequently, how these constraints influence interbacterial antagonism has not been investigated. Here, I will discuss how proteome allocation constraints can re-contextualize our existing understanding of the costs of both deploying and resisting attacks and how investigation of these constraints may further our understanding of interbacterial antagonism.
Increased intestinal colonization by adherent-invasive Escherichia coli (AIEC) has been implicated in Crohn's disease, an inflammatory bowel disease characterized by abnormal immune responses and chronic inflammation in the gastrointestinal tract. LPS-Toll-like receptor 4 signalling may contribute to AIEC-associated intestinal inflammation. Here, we show that disruption of rfaG-dependent cell envelope biogenesis is associated with reduced type 1 fimbrial expression, epithelial invasion and intestinal colonization in the AIEC strain LF82. Deletion of rfaG, a gene involved in cell envelope biogenesis, resulted in reduced type 1 fimbrial expression and activity, accompanied by reduced transcription of fim genes. The rfaG mutant also exhibited impaired invasion of epithelial cells compared with the parental LF82 strain. Finally, the rfaG mutant showed reduced competitive colonization in the murine intestinal tract, whereas chromosomal complementation with rfaG restored this colonization defect. Inducible expression of type 1 fimbriae also partially reversed the reduced colonization at the early stage of infection, raising the possibility that additional rfaG-dependent mechanisms, independently of type 1 fimbrial expression, contribute to intestinal colonization. These findings suggest that rfaG-dependent cell envelope biogenesis contributes to the coordinated expression of surface-associated virulence factors associated with intestinal colonization in AIEC.
Group B Streptococcus (GBS) is a leading cause of neonatal sepsis and meningitis worldwide. Capsular polysaccharide (CPS) is a major virulence factor that aids GBS in colonizing the gastrointestinal (GI) and vaginal tracts, surviving in whole blood and evading the immune system. CPS is also the basis for a GBS CPS-protein conjugate vaccine candidate. In addition to its function on the surface of the bacteria, CPS can also be released and shed into the environment. In Streptococcus pneumoniae , shed CPS has been shown to absorb antimicrobial peptides and CPS-specific antibodies, allowing the bacteria to evade the innate immune system and potentially limiting vaccine protection. In this work, we show that CPS shedding occurs in ~75% of GBS isolates that we screened from a large library of clinical strains. We then identified ypmS as a genetic determinant of CPS shedding using an indexed transposon library. We used a clean in-frame deletion mutant of ypmS to assess the importance of shedding in GBS colonization and immune evasion. We explored the role of ypmS in colonization using murine models of GI and vaginal co-colonization and showed that the wild-type strain outcompeted the Δ ypmS mutant in both scenarios. We also assessed the effect of shed capsule on opsonophagocytosis. Free CPS was protective against bacterial killing in an opsonophagocytic killing assay using CPS-specific antibodies. With this work, we have identified a novel genetic cause of CPS shedding and shown its contribution to GBS colonization fitness and immune evasion.
For the clinical treatment of infections with Enterobacteriaceae , the development of resistance to last-resort antimicrobials like colistin is of concern, as it limits treatment options. Since 2015, ten families of mobile colistin resistance ( mcr ) genes have been discovered; however, our understanding of MCR remains limited because (i) mcr-1 is the most studied variant, (ii) mcr variants are rarely compared and (iii) mcr variants are primarily studied in Escherichia coli , even though studies have shown that mcr variants can confer significantly different phenotypes and that lipid A (i.e. the target of colistin and mcr- modification) structurally differs between bacterial species. To fill this gap, we examined how mcr-3 and mcr-9 , as two less frequently studied mcr variants, impact the colistin resistance of laboratory-adapted and real-world (i.e. clinical or food) isolates of E. coli , Salmonella enterica and Klebsiella pneumoniae . We found that while mcr-3 consistently conferred colistin resistance to all tested strains, mcr-9 only conferred resistance to two strains. The fold-change in colistin minimum inhibitory concentrations was significantly impacted by both the bacterial species and the mcr variant but not by whether a strain was a laboratory-adapted or real-world isolate. Overall, our results suggest that while laboratory-adapted strains may provide a good estimate of mcr -mediated colistin resistance of real-world isolates, findings of mcr -mediated phenotypes in one bacterial species should not be extrapolated to another.
Acidic, metal-contaminated environments harbour specialized microbial consortia adapted to extreme stress. We examined an environmental Euglena mutabilis culture naturally associated with Talaromyces and Acidiphilium acidophilum and exposed it to cadmium (Cd). Integrated transcriptomic, hormonal, structural and taxonomic analyses revealed a coordinated Cd-tolerance strategy. RNA sequencing showed differential regulation of metal transporters consistent with a shift from Cd uptake to intracellular sequestration. Transmission electron microscopy confirmed Cd compartmentalization within chloroplasts and increased paramylon granules. Cd exposure suppressed light-harvesting complex genes and formate/nitrite transporters while maintaining core photosynthetic function. Hormone profiling indicated strong repression of bioactive auxin and cytokinin (CK) free bases, alongside accumulation of CK nucleotides and downregulation of CK biosynthetic and activation genes. Metagenomics revealed Cd-driven enrichment of Talaromyces and Acidiphilium, implicating them in detoxification and stress support. Together, these responses highlight early Cd uptake followed by chloroplast-based detoxification, metabolic buffering via paramylon, hormonal downregulation of growth and community-mediated resilience.
The type VI secretion system (T6SS) is a molecular nanomachine that provides Gram-negative bacteria with competitive advantages during host colonization and interbacterial interactions. Citrobacter rodentium, a mouse-specific attaching and effacing pathogen, employs a T6SS encoded by the cts1 locus to outcompete the gut microbiota and establish infection. However, under standard laboratory growth conditions, cts1 expression remains silent and the regulatory mechanisms governing its activation are unknown. Here, we investigated the regulation of cts1, which is organized into two divergent gene clusters, cts1L and cts1R. Transcriptional fusions of the cts1L-cts1R intergenic region in both orientations were inactive in wild-type C. rodentium, but became active in the absence of the global regulator H-NS. Systematic deletion and site-directed mutagenesis of this region identified four promoter regions, one driving cts1L transcription and three directing cts1R expression (P1, P2 and P3). Additional promoters were identified within the cts1R cluster. All cts1 promoters were differentially repressed by H-NS. Moreover, expression of the response regulator CpxR from a low-copy plasmid enhanced promoter activity in the Δhns background. Electrophoretic mobility shift assays showed that H-NS binds the cts1 regulatory region directly, whereas phosphorylated CpxR bound the internal cts1R promoters but not the intergenic P1-P3 regions, suggesting that CpxR activates the latter indirectly. Together, these findings demonstrate that cts1 expression is controlled by a complex hierarchical regulatory network in which H-NS acts as the primary repressor and CpxR contributes to activation only once H-NS-mediated repression is relieved, in a context-dependent manner. This multifactorial regulation likely involves additional, yet-unidentified factors that fine-tune cts1 expression in response to environmental cues.
Industrial adoption of microbial cell-free protein synthesis (CFPS), an in vitro protein production platform that uses transcription and translation machinery from lysed cells, remains limited despite sustained technical progress across the field. This perspective positions microbial CFPS within a manufacturing context, focusing on its current capabilities, the limits of those capabilities and the concrete steps required for industrial adoption. In CFPS, the absence of cell growth constraints supports rapid protein production, simplified upstream workflows and direct control over reaction conditions, while also lending itself to formats such as lyophilized reagents for storage and transport. Beyond basic protein expression, microbial CFPS is increasingly used as an adaptable production environment in which reaction composition can be tuned, accessory enzymatic functions can be introduced and post-translational modification strategies can be engineered to better align with product requirements. The expanding range of microbial chassis further broadens the design space, allowing platform choice to be guided by functional, regulatory and deployment considerations rather than yield alone. However, increased capability has not yet translated into routine manufacturing use, and persistent barriers include reproducibility, vulnerable supply chains, limited scale-up practice, downstream purification suitable for therapeutic products and the absence of CFPS-specific quality and regulatory expectations. Overall, microbial CFPS is best evaluated not as a replacement for existing biomanufacturing but as a complementary manufacturing paradigm whose industrial relevance will depend on establishing standardization, economics and regulatory readiness.
The type III secretion system (T3SS) is a virulence mechanism commonly used by Gram-negative bacterial pathogens to deliver virulence proteins, known as effectors, into infected cells. The T3SS secretes a range of different substrates: first the needle subunits, then the translocon pore components and finally a pathogen-specific range of effector proteins. Each of these classes of substrates interacts with a corresponding class of bacterial chaperones, which are required for their efficient secretion. The requirement for these chaperones has been attributed to multiple functions, including preventing premature substrate activity, maintaining substrate stability in the bacterial cytoplasm and mediating substrate targeting and secretion hierarchy. Here, we bring together what is known about the function of T3SS chaperones in a range of different bacterial pathogens. Through analysis of the conservation of chaperone sequence and structure, we discuss how these proteins interact with and support the secretion of diverse substrates. Finally, we evaluate the extent to which chaperones are universally required for effector secretion.
Antibiotic resistance and the microbiome are two of the most prominent and highly active research areas currently in microbiology. However, these studies are commonly siloed. Research into antibiotic resistance often takes a highly pathogenic-centric view, and microbiome studies typically assess changes in community composition at the genus or species level, rather than at the level of small changes in bacterial genotype that often underpin rapid and significant changes in antibiotic resistance. One of the major mechanisms of antibiotic resistance evolution is via the acquisition of de novo resistance mutations, spontaneous mutations that occur randomly and provide a selective advantage in the presence of antibiotics. In this perspective, we address how interactions within the microbiome can shape the emergence and spread of de novo resistance mutations. We outline existing theoretical and empirical support for how microbial interactions have the potential to influence (i) the probability of de novo resistance mutations emerging, (ii) the fitness costs associated with new resistance mutations and (iii) the long-term selection against resistance and the ability of resistant mutants to transmit to new sites. Existing evolutionary theory may help us predict how microbial interactions will impact the probability of resistance mutations emerging, through understanding how microbial communities will impact pathogen population size, mutation rates and the supply of genetic variation. While a number of these links are simple and intuitive, there is a need for empirical data to understand how the complexity of interactions that exist within a microbial community at any one time come together to shape the evolution of antibiotic resistance. Future research in this field has the potential to inform the development of novel strategies to combat antibiotic resistance based on manipulating microbial interactions.
Respiratory bacterial infections remain a major cause of morbidity and mortality worldwide, and their treatment is increasingly complicated by antimicrobial resistance. Mānuka honey has attracted interest as a potential alternative antimicrobial agent, although the extent to which antibacterial activity varies with Unique Mānuka Factor (UMF™) grade and the relative contribution of methylglyoxal (MGO) remain unclear. In this study, the antimicrobial activity of five UMF™ grades of Mānuka honey (5+, 10+, 12+, 15+ and 20+) was evaluated against clinically relevant respiratory pathogens: methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Klebsiella pneumoniae and Pseudomonas aeruginosa . MICs were determined using broth microdilution assays. To assess the contribution of osmotic stress, bacterial growth responses were compared with a sugar solution matched to the carbohydrate composition of honey. In addition, MGO-matched control solutions were prepared to examine the contribution of MGO to antimicrobial activity. All Mānuka honey samples inhibited bacterial growth, and MIC values decreased with increasing UMF™ grade. MSSA and MRSA were the most susceptible organisms, whereas higher concentrations were required to inhibit the Gram-negative species. Mānuka honey consistently inhibited bacterial growth more strongly than the matched sugar control, indicating that osmotic effects alone do not explain its antimicrobial activity. Although MGO-matched solutions exhibited antibacterial activity, they were generally less potent than the corresponding whole honey samples. These findings demonstrate that Mānuka honey exhibits UMF-dependent antimicrobial activity against respiratory pathogens and suggest that its antibacterial effects arise from the combined action of MGO and additional components within the honey matrix
Mathematical models are increasingly used to infer traits, interactions and functional dynamics of microbial systems. One common example is a rate-based ordinary differential equation model parameterized with microbial traits. However, fitting such models with associated parameters to data requires a principled approach to extract information from time series while accounting for prior knowledge and measurement noise. These principles often remain implicit and not necessarily well defined. Here, we make the implicit, explicit: introducing Bayesian inference of ecological models for microbial time series, including three detailed case studies of algal population dynamics that follow a birth-death process. Complementing this primer, we provide an online tutorial on Bayesian inverse modelling with cross-programming language support via Python (PyMC) and Julia (Turing). By connecting theory, code, data and a series of hands-on educational modules, this primer aims to bring the utility of Bayesian learning to the broader microbial ecology research community.
Streptomyces are filamentous, spore-forming members of the Actinomycetota, renowned for their capacity to produce chemically diverse, specialized metabolites with medically important properties. Traditionally, Streptomyces have been viewed as soil-dwelling microbes, and their roles in soil ecology, plant health and plant disease have been extensively studied. However, advances in metagenomic sequencing and molecular approaches have greatly expanded our ability to investigate interkingdom interactions between Streptomyces and more complex organisms, including animals. In recent years, a growing body of work has revealed diverse and often intimate associations between Streptomyces and members of the Animalia. These include interactions with microfauna such as nematodes (Nematoda), insects (Insecta), including bees and ants, mammals such as bats (Chiroptera) and humans (Homo sapiens). This review consolidates our current knowledge of Streptomyces - animal interactions, with a particular focus on chemical ecology and the roles of specialized metabolites in shaping these relationships. This work highlights the emerging body of work investigating the role of Streptomyces ecology beyond soil ecosystems and draws attention to the importance of exploring non-traditional niches, including animal-associated microbiomes, to deepen our understanding of microbial-animal interactions and to expand opportunities for natural product discovery.
Rhizobacteria play a central role in supporting plant growth, contributing to nutrient acquisition, stress tolerance and disease suppression. Harnessing and improving rhizosphere microbial communities therefore represents a promising avenue towards more sustainable agriculture. Recent advances in microbiome ecology and synthetic biology have enabled the rational design of microbial consortia. Synthetic communities are widely used as tractable models to study ecological interactions and are increasingly explored as biofertilizers and biocontrol agents. Here, we define engineered microbial communities (EngComs) as microbial consortia augmented with strains carrying synthetic genetic circuits. These systems extend SynCom approaches by enabling programmable functions, such as intercellular communication, division of labour, biosensing and controlled nutrient mobilization, ultimately improving functional stability in complex environments. Beyond bacteria-bacteria interactions, we highlight emerging strategies to engineer plant-microbe interfaces through synthetic signalling pathways and multi-input genetic circuits that enable context-dependent responses. Despite this progress, the engineering of rhizobacteria for real soil environments remains at an early stage. Most systems are still characterized in simplified or artificial conditions, and key challenges persist, including environmental complexity, genetic stability, biocontainment and regulatory constraints. Addressing these limitations will be essential to translate engineered functions from laboratory settings to the field. Overall, continued integration of synthetic biology with ecological and biophysical understanding of the rhizosphere will pave the way for programmable plant-microbe systems, offering new opportunities to enhance crop productivity while reducing environmental impact.
Antibiotic resistance of Gram-positive bacteria poses a major clinical challenge. Daptomycin, a lipopeptide antibiotic, is a treatment option for drug-resistant organisms. However, there have been clinical reports of daptomycin resistance and in vitro reports of daptomycin tolerance that can result in treatment failure. Daptomycin possesses a fatty acid tail that inserts into the membrane, and membrane fatty acid content can influence its binding. Given that, we examined the potential of fatty acids to enhance daptomycin activity against enterococcal strains. We demonstrate that the saturated fatty acids myristic and palmitic acids increase bacterial susceptibility to the antibiotic, even for a daptomycin-resistant clinical isolate. These effects were highly specific with no impact on daptomycin susceptibility of Staphylococcus aureus. Growth of enterococci with myristic or palmitic acid reduced membrane fluidity, potentially promoting tighter daptomycin insertion. However, we noted no difference in membrane permeability for cells treated with fatty acids and daptomycin. While cells exposed to saturated fatty acids experience greater membrane depolarization, the lack of consistent, significant time-dependent changes upon daptomycin treatment suggests that depolarization alone does not fully explain the increased sensitivity. A strain that is unable to incorporate exogenous fatty acids into phospholipids was insensitive to the effects of saturated fatty acids on its susceptibility to daptomycin, highlighting the necessity of fatty acid incorporation onto lipid headgroups for potentiation. Overall, our data suggest that the incorporation of specific saturated fatty acids within the enterococcal membrane likely disrupts lipid composition, membrane organization and cellular envelope homeostasis, ultimately contributing to enhanced daptomycin susceptibility.
Antibiotic resistance remains one of the most pressing challenges in modern microbiology. Decades of research have focused on identifying resistance genes and molecular mechanisms in opportunistic pathogens. Yet bacterial survival under antibiotic stress is often shaped by broader physiological processes that are not always considered within the conventional frameworks associated with antibiotic resistance. In this perspective, I argue that bacterial metabolism plays a broader and more integrative role in shaping antibiotic resistance than is often appreciated. I discuss how metabolic networks and core housekeeping genes may serve as underlying drivers of bacterial survival and adaptation during antibiotic exposure. Integrating metabolic context into resistance research may reveal new evolutionary insights and highlight previously unrecognized vulnerabilities in bacterial physiology. Indeed, viewing antibiotic resistance through the lens of metabolism may ultimately help guide the development of more effective, 'metabolism-aware' antimicrobial strategies.
Antimicrobial resistance is an increasing threat to global health. However, there is a limited set of antibiotics that are effective against drug-resistant Gram-negative bacteria like Pseudomonas aeruginosa. One strategy to enhance the efficacy and longevity of existing antibiotics is by combining them with non-traditional antimicrobial adjuvants. Here, we examined if the host-derived antimicrobial lipid sphingosine could enhance the efficacy of a panel of antibiotics against P. aeruginosa in vitro. We found that sphingosine displayed strong synergy with the polymyxin antibiotics, polymyxin B and colistin, to inhibit growth of and kill P. aeruginosa but did not significantly alter the efficacy of other tested antibiotic classes. The addition of sphingosine reduced the MIC of polymyxin B and colistin from 0.5 to 0.031 µg ml-1 and 8 to 0.5 µg ml-1, respectively. This combination of sphingosine and polymyxin B synergized to inhibit the growth and survival of Klebsiella pneumoniae, as well as clinically isolated of P. aeruginosa. In addition to sphingosine, we found that the sphingoid bases sphinganine (dihydrosphingosine) and phytosphingosine also enhanced the activity of polymyxins. Overall, these findings demonstrate that sphingosine is a potent adjuvant for polymyxins and that the sphingosine-polymyxin combination is capable of killing P. aeruginosa and K. pneumoniae while using relatively low concentrations of polymyxin. This study may help in the development of new antimicrobial therapies for the treatment of Gram-negative bacterial infections.
The initiation reaction of fatty acid synthesis in Enterococcus faecalis is catalysed by the FabH 3-ketoacyl-acyl carrier protein (ACP) synthase III. Deletion of the fabH gene seriously impaired growth but failed to fully block de novo fatty acid synthesis. We report that exogenous medium-chain unsaturated fatty acids restore normal growth of a ∆fabH strain and upon elongation provide substrates for synthesis of functional membrane phospholipid bilayers. Entry of these acids into the elongation pathway was aided by increased expression of the AcpA ACP of fatty acid synthesis and/or of the phosphate: ACP acyltransferase PlsX. The efficacy of incorporation of these acids increased with increasing chain length. Coordinate overexpression of E. faecalis AcpA and PlsX or expression of the Lactococcus lactis PlsX allowed elongation of the short-chain octanoic acid to provide unsaturated fatty acids for growth of the ∆fabH strain. We also tested the abilities of the two long-chain β-ketoacyl-ACP synthases, FabO and FabF, in elongation of exogenous unsaturated fatty acids.
Vibrio pectenicida is a marine Gram-negative, facultatively anaerobic, rod-shaped bacterium that exists in coastal waters. First described in 1998, these bacteria are associated with deadly disease outbreaks including sea star wasting disease (SSWD) and high mortality in larval scallops in hatcheries. Multiple strain types exist, and V. pectenicida A365 is associated with mortality in scallop larvae due to vibrio haemocyte-killer toxin. V. pectenicida strain FHCF-3 is associated with SSWD which causes sea stars to 'melt' to death. Current research efforts focus on isolating potential new strains to determine if they are causing disease and how this bacterium may influence other marine invertebrates.
