The CRISPR-associated protein 9 (Cas9) produced by disease-associated strains of contributes to full virulence, including immune evasion and bacterial survival inside eukaryotic cells. In this work, we explored the role of Cas9 (CjeCas9) in cell envelope integrity, antibiotic resistance, intracellular survival inside Caco-2 intestinal epithelial cells and Toll-like receptor 2 (TLR-2) activation. We show that CjeCas9 modulates the permeability of the cell envelope, sialylated lipooligosaccharide expression and susceptibility to ciprofloxacin, the most commonly prescribed antibiotic to treat infections. Moreover, we reveal that WT production of CjeCas9 increased intracellular survival of inside Caco-2 intestinal epithelial cells by a factor of 550 compared to the respective gene deletion mutant and that intracellular survival was associated with the activation of TLR-2. In conclusion, we established that CjeCas9 modulates (intracellular) virulence traits, including intracellular survival.

To develop a low-cost, environmentally friendly taurine fermentation method for sustainable marine fish culture using feed derived from photosynthetically produced agricultural products, it is crucial to study cysteine sulfinic acid decarboxylase (CSAD), a key enzyme in the taurine biosynthetic pathway in applicable microorganisms. In this study, a method was devised to screen for CSAD genes using growth as an indicator, based on sulfur assimilation following the decarboxylation of l-cysteic acid, a taurine precursor compound. The used has a double deletion mutation of (sulfate/thiosulfate ABC transporter) and (FMNH-dependent alkanesulfonate monooxygenase) genes. If needed, an additional defect in enzyme genes, such as (adenylyl-sulfate kinase), which participates in the pathway reducing sulfate to sulfite, is also introduced. Using this method, it was demonstrated that the glutamic acid decarboxylase gene from possesses CSAD activity. The identified decarboxylase was further confirmed to act on l-cysteine sulfinic acid. Additionally, two observations made during method refinement to reduce background growth in screening are discussed: that SsuD is involved in sulfur assimilation from an unknown sulfur compound and that certain (mechanosensitive channel) missense mutations enable external sulfate above a specific concentration to enter the cell.

We determined the mechanism of resistance to seven chemical series with potent activity against . Resistant mutants were isolated against the aminothiazoles, phenylhydrazones, 8-hydroxyquinolines, nitazoxanides, phenyl alkylimidazoles, morpholino thiophenes and trifluoromethyl pyrimidinones. We demonstrated that mutations in several components of the Esx-3 type VII secretion system (EccA3, EccB3, EccC3 and EccD3) conferred resistance to these disparate scaffolds. We conclude that mutations in Esx-3 are a common mechanism of resistance to anti-tubercular agents, which may have clinical relevance for new drugs.

On 4 June 2025, the MHRA published 'Regulatory considerations for therapeutic use of bacteriophages in the UK'. This was in response to recommendations made by the House of Commons Science, Innovation and Technology Committee Inquiry into the 'The Antimicrobial Potential of Bacteriophages'. The MHRA Regulatory Considerations for phage therapeutic products (PTPs) outlines the relevant regulatory route and requirements to use PTPs as licensed or unlicensed medicines. While this guidance provides the necessary information, it is recognized that regulatory information can be inaccessible to academic and small- to medium-sized enterprise developers who are often unfamiliar with the language, terminology and location of such information. The MHRA, in consultation with the Innovate UK Phage Innovation Network, has therefore developed this interpretation to help PTP developers understand what the guidance is saying, and what evidence is required for regulatory assessment of a marketing authorization application. Examples have been included throughout to provide context and as an aid to understanding.

Artificial intelligence (AI) and machine learning (ML) are reshaping microbiology, enabling rapid antibiotic discovery, resistance prediction and clinical diagnostics. For microbiologists, the goal is not to build new algorithms but to recognize when ML is appropriate, how to prepare data and how to interpret outputs responsibly. This primer takes that practical stance - driving the ML car rather than rebuilding the engine. At a high level, ML learns from complex patterns, often noisy data. In antibiotic discovery, ML models help identify compounds in biological data and design new ones from scratch using generative AI. In microbiome studies, where measurements are compositional, sparse and often confounded, ML helps uncover community structure and link taxa or functions to phenotypes. In pathogen genomics, supervised models map sequence-derived features (e.g. k‑mers, SNPs and gene presence/absence) to outcomes such as species identity, antimicrobial susceptibility or MIC. Unsupervised learning supports exploration, including clustering, latent gradients and dimensionality reduction for visualization. Across these settings, success hinges less on exotic architectures than on sound problem framing, careful preprocessing and experimental validation.

Antimicrobial resistance is a massive threat, but developing a new antibiotic can take decades. That time could be drastically reduced if we were able to anticipate desirable properties of a chemical, such as its potential to target specific bacterial compartments. This would provide the opportunity to prioritize the development of molecules that target, for instance, the cell membrane, as this does not rely on transporters and usually results in a fast-acting bactericidal effect. We used flow cytometry and a set of fluorophores together with a group of antibiotics to discriminate between antimicrobials acting on cell membrane versus intracellularly against two Gram-negative bacteria, and . We then chose Rhodamine 123 as a fluorescent marker to screen a commercial library of chemical compounds. Using flow cytometry, several drugs present in the Prestwick library were observed to have cytotoxic effects at 1 µM final concentration towards . This was confirmed with growth inhibitory assays in both and for pantoprazole, theophylline and zoledronic acid. This represents an approach to the large-scale screening of small molecules with the potential to deliver fast-acting molecules that target cell membranes in Gram-negative bacteria.

is a white-rot wood-degrading basidiomycete of ecological, biotechnological and medicinal interest. In addition to its role in lignin degradation, it produces bioactive metabolites with reported antimicrobial and antioxidant activities. However, the mechanisms of iron acquisition, including siderophore-mediated pathways, remain poorly characterized in species. Improved understanding of these systems is essential to elucidate their contributions to fungal physiology, secondary metabolism and ecological adaptation. In this study, the genome of was sequenced for the first time and screened for genes that may be involved in the production of secondary metabolites. A gene cluster was identified as potentially involved in iron uptake. In particular, genes related to non-ribosomal peptide synthetases were detected next to a gene encoding a monooxygenase and indicated a potential hydroxamate-family siderophore. Liquid chromatography (LC)-ES-MS analysis of secondary metabolites secreted by into the growth medium under iron-limiting conditions revealed a group of previously undescribed siderophores. Genome and MS/MS analysis suggested that these structures might be related to the coprinoferrin family of siderophores. Aside from siderophores, the genome and LC-MS analysis revealed to be a prolific producer of a variety of triterpenoids and sesquiterpenoids, in agreement with previous findings. This is the first description of the genome sequence of and its siderophores.

Oligopeptide transporters are important proteins in several lactic acid bacteria (LAB) that facilitate the transport of oligopeptides, the primary nitrogen source for growth in milk. Although the proteolytic mechanisms are well understood in some LAB species, limited research has been conducted on the peptide transport systems of (formerly ) strain Shirota (LcS), particularly its oligopeptide transporters. This study investigated the nitrogen uptake mechanism of LcS, a probiotic lactic acid bacterium, by generating gene knockout (KO) strains of two oligopeptide transporters, Opp and Dpp. Consequently, the disruption of these genes eliminated the ability of the bacterium to grow in milk, identifying Opp and Dpp as the primary oligopeptide transporters in LcS. Growth in a leucine-free chemically defined medium with a Leu-containing peptide as the sole nitrogen source indicated that Opp and Dpp transport peptides of 4-8 and 3-7 residues, respectively. To our knowledge, this study provides the first experimental evidence of oligopeptide transporters in capable of transporting peptides up to eight residues long. Analysis of KO strains targeting OppA or DppA to identify other oligopeptide-binding proteins (OBPs) within each oligopeptide transporter operon that may influence substrate specificity revealed that OppA is the only OBPs in Opp. However, DppA and DppA, encoded at chromosomal locations distant from the Dpp operon, may function as subunits constituting Dpp and DppA. These findings enhance our understanding of nitrogen source utilization in lactobacilli and might inform future strategies to optimize nitrogen sources for LcS and improve culture technology for LcS-based products.

Phage therapy is a promising treatment for multidrug-resistant bacterial infections. Due to their high host specificity, phages must be matched to the target clinical strains. Efficiently identifying appropriate phages and producing sufficient titres for clinical use requires comprehensive phage libraries and multiple propagation hosts. An idealized system would use a highly promiscuous bacterial host to isolate a broader range of phages and streamline optimized phage production. Anti-phage defences constrain bacterial host promiscuity, such as restriction-modification systems that recognize and cleave foreign DNA. Here, the type I restriction endonuclease, HsdR, was deleted from PAO1 to make a more promiscuous phage isolation and propagation host. Removal of this endonuclease more than doubled the efficiency of phage propagation on solid media, improved yields from hard-to-propagate phages in liquid bulk-ups and yielded seven times more phages from freshwater samples than wild-type PAO1 - an important step in producing an optimized strain for isolating and propagating phages for clinical phage therapy.

Post-transcriptional regulation can be mediated by small regulatory RNAs (sRNAs) in bacteria, which can act by base-pairing to a target mRNA. The discovery and mechanistic validation of base-pairing sRNAs in multidrug-resistant has been hampered by the lack of genetic tools to assess RNA-RNA interactions. Here, we created two compatible plasmids for , which addresses this need. The newly designed plasmids validated the known Aar sRNA- mRNA and a new interaction of sRNA44 and the mRNA of the biofilm-associated protein Bap. The new plasmid system should accelerate the mechanistic characterization of sRNAs in .

Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.

Cellular proliferation relies on the successful coordination and completion of genome replication and segregation. To help achieve this, many bacteria utilize regulatory pathways that ensure DNA replication initiation only occurs once per cell cycle. When dysregulated, loss of DNA replication control can have severe consequences. In , it has been established that hyper-initiation of DNA synthesis leads to pleiotropic genome instability and cell death. Therefore, targeting DNA replication initiation proteins to promote hyper-initiation may be an approach to generate novel antimicrobials. However, the pathways and potential consequences of replication hyper-initiation in Gram-positive species remain enigmatic. To address this question, we devised genetic systems to artificially induce hyper-initiation in the model organism and the pathogen . In both species, hyper-initiation elicited cellular degeneration culminating in growth inhibition by cell death. During this process in , temporal analyses revealed the early onset of the DNA damage response, followed by membrane depolarization and cell lysis. This phenotype could be suppressed by removing pathways that repair damaged DNA, suggesting that cell death is a consequence of conflicts between DNA replication and repair. In cells quickly accumulated striking morphological changes associated with rapid loss of chromosomal DNA and death via a lysis-independent pathway. Moreover, inducing hyper-initiation in was observed to decrease bacterial survival during infection of murine macrophages. Taken together, the data suggest that stimulating initiation of bacterial DNA synthesis could be an alternative approach to inhibiting microbial growth, particularly in combination with compounds that inhibit or poison DNA repair, akin to cancer therapies.

Lipooligosaccharides (LOSs) are polar glycolipids found in the cell envelope of many pathogenic mycobacteria. Here, we show that LOS transport in requires , a member of the resistance-nodulation-division family of membrane proteins. Deletion of resulted in a rough colony morphology and increased hydrophobicity. The △ mutant accumulated three of the biosynthesis intermediates of LOSs (LOS-I, LOS-II and LOS-III) intracellularly and failed to produce the final product, LOS-IV, suggesting that final glycosylation of LOS-III to yield LOS-IV occurs extracellularly after LOS-III export. structural analysis of the MmpL12 suggests that it is a proton-driven transporter that shares very similar organization with other subclass 1 MmpLs (MmpL1, 2, 4-8 and 9-10), featuring a large periplasmic loop (PD3 domain) which is predicted to form a large coiled coil that may be involved in the trimerization of this subset of MmpL transporters. Furthermore, the long C-terminal extension domain, which is unique to MmpL12, may provide additional trimerization support and scaffold for assembly of additional LOS biosynthetic enzymes. The absence of any extracellular LOS intermediates and of LOS-IV had an impact on virulence, with the mutant strain exhibiting a larger bacterial burden in infected zebrafish embryos.

Cryptosporidiosis, caused by , poses significant health risks, particularly for children and immunocompromised individuals. Current treatments are ineffective in these vulnerable groups. This study explores the antiparasitic effects of peptoids against . Out of 14 synthetic peptidomimetics (peptoids) screened, TM9 and TM19 exhibited potent anti-cryptosporidial activity with minimal host toxicity. These findings suggest that peptoids could be a promising new therapeutic avenue for cryptosporidiosis, warranting further investigation.

is an environmentally resilient bacterium and an important cause of both acute and chronic infections in people with impaired natural barriers or immunological defences. Chronic respiratory infection with is a major cause of morbidity and mortality in people with airway diseases, including cystic fibrosis (CF) and non-CF bronchiectasis. Chronic airway infection is characterized by periods of relative stability punctuated by pulmonary exacerbations, during which times rapid bacterial outgrowth necessitates intense antimicrobial chemotherapy. The periods of stable infection can be modelled in mice by nasal instillation of airway-adapted in saline, leading to prolonged colonization of both upper airway (sinus) and lower airway (lung) environments that is not associated with symptomatic disease. Here, we use NMR metabolomics to investigate the impact of colonization on the metabolic landscape of sinuses and lungs. Lung infection led to pronounced changes in the airway metabolome, with significant depletion of glucose and myo-inositol but enrichment of glutathione (GSH), relative to uninfected lungs. Changes in the sinuses were more subtle but could be identified through dimensionality reduction approaches. The NMR spectral peaks that discriminated between infected and uninfected sinuses in partial least squares discriminant analysis included those for lactate and choline but were mostly representative of yet unidentified metabolites. These data highlight the differential impact of infection on separate airway compartments and identify undefined metabolites undergoing pronounced abundance changes during infection.

A session at the annual conference of the Microbiology Society 2025 (Liverpool) was held on gaining impact from basic and applied research, 'Beyond the lab - Turning your research into reality'. This Commentary provides a short description of the rationale behind the session, the key take-home messages, information on the speakers and key resources they shared with the microbiology community.

Seaweeds are a common and diverse component of coastal ecosystems and are known to be associated with due to faecal pollution. As a biotic substrate, beach-cast seaweed may affect bacterial physiology and thereby horizontal gene transfer (HGT). Here, we test how the presence of three distinct senescing seaweed species affects plasmid conjugation. We allow the IncP plasmid pKJK5 to conjugate while supplying a substrate of (dulse), (sea lettuce) or (serrated wrack). The three seaweed species induce distinct conjugative behaviours in : has no significant impact relative to a plastic control, the presence of results in undetectable levels of conjugation and promotes conjugation in a density-independent manner. This study highlights how biotic interactions can influence survival, HGT and antibiotic resistance in a human pathogen.

Consumers' healthy lifestyle practices have heightened the appeal of minimally processed foods, especially the fermented kind. Kefir and nabeez with numerous benefits are world-famous beverages. This study aimed to explore the enterococcal and lactococcal probiotic strains associated with these beverages. Artisanal recipes were used to make kefir and nabeez, and bacteria were isolated using classical culturing techniques. The isolates were screened based on antimicrobial potential, safety and probiotic attributes. The bacterial isolates obtained from three fermented beverages, milk kefir, water kefir and nabeez, were assessed for safety concerns, and those deemed safe were tested for antagonistic potential. Strains of (NPL1395, NPL1396 and NPL1480), (NPL1390, NPL1420 and NPL1427) and (NPL1426, NPL1428 and NPL1436) demonstrated interesting antimicrobial characteristics against food-borne pathogens. Strains from milk kefir and nabeez could tolerate strong acidic and bile stress. All strains were susceptible to lysozyme and phenol at the concentrations tested but demonstrated significant antioxidant potentials, exopolysaccharide production and bile salt hydrolase activities. Cholesterol assimilation was most significant in milk kefir and nabeez strains, which also had good adherence and biofilm formation. Statistical analysis of performance data using the principal component analysis identified strain NPL1428 as the best. It exhibited good potential to persist in the human gut based on its ability to tolerate mixtures simulating the gastrointestinal tract digestive fluids, using the static digestion model. Therefore, strain NPL1428 of traditional fermented beverage provenance has good prospects for use in probiotic product development.

KY5 was isolated from a plant-ant nest. It is primarily known for its production of the formicamycins, antibiotics with potent activity against Gram-positive pathogens including methicillin-resistant , and additionally produces an antifungal compound that inhibits multi-drug-resistant fungal pathogens including is genetically tractable using CRISPR-Cas9 gene editing, allowing for detailed analysis of the formicamycin biosynthetic gene cluster. AntiSMASH analysis predicts the genome to encode at least 45 secondary metabolite biosynthetic gene clusters, many of which appear to encode novel compounds. Current research efforts are focussing on characterising the regulation of secondary metabolism at a global level in order to switch on pathways that are not typically expressed under standard laboratory conditions with the aim of identifying novel antimicrobials.

Mezcal is a spirit obtained from the fermentation and distillation of juices obtained from different agave species. It is one of the distilled beverages with great sociocultural value in different regions of Mexico, and in recent years, it has also gained great economic importance. It is known to present differences in its flavour, thanks to the richness of compounds incorporated within the spirit, which vary according to the agave species used, the microbial population present and the processes involved in its manufacture. This variety reflects the richness of local traditions and the craftsmanship behind its production. The main objective of the present work was to explore parameters that could impact fungal and bacterial diversity. The microbiome of bacteria and yeasts present in fermentations in the same distillery, in two different years and with three different agave species was investigated by metataxonomic analysis obtained from the sequencing of regions V3-V4 for bacteria and ITS1 for yeasts. The results showed that the dominant fungal genera in the fermentations correspond to non- yeasts ( and ). A major finding was that was not the dominant yeast in any of the 15 fermentations characterized. The dominant bacteria belong to the groups of lactic acid bacteria and acetic acid bacteria. The statistical analysis of the alpha and beta diversities shows that the main statistical differences are seen in the year of fermentation rather than in the species of agave used. Finally, the microbial consortium was composed of the same genera during the different fermentations studied; the fundamental difference was the dominant genus in each fermentation.