Microbiology

Pseudolysogeny is a latent state in which phage development is delayed after infection and has been proposed to promote phage persistence under unfavorable conditions. Virulent phage T3 has been reported to establish pseudolysogeny after infecting starved E. coli, then resume lytic replication following transfer to nutrient-rich media, a phenotype linked to the T3 SAMase gene. Here, we revisited the findings of Krueger et al. (1975) to test pseudolysogeny in T3 and examine phage propagation under nutrient-limited conditions. Both T3 and T7 showed impaired propagation under nutrient limitation, with the most stringent conditions causing substantial losses in recoverable infective centers. T3 was modestly more resilient than T7 under these conditions, but we were unable to reproduce the reported phenotype in which T3 remained latent while T7 replicated normally. Supplementation of minimal medium with small amounts of LB supported propagation of both phages, and a repeat experiment designed to more closely match the historical protocol, including post-adsorption reduction of extracellular phage carryover, likewise failed to reveal a T3-specific pseudolysogenic state. Together, our results indicate that, in this experimental system, phage propagation dynamics are more consistently explained by nutrient conditions and media switching than by starvation prior to infection. These findings suggest that the previously reported T3 pseudolysogeny phenotype may depend on additional environmental or methodological factors and underscore the importance of revisiting historically reported phage behaviors using modern controls.

The RND efflux pump FemT encoded by SAUSA300_2213 of Staphylococcus aureus USA300 is co-transcribed with femX which has an essential role in synthesizing the Lipid II precursor of peptidoglycan. Anticipating that this arrangement reflects a critical accessory role for femT, we constructed USA300{Delta}femT to assess its function. Although growth of USA300{Delta}femT in tryptic soy broth (TSB) was not impaired, transcriptomic data revealed a mild cellular stress response, accompanied by reduced expression of ohyA and crt genes involved in fatty acid metabolism and carotenoid lipid synthesis respectively. Accordingly, USA300{Delta}femT exhibited impaired growth on exposure to saturated and unsaturated fatty acids, and exposure to subinhibitory 50 {micro}M palmitic acid promoted accumulation of reactive oxygen species, reduced respiratory activity, and altered membrane function and morphology. The transcriptome of cells grown under this condition revealed strongly attenuated expression of ohyA and crt, and several genes required for oxidative and anaerobic respiration, concomitant with strongly enhanced expression of several stress response pathways. Cellular metabolites were also profoundly altered. Finally, lipidomic analysis of USA300{Delta}femT exposed to oleic acid revealed increased incorporation of oleic acid into phosphatidylglycerol, accompanied by a significant reduction in undecaprenol C55 lipid carrier, and respiratory quinones MK-7 and MK-8. Our data are consistent with a role for FemT in maintaining cellular lipid homeostasis by promoting efflux of isoprenoid and carotenoid lipids that are prone to oxidative damage, including C55 and menaquinones that undergo cyclic reactions in peptidoglycan synthesis and electron transport. IMPORTANCEThe FemT efflux pump of S. aureus is co-expressed in an operon with femX encoding an essential enzyme needed to complete the synthesis of peptidoglycan precursor Lipid II. Although this alluded to a specific role for FemT in supporting peptidoglycan synthesis, our data are instead consistent with a general role in efflux of cellular isoprenoids and carotenoid lipids that are susceptible to oxidation during routine cellular functions. Consequently, S. aureus became strongly dependent on FemT function when exogenous host-derived fatty acids were being actively metabolized. This represents a significant advance in our understanding of the role of an RND efflux pump in supporting routine growth-related functions of S. aureus and exposes a function that could be targeted to impair S. aureus growth on exposure to host-derived fatty acids.

People with intellectual disabilities and their communities are rarely involved in setting priorities for research. Our study addressed this gap through consultations with the UK communities of three genetic syndromes in which intellectual disabilities are common: Down syndrome (DS), Fragile X syndrome (FXS) and Williams syndrome (WS). The study aimed to provide an understanding of (1) the views of the DS, FXS and WS communities on current UK research; (2) their priorities for future research; and (3) participants views of engaging with UK research. We conducted focus group discussions with 39 community members including: children and adults with DS, FXS and WS; parent/carers of people with DS, FXS and WS; practitioners and researchers who work with these communities. Our study was carried out in collaboration with a Steering Group and two Advisory Groups of DS, FXS and WS community members. We identified three themes. First, participants shared their dissatisfaction with the current research landscape and wanted a more balanced landscape, with more research with direct application to the daily lives of people with DS, FXS and WS. Second, community members emphasised the importance of translating research into practice, advocating for better access to research and more meaningful participation to research of individuals with lived experience. Third, our study not only identified what should be the focus of future research on DS, FXS and WS, but also how researchers should conduct their research. Whilst including children in our sample was a strength, there were some limitations to the diversity of our sample; children with FXS were not represented and gender, ethnic and geographic diversity could have been broader. Nevertheless, we hope that our findings will change the future of research in this field so that research carried out in the name of individuals with intellectual disabilities such as DS, FXS and WS, is of direct use to these communities.

Pseudomonas aeruginosa is an opportunistic pathogen that often adopts persistent phenotypes -- such as biofilm formation-- that are associated with chronic infections including those observed in the cystic fibrosis (CF) lung Recently, highly effective modulator therapy (HEMT) such as elexacaftor/tezacaftor/ivacaftor (ETI) has significantly improved the quality of life of people with CF (pwCF). Yet a potential direct impact of ETI on the physiology of P. aeruginosa during growth to a remodeled CF lung environment has remained unexplored. To address this, we conducted an experimental evolution using P. aeruginosa PA14 grown in CF-like conditions in the presence or absence of ETI. We observed a marked reduction in biofilm formation and in the number of small colony variants (SCVs) for P. aeruginosa populations evolved under ETI treatment. Also, sequencing of specific evolved clones exhibiting distinct morphotypes revealed two major observations: (i) P. aeruginosa-evolved communities exposed to ETI retained a wild type-like morphotype and, (ii) P. aeruginosa populations evolved in the absence of ETI adopted a SCV-like phenotype with mutations acquired in the Wsp chemosensory pathway. Furthermore, analysis of evolved populations revealed that ETI treatment likely modulates c-di-GMP pools by driving mutations in an enzyme catalyzing the degradation of this second messenger. Overall, our work suggests that ETI has the potential to hinder the acute to chronic biofilm transition of P. aeruginosa thereby limiting the emergence of variants typically associated with long-term CF lung colonization.

Chronic Pseudomonas aeruginosa lung infections are becoming harder to treat due to global escalation of antimicrobial resistance (AMR). Bacteriophage (phage) therapy has emerged as a promising adjunct to conventional antibiotics, especially in chronic lung infections such as those seen in cystic fibrosis (CF). However, phage monotherapy may be limited by the emergence of phage-resistant bacterial populations and there remains limited preclinical evidence evaluating both antimicrobial efficacy and host safety in physiologically relevant human airway models. Here, we evaluated the safety and antimicrobial activity of Kara-mokiny 3, a myovirus bacteriophage, alone and in combination with subinhibitory concentrations of tobramycin using fully differentiated paediatric primary airway epithelial cells (pAECs) cultured at the air-liquid interface (ALI). Kara-mokiny 3 rapidly reduced P. aeruginosa viability and exhibited synergistic activity with tobramycin, resulting in significantly greater bacterial killing than either treatment alone. Importantly, phage treatment replicated efficiently in the presence of its bacterial host while preserving epithelial morphology, mucin production and epithelial barrier architecture., without inducing cytotoxicity or excessive IL-6 and IL-8 inflammatory responses. These findings demonstrate that phage-antibiotic combination therapy can enhance antimicrobial activity while maintaining epithelial safety in a physiologically relevant human airway model. This study represents one of the first comprehensive evaluations of phage-antibiotic combination therapy in differentiated primary airway epithelial cultures, providing important preclinical evidence supporting the development of personalised phage-based therapies for the treatment of MDR pulmonary infections. ImportanceThe rise of MDR P. aeruginosa has created an urgent need for alternative treatment strategies for chronic lung infections. Although phage therapy is receiving increasing clinical attention, there is limited evidence evaluating its safety and efficacy in physiologically relevant human airway models. Using differentiated primary airway epithelial cultures, we demonstrate that a phage-antibiotic combination reduces bacterial burden without compromising epithelial integrity and toxicity or excessive inflammatory responses. These findings provide translational evidence supporting phage-antibiotic combination therapy and highlight the value of primary airway epithelial models for the preclinical assessment of emerging antimicrobial interventions, supporting the translation of personalised phage therapies.

Onion tissues produce antimicrobial thiosulfinates after tissue damage and cellular decompartmentalization. Burkholderia gladioli pv. alliicola (Bga), a common onion pathogen, encodes a thiosulfinate tolerance gene (TTG) cluster that protects the bacterium during thiosulfinate exposure. Previous work showed that the TTG cluster contributes to foliar infection but has little effect on infection of onion bulb tissue. To further examine Bga-thiosulfinate interactions in foliar and bulb tissues, we used a thiosulfinate-responsive PaltR-Lux reporter strain to determine when and where Bga encounters thiosulfinates. In leaves, Bga-induced necrosis was associated with de-repression of the PaltR-Lux reporter and coincided with a contribution of the TTG cluster to bacterial population size, indicating thiosulfinate exposure during foliar infection. In contrast, TTG mutants and wild-type (WT) strains showed similar growth in scales, and PaltR-Lux signal declined as scale necrosis progressed, suggesting limited thiosulfinate exposure during bulb colonization. However, when necrosis was induced by the non-native toxin pantaphos, PaltR-Lux was de-repressed and recovery of the TTG mutant was reduced. These results indicate that Bga encounters thiosulfinates during foliar infection but largely avoids exposure during bulb infection. Preconditioning the TTG mutant in onion scale tissue did not alter its thiosulfinate sensitivity in vitro, arguing against an infection-associated thiosulfinate exclusion mechanism. In contrast, partial rescue of the TTG mutant by the WT strain in zone-of-inhibition co-plating assays suggests extracellular thiosulfinate detoxification. Together, these findings indicate that Bga detoxifies thiosulfinates released during bulb necrosis, limiting thiosulfinate exposure during onion bulb infection. The molecular basis for detoxification and tissue specificity remain unresolved.

S. mutans and V. parvula cooperate in dental plaque to assemble a healthy biofilm and are associated with increased caries risk. S. mutans produces lactic acid from carbohydrates resulting in a final biofilm pH[~]4, while V. parvula metabolizes lactate to acetic and propionic acids resulting in pH[~]5. This process results in healthier biofilms that still generate a pH capable of demineralizing tooth surfaces (pH<5.5). The purpose of this study was to identify V. parvula genes required for deacidification of S. mutans biofilms and determine whether the ability of V. parvula to deacidify S. mutans biofilms correlates with enhanced biofilm health. Using transposon mutagenesis in V. parvula we identified several genes required for deacidification of S. mutans biofilms. These included numerous V. parvula transposon mutations in the previously unstudied lutABC lactate utilization operon. To assess biofilm health, S. mutans in the presence of various V. parvula mutants were stained with a LIVE/DEAD stain and imaged by fluorescence microscopy. An intact lutABC operon was required to enhance biofilm health, as demonstrated by plasmid-based complementation of a lutB transposon mutant. Transposon insertions in other loci unrelated to deacidification had no impact on biofilm health. Addition of HEPES buffer at the time of S. mutans biofilm assembly prevented full acidification of the biofilm and resulted in improved biofilm health, even without the addition of V. parvula. Finally, we found V. parvula can use either nitrate or fumarate as a final ETC electron acceptor during lactate utilization. In all, we found the lutABC lactate utilization operon of V. parvula is critical for the ability of V. parvula to deacidify S. mutans biofilms and promote biofilm health. Interfering with this pathway would interrupt the mutually beneficial relationship between S. mutans and V. parvula that leads to their co-association in caries, root caries, and early childhood caries.

Anaerobic gut fungi (AGF) are central to the degradation of plant material in the digestive systems of herbivores. However, how their environment influences their colonisation and degradation of complex biomass is unclear. Here, cellulose filter paper was used as a simplified model of the plant cell wall to investigate how the presence of free sugars in the rumen can affect AGF growth and degradative responses of phylogenetically distinct AGF isolates. From this, galactose was revealed to be inhibitory to both Neocallimastix frontalis and Caecomyces communis, and mannose inhibitory to C. communis. Complete inhibition of C. communis growth was conserved when galactose and mannose were added in their polymeric forms, whereas in contrast, N. frontalis growth was unaffected. This indicates, depending on the AGF isolate, the presence of free sugars and their polymeric form may influence AGF growth through regulatory and metabolic interactions - even if the sugar cannot be utilised for growth as the sole substrate. Collectively, this work highlights the functional diversity in AGF carbohydrate responses and the need for greater understanding of their metabolic regulation for applications in lignocellulosic bioconversion and ruminant nutrition.

Myxococcus xanthus bacteria form aggregates when starved on solid surfaces and some cells differentiate into spores. Studies of mutants in monoculture have advanced knowledge of this multi-cellular developmental process, but our understanding of the genetic determinants is incomplete. To assess gene function genomewide, we generated a pool of barcoded transposon insertion mutants, subjected it to starvation, and separated developmental samples into non-aggregated cells, aggregated cells, and spores. We also subjected our pool to chemically-induced unicellular sporulation. Evaluation of changes in the abundance of mutants in samples allowed identification of 200 genes in which insertions reproducibly caused distinct patterns of depletion and/or accumulation over time. Many of these genes have well-established roles in development, validating our approach, while many others have not previously been associated with development. Genes involved in type IV pili (T4P)-dependent motility were more important than gliding motility genes for aggregation and sporulation in the mixed population. Although exopolysaccharide (EPS) synthesis genes are required for aggregation in monoculture, most were dispensable for aggregation in our pool, consistent with EPS sharing between cells, yet these genes were required cell-autonomously for efficient sporulation. Genes for positive regulators of EPS synthesis were important for aggregation as well as sporulation, suggesting functions beyond EPS production. Insertions in several novel genes impaired both starvation- and chemically-induced sporulation. Many genes increased the efficiency of starvation-induced sporulation. Some of these mutants, which we call "developmental winners", are novel cheaters. Our results demonstrate the power of using the newly-created mutant library to elucidate M. xanthus biology.

O_LIVitamin B12, an essential micronutrient for many microalgae and humans, is synthesised only by certain prokaryotes. B12 is a complex tetrapyrrole that can exist in many forms (vitamers), some more bioavailable than others. Some microalgae are able to interconvert, or remodel, different B12 vitamers. As microalgae are important primary producers, it is crucial to understand how diverse microalgae acquire, utilise, and remodel this micronutrient. C_LIO_LIThrough the development of a novel algal bioassay for B12 quantification that distinguishes between B12 vitamers with different lower axial ligands, and the generation of targeted knock-out lines, we characterised the role of proteins involved in algal B12 uptake and remodelling. C_LIO_LIWe found that the previously characterised protein CoBalamin-Acquisition protein 1 (CBA1) is also necessary for the acquisition of pseudocobalamin, a less bioavailable form of B12. In addition, we provide the first experimental evidence that COBT is required for Chlamydomonas reinhardtii to remodel B12. C_LIO_LIWe apply the algal B12 bioassay to show that the edible alga Chlorella vulgaris can accumulate pseudocobalamin but is unable to remodel it, highlighting the need for thorough investigation of the metabolic requirements and capabilities of microalgae, especially given the growing interest in microalgae-based food additives. C_LI

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that colonizes a significant proportion of humans, contains numerous virulence factors promoting infection, and continues to threaten human lives and burden healthcare systems globally. Many MRSA virulence factors are known to be either secreted or anchored on the outer leaflet of the cell surface. Although many virulence factors have been studied intensively in MRSA, there remains a significant proportion of secreted and surface proteins that are unstudied for their potential as virulence factors. We began with identifying proteins secreted from MRSA in axenic culture using an unbiased mass-spectrometry based approach. 2 secreted proteins thus identified mapped to an operon of 6 genes, SAUSA300_1739 to SAUSA300_1744. Mutation of each of the individual genes in the operon resulted in attenuation in a mouse model of subcutaneous infection. We demonstrate that two genes in the operon, SAUSA300_1739, and SAUSA300_1740, encode nucleases with DNase activity. Genetic analysis of the SAUSA300_1739 to SAUSA300_1744 operon across several Staphylococcus aureus strains indicate that the operon is highly conserved, highlighting its importance for virulence.

Human-targeted non-antibiotic drugs are increasingly recognized for their intrinsic antibacterial activity, yet Gram-negative pathogens such as Acinetobacter baumannii exhibit substantial tolerance to these compounds. This tolerance is largely attributed to restricted outer membrane permeability and the activity of multidrug efflux systems. While Resistance Nodulation Division (RND) transporters have been extensively studied, the contribution of individual Major Facilitator Superfamily (MFS) transporters to non-antibiotic drug tolerance remains poorly understood. Here, we investigated H0N29_04330, designated Drug Resistance Transporter A (DrtA), a Bcr/CflA subfamily MFS transporter, to define its substrate specificity and contribution to antibiotic and non-antibiotic drug tolerance. DrtA was highly conserved across the A. calcoaceticus-baumannii complex and exhibited broad substrate specificity when heterologously expressed in an efflux-deficient Escherichia coli background, conferring resistance to benzalkonium, ethidium bromide, phenicols, and the antineoplastic agent mitomycin C. Intriguingly, drtA expression increased E. coli susceptibility to the antifolate compounds methotrexate and aminopterin, suggesting that DrtA may recognize folate-related metabolites rather than function as a dedicated antifolate transporter. In contrast, loss of drtA in its native A. baumannii host primarily impaired tolerance to mitomycin C, highlighting a context-dependent physiological role influenced by the extensive functional redundancy among A. baumannii efflux systems. Site-directed mutagenesis further identified M18 and the membrane-embedded protonatable residue D26 as critical determinants of DrtA transport activity and substrate recognition. Together with previous characterization of CraA, our findings demonstrate that Bcr/CflA subfamily MFS transporters contribute to protection against structurally diverse human-targeted compounds and expand the functional landscape of efflux-mediated intrinsic tolerance beyond conventional antibiotic resistance.

Anaerobic gut fungi (AGF) are key degraders of plant biomass in ruminants, yet there is limited knowledge of how AGF respond to mixtures of plant-derived sugars. Here, we assessed monosaccharide and disaccharide utilisation by Neocallimastix frontalis CoB3, Caecomyces communis SHB, and Piromyces edwardsiae SHC, which are abundant in the rumen microbiome. While all AGF isolates shared a core set of sugars that supported growth, they had different hierarchies of uptake. Co-substrate experiments using glucose and lignocellulose-derived sugars revealed species-specific responses, with N. frontalis displaying a novel concentration-dependent co-utilisation of glucose and mannose, whereas growth of P. edwardsiae was inhibited under the same conditions, and C. communis exhibited growth inhibition in glucose and xylose co-substrate cultures. Together, these findings demonstrate functional diversity in monosaccharide and disaccharide metabolism amongst the AGF investigated here. Understanding such sugar utilisation phenotypes provides a foundation for evaluating AGF isolate suitability for lignocellulosic biomass valorisation.

The emergence of antibiotic tolerance in Staphylococcus aureus reduces antibiotic efficacy by allowing bacterial survival despite prolonged antibiotic exposure, the molecular basis of which remains poorly understood. Moreover, the phenotypic indistinguishability of tolerant isolates in antimicrobial susceptibility testing impedes effective diagnosis and therapy. Increased concentration of the second-messenger, cyclic-di-AMP (CDA), has recently been implicated in tolerance to {beta}-lactams as well as other cell-wall-reactive antibiotics. Using the ScanLag assay, Tolerance-Disk test, and traditional methodologies and employing isogenic mutagenized strains, we demonstrate that loss of GdpP function, a phosphodiesterase that hydrolyzes CDA, confers tolerance specifically to {beta}-lactam antibiotics independent of their class. The extent of {beta}-lactam tolerance correlated directly with the intracellular CDA concentration and inversely with the inhibition of bacterial cell-wall synthesis. {Delta}gdpP mutants caused higher mortality than wild-type strains in the Galleria mellonella infection model upon {beta}-lactam treatment, suggesting GdpP-mediated tolerance could lead to {beta}-lactam treatment failure. Large-scale within-host evolution analysis demonstrated that MRSA and MSSA strains isolated from patients acquire GdpP loss-of-function mutations during invasive infections but not during nasal carriage. Overall, this study highlights the clinical relevance of gdpP mutations, frequently selected in persistent S. aureus infections, as key mediators that could promote treatment failure due to {beta}-lactam tolerance.

Enterococcus faecalis is an opportunistic pathogen and facultative anaerobe that primarily relies on fermentative metabolism to colonize a wide range of aerobic and anaerobic environments. In the presence of exogenous heme, E. faecalis can assemble a minimal electron transport chain consisting of membrane-associated primary dehydrogenases, demethylmenaquinone, and the terminal cytochrome bd oxidase (CydAB). This respiratory chain is thought to generate a proton motive force to drive ATP synthesis via the F-type ATP synthase, thereby improving energy conservation under aerobic conditions. However, a cytosolic NADH oxidase (Nox) also consumes NADH and oxygen, potentially competing with the electron transport chain for reducing equivalents and terminal electron acceptors; but the relative physiological contributions of these two oxygen-reducing pathways remain poorly understood. To define the roles of CydAB and Nox under normoxic and hypoxic conditions, we constructed {Delta}cydAB and {Delta}nox mutants. Real-time, in situ measurements revealed {Delta}cydAB had no significant effect on oxygen utilization while in the {Delta}nox it was significantly reduced; revealing Nox as the major consumer of oxygen. Semi-untargeted metabolomic analysis further revealed oxidase-specific alterations in central metabolism with the {Delta}nox causing pronounced shifts in the ATP and NADH ratios; highlighting Nox as a key determinant of intracellular redox and energy homeostasis. Finally, single-cell fluorescence microscopy showed that membrane potential, a component of proton motive force, was substantially diminished only in the absence of both CydAB and Nox, or the F-type ATP synthase. These findings indicate that the F-type ATP synthase is a major generator of proton motive force, even upon aerobic growth, and demonstrate a complementary role for the electron transport chain and Nox in the bioenergetics of E. faecalis.

Pseudomonas aeruginosa remains one of the most important clinical pathogens for which new drugs are needed, due to its resistance machinery. Consequently, there is an increasing effort to develop new and effective treatments against this pathogen. We recently showed that tetrasodium ethylenediaminetetraacetic acid (tEDTA) exhibits promising antibacterial and antibiofilm activity against P. aeruginosa in advanced biofilm models. tEDTA is known to chelate divalent cations, with predicted effects on the outer membrane; however, a full understanding of how this kills P. aeruginosa is lacking. Also, it is currently not clear how slowly or rapidly P. aeruginosa will evolve resistance to this treatment. Using membrane disruption assays and RNA-seq, we showed that tEDTA disrupts bacterial membrane potential and permeabilises P. aeruginosa membranes. RNA-seq revealed the significant upregulation of genes involved in the transport of iron, phosphate, potassium, and magnesium ion. The arnABCD operon which is involved in lipid A biosynthesis was also upregulated. Using a 7-day evolutionary ramp approach, we showed that P. aeruginosa could not evolve resistance to tEDTA under strong selection. Lastly, we carried out a cytotoxicity assay with Human Epithelial type 2 (HEp-2) cells and showed that there was reduced cytotoxicity of tEDTA compared to meropenem. This study provides good insight into the mechanism of action of tEDTA and further evidence of its potential as an alternative to antibiotics for P. aeruginosa infections.

BackgroundDetection of rare mycobacterial genotypes, including those associated with antibiotic resistance or population heterogeneity is important for diagnostic, therapeutic and research applications. This depends on efficient recovery of genomic DNA (gDNA) from sampled populations, a challenging requirement in paucibacillary clinical materials. Mycobacteria have uniquely lipid-rich, structurally robust cell envelopes which resists cell lysis by conventional methods. Here, we characterize mycobacteriophage D29-mediated lysis at the single-cell level, evaluating its utility as a biological lysis strategy for mycobacterial DNA isolation, benchmarked against the standard cetyltrimethylammonium bromide (CTAB) extraction method. MethodsConditions for mycobacteriophage D29 infection of Mycobacterium smegmatis (Msm) were established, and single-cell phage adsorption and phage-mediated lysis visualized through live-cell time-lapse fluorescence microscopy (FM). A mycobacteriophage D29-based lysis method was applied to both Msm and M. tuberculosis (Mtb), and extraction efficiencies compared with the standard CTAB method. Cell lysis efficiency was quantified by colony forming units (CFU), flow cytometry (FC) and FM; DNA yield was determined by quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR). ResultsMycobacteriophage D29 adsorption was observed at the poles and septa of individual mycobacterial cells. Phage infection was associated with loss of cytoplasmic green fluorescence protein (GFP) reporter protein, with uptake of a cell death marker propidium iodide (PI). Mycobacteriophage D29 infection resulted in a marked loss of cell viability, with >6log10 reduction in CFU, and cell lysis efficiencies calculated as 93.3% (FC) and 96.8% (FM). Molecular quantification (qPCR and ddPCR) indicated that the mycobacteriophage-based lysis achieved between 4- to 7-fold greater gDNA yields in Msm and between 3- to 12-fold greater gDNA yields in Mtb H37Ra compared with the CTAB method. Notably, gDNA extraction efficiencies in both mycobacterial species exceeded 92% in low-biomass samples containing approximately 100, 175 and 320 bacilli. ConclusionThese results demonstrate the utility of the mycobacteriophage D29-based method for improved DNA extraction yields from mycobacteria through direct lysis of individual bacilli, with performance suited to low-biomass samples. SummaryRecovering genomic DNA (gDNA) from low numbers of mycobacteria is a persistent bottleneck for diagnostics and genomic studies, because the lipid-rich mycobacterial envelope resists conventional lysis. Here we show that mycobacteriophage D29 provides an efficient, biologically selective route to mycobacterial DNA. Leveraging single-cell live imaging, we reveal that phage D29 adsorbs preferentially at the poles and septa of individual cells, and that infection is heterogeneous and asynchronous, progressing from envelope permeabilization to loss of viability. Applied as an extraction method and benchmarked against the standard cetyltrimethylammonium bromide (CTAB) protocol, phage D29-mediated lysis recovered 4- to 7-fold more gDNA in Mycobacterium smegmatis (Msm) and 3- to 12-fold more in Mycobacterium tuberculosis (Mtb). Critically, extraction efficiency exceeded 92% in both species in low-biomass samples of approximately 100, 175 and 320 bacilli, where CTAB performed poorly (<20% efficiency). These findings support phage-mediated lysis as a quantitative, near-complete DNA-recovery method that outperforms conventional extraction precisely in the paucibacillary regime of greatest clinical relevance and demonstrate the value of single-cell interrogations in building towards precision tools to engage the mycobacterial cell.

Streptococcus mutans is a key contributor to dental caries, with its capacity to form structured biofilm microcolonies being a principal component of its cariogenic potential. Yet, most mechanistic studies rely on a limited number of laboratory strains and may not capture the functional diversity present across the species. Here, we characterized a panel of phenotypically and genomically diverse S. mutans isolates to determine how strain background influences biofilm architecture, extracellular matrix accumulation, acid-associated physiology, environmental responsiveness, and antimicrobial susceptibility. Quantitative high-resolution imaging revealed extensive heterogeneity in produced biofilm microcolony size, structure, and matrix composition, demonstrating that biofilm architecture is not a uniform species-level trait. Interestingly, the commonly used reference strain UA159 displayed an intermediate phenotype related to microcolony size and biofilm organization. Human saliva further altered biofilm structure and matrix accumulation in a strain-dependent manner rather than producing a standard species-wide response. Isolates also differed in growth and retained biofilm biomass under acidic conditions, while acid accumulation within mature biofilms varied independently of average microcolony volume, demonstrating that strains that produce larger microcolonies on average were not necessarily associated with greater acid accumulation. Susceptibility to the antiseptics chlorhexidine and cetylpyridinium chloride likewise differed among isolates and could not be predicted from formed biofilm architecture alone. Together, these findings demonstrate that disease-relevant traits commonly attributed to S. mutans are distributed unevenly and only partially coupled across strain backgrounds, with biofilm spatial organization failing to serve as a dominant phenotype linking acid accumulation, acid tolerance, and antimicrobial susceptibility.

Phage D29 infects Mycobacterium smegmatis mc2 155 and has a non-canonical lysis cassette that encodes two endolysin proteins (Lysin A and Lysin B) and a single two transmembrane domain (TMD) protein, LysA2a similar to F1 cluster phage LysF1a. A 1TMD LysF1b homolog, LysA2b, is encoded by a gene found downstream of the tape measure. Exogenous expression of both LysA2 proteins in tandem is a cytotoxic to M. smegmatis. Deletion of lysA2a produces phages that are lysis competent with a 10-minute triggering delay and 30% plaque size reduction. Deletion of lysA2b results in severe lysis defects manifest by 70% reduced plaque size, delayed lysis timing and reduced burst size. Deletion of both lysA2 genes results in phages that are viable and show lysis phenotypes like the lysF1b deletion. Genetic complementation of lysA2b deleted phage with the lysF1b gene fully complements the lysis phenotypes but alters the triggering time to that of an F1 cluster phage. Energy poisons trigger lysis prematurely in all phages with lysA2 gene deletions. Lysis recovery mutants (LRM) isolated from phages lacking the lysA2b genes generate wild type plaque size and have point mutations that map to TMD1 or the C-terminal region of the lysA2a gene. LRMs isolated from phages lacking both lysA2 genes show premature lysis and have mutations that all map to residue C31 of a novel lipoprotein (gene 64). Deletion of gene 64 does not change wild type D29 lysis phenotypes or rescue the lysis defects of any of the lysA2 mutants. A fitness/competition assay shows that loss of the lysA2 genes imposes a substantial competitive fitness cost. These finding support a lysis regulatory network model where the 2TMD protein is maintained in an inactive state until activated by its cognate 1TMD lysis regulator and the lipoprotein has accessory function that may enhance lysis efficiency.

BackgroundDespite the importance of oropharyngeal gonorrhoea in transmission, suboptimal antimicrobial responses and propensity for horizontal transfer of antimicrobial resistance at this site, it remains understudied. An oropharyngeal N. gonorrhoeae controlled human infection model (CHIM) represents a promising tool to study infection and undertake translational research. MethodsA panel of five contemporary N. gonorrhoeae isolates were subject to detailed characterisation to assess antimicrobial susceptibility, in vitro infectivity, cytotoxicity and serum sensitivity to inform challenge agent selection. A method for challenge agent manufacture, including release testing, was developed and validated. FindingsAll candidate isolates were able to infect the surface of pharyngeal and cervical cells in vitro. One isolate displayed an invasive phenotype, induced higher inflammatory cytokine production and displayed elevated serum resistance and was excluded. The remaining four isolates were minimally inflammatory, did not induce cytotoxicity and were susceptible to serum killing. Three of the four isolates grew in a defined liquid medium. Together these results led to the selection of a contemporary N. gonorrhoeae isolate suitable for use in CHIM. A challenge agent manufacture workflow was established and shown to reliably and reproducibly generate doses suitable for direct inoculation in an oropharyngeal CHIM. ConclusionPhenotypic characterization of candidate N. gonorrhoeae challenge agents led to the successful identification of a contemporary isolate suitable for implementation in a novel oropharyngeal gonorrhoea CHIM. We demonstrate the feasibility of a challenge inoculum manufacturing process that aligns with international best practice guidelines.