Microbiology

Achromobacter species are emerging multidrug-resistant (MDR) pathogens in people with cystic fibrosis. Their increasing resistance has grown an interest in phage therapy as an alternative treatment strategy. However, the factors governing phage susceptibility remain poorly understood, thereby limiting the rational selection of phage candidates. Using 15 strictly lytic Achromobacter phages and 7 clinical cystic fibrosis isolates representing Achromobacter insolitus and Achromobacter xylosoxidans, we demonstrate substantial variation in infection efficiency across all 105 phage-host combinations, variation that could not be discerned from qualitative plaque assays alone. We integrated complete bacterial and phage genomes with quantitative efficiency-of-plating (EOP) assays and lineage-aware Bayesian mixed-effects modelling to show that phage infectivity in Achromobacter is governed predominantly by bacterial lineage and strain identity, accounting for 90% of total variance in log-normalised EOP, with individual strains varying substantially in permissiveness irrespective of species membership. After accounting for this lineage structure, no individual defence system, antimicrobial resistance gene class, or phage tail cluster retained a statistically significant independent or interaction association with infectivity. Together, these findings demonstrate that bacterial strain identity is the primary driver of infection outcome. Host defence systems and phage tail-associated genes remain biologically plausible contributors; their independent effect could not be resolved after accounting for lineage structure, indicating that infection outcomes are largely strain-dependent. This work shifts the question from which individual traits predict infection to how strain lineage and specific host-phage combinations jointly determine infectivity, and argues that quantitative phenotyping of individual phage-host pairs is essential for guiding phage candidate selection and supporting rational cocktail design against multidrug-resistant Achromobacter infections in cystic fibrosis. Impact statementChronic Achromobacter infections in cystic fibrosis are increasingly difficult to treat due to multidrug resistance and biofilm formation. Although phage therapy is a promising alternative, its development is limited by poorly understood and highly variable infectivity. Here, we show that infectivity within a phage host range spans a broad quantitative continuum spanning several orders of magnitude that cannot be captured by qualitative plaque assays. These infection efficiencies are primarily structured by bacterial lineage and strain identity, while the contributions of individual genomic features remain unresolved, given the current sample size. This work provides a framework for predicting phage-host compatibility and supports a shift from empirical screening toward rational, evidence-based phage selection for MDR Achromobacter infections.

Aminopeptidases are widely distributed in bacteria, but outside of a few model strains, their function is largely unexplored. Focussing on beta-alanine aminopeptidase activity, a new series of selectively-activatable, caged fluorescent probes were designed and synthesised. A beta alanine amino acid was coupled to resorufin or 7-hydroxycoumarin via a self-imolative linker, such that amino acid removal led to gain of fluorescence. These were used to probe selectivity and specificity of probe activation, against a range of priority drug-resistant pathogens. When added to bacterial growth curves run in Muller Hinton broth, these probes allowed essentially real time fluorescence measurement of activation by bacterial species, modelled on the standard microbroth dilution method. Activation was observed for all Pseudomonas aeruginosa and Burkholderia spp strains tested. Selective activation was seen for Ochrabactrum species, with the probe activated by O.anthropii (2/4 strains) but not O.intermedium and strain-specific activation was seen for some isolates of Serratia marcescens (2/4 strains). No activation was observed in any isolates of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii or Staphylococcus aureus or Eneterocccus faecium/faecalis PAO1 transposon mutants in the putative beta-alanine aminopeptidase gene (annotated as bapF or dmpA; PW3678) showed no activation of the probe in growth assays, confirming the specificity of the probe for beta-alanine aminopeptidase. Transposon mutants in other aminopeptidase genes, including those encoded by pepN, PepP and the prolyl aminopeptidase gene had no effect on probe activation in PAO1. Based on the operon structure in PA01, transposon mutants in two adjacent genes were also tested for probe activation. Mutants in both a putative transcriptional regulator (PW3674) and a predicted amino acid permease (PW3676) retained their ability to activate the beta-alanine probes with activation significantly higher than the wild type, when assessed by the total fluorescence yield after 10 hours growth. This points to both redundancy in permease function and perhaps the presence of a feedback regulatory mechanism controlling beta alanine aminopeptidase activity in P.aeruginosa. Given that the operon structure is conserved in other species, this may point to a common mechanism of beta alanine aminopeptidase function, perhaps related to exploiting beta-alanine containing peptides in certain environmental niches.

Antibiotic resistance is a growing global health crisis, yet resistance is almost exclusively quantified under aerobic laboratory conditions that fail to reflect the complex microenvironments bacteria encounter during infection. Many clinically important infection sites, such as airways of individuals with cystic fibrosis or chronic wounds, are microaerobic to anoxic. To address this, we investigated how anoxia alters antibiotic resistance determinants, hypothesizing that anaerobic metabolism might change the fitness effects and selection of resistance mutations. We used experimental evolution to propagate Pseudomonas aeruginosa populations for approximately 200 generations under conditions differing in oxygen availability (oxic vs. anoxic), growth mode (biofilm vs. planktonic), and tobramycin (TOB) exposure (subinhibitory increasing to inhibitory concentrations). Subinhibitory exposure was sufficient to achieve resistance 2-4x greater than ancestral levels, with anoxic populations consistently showing higher minimum inhibitory concentrations than oxic comparisons. Resistance developed through condition-dependent genomic targets: mutations in amgS were selected in oxic populations, while fusA1 and ptsP mutations arose across all conditions. Notably, mexT mutations were nearly universally selected, particularly under anoxic or tobramycin-exposed conditions. mexT inactivation may also enhance virulence through altered quorum sensing and increased rhamnolipid production. Anoxic populations additionally exhibited significantly increased biofilm formation, some exceeding 1000% of ancestral levels, reduced twitching motility driven by type IV pilus gene mutations, and greater competitive fitness. Together, these findings demonstrate that oxygen availability shapes resistance evolution in P. aeruginosa, with the anoxic environment selecting for a more virulent, sessile, and antibiotic-resistant phenotype.

Climate-driven increases in sea surface temperature have been associated with the expansion of Vibrio species and a corresponding rise in vibriosis cases in both human populations and aquaculture systems. Coastal waters across the south of England are increasingly becoming suitable for the growth and establishment of both human- and aquaculture-associated Vibrio species, potentially increasing vulnerability to the types of infections and disease outbreaks already reported elsewhere in the world. In this study, we report the presence of a diverse and well-established Vibrio community within the Teign Estuary, (Southwest, UK), including the human-pathogenic species V. parahaemolyticus, V. cholerae (non-O1/non-O139), V. alginolyticus, and V. diabolicus, as well as the important aquaculture pathogens V. jasicida, V. aestuarianus, and V. anguillarum. We identified V. diabolicus, a species that was indistinguishable from V. alginolyticus using conventional biochemical identification methods and could only be accurately resolved by whole-genome sequencing and developed novel PCR targets to differentiate these species in the lab. Using the insect infection model Galleria mellonella, we demonstrate that environmental isolates of V. cholerae (non-O1/non-O139), V. parahaemolyticus, and V. alginolyticus possess virulence potential. We also investigated the effects of sewage effluent on the growth of Vibrio isolates from the Teign Estuary and found that sewage can preferentially promote the growth of Vibrio species. Furthermore, several Vibrio isolates were multidrug resistant and carried antimicrobial resistance genes, highlighting the potential role of environmental Vibrio populations in the Teign Estuary as reservoirs of antimicrobial resistance. Together, these findings demonstrate how rising sea surface temperatures and sewage pollution may influence the emergence, persistence, and public health and aquaculture significance of Vibrio species in UK coastal waters.

Phage therapy offers a promising solution to the antimicrobial resistance crisis. However, a major concern preventing the adoption of phage therapy is the potential for unintended consequences of phage release; both in regard to preventing the spread of phage resistance, and the proliferation of a non-endemic virus into the microbial ecosystem. Conditional replication (biocontainment) of phages through bioengineering may address these concerns, but the impact on bactericidal efficacy is unknown. Here, we created a biocontained T7 phage (T7{Delta}capsid) lacking the major structural capsid gene, gp10AB, that can only replicate on Escherichia coli strains expressing gp10AB in trans, and assessed its bactericidal efficacy compared with wild-type T7. Congruent with model predictions, T7{Delta}capsid was only able to clear a well-mixed culture of E. coli at a multiplicity of infection (MOI) of 10 or higher, whereas wild-type T7 prohibited growth at an MOI of 0.1. The reduction in efficacy was more evident in a complex structured environment within a microfluidic device, where phage success depends on its ability to penetrate a microbial niche via propagation. In this environment, T7{Delta}capsid was unable to propagate into the bacterial population and unlike wild-type T7, had no impact on the population's growth. This study shows that whilst biocontainment of phages may improve the biosafety of phage therapy, it comes at the cost of its propagation efficacy and niche penetration in relevant environments.

Type IV pili (T4P) mediate twitching motility and contribute to surface colonization, biofilm formation, and host interactions in Acinetobacter baumannii. However, the prevalence, dynamics, and diversity of twitching motility across A. baumannii populations remain poorly understood. Here, we compared twitching motility in a collection of 35 A. baumannii strains originating from clinical, environmental, and animal sources, using Pseudomonas aeruginosa PAO1 as a reference. Standardization of assay conditions revealed a strong influence of agar composition on twitching motility, with Eiken agar supporting the most robust surface translocation. Under these conditions, 14 of 35 A. baumannii isolates exhibited detectable twitching motility. Time-lapse microscopy revealed major differences between A. baumannii and P. aeruginosa. Whereas PAO1 initiated twitching within minutes after inoculation and formed characteristic multicellular rafts, motile A. baumannii strains displayed a prolonged non-motile phase before movement initiation and exhibited distinct patterns of collective organization. Two major expansion phenotypes were identified, termed Homogeneous Front (HF) and Raft-Like Front (RLF), together with Early-Onset Motility (EOM) and Delayed-Onset Motility (DOM) subgroups. Quantitative analyses further revealed substantial variation in speed, directional persistence, and migration dynamics among strains. Because a majority of isolates were non-motile, we investigated the contribution of the minor pilin FimT. Although deletion of fimT abolished twitching motility and specific substitutions modulated motility efficiency, sequence variation in FimT alone could not account for the observed phenotypic diversity. Collectively, these findings reveal extensive heterogeneity in T4P-mediated surface motility in A. baumannii and identify delayed twitching activation and distinct collective migration strategies as key features of surface colonization in this species.

Antimicrobial resistance (AMR) exacerbates bacteraemia in cancer patients, particularly in low-resource settings. At the Uganda Cancer Institute, high rates of Enterobacterales producing extended-spectrum {beta}-lactamases (ESBLs) have been reported, with DNA-based detection of bla genes limited to PCR. This study aimed to determine whether bacterial genomic DNA shipped at ambient temperature from Uganda to the UK retained sufficient quality for whole-genome sequencing (WGS), to allow in-depth genomic analyses of isolates. Genomic DNA was extracted from Gram-negative bloodstream isolates (n=77) in Uganda and shipped to the UK at ambient temperature. rpoB gene (77/77, 100%) and WGS data (72/77, 93.5%) were generated for isolates, with 66/72 (91.7%) genomes of high-quality (Escherichia coli n=34; Klebsiella spp. n=32). Bioinformatic analyses included species identification, sequence typing, SNP analysis, AMR and virulence gene profiling, and comparison with publicly available genomes of Ugandan isolates. Phenotypic-genotypic concordance was generally high: 7/77 (9.1%) isolates were misidentified by phenotypic testing, and two showed unexplained carbapenem resistance. E. coli isolates showed diverse sequence types, with high prevalence of blaCTX-M (91.2%) and blaOXA-1 (47.1%); carbapenemase genes were rare. Klebsiella isolates lacked hypermucoidy loci and displayed diverse capsule types, with a high prevalence of ESBLs. Genomic clustering suggested limited within-hospital transmission of strains. Genomic data can provide important insights into the dissemination of bacterial subclades of global concern. The widespread AMR genotypes reported here highlight the need for improved diagnostics and updated treatment guidelines for bacteraemia in Ugandan cancer patients.

Bacteria in natural environments frequently encounter nutrient limitation leading to growth arrest and must balance the potential benefits of continuing to respond to the environment by making new proteins against the costs of depleting limited resources. We previously showed that the RNA polymerase-binding regulator SutA enhances transcription of hundreds of genes during nutrient limitation in Pseudomonas aeruginosa, suggesting that it might be part of a regulatory network facilitating limited new protein synthesis. Here, we sought to expand our understanding of this network by identifying transcriptional regulators influencing sutA expression. Using northern blotting, western blotting, and reporter assays, we found that the sigma factors FliA and RpoS, and the DNA-binding regulator Lrp, impact expression from a proximal sutA promoter during the transition to stationary phase. This constellation of regulators and the dynamics of SutA expression lead us to propose that SutA is part of a regulatory network that facilitates scavenging. Scavenging includes motility toward possible nutrient sources and uptake mechanisms for these nutrients, activities which require an investment of resources but can yield important benefits during starvation. In vitro transcription experiments, proteomic analysis and reporter assays suggest that SutA directly supports new protein synthesis driven by RpoS and indirectly supports flagellar motility, perhaps by helping maintain protein biosynthetic capacity against the metabolic costs of motility. SutA expression is controlled by multiple regulatory inputs, including negative autoregulation, and the protein appears to be short-lived. These properties are consistent with a role in supporting short, controlled bursts of gene expression during nutrient limitation. Author StatementMany bacteria engage in cycles of colonising a nutrient-rich location, using the available nutrients, and then dispersing in search of a new location to colonise. While searching for new nutrients in a low-resource environment, bacteria will be starved and must coordinate resource-intensive processes such as new protein synthesis, motility, and nutrient uptake so that each crucial activity can be accomplished but none use too much of the limited pool of resources. We previously identified a regulator in Pseudomonas aeruginosa called SutA, which facilitates new protein synthesis under starvation conditions. Here, we have identified regulators of SutA expression. We find that the housekeeping sigma factor RpoD drives expression during growth, but at the entry to stationary phase, where SutA has obvious impacts on cellular physiology, the stress sigma factor RpoS, the flagellar sigma factor FliA, and the amino acid sensing transcription factor Lrp are important. Finally, we find that all cells in a nutrient-limited population express some SutA, but appear to do so in infrequent bursts, and that the protein is likely unstable. Together, these findings suggest that SutA contributes to the coordination of resource use while bacteria scavenge for new nutrients, facilitating limited amounts of new protein synthesis.

The horizontally acquired mgtC gene from Salmonella enterica confers this bacterium the abilities to survive episodes of magnesium (Mg2+) starvation, and to replicate in mammalian macrophages. The former property allows bacteria to persist in the environment through periods of Mg2+ depletion, whereas the latter allows S. enterica to overcome self-limiting intestinal colonization and cause an invasive systemic infection in susceptible mammalian hosts. Even though the biochemical function of MgtC is not completely understood, this protein is thought to function primarily by preventing the production of toxic levels of Mg2+-chelating adenosine triphosphate (ATP). In the current work, we investigated the physiological roles of mgtC homologs from an array of bacterial species, by probing the processes controlled by this gene during replication in low Mg2+ medium and in macrophages. We determined that MgtC homologs that do not participate in Pi homeostasis during Mg2+ starvation and do not promote intramacrophage replication in their resident species can partake in these processes when expressed in S. enterica. This indicates that the function of this protein is context dependent. Accordingly, we show that the physiological processes affected by S. enterica MgtC vary, depending on whether the bacteria replicate in low Mg2+ medium or inside macrophages. While these results suggest that MgtC is a regulator, they also demonstrate that horizontally acquired genes can assume different roles, depending on the genome and the biochemical context into which they are inserted. ImportanceThe mgtC gene encodes an inner membrane protein that has been horizontally acquired by multiple bacterial species, including several mammalian pathogens. In Salmonella enterica, MgtC promotes replication in mammalian macrophages and allows this bacterium to survive cytoplasmic magnesium (Mg2+) starvation. These phenotypes are thought to result from MgtCs inhibition of Pi metabolism and ATP production, which prevents the accumulation of toxic levels of Mg2+-chelating ATP and disrupts other physiological processes that are strictly dependent on Mg2+, such as ribosome assembly and translation. In the current study, we show that processes that are controlled by MgtC vary with the genetic and biochemical contexts in which this protein is expressed. While establishing a broader role for MgtC as a regulator, our findings illustrate how horizontally acquired regulatory genes can potentiate regulatory interactions, facilitating the evolution of new traits.

Genome-wide transposon insertion sequencing (Tn-seq) is a powerful tool to measure the importance of genes for growth. In this study, we applied Tn-seq to the Gram-positive model system Bacillus subtilis, and found that after growth in liquid medium the transposon library lacked transposon insertions in several genes related to lipoteichoic acid biosynthesis and cell wall teichoic acid modification. This was unexpected since these genes are not essential for normal growth. By growing the transposon library as a confluent layer of cells, and as discrete colonies, we found that these genes are only important when the transposon library is grown as a confluent layer. Apparently, growing the transposon library as a mixed population reduces the fitness of teichoic acid mutants, which was confirmed by coculturing experiments. This phenomenon can be explained when lipoteichoic acid and teichoic acid D-alanylation mutants become sensitive to secreted autologous antimicrobials and/or toxins. Extensive mutant analyses suggested that multiple autologous antimicrobials are involved. Finally, we show that the reduced fitness of teichoic acid mutants can be countered by the addition of divalent cations. These data raise several questions concerning the evolution of kin discrimination, and show that growing genome-wide mutant libraries as mixed cultures can influence library composition.

Lung transplantation (LT) is the ultimate treatment option for patients suffering from end stage cystic fibrosis (CF). Most LT-patients, colonized pre-LT by Pseudomonas aeruginosa witness colonization of their non-CF allograft within a few days or weeks post-LT, thereby compromising graft and life expectancy. How P. aeruginosa isolates adapted for years to the specific CF lung environment efficiently colonize and survive in the non-CF allograft environment remains unclear. To address this question, we collected sequential isolates from CF LT-recipients and non-CF LT-recipients and performed phenotypic and genetic analyses of pairs of early and late isolates from LT-patients. We found evidence for mutations compatible with a switch from biofilm to planktonic lifestyle as well as loss of mucoid phenotypes. Hypermutators, characteristic of chronic CF-adapted isolates, were also found in four LT-patients. Their persistence in the non-CF allograft environment suggests a continuous seeding from the sinuses. Our results suggest that in CF LT-recipients efficient colonisation by P. aeruginosa of the allograft implies both adaptation and continuous seeding from the sinuses to the lower respiratory tract.

On their own, probiotics do not entirely eliminate pathogens during infection and disease. Instead, they often serve as adjuncts that rely on distinct mechanisms to reduce the presence of harmful bacteria. Our prior research indicates that an isolated Aeromonas dhakensis strain A603 kills pathogenic Vibrio strains through two antibacterial mechanisms: the type VI secretion system (T6SS) and phenazine (AdPhen). Here, we investigate A603 as a standalone probiotic for a shrimp disease model. This study shows that A603 prevents mortality from acute hepatopancreatic necrosis disease (AHPND) in shrimp caused by pathogenic Vibrio spp. that produce the PirAB toxin. AHPND infection alters the shrimp microbiota by increasing pathogen abundance and decreasing beneficial bacterial abundance prior to death. As both a prophylactic and treatment, A603 removes pathogenic Vibrio from shrimp and reverses such alterations in the microbiota using both T6SS and AdPhen. Collectively, our findings show that A603 antibacterial mechanisms prevent AHPND.

Iron is an essential micronutrient that shapes host-pathogen interactions during infection. However, the contribution of iron to the virulence adaptation of the Enterobacter cloacae complex (ECC) remain poorly characterized. This study profiled the effects of iron on E. roggenkampii and E. asburiae clinical isolates. Growth kinetics were assessed in Luria-Bertani broth supplemented with varying iron concentrations and 5% sheep blood, and EDTA. Recovered strains were used for motility and antibiotic susceptibility assays. Phenotypic virulence trait of iron-naive and iron-recovered strains was determined using biofilm formation assays. Whole-genome sequencing was conducted to identify genetic determinants associated with iron acquisition and metabolism. Presence of iron increased bacterial growth, reduced antibiotic susceptibility, and enhanced biofilm formation. At higher iron concentrations, iron-recovered strains exhibited increased biofilm biomass, while there was a high biofilm formation with iron-naive strains at lower iron levels. Genomic analysis identified genes associated with ferrous and ferric iron transport, heme uptake, siderophore biosynthesis, and virulence-related functions, including adhesion and biofilm formation. These findings demonstrate that iron availability and prior exposure modulate ECC physiology and phenotypic traits associated with virulence, supporting a role for iron in shaping adaptive pathogenic potential. Graphical AbstractThe influence of iron metabolism on virulence adaptation of Enterobacter cloacae complex O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/737523v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@aa351eorg.highwire.dtl.DTLVardef@855345org.highwire.dtl.DTLVardef@11e0da5org.highwire.dtl.DTLVardef@11f851_HPS_FORMAT_FIGEXP M_FIG C_FIG

Probiotics produce antimicrobial peptides and small molecules that are secreted into the medium. Antimicrobial activity of cell-free supernatants can be tested using various qualitative and quantitative methods. Many of these techniques employ methods which introduce uncontrolled variables, impacting reproducibility and making comparison of reported results difficult. Here, we present a simple procedure for quantitative estimation of antimicrobial activity of cell-free supernatants which overcomes drawbacks of commonly used methods.

Hand eczema has been described as having an increased prevalence in persons with increased frequency of hand washing. This study investigated the differences in the hand microbiome of persons with and without a history of eczema and secondly the sensitivity of these microbes to commercial liquid soap as a potential trigger for eczema flares. The study identified Staphylococcus to be the most populus genus on the hands in both groups, but the distribution of species was different. Additionally, there was no difference in the number of soaps that produced zones of inhibition but there were some differences in the overall sensitivity to the different soaps tested. Overall, it was determined that liquid soap can cause bactericidal effects on some species of the commensal microbiome, but further work is required to determine if this could be the cause of hand eczema.

Background Colonisation resistance provided by the gut microbiota is a critical barrier to pathogen invasion, yet its study in vivo is constrained by the complexity and cost of vertebrate models. Here, we developed a humanised Galleria mellonella infection model by inoculating wax moth larvae with complex human faecal microbiota. 16S rRNA gene sequencing confirmed stable, reproducible establishment of a diverse human associated community across larvae over four days. Results Humanised larvae exhibited colonisation resistance against Salmonella enterica serovar Typhimurium, with mortality reduced to 20% compared to 90% in non colonised controls. To test whether prophages could overcome this barrier, we infected larvae with isogenic S. Tm strains differing in the presence of prophage P22. Infection with the P22 carrying strain resulted in a threefold higher larval mortality (60% vs. 20%), increased pathogen load, and a significant reduction in the abundance of resident E. coli. Free P22 virions were detected early after infection, indicating extensive prophage activity. Notably, P22 can neither adsorb nor lyse resident E. coli, indicating that prophage mediated invasion success did not rely on direct lysis. Instead, using high throughput metabolic profiling paired with whole genome sequencing of three replicate lineages, we found that phage activation intensified resource partitioning, accelerating functional metabolic adaptations in E. coli that significantly reduced the niche overlap between the invading pathogen and the commensal E. coli. Conclusion Our findings establish the first humanised G. mellonella model supporting complex human microbiota and provide a novel non lytic mechanism by which prophages influence species interactions. This scalable, low cost model offers a new platform to dissect pathogen phage microbiota interactions relevant to human gut ecology.

Background: Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. Results: Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. Conclusions: The E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

Background: Antimicrobial resistance (AMR) is a global health crisis, necessitating alternative antibacterial strategies. Bacteriophages (phages) offer a promising solution, and their use as a therapeutic agent relies on stringent bioinformatic characterisation using whole genome sequencing (WGS) technologies. However, phages are highly diverse, with no clear consensus on best practices concerning phage DNA extraction or sequencing platform. Efficient and repeatable DNA extraction, sequencing, and bioinformatics processes are critical for safety assessments but remain poorly defined. Additionally, the impact of sequencing platform choice and DNA extraction methods on downstream genomic analyses is not well understood. Methods: We evaluated multiple DNA extraction, library preparation, and sequencing approaches using a diverse collection of Pseudomonas phages from the PhageWA biobank. Column-based and precipitation-based DNA extraction methods were compared for DNA yield and recovery efficiency. Genome sequencing was performed using short-read (Illumina) and long-read (Oxford Nanopore Technologies) platforms, incorporating multiple library preparation kits and Nanopore basecalling models. Assemblies were assessed for completeness, quality, and sequence concordance using standardised bioinformatics pipelines, with hybrid Illumina-Nanopore assemblies used as references for comparison. Results: DNA extraction efficiency varied substantially between protocols, with the Puregene precipitation-based method yielding significantly higher DNA recovery than column-based approaches when normalised to phage titre. Illumina sequencing consistently generated complete genome assemblies, although assembly fragmentation was observed for several jumbo phages when using the SeqWell ExpressPlex 2.0 library preparation method. For Nanopore sequencing, ligation-based native barcoding libraries produced longer reads than rapid barcoding libraries, while selection of the Dorado v5.0.0 basecalling model significantly improved read quality. Genome assembly success was dependent on phage genus; native Nanopore sequencing failed to assemble several Pbunavirus genomes, likely due to modified DNA bases, but an amplification-based library preparation successfully resolved these genomes. Across successfully assembled samples, Illumina and Nanopore platforms produced highly concordant genomes with comparable completeness scores, and hybrid polishing identified only minor sequence differences. Conclusions: DNA extraction methodology, sequencing chemistry, and basecalling model selection significantly influence phage WGS outcomes. Precipitation-based DNA extraction improved DNA recovery, while both Illumina and Nanopore sequencing generated high-quality phage genomes suitable for therapeutic characterisation. Nanopore sequencing provided assemblies comparable to Illumina with minimal benefit from hybrid polishing, supporting its routine use for phage genomics. These findings provide practical guidance for phage genome characterisation workflows and contribute to the development of standardised, regulatory-grade approaches for therapeutic phage assessment.

Aims: Antimicrobial peptide (AMP) combinations have been proposed to delay resistance evolution, but it remains unclear what properties of a peptide pair determine whether a combination reduces resistance evolution relative to its component AMPs used alone. One suggested factor is mode of action, yet this has rarely been tested experimentally. In the current study we have asked whether mode of action or physicochemical similarity between peptides better predicts which combinations delay resistance. Methods: We evolved Staphylococcus aureus with six AMPs with reported membrane-targeting and intracellular-targeting activity, individually and in all 15 pairwise combinations. We quantified resistance evolution, cross-resistance and fitness costs across the full AMP panel, and performed whole-genome sequencing on 126 evolved lineages. Results: Resistance varied across AMPs and correlated with peptide chain length, not mode of action. Cross-resistance was associated with physicochemical similarity, and similar peptides selected for overlapping mutations. Most combinations reduced resistance relative to single-AMP treatment, but those whose components shared cross-resistance were less effective, channeling evolution into convergent trajectories that resolve both selective pressures at once. Notably, mode of action did not predict combination outcome. Conclusions: Cross-resistance, not mode of action, is a key factor in determining AMP combination efficacy. Physicochemical distance between peptides may serve as a practical predictor for cross-resistance, enabling selection of AMP combinations that are more likely to constrain resistance evolution.

Bacteria frequently face challenges adapting to changing environmental conditions to survive and thrive, such as shifting resource utilization. Methylotrophs capable of growth on reduced single-carbon compounds are prevalent in the phyllosphere (aerial plant surfaces), where they face continual and predictable shifts in the availability of different plant-produced carbon sources. We examined the ability of the methylotroph Methylobacterium extorquens PA1 to adapt to repeated shifts between two different carbon and energy sources: the one-carbon compound methanol and the multi-carbon organic acid succinate, both present in the phyllosphere. Evolved lineages of wild-type cells all increased their capacity for rapid transition between the carbon sources through high frequencies of loss-of-function mutations affecting a previously uncharacterized gene, named cstR for carbon source transition regulator, which encodes an orphan single-domain response receiver. Characterization showed that mutant strains were more competitive bidirectionally in the succinate-methanol transition. Though evolved populations of the {Delta}efgA and {Delta}ttmR strains, which are defective in the succinate-to-methanol transition, experienced similar phenotypic improvements in carbon-source transitions, we did not observe cstR mutations rise to prominence as extensively or frequently in these lineages. Transcriptomic work revealed loss-of-function to cstR impacted expression of genes involved in motility/chemotaxis, energy metabolism, and stress response, among others, suggesting that it coordinates responses to metabolic cues that are prevalent in certain carbon source and growth phase transitions. Loss of cstR function did not compromise exogenous formaldehyde tolerance in the {Delta}efgA and {Delta}ttmR mutants, breaking a previously described tradeoff between these two phenotypes. However, this loss did lead to defects under exposure to certain stressors, including heat, desiccation, oxidative stress agents, and particularly pH stress. Altered levels of NAD+/NADH across conditions, improved growth under acidic pH, and diminished ATP and increased mortality under heightened pH together support a model where CstR is responsible for coordinating cell signaling to manage the balance between growth and maintaining stress resilience.