ObjectiveTo identify ways of improving antimicrobial stewardship at Kakamega County Teaching and Referral Hospital (KCTRH) in western Kenya.MethodsA knowledge and awareness test was conducted among surgical ward healthcare workers to assess their understanding of how to counter antimicrobial resistance. In parallel, the global Point Prevalence Survey (PPS) was used to understand antibiotic prescription practices at the referral facility. Patient records from the adult surgical and pediatric surgical wards were examined for information on antibiotic prescription and usage. A total of 47 healthcare workers and 106 patient records were examined.ResultsHealthcare workers showed the highest competence when tested on the appropriate use of antimicrobials, with a mean score of 78.7%. However, their understanding of surveillance and monitoring was lower, with a mean score of 60.2%, followed by awareness and education (which achieved a mean score of 64.5%). The majority of prescriptions (62.3%) were for empiric treatment, with a lack of laboratory data to support targeted therapies. Prolonged antibiotic courses were noted for surgical prophylaxes with over 83% of patients being administered antibiotics for more than 1 day. The survey also revealed over-prescription of broad-spectrum agents such as ceftriaxone at 36.7%.ConclusionThere is a high use of antibiotics, but a weakness in AMR monitoring at KCTRH. Better use of laboratory testing (AMR profiles for infecting bacteria, etc) is recommended as the best way to improve patient management and deployment of antibiotics.

Pseudomonas aeruginosa is an important cause of surgical site infections (SSIs) and is characterized by extensive antimicrobial resistance and genomic plasticity. We analyzed 13 whole-genome sequences of clinical P. aeruginosa isolates recovered from SSIs at a Level 5 referral hospital in Western Kenya and integrated genomic analyses with antimicrobial susceptibility tests and phenotyping of quorum-sensing (QS). Multidrug resistance was common, with resistance observed primarily against ciprofloxacin, piperacillin, ceftazidime, and amikacin, whereas meropenem and piperacillin/tazobactam retained the greatest in vitro activity. Genomic analyses identified diverse sequence types, extensive variability in insertion sequences and genomic islands, and widespread conservation of intrinsic resistance determinants, whereas accessory antimicrobial resistance genes were infrequently detected and largely associated with genomic islands. Virulence-associated genes were predominantly chromosomal, although several showed strain-specific genomic island localizations. Functional QS analyses demonstrated that isolates with intact lasR and rhlR produced significantly higher levels of acyl-homoserine lactone signals than isolates carrying predicted loss-of-function mutations, whereas Pseudomonas quinolone signal production remained comparatively conserved. These findings provide a genomic baseline for P. aeruginosa causing SSIs in Western Kenya and highlight the importance of integrating whole-genome sequencing with phenotypic analyses to strengthen surveillance and inform antimicrobial stewardship and infection prevention strategies.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen, often associated with airway infections in the lungs of people with cystic fibrosis (CF). Biofilms of many P. aeruginosa strains, such as PAO1, produce copious quantities of filamentous Pf4 phage. High titres of Pf4 phage are associated with a decline in lung function in people living with CF. We previously showed that genes encoding Pf4 and also an unlinked hypothetical protein, PA3572, form part of the core biofilm-associated transcriptome in PAO1. Here, using reporter gene assays, we confirm that PA3572 (which we designate difP - depresses induction of filamentous Pf4 prophage) is strongly induced in biofilms of PAO1. Transcriptomic analysis of a difP deletion mutant revealed elevated expression of several Pf4 ORFs, as well as elevated expression of the cell envelope stress-associated protein, CpxP. These modulations were confirmed by quantitative reverse transcription-PCR, indicating that DifP functions to depress Pf4 gene expression. Consistent with this, Pf4 phage titres were elevated in a ΔdifP mutant, whereas over-expression of difP depressed Pf4 titres and Pf4 gene expression. This effect of difP was abolished in a cpxP mutant, indicating that DifP-dependent regulation of Pf4 production is likely linked with cell envelope stress sensing. Taken together and by contrast with most other regulators of Pf4 identified to date (which promote Pf4 production in biofilms), our data indicate that DifP plays a role in restraining Pf4 production in PAO1 biofilms.

Background The airways of people with cystic fibrosis (pwCF) are often colonized by a variety of different microbes. Although much effort has been put into cataloguing the impact of medication on the identities and abundances of these microbes, far less has been directed towards examining this from an ecological perspective, i.e., examining how medications affect the network and types of interactions between microbes. Methods In the current work, we generated an ecological model of the CF airway microbiome and examined how medications affect interactions between co-habiting airway microbiota in six pwCF. Ecological interactions were inferred from a generalized Lotka-Volterra model, and the impact of medications was determined by principal component(s) regression analysis. Results For the majority of the subjects studied, antimicrobial interventions had relatively little impact on the CF airway microbial ecology, and even appeared to stabilize ecological interactions between the microbiota. However, the microbial ecosystem in some individuals was more sensitive to external perturbations. More surprisingly, we found that some non-antimicrobial medications, and also certain carriers and excipients affect the ecosystem. Conclusions Medications affect the ecology of the CF airway microbiota. These impacts appear to be very patient-specific. We also note that some nominally non-bioactive ingredients in medications can also potentially impact the CF airway ecosystem. Our data highlight the importance of collecting patient-specific data and in employing suitable computational frameworks for disentangling medication-microbiota interactions in vivo.

Pseudomonas aeruginosa is an opportunistic human pathogen. One of the most potent virulence factors in its arsenal is the type III secretion system (T3SS). This secretion apparatus injects effector toxins directly into host cells, thereby causing cytotoxicity. The expression of all components of T3SS is regulated by a master transcriptional regulator, ExsA. The inhibition of the latter should therefore lead to the suppression of P. aeruginosa virulence. However, to date, no drugs targeting ExsA have reached the market, and only static structural models of the protein have been generated, focusing on the C-terminal domain (CTD). Here, we used μs atomistic molecular dynamics (MD) simulations to investigate the conformational dynamics of full-length ExsA bound to DNA or DNA free, investigated as monomers or dimers. Our data show how the CTD and NTD of ExsA likely interact with one another and how ExsA binds to DNA. We also analyzed the MD trajectories to predict potential druggable pocket(s) in the structure and relevant geometry. This revealed a lipid-binding pocket within the β-sheet bundle and identified two novel potentially druggable pockets at the NTD/CTD interface, which could be used in future structure-based drug discovery campaigns. Overall, a single helix-turn-helix motif seems to drive DNA recognition in each ExsA monomer and to stabilize the putative ligand-binding domain.

Pseudomonas aeruginosa is an opportunistic pathogen, commonly associated with human airway infections. Based on its amino acid sequence similarity with Pyrococcus furiosus protease I, P. aeruginosa PfpI was originally annotated as an intracellular protease. In this work, we show that PfpI is a methylglyoxalase. The X-ray crystal structure of the purified protein was solved to 1.4 Å resolution. The structural data indicated that PfpI shares the same constellation of active site residues (including the catalytic Cys112 and His113) as those seen in a well-characterized bacterial methylglyoxalase from Escherichia coli, YhbO. Using NMR, we confirmed that PfpI qualitatively converted methylglyoxal into lactic acid. Quantitation of lactate produced by the methylglyoxalase activity of PfpI yielded a kcat of 102 min-1 and a KM of 369 μM. Mutation of Cys112 and His113 in PfpI led to complete loss of methylglyoxalase activity. To investigate the functional impact of PfpI in vivo, a ΔpfpI deletion mutant was made. Quantitative proteomic analyses revealed a pattern of changes consistent with perturbation of ribosomal function, Zn2+ limitation, C1 metabolism, and glutathione metabolism. These findings are consistent with PfpI being a glutathione-independent methylglyoxalase. Previously, transposon insertion (pfpI::Tn) mutants have been reported to exhibit phenotypes associated with antibiotic resistance, motility, and the response to oxidative stress. However, the ΔpfpI mutant generated in this study displayed none of these phenotypes. Whole-genome sequencing of the previously described pfpI::Tn mutants revealed that they also contain a variety of other genetic changes that likely account for their observed phenotypes.

The bodies of macroorganisms host microbes living in multispecies communities. Sequencing approaches have revealed that different organs host different microbiota and tend to be infected by different pathogens, drawing correlations between environmental parameters at the organ level and microbial composition. However, less is known about the microscale dimension of microbial ecology, particularly during infection. In this study, we focus on the role of microscale spatial structure, studying its influence on the ecology of a polymicrobial infection of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. Although these pathogens are commonly found together in the lungs of chronically ill patients, it is unclear whether they coexist or compete and segregate in different niches. We find that, whereas P. aeruginosa quickly outcompetes C. albicans and S. aureus on large surfaces, robust spatial organization and coexistence emerges in spatially structured microenvironments. In confined spaces, slowly growing C. albicans is able to leverage rapid radial hyphal growth to conquer boundaries, where it establishes itself displacing the other pathogens. Similar outcomes are observed when the P. aeruginosa strain carries mexT-inactivating mutations, which are often found in clinical isolates. The observed spatial organization enables coexistence and potentially determines infection severity and outcomes. Our findings reveal a previously unrecognized role of mechanical forces in shaping infection dynamics, suggesting that microenvironmental structure might be a critical determinant of pathogen coexistence, virulence, and treatment outcomes. Because adaptations, such as changes in morphology, are widespread among microbes, these results are generalizable to other ecologies and environments.

Pseudomonas aeruginosa is an opportunistic pathogen, commonly associated with the airways of people with cystic fibrosis (CF) and in the wider environment too. In this work, we interrogate the International Pseudomonas Consortium Database (IPCD) to ask the question of whether CF-associated isolates display different patterns of evolutionary selection compared with environmental isolates. We do this by analysing dN /dS for each open reading frame (ORF) in the CF-associated and environmental IPCD isolates. Most ORFs displayed a pronounced signature of negative selection (i.e. the ORFs were strongly conserved). However, 373 ORFs displayed non-negative selection, and of these, 206 manifested differential signatures of selection in the CF-derived and environmental isolates. Functional analysis of the ORFs under selection pressure in the CF airways revealed a statistically significant enrichment of enzymes catalysing reactions at metabolic branchpoints. More fine-grained analyses revealed niche-specific selection pressures in individual domains and protein surfaces. Finally, we show that gene loss in the psl biosynthetic gene cluster correlates with the presence of loss-of-function mutations in the mucoidy regulator, mucA. We speculate that elevated alginate production due to these mucA mutations compensates for the loss of Psl production in these isolates.

Metal ions with well-defined coordination geometries can serve as fixed joints within self-assembled architectures, defining the relative orientations of ligands within higher-order superstructures. The exchange of ligands and metal ions between different positions is slow, involving disruption or distortion. Here we report a series of AgI12X4L6 3 × 3 metal-organic grid-like structures, where the core AgI12X4 nanocluster is in dynamic motion, with AgI ions moving between different binding sites, with concomitant conformational changes of the organic ligands, which continue to occupy well-defined positions nevertheless. The identity of the incorporated halide anion governs the activation barrier for silver ion exchange, thus enabling rate control in response to two distinct stimuli: by changing the temperature, and by exchanging one halide for another. The dynamic nanocluster within these grids thus provides a new mode of using metal ions in coordination-driven self-assembly, establishing that the mobile AgI ions behave in similar ways to Ag0 atoms in surface-bound clusters and in silver nanoparticles. The kinetic parameters determined in this work, and the techniques developed to measure them, could serve the scientific community to provide additional insight into dynamic metal nanoclusters.

The airways of people with cystic fibrosis (CF) are often co-infected by Pseudmonas aeruginosa and a variety of other co-habiting microbes; the infections are polymicrobial. P. aeruginosa isolates from the CF airways are also known to commonly acquire mutations in the quorum sensing regulator, lasR . The appearance of these lasR mutants is associated with a worsening clinical prognosis. In this work, we show that loss of lasR function has a significant impact on the stability of inter-species interactions in a polymicrobial ecosystem, and in particular on the dynamics of a common CF-associated fungus, Candida albicans . Titres of C. albicans were stable in the presence of wild type P. aeruginosa and Staphylococcus aureus . However, when wild type P. aeruginosa was replaced by a Δ lasR mutant, C. albicans titres progressively declined over time. This instability could be reversed by ectopic expression of a Type VI Secretion System effector cluster ( tsi ) in the Δ lasR mutant. We also noted that challenge of the polymicrobial cultures with a clinically-relevant combination of antibiotics (colistin and ciprofloxacin) led to a hyphal bloom of the fungus. This bloom was abolished in the Δ lasR mutant, but again, was restored by ectopic expression of the tsi cluster. Finally, we show that whereas wild type P. aeruginosa is relatively agnostic to the presence of other microbes, the Δ lasR mutant is not and undergoes substantial transcriptional reprogramming. Our data indicate that lasR has a large and previously unrecognised impact on inter-species interactions. We therefore propose that lasR is an ecological keystone gene. ### Competing Interest Statement The authors have declared no competing interest. Cystic Fibrosis Trust, https://ror.org/03xeae684, Venture and Innovation Award BBSRC

Antimicrobial resistance attracts a considerable amount of attention as it threatens the efficiency of current antibacterial treatments. Besides a more considerate use of current antibiotics to slow down the spread of antimicrobial resistance, there is ample need for new therapeutic avenues to treat already resistant strains. Here, we describe the use of a cleavable peptide-drug conjugate to target bacteria with diverse resistance strategies. The conjugate consists of three main components: a β-lactamase cleavable linker, a positively charged stapled antimicrobial peptide, and an antibiotic. The linker ensures selective cleavage and provides the prospect of lowering systemic toxicity of the conjugate. The positively charged peptide targets the negatively charged bacterial membrane, and stapling pre-organises it in a helical structure. Finally, the drug provides another, distinct mode of action to the peptide, which should overall reduce the development of resistance. A series of peptides was prepared and the most promising one was then developed into a stapled conjugate. The factors affecting the activity of this conjugate were investigated, proving cleavage by β-lactamase and superior potency compared to the non-cleavable control, as shown by its minimal inhibitory concentrations.

Pseudomonas aeruginosa is a model for the study of quorum sensing, protein secretion, and biofilm formation. Consequently, it has become one of the most intensely reviewed pathogens, with many excellent articles in the current literature focusing on these aspects of the organism's biology. Here, though, we aim to take a slightly different approach and consider some less well appreciated (but nonetheless important) factors that affect P. aeruginosa virulence. We start by reminding the reader of the global importance of P. aeruginosa infection and that the "virulome" is very niche-specific. Overlooked but obvious questions such as "what prevents secreted protein products from being digested by co-secreted proteases?" are discussed, and we suggest how the nutritional preference(s) of the organism might dictate its environmental reservoirs. Recent studies identifying host genes associated with genetic predisposition towards P. aeruginosa infection (and even infection by specific P. aeruginosa strains) and the role(s) of intracellular P. aeruginosa are introduced. We also discuss the fact that virulence is a high-risk strategy and touch on how expression of the two main classes of virulence factors is regulated. A particular focus is on recent findings highlighting how nutritional status and metabolism are as important as quorum sensing in terms of their impact on virulence, and how co-habiting microbial species at the infection site impact on P. aeruginosa virulence (and vice versa). It is our view that investigation of these issues is likely to dominate many aspects of research into this WHO-designated priority pathogen over the next decade.

The bodies of macroorganisms host microbes living in multi-species communities. Sequencing approaches have revealed that different organs host different microbiota and tend to be infected by different pathogens, drawing correlations between environmental parameters at the organ level and microbial composition. However, less is known about the microscale dimension of microbial ecology, particularly during infection. In this study, we focus on the role of microscale spatial structure, studying its influence on the ecology of a polymicrobial infection of P. aeruginosa, S. aureus and C. albicans. Although these pathogens are commonly found together in the lungs of chronically ill patients, it is unclear whether they coexist or compete and segregate in different niches. We find that, while P. aeruginosa quickly outcompetes C. albicans and S. aureus on large surfaces, robust spatial organization and coexistence emerges in microfluidic microchambers that mimic the spatial characteristics of alveoli. In these microenvironments, slowly growing C. albicans is able to leverage fast eccentric hyphal growth to conquer boundary spaces, where it establishes itself excluding the other pathogens. We show that the emerging spatial patterning is robust to changes in the virulence of the community, enabling coexistence and potentially determining infection severity and outcomes. Our findings reveal a previously unrecognized role of mechanical forces in shaping infection dynamics, suggesting that microenvironmental structure is a critical determinant of pathogen coexistence, virulence, and treatment outcomes. Because adaptations, such as changes in morphology, are widespread among microbes, these results are generalizable to other ecologies and environments. ### Competing Interest Statement The authors have declared no competing interest.

Fatty acids are a primary source of carbon for Pseudomonas aeruginosa (PA) in the airways of people with cystic fibrosis (CF). Here, we use tandem mass-tag proteomics to analyse the protein expression profile of a CF clinical isolate grown on different fatty acids. Two fatty acyl-CoA dehydrogenases (designated FadE1 and FadE2) are strongly induced during growth on fatty acids. FadE1 displays a strong preference for long-chain acyl-CoAs, whereas FadE2 exclusively utilizes medium-chain acyl-CoAs. Structural analysis of the enzymes enables us to identify residues comprising the substrate selectivity filter in each. Engineering these residues enables us to invert the substrate specificity of each enzyme. Mutants in fadE1 displayed impaired virulence in an infection model, and decreased growth on long chain fatty acids. The unique features of the substrate binding pocket enable us to identify an inhibitor that is differentially active against FadE1 and FadE2.

Fluorescent pseudomonads catabolize purines via uric acid and allantoin, a pathway whose end-product is glyoxylate. In this work, we show that in Pseudomonas aeruginosa strain PAO1, the ORFs PA1498-PA1502 encode a pathway that converts the resulting glyoxylate into pyruvate. The expression of this cluster of ORFs was stimulated in the presence of allantoin, and mutants containing transposon insertions in the cluster were unable to grow on allantoin as a sole carbon source. The likely operonic structure of the cluster is elucidated. We also show that the purified proteins encoded by PA1502 and PA1500 have glyoxylate carboligase (Gcl) and tartronate semialdehyde (TSA) reductase (GlxR) activity, respectively, in vitro. Gcl condenses two molecules of glyoxylate to yield TSA, which is then reduced by GlxR to yield d-glycerate. GlxR displayed much greater specificity (k cat/KM) for Gcl-derived TSA than it did for the TSA tautomer, hydroxypyruvate. This is relevant because TSA can potentially spontaneously tautomerize to yield hydroxypyruvate at neutral pH. However, kinetic and [1H]-NMR evidence indicate that PA1501 (which encodes a putative hydroxypyruvate isomerase, Hyi) increases the rate of the Gcl-catalysed reaction, possibly by minimizing the impact of this unwanted tautomerization. Finally, we use X-ray crystallography to show that apo-GlxR is a configurationally flexible enzyme that can adopt two distinct tetrameric assemblies in vitro.

The 2-methylcitrate cycle and the glyoxylate cycle are central metabolic pathways in Pseudomonas aeruginosa, enabling the organism to utilize organic acids such as propionate and acetate during infection. Here, we show that these cycles are linked through enzymatic redundancy, with isocitrate lyase (AceA) exhibiting secondary 2-methylisocitrate lyase activity. Furthermore, we use a combination of structural analyses, enzyme kinetics, metabolomics, and targeted mutation of PrpBPa to demonstrate that whereas loss of PrpB function impairs growth on propionate, the promiscuous 2-methylisocitrate lyase activity of AceA compensates for this by mitigating the accumulation of toxic 2-methylcitrate cycle intermediates. Our findings suggest that simultaneous inhibition of PrpB and AceA could present a robust antimicrobial strategy to target P. aeruginosa in propionate-rich environments, such as the cystic fibrosis airways. Our results emphasize the importance of understanding pathway interconnections in the development of novel antimicrobial agents.

We describe a versatile and tuneable thiol responsive linker system using thiovinylketones, which relies on the conjugate addition-elimination mechanism of Michael acceptors for the traceless release of therapeutics. In a proof-of-principle study, we translate our findings to exhibit potent thiol-cleavable antibiotic prodrugs and antibody-drug conjugates.

Over the last two centuries, great advances have been made in microbiology as a discipline. Much of this progress has come about as a consequence of studying the growth and physiology of individual microbial species in well-defined laboratory media; so-called "axenic growth". However, in the real world, microbes rarely live in such "splendid isolation" (to paraphrase Foster) and more often-than-not, share the niche with a plethora of co-habitants. The resulting interactions between species (and even between kingdoms) are only very poorly understood, both on a theoretical and experimental level. Nevertheless, the last few years have seen significant progress, and in this review, we assess the importance of polymicrobial infections, and show how improved experimental traction is advancing our understanding of these. A particular focus is on developments that are allowing us to capture the key features of polymicrobial infection scenarios, especially as those associated with the human airways (both healthy and diseased).

In this opinion piece, we consider the meaning of the term 'wild type' in the context of microbiology. This is especially pertinent in the post- genomic era, where we have a greater awareness of species diversity than ever before. Genomic heterogeneity, in vitro evolution/selection pressures, definition of 'the wild', the size and importance of the pan- genome, gene-gene interactions (epistasis), and the nature of the 'wild- type gene' are all discussed. We conclude that wild type is an outdated and even misleading phrase that should be gradually phased out.