Quantitative reverse transcription PCR (RT- qPCR) is a popular and reliable tool for monitoring fluctuations in functional bacterial gene expression. A necessary step of the qRT- qPCR process is the use of a reference gene, which acts to distinguish between technical bias and true biological variation. Many reference genes have been defined for bacterial species; however, few studies have validated their stability across strain types and environmental test conditions. In this study of Pseudomonas aeruginosa, the expression consistency of seven commonly used reference genes (rpoD, proC, rpoS, 16S, algD, gyrA and ampC) was assessed in P. aeruginosa laboratory (PAO1) and clinical (LESB65) isolates grown in Lysogeny broth, synthetic cystic fibrosis (CF) media 2 (SCFM2) and CF lung media (CFLM) at various growth time points (2, 6, 24 and 72 h). The stability of the reference genes was then ranked using the RefFinder programme, and three differentially ranked (rpoS, 16S and ampC) were used to interpret the expression of a Pseudomonas virulence- related gene (exoS). The results showed that 16S was the only reference gene that was quantifiably expressed by both P. aeruginosa strains grown in all media types at all growth times. Furthermore, analysing the expression of exoS with different reference genes significantly influenced the calculated expression of exoS in SCFM2 and CFLM. This study has identified a suitable reference gene for RT- qPCR with P. aeruginosa grown in complex respiratory- mimicking media. The results presented here also highlight the importance of validating reference gene expression under the chosen experimental conditions and increase our understanding of how pathogen biology can fluctuate across diverse conditions. Such knowledge is paramount for the development of novel therapeutics, including antimicrobials and antivirulence agents.

Opportunistic pathogens switch from a commensal to pathogenic state by sensing and responding to a variety of environmental cues, including temperature fluctuations. Minor temperature oscillations can alert the pathogen to a changing niche ecosystem, necessitating efficient sensing and rapid integration to trigger behavioral change. This is typically achieved through master regulators, dictating pleiotropic phenotypes. Here, we uncover a pivotal role of minor temperature shifts in transition of Streptococcus pneumoniae (SPN) from commensal to virulent lifestyles, mediated via an RNA thermosensing (RNAT) element within the untranslated region of the global regulator CiaR. By positively regulating the expression of the surface adhesin, Phosphorylcholine (PCho), in response to elevated temperature, CiaR potentiates pneumococcal infection. Engineering the RNAT structure to create translation restrictive or permissive versions allowed us to demonstrate how modulation in expression of CiaR could alter pneumococcal invasion capability, influencing infection outcomes. Moreover, intranasal administration of PCho mitigated SPN-induced bacteraemic pneumonia. Since a majority of opportunistic respiratory bacterial pathogens decorate their surface with PCho, this signaling arm could be exploited for anti-infective interventions.

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not reflect the environmental conditions of the hostile lung niche. Current reductionist testing conditions can lead to the progression of compounds along a preclinical pipeline without evidence of their activity under cystic fibrosis lung niche-appropriate conditions. Several approaches used to study traditional antimicrobials may not be suitable for antibiotic alternatives, including anti-virulence therapeutics like anti-quorum sensing agents and siderophore inhibitors. This protocol documents an aggregate biofilm model of Pseudomonas aeruginosa to compare resistance and infection-relevant gene expression in single-species and multi-species cultures (Staphylococcus aureus and Candida albicans), examining colony-forming unit (CFU) reductions and changes in gene expression, using algD as an exemplar. The model was optimized for small, static volumes of bacterial cultures to allow the study of novel compounds in the discovery phase of the drug development pipeline, where compound quantities may be limited. Single-species P. aeruginosa biofilms were formed in Synthetic Cystic Fibrosis Medium 2 (SCFM2) for 24 h before treatment with meropenem at different concentrations (1, 16, and 256 µg/mL) for a further 24 h. Polymicrobial biofilms were established by growing Staphylococcus aureus and Candida albicans together in SCFM2, then inoculating with P. aeruginosa for an additional 24 h and treating with meropenem. The lack of a direct connection between compound efficacy measures in pre-clinical testing and clinical trial results has cast doubt on the applicability of current laboratory screening tools. This model allows us to understand the impact of relevant factors on P. aeruginosa gene expression, including genes contributing to resistance and virulence, thereby bridging this gap.

Streptococcus pneumoniae (the pneumococcus) causes a range of life-threatening diseases including pneumonia, sepsis and meningitis. Despite widespread vaccine deployment, pneumococcal disease remains a leading cause of global mortality. The human nasopharynx is its principal ecological niche, and all pneumococcal disease originates from this reservoir of organisms. Acute infections are, however, an evolutionary dead-end for the pneumococcus. What sustains the pneumococcus within human populations are cycles of colonisation and transmission. To persist within the nasopharynx, it must overcome the physical and immunological barriers established by the host while acquiring sufficient nutrients to proliferate in the face of competing airway microbes. Here, we outline the metabolic, microbial, and immunological challenges of colonisation, and the often-competing demands of transmission, which together have shaped the pneumococcus into one of the most formidable human pathogens.

Antimicrobial resistance is a growing threat to human health and agriculture. Sulfur-containing compounds and elemental sulfur have a long history of use as antimicrobials, but challenges related to solubility and formulation have limited their broad application. Recent advances in sulfur polymer chemistry have enabled the development of novel sulfur-rich materials with antimicrobial activity. However, most of these materials are water-insoluble, limiting their use in biomedical and agricultural applications. Here, we report the synthesis of a linear poly(trisulfide) via photochemical ring-opening polymerization of a cyclic trisulfide monomer bearing a carboxylic acid. Deprotonation of the carboxylic acid renders the poly(trisulfide) fully water soluble, with concomitant chain scission via S-S cleavage. The resulting poly(trisulfide) oligomers exhibited potent antifungal activity against Candida albicans (MIC < 8 µg/mL), and stronger inhibition of the Gram-positive bacterium Staphylococcus aureus (MIC = 16 µg/mL) compared to the Gram-negative Escherichia coli (MIC > 512 µg/mL). In control experiments, the monomer did not show potent antifungal or antibacterial activity. This work demonstrates a simple and controllable synthesis of a poly(trisulfide) species that serves as a prodrug: treatment with NaOH converts the polymer to water-soluble oligomers with antimicrobial activity. This approach is a new direction for biological applications of sulfur polymers and a new strategy to address antimicrobial resistance.

Streptococcus pneumoniae colonizes human airways, where it acquires sugars from glycosylated mucins using glycoside hydrolases and sugar transport systems. This study identifies widespread nucleotide sequence variation in the promoter of a pneumococcal operon encoding a glycan scavenging system. We identify 78 promoter sequence patterns across 21,155 genomes, with variation clustered within a stretch of adenines, where mutations accumulate via strand slippage during DNA replication. Promoter mutations influence operon transcription, and multiple promoter patterns are co-identified during single-carriage episodes, suggesting that heterogeneous gene expression provides population-level benefits. In a mouse nasopharyngeal colonization model, promoter mutations arise and undergo selection, with nucleotide insertion promoting gene expression and prolonging carriage longevity. Pre-existing immunity confers resistance to colonization by strains carrying single promoter patterns but does not protect against mixed infections with otherwise isogenic strains differing in promoter sequence. Promoter region sequence variation offers an evolutionary strategy for exploration of phenotypic space to maximize fitness within-host.

The rise in antifungal resistance and changing epidemiology underscores the need to improve antifungal susceptibility testing. This study tested the standard medium RPMI1640 and healthy lung and sinus media against Aspergillus, Mucor, and Candida species. Candida showed greater variability in minimum inhibitory concentrations (MICs) compared with molds, with higher MICs in respiratory tract-mimicking media than in RPMI1640. Aspergillus terreus was the most affected mold, showing higher MICs for certain antifungals in these media.

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

Sulfur polymers, prepared by inverse vulcanization using elemental sulfur and vinylic monomers, are emerging functional materials of current research interest; however, sulfur polymers suffer from limited water solubility due to the hydrophobic nature of conventional comonomers and sulfur. Herein, the preparation of ionic liquid (IL)-containing sulfur polymers are reported using the hydrophilic ionic liquid, 1-allyl-3-vinylimidazolium chloride (AVImCl) as a comonomer. The introduction of IL significantly enhances the hydrophilicity of sulfur polymers, enabling them to dissolve in water. Benefiting from the thorough contact with aqueous mercury ions, the resultant sulfur polymer possesses high uptake capacity (436 mg g-1). After binding mercury, a coordination complex is formed and precipitated. The charged sulfur polymers gain a new application in demulsification. The polymer quickly breaks oil-in-water (O/W) emulsions through anion exchange between Cl- of the polymer and dodecylbenzenesulfonate (DBS-) of the surfactant. In addition, the polymer has a growth inhibitory effect against Staphylococcus aureus. The integration of IL and elemental sulfur provides a novel approach to modifying the wettability of sulfur polymers. Also, this novel water-soluble IL-containing sulfur polymer, with mercury capture, demulsification, and antibacterial activity, can be considered as a multifunctional material in practical water purification. An inverse vulcanised sulfur polymer is prepared using ionic liquid precursors, giving it increased hydrophilicity, solubility in water, and applications in demulsification, mercury capture, and antibacterial activity.image

Rationale Lung function (FEV1) and quality of life (QoL) are key outcomes in most interventional clinical trials conducted in people living with cystic fibrosis. However, no robust pre-clinical surrogates for FEV1 and QoL exist. The precise physiological mechanisms leading to treatment-related improvements in these outcomes are incompletely understood. In this post-hoc analysis we explored the relationship between changes in the sputum proteome and these outcomes with the aim of identifying translational biomarkers. Methods Paired sputum samples collected during the AZTEC-CF study ([NCT02894684][1]) pre and post 14 days of antibiotic treatment for an acute pulmonary exacerbation were included. Samples were analysed using in vitro Mesoscale Discovery (MSD) assays and by nano LC-MS/MS. Peptide identification and quantification was performed and the log-fold change for individual proteins and relationships between protein change and changes in FEV1 and QoL were evaluated. Results Distinct patterns were found between proteins that correlated with FEV1 and those that correlated with QoL improvements. FEV1 improvement was characterised by increases in bacterially-derived proteins accompanied by decreases in proteins relating to neutrophil degranulation. Conversely, changes in QoL were associated with increases in antiprotease and antioxidant proteins. MSD analysis revealed changes in some neutrophil-associated markers significantly correlated with FEV1 improvements, but no markers significantly correlated with QoL improvements. Conclusions These results suggest changes in two key CF clinical trial outcomes (FEV1 and QoL) may be underpinned by different physiological mechanisms. Understanding these divergent mechanisms is vital to fortify optimal clinical trial design in CF and panels of biomarkers may be needed to improve translational confidence. ### Competing Interest Statement Dilip Nazareth - Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Vertex Freddy Frost - Grants or contracts from any entity: UK Health Security Agency, NIHR Patient Involvement Fund and Cystic Fibrosis Trust. Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Chiesi Ltd, Astrazeneca, Vertex. Support for attending meetings and/or travel: Chiesi Ltd, Vertex. Jo Fothergill - Grants or contracts from any entity: CF Trust - Strategic Research Centre grant (Institution), CF Foundation - Strategic Research Centre grant (Institution), Asthma + Lung UK - Project grant (Institution), EU Commission - Project grant (Institution), BBSRC - Project grant and sLoLa (Institution), Network+ - Researcher in Residence (Institution), North West Kidney Research - Project grant (Institution), NC3Rs - PhD studentship (Institution), MRC - project grant, studentship, equipment grant (Institution). Consulting fees: CARB-X (Institution), Johnson & Johnson (Personal). Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: European Food Standards Agency - Contract Writing manuscript (Personal). Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid: CF Trust grants committee (Unpaid), CF AMR Syndicate Steering Group (Unpaid), UK CF Infection Biobank Steering Group (Unpaid). Carsten Schwarz - Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: ertex, Chiesi, Pfizer, Novartis, StreamedUp, Insmed, TFF, Medialis, Viatris, Abbvie, Honorarium as speaker, advisor, and educational events. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02894684&atom=%2Fbiorxiv%2Fearly%2F2024%2F07%2F23%2F2024.07.23.604741.atom

To combat the rising global issue of antibiotic resistance, the accelerated development of novel antibiotics is essential. Current preclinical antimicrobial development yields a significant number of leads that prove unsuitable either prior to or during clinical trials. To increase the efficiency of preclinical development, relevant, standardized, accessible, and cost-effective models must be developed. Galleria mellonella (greater wax moth) larvae are widely used as an infection model to assess microbial virulence, conduct drug toxicity testing, and serve as a preliminary means of evaluating the in vivo efficacy of novel antimicrobial compounds. These infection models have greater biological relevance than many in vitro screens of comparable throughput and decrease reliance on mammalian models when used as a prescreen for antimicrobial testing. This protocol describes a standardized methodology for the optimization of G. mellonella infection models, which can be applied to bacterial species and antimicrobial therapeutics of choice. Using the WHO priority pathogen Pseudomonas aeruginosa as an exemplar, we outline steps that can be undertaken to develop a reproducible model of infection and therapeutic testing. This includes recommendations on experimental setup, sample preparation, and infection and treatment protocols. Integration of this model within preclinical antimicrobial development pipelines would decrease reliance on mammalian models, reduce the number of ineffective compounds reaching clinical trials, and ultimately increase the efficiency of preclinical antimicrobial development.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for infections in people living with chronic respiratory conditions, such as cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). Traditionally, in people with chronic respiratory disorders, P. aeruginosa infection has been managed with a combination of inhaled and intravenous antibiotic therapies. However, due in part to the prolonged use of antibiotics in these people, the emergence of multi-drug resistant P. aeruginosa strains is a growing concern. The development of anti-virulence therapeutics may provide a new means of treating P. aeruginosa lung infections whilst also combatting the AMR crisis, as these agents are presumed to exert reduced pressure for the emergence of drug resistance as compared to antibiotics. However, the pipeline for developing anti-virulence therapeutics is poorly defined, and it is currently unclear as to whether in vivo and in vitro models effectively replicate the complex pulmonary environment sufficiently to enable development and testing of such therapies for future clinical use. Here, we discuss potential targets for P. aeruginosa anti-virulence therapeutics and the effectiveness of the current models used to study them. Focus is given to the difficulty of replicating the virulence gene expression patterns of P. aeruginosa in the CF and NCFB lung under laboratory conditions and to the challenges this poses for anti-virulence therapeutic development.

The build-up of elemental sulfur waste poses problems such that only the advancement of process and product design might act as a solution. Inverse vulcanisation, a process for the generation of high sulfur content polymeric materials may be one such resolution. However, a complete understanding of how these materials form is yet to be fully agreed in this emerging field. Herein is an investigation into the understanding of 'dark sulfur' - amorphous, unreacted sulfur, not incorporated into the polymer backbone - in an attempt to understand further the formation mechanisms behind inverse vulcanisation. This research posits theories regarding polymer formation, thermal rearrangement, and the actions of OH to control the degree of product crosslinking, in relation to the quantity of sulfur unreacted into the polymer structure. The detriments and benefits of this dark sulfur in relation to application and general usage are also investigated, showing that a high content of dark sulfur may encourage planktonic bactericidal activity, while also promoting safety considerations from generated species such as hydrogen sulfide and carbon disulfide, concluded as components of this dark sulfur.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and bacteraemia and is capable of remarkable phenotypic plasticity, responding rapidly to environmental change. Pneumococcus is a nasopharyngeal commensal, but is responsible for severe, acute infections following dissemination within-host. Pneumococcus is adept at utilising host resources, but the airways are compartmentalised and those resources are not evenly distributed. Challenges and opportunities in metabolite acquisition within different airway niches may contribute to the commensal-pathogen switch when pneumococcus moves from nasopharynx into lungs. We used NMR to characterise the metabolic landscape of the mouse airways, in health and during infection. Using paired nasopharynx and lung samples from naïve animals, we identified fundamental differences in metabolite bioavailability between airway niches. Pneumococcal pneumonia was associated with rapid and dramatic shifts in the lung metabolic environment, whilst nasopharyngeal carriage led to only modest change in upper airway metabolite profiles. NMR spectra derived from the nasopharynx of mice infected with closely-related pneumococcal strains that differ in their colonisation potential could be distinguished from one another using multivariate dimensionality reduction methods. The resulting models highlighted that increased branched-chain amino acid (BCAA) bioavailability in nasopharynx is a feature of infection with the high colonisation potential strain. Subsequent analysis revealed increased expression of BCAA transport genes and increased intracellular concentrations of BCAA in that same strain. Movement from upper to lower airway environments is associated with shifting challenges in metabolic resource allocation for pneumococci. Efficient biosynthesis, liberation or acquisition of BCAA is a feature of adaptation to nasopharyngeal colonisation.

Streptococcus pneumoniae is a pathogen of global morbidity and mortality. Pneumococcal pneumonia can lead to systemic infections associated with high rates of mortality. We find that, upon pneumococcal infection, pulmonary Treg cells are activated and have upregulated TNFR2 expression. TNFR2-deficient mice have compromised Treg cell responses and highly activated IL-17A-producing γδ T cell (γδT17) responses, resulting in significantly enhanced neutrophil infiltration, tissue damage, and rapid development of bacteremia, mirroring responses in Treg cell-depleted mice. Deletion of total Treg cells predominantly activate IFNγ-T cell responses, whereas adoptive transfer of TNFR2+ Treg cells specifically suppress the γδT17 response, suggesting a targeted control of γδT17 activation by TNFR2+ Treg cells. Blocking IL-17A at early stage of infection significantly reduces bacterial blood dissemination and improves survival in TNFR2-deficient mice. Our results demonstrate that TNFR2 is critical for Treg cell-mediated regulation of pulmonary γδT17-neutrophil axis, with impaired TNFR2+ Treg cell responses increasing susceptibility to disease.

Laboratory bacteriology involves the use of high-density cultures that we often assume to be clonal but that in reality are populations consisting of multiple genotypes at various abundances. We have demonstrated that the genetic structure of a single population of a widely used Streptococcus pneumoniae strain can be substantially altered by even short-term laboratory handling and culture and that, over time, this can lead to changes in virulence characteristics.

Self-activating crosslinkers were used to create inverse vulcanised polymers with improved properties via method optimised dispersion polymerisation, and were also used alongside other comonomers to enhance the product polymer's properties.