Use of Antiviral Agents and other Therapies for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic led to a remarkably rapid development of a range of effective prophylactic vaccines, including new technologies that had not previously been approved for human use. In contrast, the development of new small molecule antiviral therapeutics has taken years to produce the first approved drugs specifically targeting severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), with the intervening years filled with attempts to repurpose existing drugs and the development of biological therapeutics. This review will discuss the reasons behind this variation in timescale and provide a survey of the many new treatments that are progressing through the clinical pipeline.

Thermoresponsive Polymer-Antibiotic Conjugates Based on Gradient Copolymers of 2-Oxazoline and 2-Oxazine.

A polymer-antibiotic conjugate with thermoresponsive properties near body temperature is presented. The backbone polymer is a copolymer of 2-n-propyl-2-oxazine (PropOzi) and methoxycarbonylethyl-2-oxazoline (C2MestOx) which is conjugated with the broad-spectrum antibiotic, cefazolin, via modification of the methyl ester group of C2MestOx. The resulting polymer-antibiotic conjugate has a cloud point temperature near body temperature, meaning that it can form a homogenous solution if cooled, but when injected into a skin-mimic at 37 °C, it forms a drug depot precipitate. Cleavage of the ester linker leads to quantitative release of the pristine cefazolin (with some antibiotic degradation observed) and redissolution of the polymer. When Escherichia coli were treated with polymer-antibiotic conjugate total clearance is observed within 12 h. The power of this approach is the potential for localized antibiotic delivery, for example, at a specific tissue site or into infected phagocytic cells.

Nitroxide Functionalized Antibiotics Are Promising Eradication Agents against Staphylococcus aureus Biofilms.

Treatment of biofilm-related Staphylococcus aureus infections represents an important medical challenge worldwide, as biofilms, even those involving drug-susceptible S. aureus strains, are highly refractory to conventional antibiotic therapy. Nitroxides were recently shown to induce the dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial biofilms remains unknown. Here, we demonstrate that the biofilm dispersal activity of nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Coadministration of the nitroxide CTEMPO (4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl) with ciprofloxacin significantly improved the biofilm eradication activity of the antibiotic against S. aureus Moreover, covalently linking the nitroxide to the antibiotic moiety further reduced the ciprofloxacin minimal biofilm eradication concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids could penetrate S. aureus biofilms and enter cells localized at the surface and base of the biofilm structure. No toxicity to human cells was observed for the nitroxide CTEMPO or the nitroxide-antibiotic hybrids. Taken together, our results show that nitroxides can mediate the dispersal of Gram-positive biofilms and that dual-acting biofilm eradication antibiotics may provide broad-spectrum therapies for the treatment of biofilm-related infections.

Synthesis and Evaluation of Ciprofloxacin-Nitroxide Conjugates as Anti-Biofilm Agents.

As bacterial biofilms are often refractory to conventional antimicrobials, the need for alternative and/or novel strategies for the treatment of biofilm related infections has become of paramount importance. Herein, we report the synthesis of novel hybrid molecules comprised of two different hindered nitroxides linked to the piperazinyl secondary amine of ciprofloxacin via a tertiary amine linker achieved utilising reductive amination. The corresponding methoxyamine derivatives were prepared alongside their radical-containing counterparts as controls. Subsequent biological evaluation of the hybrid compounds on preformed P. aeruginosa flow cell biofilms divulged significant dispersal and eradication abilities for ciprofloxacin-nitroxide hybrid compound 10 (up to 95% eradication of mature biofilms at 40 µM). Importantly, these hybrids represent the first dual-action antimicrobial-nitroxide agents, which harness the dispersal properties of the nitroxide moiety to circumvent the well-known resistance of biofilms to treatment with antimicrobial agents.

Siderophore conjugates to combat antibiotic-resistant bacteria.

Antimicrobial resistance (AMR) is a global threat to society due to the increasing emergence of multi-drug resistant bacteria that are not susceptible to our last line of defence antibiotics. Exacerbating this issue is a severe gap in antibiotic development, with no new clinically relevant classes of antibiotics developed in the last two decades. The combination of the rapidly increasing emergence of resistance and scarcity of new antibiotics in the clinical pipeline means there is an urgent need for new efficacious treatment strategies. One promising solution, known as the 'Trojan horse' approach, hijacks the iron transport system of bacteria to deliver antibiotics directly into cells - effectively tricking bacteria into killing themselves. This transport system uses natively produced siderophores, which are small molecules with a high affinity for iron. By linking antibiotics to siderophores, to make siderophore antibiotic conjugates, the activity of existing antibiotics can potentially be reinvigorated. The success of this strategy was recently exemplified with the clinical release of cefiderocol, a cephalosporin-siderophore conjugate with potent antibacterial activity against carbapenem-resistant and multi-drug resistant Gram-negative bacilli. This review discusses the recent advancements in siderophore antibiotic conjugates and the challenges associated with the design of these compounds that need to be overcome to deliver more efficacious therapeutics. Potential strategies have also been suggested for new generations of siderophore-antibiotics with enhanced activity.

Repeat-Unit Elongations To Produce Bacterial Complex Long Polysaccharide Chains, an O-Antigen Perspective.

The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway. This pathway produces repeat-units of mostly 3-8 sugars on the cytosolic face of the cytoplasmic membrane that is translocated by Wzx flippase to the periplasmic face and polymerized by Wzy polymerase to give long-chain polysaccharides. The Wzy polymerase is a highly diverse integral membrane protein typically containing 10-14 transmembrane segments. Biochemical evidence confirmed that Wzy polymerase is the sole driver of polymerization, and recent progress also began to demystify its interacting partner, Wzz, shedding some light to speculate how the proteins may operate together during polysaccharide biogenesis. However, our knowledge of how the highly variable Wzy proteins work as part of the O-antigen processing machinery remains poor. Here, we discuss the progress to the current understanding of repeat-unit polymerization and propose an updated model to explain the formation of additional short chain O-antigen polymers found in the lipopolysaccharide of diverse Gram-negative species and their importance in the biosynthetic process.

Metals to combat antimicrobial resistance.

Bacteria, similar to most organisms, have a love-hate relationship with metals: a specific metal may be essential for survival yet toxic in certain forms and concentrations. Metal ions have a long history of antimicrobial activity and have received increasing attention in recent years owing to the rise of antimicrobial resistance. The search for antibacterial agents now encompasses metal ions, nanoparticles and metal complexes with antimicrobial activity ('metalloantibiotics'). Although yet to be advanced to the clinic, metalloantibiotics are a vast and underexplored group of compounds that could lead to a much-needed new class of antibiotics. This Review summarizes recent developments in this growing field, focusing on advances in the development of metalloantibiotics, in particular, those for which the mechanism of action has been investigated. We also provide an overview of alternative uses of metal complexes to combat bacterial infections, including antimicrobial photodynamic therapy and radionuclide diagnosis of bacterial infections.

A Buried Water Network Modulates the Activity of the Escherichia coli Disulphide Catalyst DsbA.

The formation of disulphide bonds is an essential step in the folding of many proteins that enter the secretory pathway; therefore, it is not surprising that eukaryotic and prokaryotic organisms have dedicated enzymatic systems to catalyse this process. In bacteria, one such enzyme is disulphide bond-forming protein A (DsbA), a thioredoxin-like thiol oxidase that catalyses the oxidative folding of proteins required for virulence and fitness. A large body of work on DsbA proteins, particularly Escherichia coli DsbA (EcDsbA), has demonstrated the key role that the Cys30-XX-Cys33 catalytic motif and its unique redox properties play in the thiol oxidase activity of this enzyme. Using mutational and functional analyses, here we identify that a set of charged residues, which form an acidic groove on the non-catalytic face of the enzyme, further modulate the activity of EcDsbA. Our high-resolution structures indicate that these residues form a water-mediated proton wire that can transfer protons from the bulk solvent to the active site. Our results support the view that proton shuffling may facilitate the stabilisation of the buried Cys33 thiolate formed during the redox reaction and promote the correct direction of the EcDsbA-substrate thiol-disulphide exchange. Comparison with other proteins of the same class and proteins of the thioredoxin-superfamily in general suggest that a proton relay system appears to be a conserved catalytic feature among this widespread superfamily of proteins. Furthermore, this study also indicates that the acidic groove of DsbA could be a promising allosteric site to develop novel DsbA inhibitors as antibacterial therapeutics.

Perforated intravenous catheter design is acceptable for the administration of contrast-enhanced computed tomography administration in cancer patients: Results of a pilot randomised controlled trial.

BACKGROUND: Optimising first time success of peripheral intravenous catheter (PIVC) insertion and reducing intravenous (IV) complications in cancer patients undergoing contrast-enhanced computed tomography (CT) is vital to ensure vascular access preservation and diagnostic accuracy. The aim of this study was to test the feasibility of a randomised controlled trial (RCT) evaluating a novel perforated PIVC compared to a standard PIVC. METHODS: A single centre, parallel-group, pilot RCT was conducted between March and May 2020. Adult participants diagnosed with cancer were randomised to a non-perforated PIVC (standard care) or a PIVC with a novel perforated design (intervention) for the administration of IV contrast. There were two primary outcomes: (1) feasibility of an adequately powered RCT with pre-established criteria; and (2) all-cause PIVC failure. Secondary outcomes included: first insertion success, modes of PIVC failure, dwell time, contrast injection parameters (volume and injection rate), contrast enhancement, radiographer satisfaction and adverse events. RESULTS: Feasibility outcomes were met, except for eligibility (⩾90%) and recruitment (⩾90%). In total, 166 participants were screened, 128 (77%) were eligible and of these 101/128 (79%) were randomised; 50 to standard care and 51 to intervention. First time insertion rate was 94% (47/50) in standard care and 90% (46/50) in intervention. The median dwell time was 37 minutes (interquartile range (IQR): 25-55) in standard care and 35 minutes (IQR: 25-60) in the intervention group. There was one PIVC failure, a contrast media extravasation, in the intervention group (1/51; 2%). The desired contrast injection rate was not achieved in 4/101 (4%) of participants; two from each group. Radiographers were satisfied with the contrast flow rate. CONCLUSIONS: This pilot RCT suggests perforated PIVCs provide expected flow rate, with no evidence of differences in contrast enhancement to non-perforated PIVCs. The feasibility of conducting a larger powered RCT was demonstrated.

Combination Therapies for Biofilm Inhibition and Eradication: A Comparative Review of Laboratory and Preclinical Studies.

Microbial biofilms are becoming increasingly difficult to treat in the medical setting due to their intrinsic resistance to antibiotics. To combat this, several biofilm dispersal agents are currently being developed as treatments for biofilm infections. Combining biofilm dispersal agents with antibiotics is emerging as a promising strategy to simultaneously disperse and eradicate biofilms or, in some cases, even inhibit biofilm formation. Here we review studies that have investigated the anti-biofilm activity of some well-studied biofilm dispersal agents (e.g., quorum sensing inhibitors, nitric oxide/nitroxides, antimicrobial peptides/amino acids) in combination with antibiotics from various classes. This review aims to directly compare the efficacy of different combination strategies against microbial biofilms and highlight synergistic treatments that warrant further investigation. By comparing across studies that use different measures of efficacy, we can conclude that treating biofilms in vitro and, in some limited cases in vivo, with a combination of an anti-biofilm agent and an antibiotic, appears overall more effective than treating with either compound alone. The review identifies the most promising combination therapies currently under development as biofilm inhibition and eradication therapies.

Isothiazolone-Nitroxide Hybrids with Activity against Antibiotic-Resistant Staphylococcus aureus Biofilms.

Isothiazolones are widely used as biocides in industrial processing systems and personal care products, but their use to treat infections in humans has been hampered by their inherent cytotoxicity. Herein, we report a strategy to alleviate isothiazolone toxicity and improve antibacterial and antibiofilm potency by functionalization with a nitroxide moiety. Isothiazolone-nitroxide hybrids 6 and 22 were prepared over three steps in moderate yields (58 and 36%, respectively) from (Z)-3-(benzylsulfanyl)-propenoic acid. Hybrid 22 displayed better activity (minimum inhibitory concentration (MIC) = 35 µM) than the widely used methylisothiazolinone (MIT 1, MIC = 280 µM) against methicillin-susceptible Staphylococcus aureus (MSSA). Hybrid 22 was even more active against drug-resistant strains, such as vancomycin-resistant Staphylococcus aureus (VRSA, MIC = 8.75 µM) over MIT 1 (MIC = 280 µM). The enhanced antibacterial activity of hybrid 22 over MIT 1 was retained against established MSSA and VRSA biofilms, with minimum biofilm eradication concentration (MBEC) values of 35 and 70 µM, respectively, for 22 (the MBEC value for MIT 1 against both strains was ≥280 µM). No toxicity was observed in human epithelial T24 cells treated with hybrid 22 in concentrations up to 560 µM using a lactate dehydrogenase assay.

Loss of ß-Ketoacyl Acyl Carrier Protein Synthase III Activity Restores Multidrug-Resistant Escherichia coli Sensitivity to Previously Ineffective Antibiotics.

Antibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, 9 out of 12 are Gram-negative, with four being of "critical priority." One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes, making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial type II fatty acid synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic-susceptible and antibiotic-resistant clinical isolates. The fabH gene encodes the ß-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multidrug-resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens. IMPORTANCE Gram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor toward antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids, and their synthesis constitutes an attractive antimicrobial target, as it follows distinct pathways in prokaryotes and eukaryotes. Here, we identified a component of fatty acid synthesis, FabH, as a gate-keeper of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are resensitized to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

A high-throughput cell-based assay pipeline for the preclinical development of bacterial DsbA inhibitors as antivirulence therapeutics.

Antibiotics are failing fast, and the development pipeline remains alarmingly dry. New drug research and development is being urged by world health officials, with new antibacterials against multidrug-resistant Gram-negative pathogens as the highest priority. Antivirulence drugs, which inhibit bacterial pathogenicity factors, are a class of promising antibacterials, however, their development is stifled by lack of standardised preclinical testing akin to what guides antibiotic development. The lack of established target-specific microbiological assays amenable to high-throughput, often means that cell-based testing of virulence inhibitors is absent from the discovery (hit-to-lead) phase, only to be employed at later-stages of lead optimization. Here, we address this by establishing a pipeline of bacterial cell-based assays developed for the identification and early preclinical evaluation of DsbA inhibitors, previously identified by biophysical and biochemical assays. Inhibitors of DsbA block oxidative protein folding required for virulence factor folding in pathogens. Here we use existing Escherichia coli DsbA inhibitors and uropathogenic E. coli (UPEC) as a model pathogen, to demonstrate that the combination of a cell-based sulfotransferase assay and a motility assay (both DsbA reporter assays), modified for a higher throughput format, can provide a robust and target-specific platform for the identification and evaluation of DsbA inhibitors.

Antivirulence DsbA inhibitors attenuate Salmonella enterica serovar Typhimurium fitness without detectable resistance.

Inhibition of the DiSulfide Bond (DSB) oxidative protein folding machinery, a major facilitator of virulence in Gram-negative bacteria, represents a promising antivirulence strategy. We previously developed small molecule inhibitors of DsbA from Escherichia coli K-12 (EcDsbA) and showed that they attenuate virulence of Gram-negative pathogens by directly inhibiting multiple diverse DsbA homologues. Here we tested the evolutionary robustness of DsbA inhibitors as antivirulence antimicrobials against Salmonella enterica serovar Typhimurium under pathophysiological conditions in vitro. We show that phenylthiophene DsbA inhibitors slow S. Typhimurium growth in minimal media, phenocopying S. Typhimurium isogenic dsbA null mutants. Through passaging experiments, we found that DsbA inhibitor resistance was not induced under conditions that rapidly induced resistance to ciprofloxacin, an antibiotic commonly used to treat Salmonella infections. Furthermore, no mutations were identified in the dsbA gene of inhibitor-treated S. Typhimurium, and S. Typhimurium virulence remained susceptible to DsbA inhibitors. Our work demonstrates that under in vitro pathophysiological conditions, DsbA inhibitors can have both antivirulence and antibiotic action. Importantly, our finding that DsbA inhibitors appear to be evolutionarily robust offers promise for their further development as next-generation antimicrobials against Gram-negative pathogens.

Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities.

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound α-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35, 21-39.

An in vitro Reconstructed Human Skin Equivalent Model to Study the Role of Skin Integration Around Percutaneous Devices Against Bacterial Infection.

Percutaneous devices are a key technology in clinical practice, used to connect internal organs to external medical devices. Examples include prosthesis, catheters and electrical drivelines. Percutaneous devices breach the skin's natural barrier and create an entry point for pathogens, making device infections a widespread problem. Modification of the percutaneous implant surface to increase skin integration with the aim to reduce subsequent infection is attracting a great deal of attention. While novel surfaces have been tested in various in vitro models used to study skin integration around percutaneous devices, no skin model has been reported, for the study of bacterial infection around percutaneous devices. Here, we report the establishment of an in vitro human skin equivalent model for driveline infections caused by Staphylococcus aureus, the most common cause of driveline-related infections. Three types of mock drivelines manufactured using melt electrowriting (smooth or porous un-seeded and porous pre-seeded with human fibroblasts) were implanted in human skin constructs and challenged with S. aureus. Our results show a high and stable load of S. aureus in association with the skin surface and no signs of S. aureus-induced tissue damage. Furthermore, our results demonstrate that bacterial migration along the driveline surface occurs in micro-gaps caused by insufficient skin integration between the driveline and the surrounding skin consistent with clinical reports from explanted patient drivelines. Thus, the human skin-driveline infection model presented here provides a clinically-relevant and versatile experimental platform for testing novel device surfaces and infection therapeutics.

Bacterial Biofilm Eradication Agents: A Current Review.

Most free-living bacteria can attach to surfaces and aggregate to grow into multicellular communities encased in extracellular polymeric substances called biofilms. Biofilms are recalcitrant to antibiotic therapy and a major cause of persistent and recurrent infections by clinically important pathogens worldwide (e.g., Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus). Currently, most biofilm remediation strategies involve the development of biofilm-inhibition agents, aimed at preventing the early stages of biofilm formation, or biofilm-dispersal agents, aimed at disrupting the biofilm cell community. While both strategies offer some clinical promise, neither represents a direct treatment and eradication strategy for established biofilms. Consequently, the discovery and development of biofilm eradication agents as comprehensive, stand-alone biofilm treatment options has become a fundamental area of research. Here we review our current understanding of biofilm antibiotic tolerance mechanisms and provide an overview of biofilm remediation strategies, focusing primarily on the most promising biofilm eradication agents and approaches. Many of these offer exciting prospects for the future of biofilm therapeutics for a large number of infections that are currently refractory to conventional antibiotics.

Profluorescent Fluoroquinolone-Nitroxides for Investigating Antibiotic⁻Bacterial Interactions.

Fluorescent probes are widely used for imaging and measuring dynamic processes in living cells. Fluorescent antibiotics are valuable tools for examining antibiotic⁻bacterial interactions, antimicrobial resistance and elucidating antibiotic modes of action. Profluorescent nitroxides are 'switch on' fluorescent probes used to visualize and monitor intracellular free radical and redox processes in biological systems. Here, we have combined the inherent fluorescent and antimicrobial properties of the fluoroquinolone core structure with the fluorescence suppression capabilities of a nitroxide to produce the first example of a profluorescent fluoroquinolone-nitroxide probe. Fluoroquinolone-nitroxide (FN) 14 exhibited significant suppression of fluorescence (>36-fold), which could be restored via radical trapping (fluoroquinolone-methoxyamine 17) or reduction to the corresponding hydroxylamine 20. Importantly, FN 14 was able to enter both Gram-positive and Gram-negative bacterial cells, emitted a measurable fluorescence signal upon cell entry (switch on), and retained antibacterial activity. In conclusion, profluorescent nitroxide antibiotics offer a new powerful tool for visualizing antibiotic⁻bacterial interactions and researching intracellular chemical processes.

Eradicating uropathogenic Escherichia coli biofilms with a ciprofloxacin-dinitroxide conjugate.

Urinary tract infections (UTIs) are amongst the most common and prevalent infectious diseases worldwide, with uropathogenic Escherichia coli (UPEC) reported as the main causative pathogen. Fluoroquinolone antibiotics are commonly used to treat UTIs but for infections involving UPEC biofilms, which are commonly associated with catheter use and recurrent episodes, ciprofloxacin is often ineffective. Here we report the development of a ciprofloxacin-dinitroxide (CDN) conjugate with potent UPEC biofilm-eradication activity. CDN 11 exhibited a 2-fold increase in potency over the parent antibiotic ciprofloxacin against UPEC biofilms. Moreover, CDN 11 resulted in almost complete UPEC biofilm cell eradication (99.7%) at concentrations as low as 12.5 µM, and significantly potentiated ciprofloxacin's biofilm-eradication activity against UPEC upon co-administration. The biofilm-eradication activity of CDN 11 highlights the potential of nitroxide functionalized antibiotics as a promising strategy for the treatment of biofilm-related UTIs.

Moraxella catarrhalis NucM is an entry nuclease involved in extracellular DNA and RNA degradation, cell competence and biofilm scaffolding.

Moraxella catarrhalis is a host-adapted bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease. This study characterises the conserved M. catarrhalis extracellular nuclease, a member of the ßßα metal finger family of nucleases, that we have named NucM. NucM shares conserved sequence motifs from the ßßα nuclease family, including the DRGH catalytic core and Mg2+ co-ordination site, but otherwise shares little primary sequence identity with other family members, such as the Serratia Nuc and pneumococcal EndA nucleases. NucM is secreted from the cell and digests linear and circular nucleic acid. However, it appears that a proportion of NucM is also associated with the cell membrane and acts as an entry nuclease, facilitating transformation of M. catarrhalis cells. This is the first example of a ßßα nuclease in a Gram negative bacteria that acts as an entry nuclease. In addition to its role in competence, NucM affects cell aggregation and biofilm formation by M. catarrhalis, with ΔnucM mutants having increased biofilm biomass. NucM is likely to increase the ability of cells to survive and persist in vivo, increasing the virulence of M. catarrhalis and potentially affecting the behaviour of other pathogens that co-colonise the otorhinolaryngological niche.