Preclinical Evaluation of Nitroxide-Functionalised Ciprofloxacin as a Novel Antibiofilm Drug Hybrid for Urinary Tract Infections.

Urinary tract infections (UTIs) are the second most common bacterial infection with high recurrence rates and can involve biofilm formation on patient catheters. Biofilms are inherently tolerant to antimicrobials, making them difficult to eradicate. Many antibiofilm agents alone do not have bactericidal activity; therefore, linking them to antibiotics is a promising antibiofilm strategy. However, many of these hybrid agents have not been tested in relevant preclinical settings, limiting their potential for clinical translation. Here, we evaluate a ciprofloxacin di-nitroxide hybrid (CDN11), previously reported to have antibiofilm activity against uropathogenic Escherichia coli (UPEC) strain UTI89 in vitro, as a potential UTI therapeutic using multiple preclinical models that reflect various aspects of UTI pathogenesis. We report improved in vitro activity over the parent drug ciprofloxacin against mature UTI89 biofilms formed inside polyethylene catheters. In bladder cell monolayers infected with UTI89, treatment with CDN11 afforded significant reduction in bacterial titers, including intracellular UPEC. Infected mouse bladders containing biofilm-like intracellular reservoirs of UPEC UTI89 showed decreased bacterial loads after ex vivo bladder treatment with CDN11. Activity for CDN11 was reported across different models of UTI, showcasing nitroxide-antibiotic hybridization as a promising antibiofilm approach. The pipeline we described here could be readily used in testing other new therapeutic compounds, fast-tracking the development of novel antibiofilm therapeutics.

Bladder-draining lymph nodes support germinal center B cell responses during urinary tract infection in mice.

Bacterial urinary tract infections (UTIs) are both common and exhibit high recurrence rates in women. UTI healthcare costs are increasing due to the rise of multidrug-resistant (MDR) bacteria, necessitating alternative approaches for infection control. Here, we directly observed host adaptive immune responses in acute UTI. We employed a mouse model in which wild-type C57BL/6J mice were transurethrally inoculated with a clinically relevant MDR UTI strain of uropathogenic Escherichia coli (UPEC). Firstly, we noted that rag1-/- C57BL/6J mice harbored larger bacterial burdens than wild-type counterparts, consistent with a role for adaptive immunity in UTI control. Consistent with this, UTI triggered in the bladders of wild-type mice early increases of myeloid cells, including CD11chi conventional dendritic cells, suggesting possible involvement of these professional antigen-presenting cells. Importantly, germinal center B cell responses developed by 4 weeks post-infection in bladder-draining lymph nodes of wild-type mice and, although modest in magnitude and transient in nature, could not be boosted with a second UTI. Thus, our data reveal for the first time in a mouse model that UPEC UTI induces local B cell immune responses in bladder-draining lymph nodes, which could potentially serve to control infection.

Simple and Efficient Synthesis of 3-Aryl-2-oxazolidinone Scaffolds Enabling Increased Potency toward Biofilms.

Infectious diseases caused by bacterial pathogens are a leading cause of mortality worldwide. In particular, recalcitrant bacterial communities known as biofilms are implicated in persistent and difficult to treat infections. With a diminishing antibiotic pipeline, new treatments are urgently required to combat biofilm infections. An emerging strategy to develop new treatments is the hybridization of antibiotics. The benefit of this approach is the extension of the useful lifetime of existing antibiotics. The oxazolidinones, which include the last resort antibiotic linezolid, are an attractive target for improving antibiofilm efficacy as they present one of the most recently discovered classes of antibiotics. A key step in the synthesis of new 3-aryl-2-oxazolidinone derivatives is the challenging formation of the oxazolidinone ring. Herein we report a direct synthetic route to the piperazinyl functionalized 3-aryl-2-oxazolidinone 17. We also demonstrate an application of these piperazine molecules by functionalizing them with a nitroxide moiety as a strategy to extend the useful lifetime of oxazolidinones and improve their potency against Methicillin-resistant Staphylococcus aureus (MRSA) biofilms. The antimicrobial susceptibility of the linezolid-nitroxide conjugate 11 and its corresponding methoxyamine derivative 12 (a control for biofilm dispersal) was assessed against planktonic cells and biofilms of MRSA. In comparison to linezolid and our lead compound 10 (a piperazinyl oxazolidinone derivative), the linezolid-nitroxide conjugate 11 displayed a minimum inhibitory concentration that was 4-16-fold higher. The opposite effect was seen in biofilms where the linezolid-nitroxide hybrid 11 was >2-fold more effective (160 µg/mL versus >320 µg/mL) in eradicating MRSA biofilms. The methoxyamine derivative 12 performed on par with linezolid. The drug-likeness of the compounds was also assessed, and all compounds were predicted to have good oral bioavailability. Our piperazinyl oxazolidinone derivative 10 was confirmed to be lead-like and would be a good lead candidate for future functionalized oxazolidinones. The modification of antibiotics with a dispersal agent appears to be a promising approach for eradicating MRSA biofilms and overcoming the antibiotic resistance associated with the biofilm mode of growth.

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.

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.

The Wzi outer membrane protein mediates assembly of a tight capsular polysaccharide layer on the Acinetobacter baumannii cell surface.

Identification of novel therapeutic targets is required for developing alternate strategies to treat infections caused by the extensively drug-resistant bacterial pathogen, Acinetobacter baumannii. As capsular polysaccharide (CPS) is a prime virulence determinant required for evasion of host immune defenses, understanding the pathways for synthesis and assembly of this discrete cell-surface barrier is important. In this study, we assess cell-bound and cell-free CPS material from A. baumannii AB5075 wildtype and transposon library mutants and demonstrate that the Wzi outer membrane protein is required for the proper assembly of the CPS layer on the cell surface. Loss of Wzi resulted in an estimated 4.4-fold reduction in cell-associated CPS with a reciprocal increase in CPS material shed in the extracellular surrounds. Transmission electron microscopy revealed a disrupted CPS layer with sparse patches of CPS on the external face of the outer membrane when Wzi function was lost. However, this genotype did not have a significant effect on biofilm formation. Genetic analysis demonstrated that the wzi gene is ubiquitous in the species, though the nucleotide sequences were surprisingly diverse. Though divergence was not concomitant with variation at the CPS biosynthesis K locus, an association between wzi type and the first sugar of the CPS representing the base of the structure most likely to interact with Wzi was observed.