BACKGROUND:Catheter-associated urinary tract infections (CAUTI) are among the most prevalent healthcare-associated infections and an important site for development and spread of antimicrobial resistance. Although CAUTI are frequently polymicrobial, the majority of research focuses on individual pathogens in monoculture, largely due to a lack of representative and tractable models. AIMS:The aim of this study was to develop a tractable and reproducible model of polymicrobial CAUTI. METHODS:Here we describe the use of an in vitro model of the catheterized urinary tract to generate polymicrobial communities encompassing common uropathogens (Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Proteus mirabilis), in an environment representative of the catheterized urinary tract. RESULTS AND CONCLUSIONS:We show that our system establishes stable and reproducible polymicrobial communities and facilitates analysis across both planktonic and biofilm lifestyles. We confirm that polymicrobial biofilms in this system display distinct population dynamics compared to planktonic populations and modulate the impact of ciprofloxacin treatment by protecting the most susceptible community members. In addition, we demonstrate the capacity of P. mirabilis to encrust and block catheters when part of a polymicrobial community and confirm that thioridazine treatment remains effective at inhibiting catheter blockage under these conditions.

We report a novel Efflux Resistance Breaker (ERB) strategy for designing antibiotics intrinsically resistant to efflux, using fluoroquinolones as a model class. ERB-modified fluoroquinolones showed enhanced intracellular accumulation and markedly improved antibacterial activity, with up to 512-fold reduction in MIC (MIC90 0.03-2 μg/mL) across multidrug-resistant bacteria. Lead compounds KSN-L22 (46) and BL-7 (50) demonstrated potent activity against MRSA, Streptococcus pneumoniae (including MDR and PRSP), Enterococcus faecalis and E. faecium (VanA, VanB and VanD), as well as Acinetobacter baumannii and Escherichia coli. The compounds inhibited both wild-type and S84L mutant DNA gyrase (IC50 ∼ 3.8 μg/mL) and achieved a > 4-log bacterial load reduction in a murine thigh infection model at oral doses of 50 mg/kg. Favorable oral and intravenous PK/PD profiles, absence of toxicity at 1200 mg/kg/day, and no hERG, CYP450, or off-target liabilities were observed. ERB technology provides a promising strategy for designing antibiotics that are intrinsically less susceptible to efflux.

The rise of multidrug-resistant Gram-positive pathogens necessitates new antibacterial agents with mechanisms that are distinct from those of existing therapies. Here, we report the antibacterial activity and mechanistic characterization of two Strathclyde minor groove binders (S-MGBs), S-MGB-234 and S-MGB-235, synthetic DNA binding molecules designed to disrupt essential bacterial processes. These compounds displayed potent in vitro activity against clinically relevant Gram-positive pathogens, including Staphylococcus aureus and Enterococcusspp. However, potency was less pronounced against Enterococcus faecalis. Activity was retained against drug-resistant strains, and reduced susceptibility emerged more slowly during serial passaging than that observed for gentamicin under the conditions tested. Whole-genome sequencing of reduced susceptibility mutants did not identify mutations in canonical DNA targets but instead revealed recurring changes in genes associated with the cell envelope, including norA, fmtA, and cozEb, suggesting that envelope-mediated effects influence compound access rather than direct target modification. Biophysical assays demonstrate strong interactions with AT-rich oligonucleotides and gDNA, consistent with the DNA binding properties previously reported for the S-MGB class. Together, these findings demonstrate that S-MGBs represent a promising class of DNA-targeting antibacterials and provide initial evidence that reduced susceptibility emerges through heterogeneous mechanisms that do not involve obvious modifications of DNA targets.

Clade Ib mpox is a newly emerged strain of the mpox virus (MPXV). The antiviral efficacy of 12 different therapeutic drugs was evaluated, in vitro using a live-virus, foci reduction assay, against MPXV clade Ib. We report that antiviral activity is retained against clade Ib with inhibitory concentrations (required to reduce the viral foci count by 50% (IC50)) of 0.025 ± 0.018 and 43.8 ± 15.2 μM for tecovirimat and cidofovir, respectively. These values are not significantly different from those observed for clade IIb, when measured in the same foci reduction assay (IC50 values of 0.010 ± 0.02 and 15.7 ± 14.3 μM for tecovirimat and cidofovir, respectively). Activity was also demonstrated for other antivirals, with the IC50 of the active metabolites of molnupiravir (EIDD-1931; 4.47 ± 1.72 μM) and remdesivir (GS-441524; 11.8 ± 6.43 μM) and with other licensed antivirals such as ribavirin (34.4 ± 10.3 μM) and baloxavir marboxil (22.6 ± 10.5 μM). In contrast, no inhibitory activity was observed with acyclovir, L-valacyclovir hydrochloride or favipiravir (IC50 >100 μM). Interestingly, the anti-parasitic drugs nitazoxanide, mefloquine hydrochloride and chloroquine diphosphate, showed inhibitory activity against the clade Ib virus, with IC50 values of 14.5 ± 3.41, 5.37 ± 1.37 and 24.7 ± 2.38 μM, respectively. This study shows that several therapeutics, including several licensed antivirals, may offer alternative treatment options for mpox clade Ib. ### Competing Interest Statement The authors have declared no competing interest.

Proteus mirabilis is a frequent cause of catheter-associated urinary tract infection and often exhibits high tolerance to chlorhexidine (CHD), a biocide used widely in healthcare settings. We previously demonstrated that inactivation of the smvR repressor (leading to overexpression of the smvA efflux system), truncation of the MltA-interacting protein MipA and aspects of lipopolysaccharide (LPS) structure modulate CHD susceptibility in this organism. However, the prevalence of these mechanisms among P. mirabilis clinical isolates, the conditions under which they can be acquired and their impact on susceptibility to other cationic biocides require further study. Through phenotypic and genomic analysis of a panel of 78 P. mirabilis clinical isolates, we have confirmed that deleterious mutations in smvR commonly arise in P. mirabilis and are significantly associated with reduced susceptibility to CHD and other cationic biocides. Mutations in mipA were also associated with CHD tolerance. Conversely, mutations in smvA and the rppA response regulator (which governs lipid A modifications that alter LPS surface charge) were associated with increased susceptibility to several biocides. Several isolates harbouring smvR mutations displayed incongruous phenotypes, exhibiting relatively modest CHD tolerance, which could not be accounted for by co-occurring mutations in smvA and rppA or defects in LPS (as assessed by polymyxin B susceptibility). Further analysis of these isolates revealed mutations in the LPS core biosynthesis gene waaG, leading to LPS truncation from the inner core region. Directed evolution experiments further reinforced the importance of smvR inactivation in biocide adaptation in P. mirabilis and demonstrated that relevant mutations can be selected for by exposure to CHD concentrations up to four times lower than the minimum inhibitory concentration. Taken together, these results expand our understanding of mechanisms underlying tolerance to cationic biocides in this species and provide evidence for common mechanisms of cationic biocide tolerance.

Efflux, mediated by a series of multidrug efflux pumps, is a major contributor to antibiotic resistance in Gram-negative bacteria. Efflux pump inhibitors (EPIs), which can block efflux, have the potential to be used as adjuvant therapies to resensitize bacteria to existing antibiotics. In this study, 36 quinoline-based compounds were synthesized as potential EPIs targeting resistance nodulation division (RND) family pumps in the multidrug-resistant pathogen Acinetobacter baumannii. In A. baumannii strains with overexpressed AdeFGH (chloramphenicol-adapted) and AdeABC (AYE, Ab5075-UW), these compounds enhanced Hoechst dye accumulation, indicating general efflux inhibition, and potentiated chloramphenicol, which is an AdeG substrate. The research focused on two generations of quinoline compounds, with modifications at the C-7 position of first-generation compounds to improve hydrophobic interactions with the Phe loop in the AdeG efflux pump, to generate second-generation compounds. The modified quinolines showed strong pump inhibition and significant chloramphenicol potentiation, with MIC reductions of 4- to 64-fold. Notably, compounds 1.8 and 3.8 exhibited the highest inhibitory activity, while compounds 1.3 and 3.3 showed up to 64-fold potentiation, highlighting the importance of specific structural features at the C-7 position for efflux pump inhibition. The study also revealed selective inhibition of AdeFGH over AdeABC, with no potentiation observed for gentamicin, showing the specificity of these quinoline-based inhibitors. Importantly, the compounds showed no toxicity in a Galleria mellonella model at a dose level of 20 mg/kg, highlighting their suitability as potential antibiotic adjuvants for combating bacterial resistance.

Pyrrolobenzodiazepines (PBDs) containing C8-linked aliphatic heterocycles have been developed as a new class of potent antibacterial compounds. They are active against multidrug resistant Gram-negative pathogens, including Klebsiella pneumoniae. When isolates were exposed to PBDs, they acquired resistance, with significant increases in inhibitory concentrations. Resistant strains showed mutations in genes associated with resistance to albicidin, specifically tsx and merR-family regulator albA. Heterologous expression of AlbA in E. coli and introducing the L120Q AlbA resistance-mediating modification into the genome of a sensitive K. pneumoniae strain conferred PBD and albicidin resistance. Proteomic analysis of the resistant strains showed elevated AlbA protein levels compared to isogenic wild-type strains. Crystallographic studies with the antibiotic binding domain of AlbA show binding of KMR-14-14 to the same groove shown to bind albicidin. Given the parallels between these two structurally unrelated compound classes, AlbA may offer resistance to further antibiotics and should be considered in future antibiotic discovery.

OBJECTIVES:Evidence-based antibiotic prescribing for urinary tract infections (UTIs) would increase treatment success and improve antibiotic stewardship. Current antimicrobial susceptibility tests (AST) are time-consuming. A novel phenotypic impedance-based Fast AST (iFAST) measures changes in the electrical phenotype of single bacteria in response to antibiotic exposure. Suitability of this technology for UTI causing bacteria was investigated. METHODS:Fifty-eight strains of Escherichia coli and Klebsiella pneumoniae were exposed to EUCAST breakpoint concentrations of UTI antibiotics. Following a two-hour exposure, the % cell count compared to unexposed control populations were compared and susceptibility deduced. Results were compared to gold standard broth microdilution (BMD) AST results. Susceptibility thresholds were clinically evaluated. Strain-antibiotic combinations with a minimum inhibitory concentration (MIC) on or one doubling dilution above the breakpoint were exposed to doubling dilutions of antibiotics and measured on iFAST to determine an electrical MIC. RESULTS:100% correlation was obtained for all eight antibiotics against laboratory strains, when allowing for the inherent 2-fold variability of the BMD MIC measurement, within a five-hour test. Clinical evaluation showed concordance in at least 74 out of 80 tests. Electrical MICs showed broad equivalence with classical MICs. CONCLUSIONS:iFAST has potential as an accurate and rapid AST for UTI causing Enterobacterales.

The growing threat of multidrug-resistant Klebsiella pneumoniae, coupled with its role in gut colonisation, has intensified the search for new treatments, including bacteriophage therapy. Despite increasing documentation of Klebsiella-targeting phages, clinical applications remain limited, with key phage–bacteria interactions still poorly understood. A major obstacle is fragmented access to well-characterised phage–bacteria pairings, restricting the collective advancement of therapeutic and mechanistic insights. To address this gap, we created the Klebsiella Phage Collection (KlebPhaCol), an open resource comprising 52 phages and 74 Klebsiella isolates, characterised at phenotypic and genomic levels. These phages span six families—including a novel family, Felixviridae, associated with the human gut—and target 20 sequence types (including ST258, ST11, and ST14) and 19 capsular-locus types (including KL1 and KL2), across 6 Klebsiella species. Freely accessible at www.klebphacol.org, KlebPhaCol invites the scientific community to both use and contribute to this resource, fostering collaborative research and a deeper understanding of Klebsiella-phage interactions beyond therapeutic use.

Severe coronavirus disease 2019 (COVID-19) patients who require hospitalization are at high risk of invasive pulmonary mucormycosis. Amphotericin B (AmB), which is the first-line therapy for invasive pulmonary mucormycosis, has been shown to promote or inhibit replication of a spectrum of viruses. In this study, we first predicted that AmB and nystatin had strong interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins using in silico screening, indicative of drugs with potential therapeutic activity against this virus. Subsequently, we investigated the impact of AmB, nystatin, natamycin, fluconazole, and caspofungin on SARS-CoV-2 infection and replication in vitro. Results showed that AmB and nystatin actually increased SARS-CoV-2 replication in Vero E6, Calu-3, and Huh7 cells. At optimal concentrations, AmB and nystatin increase SARS-CoV-2 replication by up to 100- and 10-fold in Vero E6 and Calu-3 cells, respectively. The other antifungals tested had no impact on SARS-CoV-2 infection in vitro. Drug kinetic studies indicate that AmB enhances SARS-CoV-2 infection by promoting viral entry into cells. Additionally, knockdown of genes encoding for interferon-induced transmembrane (IFITM) proteins 1, 2, and 3 suggests AmB enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of the IFITM3 protein. This study further elucidates the role of IFITM3 in viral entry and highlights the potential dangers of treating COVID-19 patients, with invasive pulmonary mucormycosis, using AmB.IMPORTANCEAmB and nystatin are common treatments for fungal infections but were predicted to strongly interact with SARS-CoV-2 proteins, indicating their potential modulation or inhibition against the virus. However, our tests revealed that these antifungals, in fact, enhance SARS-CoV-2 infection by facilitating viral entry into cells. The magnitude of enhancement could be up to 10- or 100-fold, depending on cell lines used. These findings indicate that AmB and nystatin have the potential to enhance disease when given to patients infected with SARS-CoV-2 and therefore should not be used for treatment of fungal infections in active COVID-19 cases.

Klebsiella pneumoniae is designated as one of six priority ESKAPE pathogens by the World Health Organisation (WHO). It is the causative agent of a number of serious infections, including pneumonia, and worryingly strains are known to have resistance to the four major antibiotic classes. Pyrrolobenzodiazepines (PBDs) with a C8-linked aliphatic heterocycle have been developed as a new class of potent antibacterial compounds. They are active against multidrug resistant (MDR) Gram-negative pathogens, including K. pneumoniae. When K. pneumoniae isolates were exposed to PBDs, they acquired resistance, with an increase in minimum inhibitory concentration (MIC) from 1-4 µg/mL to >32 µg/mL. Resistant strains showed mutations in genes associated with resistance to the phytotoxin albicidin, specifically tsxand merR-family regulator albA. Heterologous expression of AlbA in Escherichia coli, and introduction of a proposed resistance-mediating single-nucleotide polymorphism (SNP) (AlbA L120Q) into the genome of a sensitive K. pneumoniae strain confers both PBD and albicidin resistance. Given the parallels between these two structurally unrelated compound classes, these mechanisms may offer resistance to further antibiotics in K. pneumoniae and should be considered in future antibiotic discovery.

Aims We aimed to identify mechanisms underlying the tolerance of Proteus mirabilis-a common cause of catheter associated urinary tract infection-to the clinically used biocides chlorhexidine (CHD) and octenidine (OCT).Methods and results We adapted three clinical isolates to grow at concentrations of 512 mu g ml-1 CHD and 128 mu g ml-1 OCT. Genetic characterization and complementation studies revealed mutations inactivating the smvR repressor and increasing smvA efflux expression were associated with adaptation to both biocides. Mutations in mipA (encoding the MltA interacting protein) were less prevalent than smvR mutations and only identified in CHD adapted populations. Mutations in the rppA response regulator were exclusive to one adapted isolate and were linked with reduced polymyxin B susceptibility and a predicted gain of function after biocide adaptation. Biocide adaptation had no impact on crystalline biofilm formation.Conclusions SmvR inactivation is a key mechanism in both CHD and OCT tolerance. MipA inactivation alone confers moderate protection against CHD, and rppA showed no direct role in either CHD or OCT susceptibility.

The growing threat of multidrug-resistant Klebsiella pneumoniae, coupled with its role in gut colonisation, has intensified the search for new treatments, including bacteriophage therapy. Despite increasing documentation of Klebsiella-targeting phages, clinical applications remain limited, with key phage-bacteria interactions still poorly understood. A major obstacle is fragmented access to well-characterised phage-bacteria pairings, restricting the collective advancement of therapeutic and mechanistic insights. To address this gap, we created the Klebsiella Phage Collection (KlebPhaCol), an open-source resource comprising 53 phages and 74 Klebsiella isolates, all fully characterised. These phages span five families - including a novel order, Felixvirales, associated with the human gut - and target 27 sequence types (including ST258, ST11, ST14) and 28 capsular-locus types (including KL1 and KL2), across six Klebsiella species. Freely accessible at www.klebphacol.org, KlebPhaCol invites the scientific community to both use and contribute to this resource, fostering collaborative research and a deeper understanding of Klebsiella-phage interactions beyond therapeutic use. ### Competing Interest Statement The authors have declared no competing interest.

Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin—a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies.

The development of new therapies against SARS-CoV-2 is required to extend the toolkit of intervention strategies to combat the global pandemic. In this study, hyperimmune plasma from sheep immunised with whole spike SARS-CoV-2 recombinant protein has been used to generate candidate products. In addition to purified IgG, we have refined candidate therapies by removing non-specific IgG via affinity binding along with fragmentation to eliminate the Fc region to create F(ab′) 2 fragments. These preparations were evaluated for in vitro activity and demonstrated to be strongly neutralising against a range of SARS-CoV-2 strains, including Omicron B2.2. In addition, their protection against disease manifestations and viral loads were assessed using a hamster SARS-CoV-2 infection model. Results demonstrated protective effects of both IgG and F(ab′) 2 , with the latter requiring sequential dosing to maintain in vivo activity due to rapid clearance from the circulation.

The 2',5'- oligoadenylate synthetase (OAS) – ribonuclease L (RNAseL) - phosphodiesterase 12 (PDE12) pathway is an essential interferon-induced effector mechanism against RNA virus infection. Inhibition of PDE12 leads to selective amplification of RNAseL activity in infected cells. We aimed to investigate PDE12 as a potential pan-RNA virus antiviral drug target and develop PDE12 inhibitors that elicit antiviral activity against a range of viruses. A library of 18 000 small molecules was screened for PDE12 inhibitor activity using a fluorescent probe specific for PDE12. The lead compounds (CO-17 or CO-63) were tested in cell-based antiviral assays using encephalomyocarditis virus (EMCV), hepatitis C virus (HCV), dengue virus (DENV), West Nile virus (WNV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro. Cross reactivity of PDE12 inhibitors with other PDEs and in vivo toxicity were measured. In EMCV assays, CO-17 potentiated the effect of IFNα by 3 log10. The compounds were selective for PDE12 when tested against a panel of other PDEs and non-toxic at up to 42 mg kg−1 in rats in vivo. Thus, we have identified PDE12 inhibitors (CO-17 and CO-63), and established the principle that inhibitors of PDE12 have antiviral properties. Early studies suggest these PDE12 inhibitors are well tolerated at the therapeutic range, and reduce viral load in studies of DENV, HCV, WNV and SARS-CoV-2 in human cells and WNV in a mouse model.

The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5–67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as “resistant” to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.