Objectives : Farm animals exposed to excessive amounts of antibiotics are reservoirs for antimicrobial-resistance genes and multidrug-resistant bacteria. The goal of this study was to investigate the adjuvant activity of a previously synthesized, proline-rich lipopeptide called C12-PRP in combination with various antibiotics against multidrug-resistant E. coli isolated from chicken and cattle. Methods : The microbroth dilution method was used to determine the minimum inhibitory concentration of 10 antibiotics against four E. coli isolates. The adjuvant activity of C12-PRP was then tested in combination with the same antibiotics using a checkerboard assay. The best synergistic antibiotic-C12-PRP combinations were tested with the most resistant E. coli isolate using the Galleria mellonella insect infection model. A fluorescence-based membrane permeabilization assay was used to investigate a possible mode of action for C12-PRP. Results : The four E. coli isolates showed varying resistance to the antibiotics (0.008 µg/mL to ≥256 µg/mL), and overall, the addition of C12-PRP lowered the minimum inhibitory concentration. The two most-potent antibiotic-C12-PRP combinations were with novobiocin and erythromycin showing a susceptibility increase of 128-fold and up to 32–fold, respectively. G. mellonella survival was significantly improved when C12-PRP was included with novobiocin or erythromycin during treatment of infected larvae. Membrane permeabilization of an E. coli isolate was markedly higher in the presence of C12-PRP. Conclusions : We demonstrated the synergistic ability of C12-PRP to enhance antibiotic activity and reduce the virulence of the veterinary E. coli isolates, strengthening the potential promise of this adjuvant candidate as a therapeutic agent for multidrug-resistant bacteria.

The prevalence of antimicrobial resistance (AMR) necessitates the development of alternative therapeutic options, particularly against critical priority Gram-negative pathogens. The utilization of antibiotic adjuvants or potentiators is an advantageous strategy that targets bacterial resistance mechanisms, thereby augmenting the activity of an antibiotic used in combination. Among these, outer membrane (OM) permeabilizers are a promising class of adjuvants which compromise the OM barrier unique to Gram-negative bacteria. This review focuses on the emerging role of polymyxins and aminoglycosides – two structurally distinct antibiotics with different modes of action, but share the ability to interact with the bacterial OM. Here, we explore the design, modification, and application of polymyxin- and aminoglycoside-based OM permeabilizers, highlighting their potential against resistant Gram-negative infections.

Cefepime–taniborbactam (FEP–TAN) and meropenem–xeruborbactam (MEM–XER) are β-lactam–β-lactamase inhibitor (BL–BLI) combinations currently in development and both projected to treat metallo-β-lactamase (MBL)-producing Gram-negative pathogens.

The widespread emergence of multidrug-resistant (MDR) Gram-negative bacteria prompted the reintroduction of polymyxins in the clinic despite their adverse effects. Ongoing research is primarily focused on the development of non-nephrotoxic and -neurotoxic polymyxins as not only standalone agents but also as potentiators that enhance the activity of a partner antibiotic. Safer derivatives of polymyxin B3, a minor component of polymyxin B, were synthesized and utilized as a potentiator of multiple antibiotics. Compound 1, consisting of Dap residues, was nontoxic to kidney cells and is a promising outer membrane permeabilizer that synergized with six different classes of antibiotics against MDR Gram-negative bacteria. Compound 1 extended the activity spectrum of rifampicin, zoliflodacin, and pristinamycin by lowering the minimum inhibitory concentrations of these antibiotics below their interpretative susceptibility breakpoints in MDR Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. Notably, the novel combination of zoliflodacin, a first-in-class antibiotic in phase III trials for gonorrhea, and compound 1 exhibited potent bactericidal activity in MDR P. aeruginosa and A. baumannii.

OBJECTIVES:CANWARD is a Canadian Antimicrobial Resistance Alliance (CARA)/Health Canada partnered national surveillance study established in 2007 to annually assess the in vitro activities of commonly tested and recently approved antimicrobial agents for bacterial pathogens isolated from patients receiving care in Canadian hospitals. METHODS:In total, 34 155 Gram-negative pathogens were tested using the CLSI reference broth microdilution method. RESULTS:In total, 39.4%, 37.1%, 17.7% and 5.8% of isolates tested were from respiratory, blood, urine and wound specimens, respectively; 31.1%, 23.9%, 19.0%, 18.5% and 7.5% of isolates were from patients in medical wards, emergency rooms, ICUs, hospital clinics and surgical wards. In total, 51.8% of isolates were from male patients; and 10.1%, 40.7% and 49.2% of isolates were from patients aged ≤17, 18-64 and ≥65 years. The most common Gram-negative pathogens received were: Escherichia coli (34.9%), Pseudomonas aeruginosa (17.5%) and Klebsiella pneumoniae (11.6%). An ESBL-producing phenotype was identified in 8.4% of E. coli and 5.6% of K. pneumoniae isolates. Percent susceptible values for E. coli included: 100% for meropenem/vaborbactam and imipenem/relebactam; >99% for ceftazidime/avibactam, meropenem and ceftolozane/tazobactam; 95.6% for piperacillin/tazobactam; and 73.9% for ciprofloxacin. Percent susceptible values for K. pneumoniae included: >99% for meropenem/vaborbactam, imipenem/relebactam, ceftazidime/avibactam and meropenem; 97.4% for ceftolozane/tazobactam; 91.9% for piperacillin/tazobactam; and 87.0% for ciprofloxacin. Percent susceptible values for P. aeruginosa included: 96.6% for ceftolozane/tazobactam; 92.6% for ceftazidime/avibactam; 92.0% for imipenem/relebactam; 79.9% for piperacillin/tazobactam; ceftazidime 78.1%; 78.0% for meropenem; and 68.6% for ciprofloxacin. CONCLUSIONS:The CANWARD surveillance study has provided 17 years of reference antimicrobial susceptibility testing data.

This study reports the synthesis, cytotoxic evaluation, and mechanistic insights of an amphiphilic triamino glycosylated antitumor ether lipid (GAEL). A series of aryl-substituted tricationic d-galacto-GAELs were synthesized to mimic cationic amphiphilic drug (CAD)-like structural characteristics. Among the series, the quinoline-bearing triamino GAEL (compound 17) exhibited the highest cytotoxicity in 2D cultures against drug-sensitive and drug-resistant ovarian, breast, pancreatic, liver, prostate, and brain cancer cells, completely eliminating all cells, whereas cisplatin and doxorubicin were less effective. GAEL 17 also demonstrated superior efficacy in an SK-OV-3 3D tumor spheroid model, fully disintegrating spheroids and inducing cell death at concentrations ≥25 μM. In contrast, doxorubicin reduced viability but did not eradicate spheroids at 50 μM, likely due to slower drug action or limited penetration over 48 h exposure. GAEL 17 retained caspase-independent, non-apoptotic cell death. LysoTracker assay indicated lysosomal disruption, while LipidTOX staining showed dose-dependent fluorescence, consistent with CAD-like lipid accumulation.

Multicomponent therapy combining antibiotics with enhancer molecules known as adjuvants is an emerging strategy to combat antimicrobial resistance. Niclosamide is a clinically relevant anthelmintic drug with potential to be repurposed for its inherent antibacterial activity against Gram‐positive bacteria and its ability to potentiate the antibacterial activity of colistin against susceptible and resistant Gram‐negative bacteria. Herein, sulfonamide analogs of niclosamide were prepared and found to enhance colistin activity against Gram‐negative bacteria. The ability of niclosamide and the new sulfonamide analogs to synergize with bacitracin against vancomycin‐resistant Enterococcus faecium was also discovered.

Taniborbactam (formerly known as VNRX-5133) is a novel bicyclic boronate β-lactamase inhibitor of serine β-lactamases (SBLs) [Ambler classes A, C, and D] and metallo-β-lactamases (MBLs) [Ambler class B], including NDM and VIM, but not IMP. Cefepime–taniborbactam is active in vitro against most isolates of carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA), including both carbapenemase-producing and carbapenemase-non-producing CRE and CRPA, as well as against multidrug-resistant (MDR), ceftazidime-avibactam-resistant, meropenem-vaborbactam-resistant, and ceftolozane-tazobactam-resistant Enterobacterales and P. aeruginosa. The addition of taniborbactam to cefepime resulted in a > 64-fold reduction in MIC90 compared with cefepime alone for a 2018–2021 global collection of > 13,000 clinical isolates of Enterobacterales. In the same study, against > 4600 P. aeruginosa, a fourfold MIC reduction was observed with cefepime–taniborbactam, compared with cefepime alone. Whole genome sequencing studies have shown that resistance towards cefepime–taniborbactam in Enterobacterales arises due to the presence of multiple resistance mechanisms, often in concert, including production of IMP, PBP3 alterations, permeability (porin) defects, and upregulation of efflux pumps. In P. aeruginosa, elevated cefepime–taniborbactam MICs are also associated with the presence of multiple, concurrent mechanisms, most frequently IMP, PBP3 mutations, and upregulation of efflux pumps, as well as AmpC (PDC) overexpression. The pharmacokinetics of taniborbactam are dose proportional, follow a linear model, and do not appear to be affected when combined with cefepime. Taniborbactam’s approximate volume of distribution (Vd) at steady state is 20 L and the approximate elimination half-life (t½) is 2.3 h, which are similar to cefepime. Furthermore, like cefepime, taniborbactam is primarily cleared renally, and clearance corresponds with renal function. Pharmacodynamic studies (in vitro and in vivo) have reported that cefepime–taniborbactam has bactericidal activity against various β-lactamase-producing Gram-negative bacilli that are not susceptible to cefepime alone. It has been reported that antimicrobial activity best correlated with taniborbactam exposure (area under the curve). A phase III clinical trial showed that cefepime–taniborbactam (2 g/0.5 g administered as an intravenous infusion over 2 h) was superior to meropenem for the treatment of complicated urinary tract infection (cUTI), including acute pyelonephritis, caused by Enterobacterales species and P. aeruginosa while demonstrating similar safety compared with meropenem. The safety and tolerability of taniborbactam and cefepime–taniborbactam has been reported in one pharmacokinetic trial, and in two pharmacokinetic trials and one phase III clinical trial, respectively. Cefepime–taniborbactam appears to be well tolerated in both healthy subjects and patients. Headache and gastrointestinal upset are the most common drug-related adverse effects associated with cefepime–taniborbactam use. Cefepime–taniborbactam will likely have a role in the treatment of infections proven or suspected to be caused by MDR Gram-negative bacteria, including Enterobacterales and P. aeruginosa. In particular, it may be useful in the treatment of infections caused by isolates that harbor an MBL (NDM, VIM) enzyme, although further clinical data are needed. Additional safety and efficacy studies may support indications for cefepime–taniborbactam beyond cUTI.

The development of techniques to detect the presence of resistance in pathogens are urgently needed to face the deadly spread of multi-drug-resistant bacteria. The present work presents the electroanalytical quantification of tobramycin (TOB) retention in susceptible and resistant bacterial strains of Escherichia coli. The electrochemical characterization of TOB demonstrates the suitability of electrochemistry for drug detection. Differential pulse voltammetry (DPV) parameters were optimized by full factor experimental designs, which increased two-times the electrochemical current response, improving the overall sensitivity of the method. The developed assay was able to differentiate between resistant and susceptible E. coli strains within 15 min. The demonstrated methodology is expected to be applicable to both drug efflux-mediated and drug uptake inhibition-mediated resistant bacteria. Because these two mechanisms represent the most predominant reasons for drug resistance in bacteria, the reported method has a strong potential to be a reliable, fast, and cost-efficient alternative for antibiotic resistance detection. (c) 2024 The Electrochemical Society ("ECS"). Published on behalf of ECS by IOP Publishing Limited. All rights, including for text and data mining, AI training, and similar technologies, are reserved.

Escherichia coli are Gram-negative bacteria that are commonly found in the intestines of many different animals, including humans. E. coli can cause severe infections to humans if a foreign strain is ingested. Antibiotic resistance is a global problem for treating E. coli infections. 1 A common class of drugs for treating Gram-negative infections are aminoglycosides, such as tobramycin (TOB). The presented work focuses on the quantification of cellular drug uptake to draw conclusions about the resistance phenotype in bacteria. This procedure is applicable to pathogens that are tobramycin-resistant either because of drug efflux or because they exhibit reduced membrane permeability. TOB is an electroactive molecule which is quantitatively detected directly using electroanalytical chemistry. Herein, both susceptible and resistant strains of E. coli were exposed to TOB and their ability to retain the drug is quantified using differential pulse voltammetry. The presented method successfully determines drug resistance in E. coli using electrochemistry. References: Poirel, L. et al. Antimicrobial Resistance in Escherichia coli. Microbiology Spectrum 6 , (2018).

We describe the structure-activity relationship studies of galactose-based glycosylated antitumor ether lipids (GAELs) by installing amine groups at different positions of galactose and the glycerol backbone. Different dibasic and tribasic analogues of galacto-GAELs were synthesized and tested against a panel of human epithelial cancer cell lines. A β-anomeric triamino galactose scaffold, was the most active compound of the series and displayed CC50 in the range of 2.6 ± 0.2 μM to 6.5 ± 0.1 μM against various epithelial cancer cell lines. This compound exhibited superior activity to kill cancer cells than cisplatin. The hit GAEL compound did not induce caspase activation and therefore, the cell-killing effect does not occur due to caspase-mediated apoptosis. This observation is in line with the previously reported GAEL prototypes.

Pseudomonas aeruginosa is an opportunistic critical 'priority 1' Gram-negative bacterium that poses a severe threat to public healthcare due to rising antibiotic resistance. Particularly, low membrane permeability and overexpression of efflux pumps in P. aeruginosa lead to intrinsic resistance that compromises the antibacterial activity of antibiotics. The broad-spectrum antibiotics class, tetracyclines, are rarely used to treat P. aeruginosa infections. In the present study, we describe a series of tobramycin-ciprofloxacin (TOB-CIP) conjugates in which the carboxylic acid of ciprofloxacin is linked to the aminoglycoside tobramycin using various tethers thereby generating a cationic amphiphile. The emerging amphiphilic conjugates potentiate tetracycline antibiotics including minocycline, doxycycline, tigecycline, and eravacycline against multidrug-resistant P. aeruginosa isolates. The structure-activity relationship investigation indicates that the flexible hydrophobic C12 carbon-chain linker in TOB-CIP conjugate 1a is an optimal potentiator of tetracyclines against tetracycline-resistant and -susceptible strains of P. aeruginosa. Furthermore, conjugate 1a consistently synergized with the 3rd generation tetracycline, eravacycline, in P. aeruginosa PAO1 in the presence of up to 25% fetal bovine serum (FBS).

Colistin is primarily used as a last resort antibiotic against highly resistant Gram-negative bacteria (GNB). Rising rates of colistin resistance, however, may limit future use of this agent. The anthelmintic drug niclosamide has been shown to enhance colistin activity in combination therapy, but a detailed structure–activity relationship (SAR) for niclosamide against GNB has yet to be studied. A series of niclosamide analogs were synthesized to perform an SAR, leading to the discovery of a lead compound that displayed comparable colistin-potentiating activity to niclosamide with reduced cytotoxicity. Overall, this work provides important insights into synthetic strategies for the future development of new niclosamide derivatives and demonstrates that toxicity to mammalian cells can be reduced while maintaining colistin potentiation.

The emergence of aminoglycoside resistance has prompted the development of amphiphilic aminoglycoside derivatives which target bacterial membranes. Tobramycin and nebramine ether derivatives initially designed for this purpose were optimized and screened for their potential application as outer membrane (OM) permeabilizing adjuvants. Structure-activity relationship (SAR) studies revealed that the tobramycin benzyl ether was the most optimal OM permeabilizer, capable of potentiating rifampicin, novobiocin, vancomycin, minocycline, and doxycycline against Gram-negative bacteria. The innovative use of this compound as an adjuvant is highlighted by its ability to sensitize multidrug-resistant (MDR) Gram-negative bacteria to rifampicin and restore the susceptibility of MDR Escherichia coli to minocycline. The emergence of aminoglycoside resistance has prompted the development of amphiphilic aminoglycoside derivatives which target bacterial membranes.

Metal ions, including Fe3+, affect the target site binding of some antibiotics and control the porin- and siderophore-mediated uptake of antibiotics. Amphiphilic tobramycins are an emerging class of antibiotic potentiators capable of synergizing with multiple classes of antibiotics against Gram-negative bacteria, including Pseudomonas aeruginosa. To study how the antibiotic-potentiating effect of amphiphilic tobramycins is affected by the presence of intermolecular iron chelators, we conjugated the FDA-approved iron chelator deferiprone (DEF) to tobramycin (TOB). Three TOB-DEF conjugates differing in the length of the carbon tether were prepared and tested for antibacterial activity and synergistic relationships with a panel of antibiotics against clinical isolates of P. aeruginosa. While all TOB-DEF conjugates were inactive against P. aeruginosa, the TOB-DEF conjugates strongly synergized with outer-membrane-impermeable antibiotics, such as novobiocin and rifampicin. Among the three TOB-DEF conjugates, 1c containing a C12 tether showed a remarkable and selective potentiating effect to improve the susceptibility of multidrug-resistant P. aeruginosa isolates to tetracyclines when compared with other antibiotics. However, the antibacterial activity and antibiotic-potentiating effect of the optimized conjugate was not enhanced under iron-depleted conditions, indicating that the function of the antibiotic potentiator is not affected by the Fe3+ concentration.

Many antibiotics specific to Gram-positive bacteria like rifampicin (RIF) are inactive in Gram-negative bacteria because of outer membrane (OM) impermeability. Enhancing the OM permeability of these antibiotics with the help of OM perturbants is a promising strategy to develop new agents against Gram-negative bacteria. Here we report the synthesis and biological properties of amphiphilic tribasic galactosamines as potential RIF potentiators. Our results demonstrate that tribasic galactose-based amphiphiles potentiate RIF in multidrug-resistant Acinetobacter baumannii and Escherichia coli but not Pseudomonas aeruginosa in low salt-containing media. Under these conditions, lead compounds 20, 22 and 35 lowered the minimum inhibitory concentration of RIF by 64- to 256-fold against Gram-negative bacteria. However, the RIF-potentiating effect was reduced when bivalent Mg++ or Ca++ ions were added in the media at physiological concentrations. Overall, our results indicate that amphiphilic tribasic galactosamine-based compounds show reduced RIF-potentiating effects when compared to amphiphilic tobramycin antibiotics at physiological salt concentrations.

β-Lactam antibiotics remain one of the most effective therapeutics to treat infections caused by Gram-negative bacteria (GNB). However, since ancient times, bacteria have developed multiple resistance mechanisms toward this class of antibiotics including overexpression of β-lactamases, suppression of porins, outer membrane impermeability, overexpression of efflux pumps, and target modifications. To cope with these challenges and to extend the lifetime of existing β-lactam antibiotics, β-lactamase inhibitors are combined with β-lactam antibiotics to prevent antibiotic inactivation by β-lactamases. The combination therapy of an outer membrane permeabilizer with β-lactam antibiotics is an alternative approach to overcoming bacterial resistance of β-lactams in GNB. This approach is of particular interest for pathogens with highly impermeable outer membranes like Pseudomonas aeruginosa. Previous studies have shown that outer membrane permeabilizers can be designed by linking tobramycin and nebramine units together in the form of dimers or chimeras. In this study, we developed trimeric tobramycin and nebramine-based outer membrane permeabilizers presented on a central 1,3,5-triazine framework. The resultant trimers are capable of potentiating outer membrane-impermeable antibiotics but also β-lactams and β-lactam/β-lactamase inhibitor combinations against resistant P. aeruginosa isolates. Furthermore, the microbiological susceptibility breakpoints of ceftazidime, aztreonam, and imipenem were reached by a triple combination consisting of an outer-membrane permeabilizer/β-lactam/β-lactamase inhibitor in β-lactam-resistant P. aeruginosa isolates. Overall, our results indicate that trimeric tobramycins/nebramines can rescue clinically approved β-lactams and β-lactam/β-lactamase inhibitor combinations from resistance.

Carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) was designated as a critical priority pathogen by the World Health Organization for which new therapeutic solutions are required. With the rapid dissemination of β-lactamases in P. aeruginosa, β-lactam (BL) antibiotics are used in conjunction with β-lactamase inhibitors (BLI). The effectiveness of the BL/BLI combination could be further enhanced with the inclusion of an outer membrane (OM) permeabilizer, such as aminoglycosides and aminoglycoside-based adjuvants. Thus, the development of seven tobramycin derivatives reported herein focused on improving OM permeabilizing capabilities and reducing associated toxicity. The structure-activity relationship studies emphasized the effects of the nature of the cationic group; the number of polar head groups and positive charges; and flexibility, length, and steric bulk of the hydrophobic moiety. The optimized guanidinylated tobramycin-biphenyl derivative was noncytotoxic and demonstrated the ability to potentiate ceftazidime and aztreonam monotherapy and in dual combinations with avibactam against multidrug-resistant (MDR) and β-lactamase harboring isolates of P. aeruginosa. The triple combination of ceftazidime/avibactam plus guanidinylated tobramycin-biphenyl resulted in rapid bactericidal activity within 4-8 h of treatment, demonstrating the potential application of these guanidinylated amphiphilic tobramycin derivatives in augmenting BL/BLI combinations.

According to the World Health Organization, antibiotic resistance is a global health threat. Of particular importance are infections caused by multidrug-resistant Gram-negative bacteria including Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa for which limited treatment options exist. Multiple and simultaneously occurring resistance mechanisms including outer membrane impermeability, overexpression of efflux pumps, antibiotic-modifying enzymes, and modification of genes and antibiotic targets have made antibiotic drug development more difficult against these pathogens. One strategy to cope with these challenges is the use of outer membrane permeabilizers that increase the intracellular concentration of antibiotics when used in combination. In some circumstances, this approach can rescue antibiotics from resistance or repurpose currently marketed antibiotics. Tobramycin-based hybrid antibiotic adjuvants that combine two outer membrane-active components have been previously shown to potentiate antibiotics by facilitating transit through the outer membrane, resulting in increased antibiotic accumulation within the cell. Herein, we extended the concept of tobramycin-based hybrid antibiotic adjuvants to tobramycin-based chimeras by engineering up to three different membrane-active antibiotic warheads such as tobramycin, 1-(1-naphthylmethyl)-piperazine, ciprofloxacin, and cyclam into a central 1,3,5-triazine scaffold. Chimera 4 (TOB-TOB-CIP) consistently synergized with ciprofloxacin, levofloxacin, and moxifloxacin against wild-type and fluoroquinolone-resistant P. aeruginosa. Moreover, the susceptibility breakpoints of ceftazidime, aztreonam, and imipenem were reached using the triple combination of chimera 4 with ceftazidime/avibactam, aztreonam/avibactam, and imipenem/relebactam, respectively, against β-lactamase-harboring P. aeruginosa. Our findings demonstrate that tobramycin-based chimeras form a novel class of antibiotic potentiators capable of restoring the activity of antibiotics against P. aeruginosa.

Recurrent epithelial ovarian cancer (EOC) coincident with chemotherapy resistance remains the main contributor to patient mortality. There is an ongoing investigation to enhance patient progression-free and overall survival with novel chemotherapeutic delivery, such as the utilization of antiangiogenic medications, PARP inhibitors, or immune modulators. Our preclinical studies highlight a novel tool to combat chemotherapy-resistant human EOC. Glycosylated antitumor ether lipids (GAELs) are synthetic glycerolipids capable of killing established human epithelial cell lines from a wide variety of human cancers, including EOC cell lines representative of different EOC histotypes. Importantly, GAELs kill high-grade serous ovarian cancer (HGSOC) cells isolated from the ascites of chemotherapy-sensitive and chemotherapy-resistant patients grown as monolayers of spheroid cultures. In addition, GAELs were well tolerated by experimental animals (mice) and were capable of reducing tumor burden and blocking ascites formation in an OVCAR-3 xenograft model. Overall, GAELs show great promise as adjuvant therapy for EOC patients with or without chemotherapy resistance.