Combining commercial antibiotics with adjuvants to lower their minimum inhibitory concentration (MIC) is vital in combating antimicrobial resistance. Evaluating the ecotoxicity of such compounds is crucial due to environmental and health risks. Here, eugenol was assessed as an adjuvant for 7 commercial antibiotics against 14 pathogenic bacteria in vitro, also examining its acute ecotoxicity on various soil and water organisms (microbiota, Vibrio fischeri, Daphnia magna, Eisenia foetida, and Allium cepa). Using microdilution methods, checkerboard assays, and kinetic studies, the MICs for eugenol were determined together with the nature of its combinations with antibiotics against bacteria, some unexposed to eugenol previously. The lethal dose for the non-target organisms was also determined, as well as the Average Well Color Development and the Community-Level Physiological Profiling for soil and water microbiota. Our findings indicate that eugenol significantly reduces MICs by 75 to 98%, which means that it could be a potent adjuvant. Ecotoxicological assessments showed eugenol to be less harmful to water and soil microbiota compared to studied antibiotics. While Vibrio fischeri and Daphnia magna were susceptible, Allium cepa and Eisenia foetida were minimally affected. Given that only 0.1% of eugenol is excreted by humans without metabolism, its environmental risk when used with antibiotics appears minimal.

The number of new antibacterial agents currently being discovered is insufficient to combat bacterial resistance. It is extremely challenging to find new antibiotics and to introduce them to the pharmaceutical market. Therefore, special attention must be given to find new strategies to combat bacterial resistance and prevent bacteria from developing resistance. Two-component system is a transduction system and the most prevalent mechanism employed by bacteria to respond to environmental changes. This signaling system consists of a membrane sensor histidine kinase that perceives environmental stimuli and a response regulator which acts as a transcription factor. The approach consisting of developing response regulators inhibitors with antibacterial activity or antibiotic adjuvant activity is a novel approach that has never been previously reviewed. In this review we report for the first time, the importance of targeting response regulators and summarizing all existing studies carried out from 2008 until now on response regulators inhibitors as antibacterial agents or / and antibiotic adjuvants. Moreover, we describe the antibacterial activity and/or antibiotic adjuvants activity against the studied bacterial strains and the mechanism of different response regulator inhibitors when it\'s possible.

The rapid expansion of antibiotic-resistant bacterial diseases is a global burden on public health. It makes sense to repurpose and reposition already-approved medications for use as supplementary agents in synergistic combinations with existing antibiotics. Here, we demonstrate that the anthelmintic drug nitazoxanide (NTZ) synergistically enhances the effectiveness of the lipopeptide antibiotic polymyxin B in inhibiting gram-negative bacteria, including those resistant to polymyxin B. Mechanistic investigations revealed that nitazoxanide inhibited calcium influx and cell membrane depolarization, enhanced the affinity between polymyxin B and the extracellular membrane, and promoted intracellular ATP depletion and an increase in reactive oxygen species (ROS), thus enhancing the penetration and disruption of the Escherichia coli cell membrane by polymyxin B. The transcriptomic analysis revealed that the combination resulted in energy depletion by inhibiting both aerobic and anaerobic respiration patterns in bacterial cells. The increased bactericidal effect of polymyxin B on the E. coli ∆nuoC strain further indicates that NuoC could be a promising target for nitazoxanide. Furthermore, the combination of nitazoxanide and polymyxin B showed promising therapeutic effects in a mouse infection model infected with E. coli. Taken together, these results demonstrate the potential of nitazoxanide as a novel adjuvant to polymyxin B, to overcome antibiotic resistance and improve therapeutic outcomes in refractory infections.IMPORTANCEThe rapid spread of antibiotic-resistant bacteria poses a serious threat to public health. The search for potential compounds that can increase the antibacterial activity of existing antibiotics is a promising strategy for addressing this issue. Here, the synergistic activity of the FDA-approved agent nitazoxanide (NTZ) combined with polymyxin B was investigated in vitro using checkerboard assays and time-kill curves. The synergistic mechanisms of the combination of nitazoxanide and polymyxin B were explored by fluorescent dye, transmission electron microscopy (TEM), and transcriptomic analysis. The synergistic efficacy was evaluated in vivo by the Escherichia coli and mouse sepsis models. These results suggested that nitazoxanide, as a promising antibiotic adjuvant, can effectively enhance polymyxin B activity, providing a potential strategy for treating multidrug-resistant bacteria.

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide \'SVAP9I\' that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4-8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli\'s outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I\'s synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I\'s distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections.

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.

The emergence and widespread of multidrug-resistant Gram-negative bacteria have posed a severe threat to human health and environmental safety, escalating into a global medical crisis. Utilization of antibiotic adjuvants is a rapid approach to combat bacterial resistance effectively since the development of new antimicrobial agents is a formidable challenge. NhaA, driven by proton motive force, is a crucial secondary transporter on the cytoplasmic membrane of Escherichia coli. We found that 2-Aminoperimidine (2-AP), which is a specific inhibitor of NhaA, could enhance the activity of colistin against sensitive E. coli and reverse the resistance in mcr-1 positive E. coli. Mechanistic studies indicated that 2-AP induced dysfunction in cytoplasmic membrane through the suppression of NhaA, leading to metabolic inhibition and ultimately enhancing the sensitivity of E. coli to colistin. Moreover, 2-AP restored the efficacy of colistin against resistant E. coli in two animal infection models. Our findings reveal the potential of NhaA as a novel target for colistin adjuvants, providing new possibilities for the clinical application of colistin.

The resistance of multidrug-resistant bacteria to existing antibiotics forces the continued development of new antibiotics and antibacterial agents, but the high costs and long timeframe involved in the development of new agents renders the hope that existing antibiotics may again play a part. The \"antibiotic adjuvant\" is an indirect antibacterial strategy, but its vague concept has, in the past, limited the development speed of related drugs. In this review article, we put forward an accurate concept of a \"non-self-antimicrobial sensitisers (NSAS)\", to distinguish it from an \"antibiotic adjuvant\", and then discuss several scientific methods to restore bacterial sensitivity to antibiotics, and the sources and action mechanism of existing NSAS, in order to guide the development and further research of NSAS.

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.

BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.
METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect.
RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1).
CONCLUSIONS: This study provides the first evidence of gramine\'s therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.

Hematoma, a risk factor of implant-associated infections (IAIs), creates a Fe-rich environment following implantation, which proliferates the growth of pathogenic bacteria. Fe metabolism is a major vulnerability for pathogens and is crucial for several fundamental physiological processes. Herein, a deferiprone (DFP)-loaded layered double hydroxide (LDH)-based nanomedicine (DFP@Ga-LDH) that targets the Fe-rich environments of IAIs is reported. In response to acidic changes at the infection site, DFP@Ga-LDH systematically interferes with bacterial Fe metabolism via the substitution of Ga3+ and Fe scavenging by DFP. DFP@Ga-LDH effectively reverses the Fe/Ga ratio in Pseudomonas aeruginosa, causing comprehensive interference in various Fe-associated targets, including transcription and substance metabolism. In addition to its favorable antibacterial properties, DFP@Ga-LDH functions as a nano-adjuvant capable of delaying the emergence of antibiotic resistance. Accordingly, DFP@Ga-LDH is loaded with a siderophore antibiotic (cefiderocol, Cefi) to achieve the antibacterial nanodrug DFP@Ga-LDH-Cefi. Antimicrobial and biosafety efficacies of DFP@Ga-LDH-Cefi are validated using ex vivo human skin and mouse IAI models. The pivotal role of the hematoma-created Fe-rich environment of IAIs is highlighted, and a nanoplatform that efficiently interferes with bacterial Fe metabolism is developed. The findings of the study provide promising guidance for future research on the exploration of nano-adjuvants as antibacterial agents.