Bacterial proton motive force as an unprecedented target to control antimicrobial resistance.

Novel antibacterial therapies are urgently required to tackle the increasing number of multidrug-resistant pathogens. Identification of new antimicrobial targets is critical to avoid possible cross-resistance issues. Bacterial proton motive force (PMF), an energetic pathway located on the bacterial membrane, crucially regulates various biological possesses such as adenosine triphosphate synthesis, active transport of molecules, and rotation of bacterial flagella. Nevertheless, the potential of bacterial PMF as an antibacterial target remains largely unexplored. The PMF generally comprises electric potential (ΔΨ) and transmembrane proton gradient (ΔpH). In this review, we present an overview of bacterial PMF, including its functions and characterizations, highlighting the representative antimicrobial agents that specifically target either ΔΨ or ΔpH. At the same time, we also discuss the adjuvant potential of bacterial PMF-targeting compounds. Lastly, we highlight the value of PMF disruptors in preventing the transmission of antibiotic resistance genes. These findings suggest that bacterial PMF represents an unprecedented target, providing a comprehensive approach to controlling antimicrobial resistance.

Bismuth Drugs Reverse Tet(X)-Conferred Tigecycline Resistance in Gram-Negative Bacteria.

Antibiotic resistance has caused a serious threat to public health and human safety. Recently, the emergence of novel resistance gene tet(X4) and its variants threatens the clinical utility of tigecycline, one of the last-line antibiotics for multidrug-resistant (MDR) bacterial infections. It is highly promising to develop effective antibiotic adjuvants to restore the clinical efficacy of existing drugs and extend their life spans. Metal compounds, such as silver, have been widely used as potential antimicrobial agents for decades. However, the potentiating effect of metallo-agents on the existing antibiotics is not fully understood. Here, we found that five bismuth drugs, especially bismuth nitrate [Bi(NO3)3], commonly used in clinical treatment of stomach-associated diseases, effectively boost the antibacterial activity of tigecycline against tet(X)-positive bacteria by inhibiting the enzymatic activity of Tet(X) protein. Furthermore, the combination of Bi(NO3)3 and tigecycline prevents the development of higher-level resistance in Tet(X)-expressing Gram-negative bacteria. Using molecular docking and dynamics simulation assays, we revealed that Bi(NO3)3 can competitively bind to the active center of Tet(X4) protein, while the bismuth atom targets the Tet(X4) protein in a noncompetitive manner and changes the structure of the primary binding pocket. These two mechanisms of action both antagonize the enzymatic activity of Tet(X4) resistance protein on tigecycline. Collectively, these findings indicate the high potential of bismuth drugs as novel Tet(X) inhibitors to treat tet(X4)-positive bacteria-associated infections in combination with tigecycline. IMPORTANCE Recently, high-level tigecycline resistance mediated by tet(X4) and its variants represents a serious challenge for global public health. Antibiotic adjuvant strategy that enhances the activity of the existing antibiotics by using nonantibiotic drugs offers a distinct approach to combat the antibiotic resistance crisis. In this study, we found that bismuth drugs involve bismuth nitrate, a compound previously approved for treatment of stomach-associated diseases, remarkably potentiates tigecycline activity against tet(X)-positive bacteria. Mechanistic studies showed that bismuth drugs effectively suppress the enzymatic activity of Tet(X) resistance protein. Specifically, bismuth nitrate targets the active center of Tet(X4) protein, while bismuth binds to the resistance protein in a noncompetitive manner. Our data open up a new horizon for the treatment of infections caused by tet(X)-bearing superbugs.

Reversion of antibiotic resistance in multidrug-resistant pathogens using non-antibiotic pharmaceutical benzydamine.

Antimicrobial resistance has been a growing concern that gradually undermines our tradition treatment regimens. The fact that few antibacterial drugs with new scaffolds or targets have been approved in the past two decades aggravates this crisis. Repurposing drugs as potent antibiotic adjuvants offers a cost-effective strategy to mitigate the development of resistance and tackle the increasing infections by multidrug-resistant (MDR) bacteria. Herein, we found that benzydamine, a widely used non-steroidal anti-inflammatory drug in clinic, remarkably potentiated broad-spectrum antibiotic-tetracyclines activity against a panel of clinically important pathogens, including MRSA, VRE, MCRPEC and tet(X)-positive Gram-negative bacteria. Mechanistic studies showed that benzydamine dissipated membrane potential (▵Ψ) in both Gram-positive and Gram-negative bacteria, which in turn upregulated the transmembrane proton gradient (▵pH) and promoted the uptake of tetracyclines. Additionally, benzydamine exacerbated the oxidative stress by triggering the production of ROS and suppressing GAD system-mediated oxidative defensive. This mode of action explains the great bactericidal activity of the doxycycline-benzydamine combination against different metabolic states of bacteria involve persister cells. As a proof-of-concept, the in vivo efficacy of this drug combination was evidenced in multiple animal infection models. These findings indicate that benzydamine is a potential tetracyclines adjuvant to address life-threatening infections by MDR bacteria.

Benzydamine Reverses TMexCD-TOprJ-Mediated High-Level Tigecycline Resistance in Gram-Negative Bacteria.

Recently, a novel efflux pump gene cluster called tmexCD1-toprJ1 and its variants have been identified, which undermine the antibacterial activity of tigecycline, one of the last remaining options effective against multidrug-resistant (MDR) Gram-negative bacteria. Herein, we report the potent synergistic effect of the non-steroidal anti-inflammatory drug benzydamine in combination with tigecycline at sub-inhibitory concentrations against various temxCD-toprJ-positive Gram-negative pathogens. The combination of benzydamine and tigecycline killed all drug-resistant pathogens during 24 h of incubation. In addition, the evolution of tigecycline resistance was significantly suppressed in the presence of benzydamine. Studies on the mechanisms of synergism showed that benzydamine disrupted the bacterial proton motive force and the functionality of this kind of novel plasmid-encoded resistance-nodulation-division efflux pump, thereby promoting the intracellular accumulation of tigecycline. Most importantly, the combination therapy of benzydamine and tigecycline effectively improved the survival of Galleria mellonella larvae compared to tigecycline monotherapy. Our findings provide a promising drug combination therapeutic strategy for combating superbugs carrying the tmexCD-toprJ gene.

Gut microbiome alterations in high-fat-diet-fed mice are associated with antibiotic tolerance.

Antibiotic tolerance, the ability of a typically susceptible microorganism to survive extended periods of exposure to antibiotics, has a critical role in chronic and recurrent bacterial infections, and facilitates the evolution of antibiotic resistance. However, the physiological factors that contribute to the development of antibiotic tolerance, particularly in vivo, are not fully known. Despite the fact that a high-fat diet (HFD) is implicated in several human diseases, the relationship between HFD and antibiotic efficacy is still poorly understood. Here, we evaluated the efficacy of multiple clinically relevant bactericidal antibiotics in HFD-fed mice infected with methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli. We found that HFD-fed mice had higher bacterial burdens and these bacteria displayed lower susceptibility to bactericidal antibiotic treatment compared with mice that were fed a standard diet, while microbiota-depleted standard-diet- or HFD-fed mice showed similar susceptibility. Faecal microbiota transplantation from HFD-fed mice impaired antibiotic activity in mice fed a standard diet, indicating that alteration of the gut microbiota and related metabolites in HFD-fed mice may account for the decreased antibiotic activity. 16S rRNA sequencing and metabolomics analysis of faecal samples revealed decreased microbial diversity and differential metabolite profiles in HFD-fed mice. Notably, the tryptophan metabolite indole-3-acetic acid (IAA) was significantly decreased in HFD-fed mice. Further in vitro studies showed that IAA supplementation inhibited the formation of bacterial persisters and promoted the elimination of persisters in combination with antibiotic treatment, potentially through the activation of bacterial metabolic pathways. In vivo, the combination of IAA and ciprofloxacin increased the survival rate of HFD-fed mice infected with MRSA persisters. Overall, our data reveal that a HFD has an antagonistic effect on antibiotic treatment in a mouse model, and this is associated with the alteration of the gut microbiota and IAA production.

Amphipathic Peptide Antibiotics with Potent Activity against Multidrug-Resistant Pathogens.

The emergence and prevalence of multidrug-resistant (MDR) bacteria have posed a serious threat to public health. Of particular concern are methicillin-resistant Staphylococcus aureus (MRSA) and blaNDM, mcr-1 and tet(X)-positive Gram-negative pathogens. The fact that few new antibiotics have been approved in recent years exacerbates this global crisis, thus, new alternatives are urgently needed. Antimicrobial peptides (AMPs) originated from host defense peptides with a wide range of sources and multiple functions, are less prone to achieve resistance. All these characteristics laid the foundation for AMPs to become potential antibiotic candidates. In this study, we revealed that peptide WW307 displayed potent antibacterial and bactericidal activity against MDR bacteria, including MRSA and Gram-negative bacteria carrying blaNDM-5, mcr-1 or tet(X4). In addition, WW307 exhibited great biofilm inhibition and eradication activity. Safety and stability experiments showed that WW307 had a strong resistance against various physiological conditions and displayed relatively low toxicity. Mechanistic experiments showed that WW307 resulted in membrane damage by selectively targeting bacterial membrane-specific components, including lipopolysaccharide (LPS), phosphatidylglycerol (PG), and cardiolipin (CL). Moreover, WW307 dissipated membrane potential and triggered the production of reactive oxygen species (ROS). Collectively, these results demonstrated that WW307 represents a promising candidate for combating MDR pathogens.

Drug repurposing for next-generation combination therapies against multidrug-resistant bacteria.

Antimicrobial resistance has been a global health challenge that threatens our ability to control and treat life-threatening bacterial infections. Despite ongoing efforts to identify new drugs or alternatives to antibiotics, no new classes of antibiotic or their alternatives have been clinically approved in the last three decades. A combination of antibiotics and non-antibiotic compounds that could inhibit bacterial resistance determinants or enhance antibiotic activity offers a sustainable and effective strategy to confront multidrug-resistant bacteria. In this review, we provide a brief overview of the co-evolution of antibiotic discovery and the development of bacterial resistance. We summarize drug-drug interactions and uncover the art of repurposing non-antibiotic drugs as potential antibiotic adjuvants, including discussing classification and mechanisms of action, as well as reporting novel screening platforms. A pathogen-by-pathogen approach is then proposed to highlight the critical value of drug repurposing and its therapeutic potential. Finally, general advantages, challenges and development trends of drug combination strategy are discussed.

Thymine Sensitizes Gram-Negative Pathogens to Antibiotic Killing.

Diminished antibiotic susceptibility of bacterial pathogens is an increasingly serious threat to human and animal health. Alternative strategies are required to combat antibiotic refractory bacteria. Bacterial metabolic state has been shown to play a critical role in its susceptibility to antibiotic killing. However, the adjuvant potential of nucleotides in combination with antibiotics to kill Gram-negative pathogens remains unknown. Herein, we found that thymine potentiated ciprofloxacin killing against both sensitive and resistant-E. coli in a growth phase-independent manner. Similar promotion effects were also observed for other bactericidal antibiotics, including ampicillin and kanamycin, in the fight against four kinds of Gram-negative bacteria. The mechanisms underlying this finding were that exogenous thymine could upregulate bacterial metabolism including increased TCA cycle and respiration, which thereby promote the production of ATP and ROS. Subsequently, metabolically inactive bacteria were converted to active bacteria and restored its susceptibility to antibiotic killing. In Galleria mellonella infection model, thymine effectively improved ciprofloxacin activity against E. coli. Taken together, our results demonstrated that thymine potentiates bactericidal antibiotics activity against Gram-negative pathogens through activating bacterial metabolism, providing a universal strategy to overcome Gram-negative pathogens.

The revitalization of antimicrobial peptides in the resistance era.

The antibiotic resistance crisis is becoming incredibly thorny due to the indiscriminate employment of antibiotics in agriculture and aquaculture, such as growth promoters, and the emergence of bacteria that are capable of enduring antibiotic treatment in an endless stream. Hence, to reverse this situation, vigorous efforts should be made in the process of identifying other alternative strategies with a lower frequency of resistance. Antimicrobial peptides (AMPs), originated from host defense peptides, are generally produced by a variety of organisms as defensive weapons to protect the host from other pathogenic bacteria. The unique ability of AMPs to control bacterial infections, as well as low propensity to acquire resistance, provides the basis for it to become one of the promising antibacterial substances. Herein, we present new insights into the biological functions, structural properties, distinct mechanisms of action of AMPs and their resistance determinants. Besides, we separately discuss natural and synthetic AMPs, including their source, screening pathway and antibacterial activity. Lastly, challenges and perspectives to identify novel potent AMPs are highlighted, which will expand our understanding of the chemical space of antimicrobials and provide a pipeline for discovering the next-generation of AMPs.

Melatonin overcomes MCR-mediated colistin resistance in Gram-negative pathogens.

Background: Emergence, prevalence and widely spread of plasmid-mediated colistin resistance in Enterobacteriaceae strongly impairs the clinical efficacy of colistin against life-threatening bacterial infections. Combinations of antibiotics and FDA-approved non-antibiotic agents represent a promising means to address the widespread emergence of antibiotic-resistant pathogens. Methods: Herein, we investigated the synergistic activity between melatonin and antibiotics against MCR (mobilized colistin resistance)-positive Gram-negative pathogens through checkerboard assay and time-killing curve. Molecular mechanisms underlying its mode of action were elucidated. Finally, we assessed the in vivo efficacy of melatonin in combination with colistin against drug-resistant Gram-negative bacteria. Results: Melatonin, which has been approved for treating sleep disturbances and circadian disorders, substantially potentiates the activity of three antibiotics, particularly colistin, against MCR-expressing pathogens without enhancing its toxicity. This is evidence that the combination of colistin with melatonin enhances bacterial outer membrane permeability, promotes oxidative damage and inhibits the effect of efflux pumps. In three animal models infected by mcr-1-carrying E. coli, melatonin dramatically rescues colistin efficacy. Conclusion: Our findings revealed that melatonin serves as a promising colistin adjuvant against MCR-positive Gram-negative pathogens.

Potent Broad-Spectrum Antibacterial Activity of Amphiphilic Peptides against Multidrug-Resistant Bacteria.

The emergence and prevalence of multidrug-resistant (MDR) bacteria particularly Gram-negative bacteria presents a global crisis for human health. Colistin and tigecycline were recognized as the last resort of defenses against MDR Gram-negative pathogens. However, the emergence and prevalence of MCR or Tet(X)-mediated acquired drug resistance drastically impaired their clinical efficacy. It has been suggested that antimicrobial peptides might act a crucial role in combating antibiotic resistant bacteria owing to their multiple modes of action and characteristics that are not prone to developing drug resistance. Herein, we report a safe and stable tryptophan-rich amphiphilic peptide termed WRK-12 with broad-spectrum antibacterial activity against various MDR bacteria, including MRSA, colistin and tigecycline-resistant Escherichia coli. Mechanistical studies showed that WRK-12 killed resistant E. coli through permeabilizing the bacterial membrane, dissipating membrane potential and triggering the production of reactive oxygen species (ROS). Meanwhile, WRK-12 significantly inhibited the formation of an E. coli biofilm in a dose-dependent manner. These findings revealed that amphiphilic peptide WRK-12 is a promising drug candidate in the fight against MDR bacteria.

Correlation between Exogenous Compounds and the Horizontal Transfer of Plasmid-Borne Antibiotic Resistance Genes.

The global spread of antibiotic resistance has posed a serious threat to public healthcare and undermined decades of progress made in the fight against bacterial infections. It has been demonstrated that the lack of novel effective antibiotics and rapid spread of antibiotic resistance genes via horizontal transfer in the ecosystem are mainly responsible for this crisis. Notably, plasmid-mediated horizontal transfer of antibiotic resistance genes (ARGs) is recognized as the most dominant dissemination pathway of ARGs in humans, animals and environmental settings. Antibiotic selective pressure has always been regarded as one of the crucial contributors to promoting the dissemination of antibiotic resistance through horizontal gene transfer (HGT). However, the roles of exogenous compounds and particularly non-antibiotic drugs in the spread of ARGs are still underappreciated. In this review, we first summarize the major pathways of HGT in bacteria, including conjugation, transformation, transduction and vesiduction. Subsequently, an overview of these compounds capable of promoting the HGT is presented, which guides to the formulation of more reasonable dosing regimens and drug residue standards in clinical practice. By contrast, these compounds that display an inhibition effect on HGT are also highlighted, which provides a unique strategy to minimize the spread of ARGs. Lastly, we discuss the implementations and challenges in bringing these HGT inhibitors into clinical trials.

Cysteine Potentiates Bactericidal Antibiotics Activity Against Gram-Negative Bacterial Persisters.

PURPOSE: Bacterial metabolism regulators offer a novel productive strategy in the eradication of antibiotic refractory bacteria, particularly bacterial persisters. However, the potential of amino acids in the fight against Gram-negative bacterial persisters has not been fully explored. The aim of this study is to investigate the potentiation of amino acids to antibiotics in combating Gram-negative bacterial persisters and to reveal the underlying mechanisms of action. METHODS: Bactericidal activity of antibiotics in the absence or presence of amino acids was evaluated through detecting the reduction of bacterial CFUs. The ratio of NAD+/NADH in E. coli B2 persisters was determined using assay kit with WST-8. Bacterial respiration and ROS production were measured by the reduction of iodonitrotetrazolium chloride and fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, respectively. RESULTS: In this study, we found that cysteine possesses excellent synergistic bactericidal activity with ciprofloxacin against multiple Gram-negative bacterial persisters. Furthermore, the potentiation of cysteine was evaluated in exponential and stationary-phase E. coli ATCC 25922 and E. coli B2. Interestingly, cysteine significantly improves three bactericidal antibiotics killing against stationary-phase bacteria, but not exponential-phase bacteria, implying that the effect of cysteine correlates with the metabolic state of bacteria. Mechanistic studies revealed that cysteine accelerates the bacterial TCA cycle and promotes bacterial respiration and ROS production. These metabolic regulation effects of cysteine re-sensitive bacterial persisters to antibiotic killing. CONCLUSION: Collectively, our study highlights the synergistic bactericidal activity of bacterial metabolism regulators such as cysteine with commonly used antibiotics against Gram-negative bacterial persisters.