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976500.9354Daunorubicin resensitizes Gram-negative superbugs to the last-line antibiotics and prevents the transmission of antibiotic resistance. Although meropenem, colistin, and tigecycline are recognized as the last-line antibiotics for multidrug-resistant Gram-negative bacteria (MDR-GN), the emergence of mobile resistance genes such as bla(NDM), mcr, and tet(X) severely compromises their clinical effectiveness. Developing novel antibiotic adjuvants to restore the effectiveness of existing antibiotics provides a feasible approach to address this issue. Herein, we discover that a Food and Drug Administration (FDA)-approved drug daunorubicin (DNR) drastically potentiates the activity of last-resort antibiotics against MDR-GN pathogens and biofilm-producing bacteria. Furthermore, DNR effectively inhibits the evolution and spread of colistin and tigecycline resistance. Mechanistically, DNR and colistin combination exacerbates membrane disruption, induces DNA damage and the massive production of reactive oxygen species (ROS), ultimately leading to bacterial cell death. Importantly, DNR restores the effectiveness of colistin in Galleria mellonella and murine models of infection. Collectively, our findings provide a potential drug combination strategy for treating severe infections elicited by Gram-negative superbugs.202337235051
905010.9346Cationic Polysaccharide Conjugates as Antibiotic Adjuvants Resensitize Multidrug-Resistant Bacteria and Prevent Resistance. In recent years, traditional antibiotic efficacy has rapidly diminished due to the advent of multidrug-resistant (MDR) bacteria, which poses severe threat to human life and globalized healthcare. Currently, the development cycle of new antibiotics cannot match the ongoing MDR infection crisis. Therefore, novel strategies are required to resensitize MDR bacteria to existing antibiotics. In this study, novel cationic polysaccharide conjugates Dextran-graft-poly(5-(1,2-dithiolan-3-yl)-N-(2-guanidinoethyl)pentanamide) (Dex-g-PSS(n) ) is synthesized using disulfide exchange polymerization. Critically, bacterial membranes and efflux pumps are disrupted by a sub-inhibitory concentration of Dex-g-PSS(30) , which enhances rifampicin (RIF) accumulation inside bacteria and restores its efficacy. Combined Dex-g-PSS(30) and RIF prevents bacterial resistance in bacteria cultured over 30 generations. Furthermore, Dex-g-PSS(30) restores RIF effectiveness, reduces inflammatory reactions in a pneumonia-induced mouse model, and exhibits excellent in vivo biological absorption and degradation capabilities. As an antibiotic adjuvant, Dex-g-PSS(30) provides a novel resensitizing strategy for RIF against MDR bacteria and bacterial resistance. This Dex-g-PSS(30) research provides a solid platform for future MDR applications.202235962720
905320.9343Nordihydroguaiaretic acid reverses the antibacterial activity of colistin against MCR-1-positive bacteria in vivo/in vitro by inhibiting MCR-1 activity and injuring the bacterial cell membrane. BACKGROUND: Colistin (polymyxin E) is an effective antibiotic for the treatment of most multidrug-resistant Gram-negative bacteria. However, some bacteria, including bacterial spp. belonging to the Enterobacteriaceae family, have an acquired resistance against polymyxins, which is attributed to they possess plasmid-carried resistance genes (mcr-1 and its variants). So, there is an urgent need to develop new therapeutic strategies to target broad spectrum resistant spp. from Enterobacteriaceae family in response to the loss of the protective barrier of last-line antibiotics. Here, we report the adjuvant capacity of nordihydroguaiaretic acid (NDGA) for restoring the antibacterial activity of colistin against MCR-1-positive E. coli ZJ487 in vivo/in vitro. METHODS: A checkerboard assay, time-killing analysis, isobolograms, growth curves and inducible resistance test showed the effect of NDGA combined with colistin in vitro. TLC was used to detect the inhibitory effect of NDGA on MCR-1. Colony determination and hematoxylin and eosin (HE) staining were used to assess the synergistic effect of NDGA and colistin in mice. RESULTS: Our results showed that NDGA in combination with colistin showed a synergistic bactericidal action without inducing resistance. NDGA directly inhibited MCR-1 activity and resulted in measurable injury to the bacterial cell membrane to recover the antibacterial effect of colistin. Most importantly, NDGA in combination with colistin exhibited an in vivo synergistic effect in murine peritonitis infection models, as evidenced by the survival rate of MCR-1-positive E. coli ZJ487-infected mice which increased from 6.67 to 50.0%. CONCLUSION: Our study demonstrated that NDGA effectively rescues the efficiency of colistin against MCR-positive E. coli ZJ487 by simultaneously inhibiting both, the MCR activity and the injury to the cell membrane of bacteria.202235158237
843230.9341A 0D-2D Heterojunction Bismuth Molybdate-Anchored Multifunctional Hydrogel for Highly Efficient Eradication of Drug-Resistant Bacteria. Due to the increasing antibiotic resistance and the lack of broad-spectrum antibiotics, there is an urgent requirement to develop fresh strategies to combat multidrug-resistant pathogens. Herein, defect-rich bismuth molybdate heterojunctions [zero-dimensional (0D) Bi(4)MoO(9)/two-dimensional (2D) Bi(2)MoO(6), MBO] were designed for rapid capture of bacteria and synergistic photocatalytic sterilization. The as-prepared MBO was experimentally and theoretically demonstrated to possess defects, heterojunctions, and irradiation triple-enhanced photocatalytic activity for efficient generation of reactive oxygen species (ROS) due to the exposure of more active sites and separation of effective electron-hole pairs. Meanwhile, dopamine-modified MBO (pMBO) achieved a positively charged and rough surface, which conferred strong bacterial adhesion and physical penetration to the nanosheets, effectively trapping bacteria within the damage range and enhancing ROS damage. Based on this potent antibacterial ability of pMBO, a multifunctional hydrogel consisting of poly(vinyl alcohol) cross-linked tannic acid-coated cellulose nanocrystals (CPTB) and pMBO, namely CPTB@pMBO, is developed and convincingly effective against methicillin-resistant Staphylococcus aureus in a mouse skin infection model. In addition, the strategy of combining a failed beta-lactam antibiotic with CPTB@pMBO to photoinactivation with no resistance observed was developed, which presented an idea to address the issue of antibiotic resistance in bacteria and to explore facile anti-infection methods. In addition, CPTB@pMBO can reduce excessive proteolysis of tissue and inflammatory response by regulating the expression of genes and pro-inflammatory factors in vivo, holding great potential for the effective treatment of wound infections caused by drug-resistant bacteria.202337531599
815640.9337Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria. Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or bla(KPC) genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.202438863339
503450.9337Resensitizing carbapenem- and colistin-resistant bacteria to antibiotics using auranofin. Global emergence of Gram-negative bacteria carrying the plasmid-borne resistance genes, bla(MBL) and mcr, raises a significant challenge to the treatment of life-threatening infections by the antibiotics, carbapenem and colistin (COL). Here, we identify an antirheumatic drug, auranofin (AUR) as a dual inhibitor of metallo-β-lactamases (MBLs) and mobilized colistin resistance (MCRs), two resistance enzymes that have distinct structures and substrates. We demonstrate that AUR irreversibly abrogates both enzyme activity via the displacement of Zn(II) cofactors from their active sites. We further show that AUR synergizes with antibiotics on killing a broad spectrum of carbapenem and/or COL resistant bacterial strains, and slows down the development of β-lactam and COL resistance. Combination of AUR and COL rescues all mice infected by Escherichia coli co-expressing MCR-1 and New Delhi metallo-β-lactamase 5 (NDM-5). Our findings provide potential therapeutic strategy to combine AUR with antibiotics for combating superbugs co-producing MBLs and MCRs.202033067430
503260.9336Hijacking a small plasmid to confer high-level resistance to aztreonam-avibactam and ceftazidime-avibactam. Acquired β-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria, which also commonly carry multi-copy small plasmids. This study found that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. This study focused on aztreonam-avibactam (ATM-AVI) as this combination can be used to effectively counter almost all β-lactamases produced by bacteria, and has been recommended against carbapenem-resistant Enterobacterales. A clinical strain (085003) of carbapenem-resistant Escherichia coli was investigated, and mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L of ATM-AVI were obtained as representatives of low- and high-level resistance, respectively, by induction. Comparative genomics showed that 085003R32 and 085003R512 had a single nucleotide mutation of β-lactamase gene bla(CMY-2), encoding a novel CMY with a Thr319Ile substitution, assigned 'CMY-2R'. Cloning and enzyme kinetics were used to verify that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. Mechanisms for the discrepant resistance between 085003R32 and 085003R512 were investigated. Three tandem copies of bla(CMY-2R) were identified on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of bla(CMY-2R) on the IncP1 plasmid, but possessed anther bla(CMY-2R) on an already present 4-kb small plasmid. IS1294-mediated mobilization on to this multi-copy small plasmid increased the copy number of bla(CMY-2R) significantly, rendering higher resistance. This study shows that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.202337769749
883670.9336Identification of an anti-virulence drug that reverses antibiotic resistance in multidrug resistant bacteria. The persistent incidence of high levels of multidrug-resistant (MDR) bacteria seriously endangers global public health. In response to MDR-associated infections, new antibacterial drugs and strategies are particularly needed. Screening to evaluate a potential compound to reverse antibiotic resistance is a good strategy to alleviate this crisis. In this paper, using high-throughput screening methods, we identified that oxyclozanide potentiated tetracycline antibiotics act against MDR bacterial pathogens by promoting intracellular accumulation of tetracycline in resistant bacteria. Furthermore, mechanistic studies demonstrated that oxyclozanide could directly kill bacteria by disrupting bacterial membrane and inducing the overproduction of bacterial reactive oxygen species. Oxyclozanide effectively reduced the production of virulence proteins in S. aureus and neutralized the produced α-hemolysin, thereby effectively alleviating the inflammatory response caused by bacteria. Finally, oxyclozanide significantly reversed tetracycline resistance in animal infection assays. In summary, these results demonstrated the capacity of oxyclozanide as a novel antibiotic adjuvant, antibacterial and anti-virulence multifunctional compound to circumvent MDR bacteria and improve the therapeutic effect of persistent infections caused by MDR bacteria worldwide.202235797943
922180.9336Breaking antimicrobial resistance by disrupting extracytoplasmic protein folding. Antimicrobial resistance in Gram-negative bacteria is one of the greatest threats to global health. New antibacterial strategies are urgently needed, and the development of antibiotic adjuvants that either neutralize resistance proteins or compromise the integrity of the cell envelope is of ever-growing interest. Most available adjuvants are only effective against specific resistance proteins. Here, we demonstrate that disruption of cell envelope protein homeostasis simultaneously compromises several classes of resistance determinants. In particular, we find that impairing DsbA-mediated disulfide bond formation incapacitates diverse β-lactamases and destabilizes mobile colistin resistance enzymes. Furthermore, we show that chemical inhibition of DsbA sensitizes multidrug-resistant clinical isolates to existing antibiotics and that the absence of DsbA, in combination with antibiotic treatment, substantially increases the survival of Galleria mellonella larvae infected with multidrug-resistant Pseudomonas aeruginosa. This work lays the foundation for the development of novel antibiotic adjuvants that function as broad-acting resistance breakers.202235025730
843690.9336NIR-Activated Hydrogel with Dual-Enhanced Antibiotic Effectiveness for Thorough Elimination of Antibiotic-Resistant Bacteria. Antibiotic resistance has become a critical health crisis globally. Traditional strategies using antibiotics can lead to drug-resistance, while inorganic antimicrobial agents can cause severe systemic toxicity. Here, we have developed a dual-antibiotic hydrogel delivery system (PDA-Ag@Levo/CMCS), which can achieve controlled release of clinical antibiotics levofloxacin (Levo) and classic nanoscale antibiotic silver nanoparticles (AgNPs), effectively eliminating drug-resistant P. aeruginosa. Benefiting from the photothermal (PTT) effect of polydopamine (PDA), the local high temperature generated by PDA-Ag@Levo/CMCS can quickly kill bacteria through continuous and responsive release of dual-antibiotics to restore sensitivity to ineffective antibiotics. Moreover, AgNPs could significantly improve the efficiency of traditional antibiotics by disrupting bacterial membranes and reducing their toxicity to healthy tissues. A clever combination of PTT and drug-combination therapy can effectively eliminate biofilms and drug-resistant bacteria. Mechanism studies have shown that PDA-Ag@Levo might eliminate drug-resistant P. aeruginosa by disrupting biofilm formation and protein synthesis, and inhibit the resistance mutation of P. aeruginosa by promoting the expression of related genes, such as rpoS, dinB, and mutS. Collectively, the synergistic effect of this dual-antibiotic hydrogel combined with PTT provides a creative strategy for eliminating drug-resistant bacteria in chronic infection wounds.202539760335
5690100.9335Rapid Detection of MCR-Mediated Colistin Resistance in Escherichia coli. Colistin is one of the last-resort antibiotics for infections caused by multidrug-resistant Gram-negative bacteria. However, the wide spread of novel plasmid-carrying colistin resistance genes mcr-1 and its variants substantially compromise colistin's therapeutic effectiveness and pose a severe danger to public health. To detect colistin-resistant microorganisms induced by mcr genes, rapid and reliable antibiotic susceptibility testing (AST) is imminently needed. In this study, we identified an RNA-based AST (RBAST) to discriminate between colistin-susceptible and mcr-1-mediated colistin-resistant bacteria. After short-time colistin treatment, RBAST can detect differentially expressed RNA biomarkers in bacteria. Those candidate mRNA biomarkers were successfully verified within colistin exposure temporal shifts, concentration shifts, and other mcr-1 variants. Furthermore, a group of clinical strains were effectively distinguished by using the RBAST approach during the 3-h test duration with over 93% accuracy. Taken together, our findings imply that certain mRNA transcripts produced in response to colistin treatment might be useful indicators for the development of fast AST for mcr-positive bacteria. IMPORTANCE The emergence and prevalence of mcr-1 and its variants in humans, animals, and the environment pose a global public health threat. There is a pressing urgency to develop rapid and accurate methods to identify MCR-positive colistin-resistant bacteria in the clinical samples, providing a basis for subsequent effective antibiotic treatment. Using the specific mRNA signatures, we develop an RNA-based antibiotic susceptibility testing (RBAST) for effectively distinguishing colistin-susceptible and mcr-1-mediated colistin-resistant strains. Meanwhile, the detection efficiency of these RNA biomarkers was evidenced in other mcr variants-carrying strains. By comparing with the traditional AST method, the RBAST method was verified to successfully characterize a set of clinical isolates during 3 h assay time with over 93% accuracy. Our study provides a feasible method for the rapid detection of colistin-resistant strains in clinical practice.202235616398
9808110.9333Understanding Recent Developments in Colistin Resistance: Mechanisms, Clinical Implications, and Future Perspectives. Colistin resistance, driven by chromosomal mutations and the spread of plasmid-mediated MCR genes, has emerged as a critical challenge in combating multidrug-resistant Gram-negative bacteria. This resistance compromises the efficacy of colistin, leading to higher treatment failure rates, prolonged hospitalizations, and increased mortality. Recent studies have highlighted key mechanisms, including lipid A modifications, that enable bacteria to evade colistin's effects. The global spread of MCR genes exacerbates the issue, underlining the need for improved diagnostics and rapid detection of resistant strains to prevent adverse patient outcomes. To combat this growing threat, a multifaceted approach is essential, involving enhanced antimicrobial stewardship, stricter infection control measures, and continued research into alternative therapies and diagnostic methods. Collaborative efforts from researchers, healthcare providers, policymakers, and the pharmaceutical industry are crucial to preserving colistin's effectiveness and mitigating the broader impact on public health.202541148650
9746120.9331Fluoroamphiphilic polymers exterminate multidrug-resistant Gram-negative ESKAPE pathogens while attenuating drug resistance. ESKAPE pathogens are a panel of most recalcitrant bacteria that could "escape" the treatment of antibiotics and exhibit high incidence of drug resistance. The emergence of multidrug-resistant (MDR) ESKAPE pathogens (particularly Gram-negative bacteria) accounts for high risk of mortality and increased resource utilization in health care. Worse still, there has been no new class of antibiotics approved for exterminating the Gram-negative bacteria for more than 50 years. Therefore, it is urgent to develop novel antibacterial agents with low resistance and potent killing efficacy against Gram-negative ESKAPE pathogens. Herein, we present a class of fluoropolymers by mimicking the amphiphilicity of cationic antimicrobial peptides. Our optimal fluoroamphiphilic polymer (PD(45)HF(5)) displayed selective antimicrobial ability for all MDR Gram-negative ESAKPE pathogens, low resistance, high in vitro cell selectivity, and in vivo curative efficacy. These findings implied great potential of fluoroamphiphilic cationic polymers as promising antibacterial agents against MDR Gram-negative ESKAPE bacteria and alleviating antibiotic resistance.202439196947
9099130.9328Small molecule downregulation of PmrAB reverses lipid A modification and breaks colistin resistance. Infections caused by multi-drug resistant bacteria, particularly Gram-negative bacteria, are an ever-increasing problem. While the development of new antibiotics remains one option in the fight against bacteria that have become resistant to currently available antibiotics, an attractive alternative is the development of adjuvant therapeutics that restore the efficacy of existing antibiotics. We report a small molecule adjuvant that suppresses colistin resistance in multidrug resistant Acinetobacter baumannii and Klebsiella pneumoniae by interfering with the expression of a two-component system. The compound downregulates the pmrCAB operon and reverses phosphoethanolamine modification of lipid A responsible for colistin resistance. Furthermore, colistin-susceptible and colistin-resistant bacteria do not evolve resistance to combination treatment. This represents the first definitive example of a compound that breaks antibiotic resistance by directly modulating two-component system activity.201424131198
8192140.9328Resisting the Heat: Bacterial Disaggregases Rescue Cells From Devastating Protein Aggregation. Bacteria as unicellular organisms are most directly exposed to changes in environmental growth conditions like temperature increase. Severe heat stress causes massive protein misfolding and aggregation resulting in loss of essential proteins. To ensure survival and rapid growth resume during recovery periods bacteria are equipped with cellular disaggregases, which solubilize and reactivate aggregated proteins. These disaggregases are members of the Hsp100/AAA+ protein family, utilizing the energy derived from ATP hydrolysis to extract misfolded proteins from aggregates via a threading activity. Here, we describe the two best characterized bacterial Hsp100/AAA+ disaggregases, ClpB and ClpG, and compare their mechanisms and regulatory modes. The widespread ClpB disaggregase requires cooperation with an Hsp70 partner chaperone, which targets ClpB to protein aggregates. Furthermore, Hsp70 activates ClpB by shifting positions of regulatory ClpB M-domains from a repressed to a derepressed state. ClpB activity remains tightly controlled during the disaggregation process and high ClpB activity states are likely restricted to initial substrate engagement. The recently identified ClpG (ClpK) disaggregase functions autonomously and its activity is primarily controlled by substrate interaction. ClpG provides enhanced heat resistance to selected bacteria including pathogens by acting as a more powerful disaggregase. This disaggregase expansion reflects an adaption of bacteria to extreme temperatures experienced during thermal based sterilization procedures applied in food industry and medicine. Genes encoding for ClpG are transmissible by horizontal transfer, allowing for rapid spreading of extreme bacterial heat resistance and posing a threat to modern food production.202134017857
9772150.9328Naringenin Microsphere as a Novel Adjuvant Reverses Colistin Resistance via Various Strategies against Multidrug-Resistant Klebsiella pneumoniae Infection. The efficacy of colistin, the last option against multidrug-resistant (MDR) Gram-negative bacteria, is severely threatened by the prevalence of plasmid- or chromosome-mediated colistin resistance genes. Herein, naringenin has dramatically restored colistin sensitivity against colistin-resistant Klebsiella pneumoniae infection without affecting bacterial viability, inducing resistance and causing obvious cell toxicity. Mechanism analysis reveals that naringenin potentiates colistin activity by multiple strategies including inhibition of mobilized colistin resistance gene activity, repression of two-component system regulation, and acceleration of reactive oxygen species-mediated oxidative damage. A lung-targeted delivery system of naringenin microspheres has been designed to facilitate naringenin bioavailability, accompanied by an effective potentiation of colistin for Klebsiella pneumoniae infection. Consequently, a new recognition of naringenin microspheres has been elucidated to restore colistin efficacy against colistin-resistant Gram-negative pathogens, which may be an effective strategy of developing potential candidates for MDR Gram-negative bacteria infection.202236530172
639160.9327A Single Residue within the MCR-1 Protein Confers Anticipatory Resilience. The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz., the two-component signal transduction system CpxRA (conjugative pilus expression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers.202337071007
5033170.9326A novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Imipenem-resistant Escherichia coli strains represent a growing public health concern, posing a threat due to their resistance to last-resort antibiotics. Here, we present the discovery of the Imipenem-Linked Resistance Gene VIN (ILR-VIN) within E. coli isolates from Vietnam, revealing its absence in non-resistant E. coli and local bacterial species. ILR-VIN constitutes a previously unrecognized genetic element potentially linked to Imipenem resistance, with notable prevalence in Vietnamese E. coli strains.We conducted an in-depth examination of the genetic basis of Carbapenem resistance in E. coli strains causing urinary tract infections. In a set of 47 UTI strains, we identified five displaying Imipenem resistance, with four of them carrying known resistance genes. Interestingly, ECV219, despite exhibiting Imipenem resistance, lacked known resistance genes, suggesting an unreported resistance mechanism. Comparative genetic analysis revealed distinct genes in ECV219, indicating a novel Imipenem resistance gene. To assess its function, we conducted transformation experiments in E. coli Rosetta™(DE3)pLysS and performed bioinformatics analyses using BLASTp, InterProScan, and Pfam to characterize the gene's structure and potential functions.Our study identifies ILR-VIN as a novel gene linked to Imipenem resistance in E. coli isolate lacking known Imipenem-resistance genes. Experimental evidence confirmed that ILR-VIN expression enhances bacterial survival under Imipenem stress, providing direct evidence of its role in resistance. This discovery highlights the importance of ongoing research into antibiotic resistance genes to develop effective treatment strategies against antibiotic-resistant bacterial infections.202540097570
5691180.9326Rapid and Accurate Antibiotic Susceptibility Determination of tet(X)-Positive E. coli Using RNA Biomarkers. The emergence and prevalence of novel plasmid-mediated tigecycline resistance genes, namely, tet(X) and their variants, pose a serious threat to public health worldwide. Rapid and accurate antibiotic susceptibility testing (AST) that can simultaneously detect the genotype and phenotype of tet(X)-positive bacteria may contribute to the deployment of an effective antibiotic arsenal, mortality reduction, and a decrease in the use of broad-spectrum antimicrobial agents. However, current bacterial growth-based AST methods, such as broth microdilution, are time consuming and delay the prompt treatment of infectious diseases. Here, we developed a rapid RNA-based AST (RBAST) assay to effectively distinguish tet(X)-positive and -negative strains. RBAST works by detecting specific mRNA expression signatures in bacteria after short-term tigecycline exposure. As a proof of concept, a panel of clinical isolates was characterized successfully by using the RBAST method, with a 3-h assay time and 87.9% accuracy (95% confidence interval [CI], 71.8% to 96.6%). Altogether, our findings suggest that RNA signatures upon antibiotic exposure are promising biomarkers for the development of rapid AST, which could inform early antibiotic choices. IMPORTANCE Infections caused by multidrug-resistant (MDR) Gram-negative pathogens are an increasing threat to global health. Tigecycline is one of the last-resort antibiotics for the treatment of these complicated infections; however, the emergence of plasmid-encoded tigecycline resistance genes, namely, tet(X), severely diminishes its clinical efficacy. Currently, there is a lack of rapid and accurate antibiotic susceptibility testing (AST) for the detection of tet(X)-positive bacteria. In this study, we developed a rapid and robust RNA-based antibiotic susceptibility determination (RBAST) assay to effectively distinguish tet(X)-negative and -positive strains using specific RNA biomarkers in bacteria after tigecycline exposure. Using this RBAST method, we successfully characterized a set of clinical strains in 3 h. Our data indicate that the RBAST assay is useful for identifying tet(X)-positive Escherichia coli.202134704829
9051190.9326Chlorogenic acid inhibits virulence and resistance gene transfer in outer membrane vesicles of carbapenem-resistant Klebsiella pneumoniae. INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae (CRKp) infection poses a significant global public health challenge, with the misuse of antibiotics further contributing to the development of resistance and triggering harmful inflammatory responses. Outer membrane vesicles (OMVs) released by CRKp under sub-lethal concentration of MEM pressure (KOMV-MEM) exhibit enhanced virulence and greater efficiency in transferring resistance genes. METHODS: We investigated the inhibitory effects of chlorogenic acid (CA) on KOMV-MEM characteristics and its protective role in KOMV-MEM infected mice. Based on LC-MS proteomic analysis of vesicles, we screened for potential targets of KOMV-MEM in promoting macrophage (MØ) pyroptosis pathways and inducing resistance gene transfer. Subsequently, computational predictions and experimental validation were performed to determine how CA regulates these mechanisms. RESULTS: This study confirmed that, under MEM pressure, the exacerbated infection levels in CRKp-inoculated mice are attributable to the high virulence of KOMV-MEM. Computational and experimental results demonstrated that CA inhibits pyroptosis by reducing MØ capture of KOMV-MEM through blocking the interaction between GroEL and LOX-1. Furthermore, CA prevents the spread of resistance genes by disrupting the conjugation and transfer processes between KOMV-MEM and recipient bacteria. Finally, in vitro and in vivo assays showed that CA inhibits KOMV-MEM resistance enzymes, thereby preventing the hydrolysis of MEM in the environment and depriving susceptible bacteria of protection. DISCUSSION: These findings provide the first confirmation that CA can inhibit both the virulence and the transmission of drug resistance in KOMV-MEM. This underscores the potential of CA treatment as a promising antimicrobial strategy against CRKp infection.202540230687