AbstractAntimicrobial Resistance Research Articles

Pathogenic E. coli from Cattle as a Reservoir of Resistance Genes to Various Groups of Antibiotics

Antimicrobial resistance has become a worldwide concern in all public health domains and reducing the spread has become a global priority. Pathogenic E. coli is responsible for a number of illnesses in humans and outbreaks in the past have been correlated with the consumption of contaminated bovine products. This is why surveillance in all the steps of production is essential. This study focused on identifying the pathogenic strains of E. coli in two large bovine abattoirs from Romania and France, and on associating them with the antimicrobial resistance patterns. A total of 250 samples from intestinal content were aseptically collected during the evisceration step of the cattle slaughtering process, from which 242 E. coli strains were isolated. Seventeen percent of all samples tested positive to at least one E. coli isolate carrying eaeA, stx1 and stx2 genes. The most prevalent genetic profile found in the E. coli strains tested was Stx1-positive and Stx2/eaeA-negative. More than 68% of the pathogenic E. coli isolated in Romania showed multi-drug resistance (MDR) and in France, the percentage was significantly lower (38%). The MDR profiles showed a high gene diversity for antibiotic resistance, which represents a great risk for environmental spread and human health. Our results indicate that in Romania, bovines can represent a reservoir for MDR E. coli and, hence, a surveillance system for antimicrobials usage in farm animals is highly needed.

An ultrasensitive microfluidic approach reveals correlations between the physico-chemical and biological activity of experimental peptide antibiotics

Antimicrobial resistance challenges the ability of modern medicine to contain infections. Given the dire need for new antimicrobials, polypeptide antibiotics hold particular promise. These agents hit multiple targets in bacteria starting with their most exposed regions—their membranes. However, suitable approaches to quantify the efficacy of polypeptide antibiotics at the membrane and cellular level have been lacking. Here, we employ two complementary microfluidic platforms to probe the structure–activity relationships of two experimental series of polypeptide antibiotics. We reveal strong correlations between each peptide’s physicochemical activity at the membrane level and biological activity at the cellular level. We achieve this knowledge by assaying the membranolytic activities of the compounds on hundreds of individual giant lipid vesicles, and by quantifying phenotypic responses within clonal bacterial populations with single-cell resolution. Our strategy proved capable of detecting differential responses for peptides with single amino acid substitutions between them, and can accelerate the rational design and development of peptide antimicrobials.

Investigating Solutions To Combat Antimicrobial Resistant Microbes

Antimicrobial resistance (AMR) is the development of resistance by pathogenic microbes to an antimicrobial agent that was previously proven to be an effective treatment. The most significant concern at this time is pathogenic bacteria as they are starting to develop resistance to antibiotics faster than other types of organisms and pose a greater threat. Bacteria are able to develop antimicrobial resistance through gene transfer or mutations in the genome of the bacterial strain. Currently, there is an urgent need for methods that can effectively treat prevalent bacterial infections. This can be achieved through either the creation of new and improved antimicrobial agents or utilizing synthetic biology at the genetic level to achieve immunity.

Screening of mcr-1 among Gram-Negative Bacteria from Different Clinical Samples from ICU Patients in Alexandria, Egypt: One-Year Study.

Antimicrobial resistance represents a global dilemma. Our present study aimed to investigate the presence of mcr-1 among different Gram-negative bacteria including Enterobacteriaceae (except intrinsically resistant to colistin) and Pseudomonas aeruginosa. Gram-negative bacterial isolates were collected from different ICUs in several Alexandria hospitals from June 2019 to June 2020. The identification of these Gram-negative isolates was made using the VITEK-2® system (BioMérieux, France). SYBR Green-based PCR was used to screen for the presence of mcr-1 using a positive control that we amplified and sequenced earlier in our pilot study. All isolates were screened for the presence of mcr-1 regardless of their colistin susceptibility. Isolates that harbored mcr-1 were tested for colistin susceptibility and for the presence of some beta-lactamase genes. Klebsiella pneumoniae isolates harboring mcr-1 were capsule typed using the wzi sequence analysis. Four hundred eighty isolates were included in this study. Only six isolates harbored mcr-1.1. Of these, four were resistant to colistin, while two (K. pneumoniae and P. aeruginosa) were susceptible to colistin. Five of the six isolates were resistant to carbapenems. They harbored blaOXA-48, and three of them co-harbored blaNDM-1. K-58 was the most often found among our K. pneumoniae harboring mcr-1.1. To our knowledge, this is the first time to report colistin susceptible P. aeruginosa and K. pneumoniae harboring the mcr-1.1 gene in Egypt. Further studies are needed to investigate the presence of the mcr genes among colistin susceptible isolates to shed more light on its significance as a potential threat.

Towards routine employment of computational tools for antimicrobial resistance determination via high-throughput sequencing.

Antimicrobial resistance (AMR) is a growing threat to public health and farming at large. In clinical and veterinary practice, timely characterization of the antibiotic susceptibility profile of bacterial infections is a crucial step in optimizing treatment. High-throughput sequencing is a promising option for clinical point-of-care and ecological surveillance, opening the opportunity to develop genotyping-based AMR determination as a possibly faster alternative to phenotypic testing. In the present work, we compare the performance of state-of-the-art methods for detection of AMR using high-throughput sequencing data from clinical settings. We consider five computational approaches based on alignment (AMRPlusPlus), deep learning (DeepARG), k-mer genomic signatures (KARGA, ResFinder) or hidden Markov models (Meta-MARC). We use an extensive collection of 585 isolates with available AMR resistance profiles determined by phenotypic tests across nine antibiotic classes. We show how the prediction landscape of AMR classifiers is highly heterogeneous, with balanced accuracy varying from 0.40 to 0.92. Although some algorithms-ResFinder, KARGA and AMRPlusPlus-exhibit overall better balanced accuracy than others, the high per-AMR-class variance and related findings suggest that: (1) all algorithms might be subject to sampling bias both in data repositories used for training and experimental/clinical settings; and (2) a portion of clinical samples might contain uncharacterized AMR genes that the algorithms-mostly trained on known AMR genes-fail to generalize upon. These results lead us to formulate practical advice for software configuration and application, and give suggestions for future study designs to further develop AMR prediction tools from proof-of-concept to bedside.

The Pathways to Create Containers for Bacteriophage Delivery.

Antimicrobial resistance is a global public health threat. One of the possible ways to solve this problem is phage therapy, but the instability of bacteriophages hinders the development of this approach. A bacteriophage delivery system that stabilizes the phage is one of the possible solutions to this problem. This study is dedicated to exploring methods to create encapsulated forms of bacteriophages for delivery. We studied the effect of proteolytic enzymes on the destruction of the polyelectrolyte microcapsule shell and revealed that protease from Streptomyces griseus was able to destroy the membrane of the microcapsule (dextran sulfate/polyarginine)3 ((DS/PArg)3). In addition, the protease decreased the activity of the bacteriophage in the second hour of incubation, and the phage lost activity after 16 h. It was found that a medium with pH 9.02 did not affect the survival of the bacteriophage or E. coli. The bacteriophages were encapsulated into polyelectrolyte microcapsules (DS/PArg)3. It was established that it is impossible to use microcapsules as a means of delivering bacteriophages since the bacteriophages are inactivated. When bacteriophages were included inside a CaCO3 core, it was demonstrated that the phage retained activity before and after the dissolution of the CaCO3 particle. From the results of this study, we recommend using CaCO3 microparticles as a container for bacteriophage delivery through the acidic stomach barrier.

From Pathophysiological Hypotheses to Case-Control Study Design: Resistance from Antibiotic Exposure in Community-Onset Infections.

Antimicrobial resistance is a global public health concern, at least partly due to the misuse of antibiotics. The increasing prevalence of antibiotic-resistant infections in the community has shifted at-risk populations into the general population. Numerous case–control studies attempt to better understand the link between antibiotic use and antibiotic-resistant community-onset infections. We review the designs of such studies, focusing on community-onset bloodstream and urinary tract infections. We highlight their methodological heterogeneity in the key points related to the antibiotic exposure, the population and design. We show the impact of this heterogeneity on study results, through the example of extended-spectrum β-lactamases producing Enterobacteriaceae. Finally, we emphasize the need for the greater standardization of such studies and discuss how the definition of a pathophysiological hypothesis specific to the bacteria–resistance pair studied is an important prerequisite to clarify the design of future studies.

Proteomic Analysis of the Colistin-resistant E. coli Clinical Isolate: Explorations of the Resistome.

Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

Reducing the Ethical Burdens of Antimicrobial Stewardship using a Social Determinants Approach.

Antimicrobial resistance is an emerging global health problem. Antimicrobial stewardship interventions attempt at regulating the prescription and use of antimicrobials so that the emergence of resistance is reduced. But antimicrobial stewardship interventions have several ethical issues such as inequity in access to antimicrobials among the poor who need them more, and limitation of the autonomy of prescribers and patients. Several upstream social determinants influence susceptibility to infections, antimicrobial prescription practices, and emergence of antimicrobial resistance. Some of these social determinants impose ethical burdens on the antimicrobial stewardship interventions. Addressing these social determinants with a public health approach will help reduce antimicrobial resistance. This article argues that the social determinants approach helps reduce the ethical burdens of antimicrobial stewardship.

Policy and strategies addressing prevention and control of antimicrobial resistance in Brazil: A scoping review protocol.

IntroductionAntimicrobial resistance (AMR) is considered one of the biggest health challenges of the 21st century. It has both social and economic consequences; therefore, timely review of public health policies that have been designed to manage AMR is essential. Brazil too has developed and implemented various polices for the prevention and control of AMR. However, till date, no study provides insights regarding the various public health policies or other programs implemented by Brazilian institutes.ObjectiveThe objective is to define a scoping review protocol of policies that were developed to address prevention and control of antimicrobial resistance in Brazil, from a human health perspective.MethodThis protocol has been registered in the Open Science Framework (DOI 10.17605/OSF.IO/EC9ZJ). Indexed literature in English, Spanish and Portuguese published till December 2020 in Lilacs, PubMed, Embase, and official websites of the Brazilian government will be reviewed. This review considers all studies identified through a comprehensive search of peer-reviewed and grey literature databases that have a reference for policies made for managing AMR in Brazil. The criteria for the scoping review will be set by two evaluators. A third evaluator will be consulted, if there is any disagreement between the two primary evaluators. A standardized form will be used for data extraction from the selected studies. The results will be presented in a tabular form with narrative abstracts related to the topics identified through the scoping review protocol. The PRISMA extension for Scoping Reviews tool will be used.

Targeted Genome Mining Reveals the Psychrophilic Clostridium estertheticum Complex as a Potential Source for Novel Bacteriocins, Including Cesin A and Estercticin A.

Antimicrobial resistance in pathogenic bacteria is considered a major public health issue necessitating the discovery of alternative antimicrobial compounds. In this regard, targeted genome mining in bacteria occupying under-explored ecological niches has the potential to reveal such compounds, including bacteriocins. In this study, we determined the bacteriocin biosynthetic potential of the psychrophilic Clostridium estertheticum complex (CEC) through a combination of genome mining and phenotypic screening assays. The genome mining was performed in 40 CEC genomes using antiSMASH. The production of bacteriocin-like compounds was phenotypically validated through agar well (primary screening) and disk diffusion (secondary screening) assays using cell free supernatants (CFS) and partially purified extracts, respectively. Stability of four selected CFS against proteolytic enzymes, temperature and pH was determined while one CFS was analyzed by HRMS and MS/MS to identify potential bacteriocins. Twenty novel bacteriocin biosynthetic gene clusters (BBGC), which were classified into eight (six lantibiotics and two sactipeptides) distinct groups, were discovered in 18 genomes belonging to C. estertheticum (n = 12), C. tagluense (n = 3) and genomospecies2 (n = 3). Primary screening linked six BBGC with narrow antimicrobial activity against closely related clostridia species. All four preselected CFS retained activity after exposure to different proteolytic, temperature and pH conditions. Secondary screening linked BBGC1 and BBGC7 encoding a lantibiotic and sactipeptide, respectively, with activity against Bacillus cereus while lantibiotic-encoding BBGC2 and BBGC3 were linked with activity against B. cereus, Staphylococcus aureus (methicillin-resistant), Escherichia coli and Pseudomonas aeruginosa. MS/MS analysis revealed that C. estertheticum CF004 produces cesin A, a short natural variant of nisin, and HRMS indicated the production of a novel sactipeptide named estercticin A. Therefore, we have shown the CEC, in particular C. estertheticum, is a source of novel and stable bacteriocins that have activities against clinically relevant pathogens.

Breaking 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.

A scoping review of factors potentially linked with antimicrobial-resistant bacteria from turkeys (iAM.AMR Project).

Antimicrobial resistance (AMR) is a complex problem that is a threat to global public health. Consumption of turkey meat may be an important source of foodborne exposure to resistant bacteria; recent outbreaks of multi-drug-resistant Salmonella Reading in Canada and the USA have implicated raw turkey products. To better understand the epidemiology of AMR in farmed turkey production, a scoping review was conducted. The objectives were to identify (1) modifiable factors potentially associated with antimicrobial-resistant Campylobacter, Enterococcus, Escherichia coli and Salmonella enterica along the farm-to-fork pathway in turkeys, and (2) data gaps with respect to factors potentially associated with AMR and Canadian commercial turkey production. A comprehensive search of the peer-reviewed literature was conducted in 2019 and updated in 2021. Thirteen references were included, reporting 36 factors. Antimicrobial use factors and their potential associations with AMR were most frequently reported (n = 15 factors; 42%), followed by biosecurity (n = 11; 31%) and management practices (n = 10; 28%). This review revealed important data gaps; no factors pertaining to S. enterica or to stages other than the farm (e.g. abattoir, retail) were identified, and only one Canadian reference was identified. These findings will inform priorities for future research and surveillance regarding turkeys and AMR.

Phytochemicals Against Drug-Resistant Bacterial Biofilms and Use of Green Extraction Solvents to Increase Their Bioactivity.

Antimicrobial resistance has become one of the major global public health issues of the twenty-first century. One of the main factors in the limited action of antimicrobials is related to the ability of microorganisms, particularly bacteria, to form biofilms. These complex and well-organized communities allow the colonizing cells to acquire survival advantages over the same cells in suspension, including antibiotic resistance. A huge percentage of bacterial infections in humans are associated with biofilms, and many of them are chronic. Therefore, there is an urgent need to develop new products effective in controlling or eradicating biofilms. Plant secondary metabolites (phytochemicals) have demonstrated their potential as antibacterials against planktonic cells and sessile communities when used alone or in synergy with other molecules. This chapter covers recent advances in the activity of phytochemicals against biofilms, particularly those formed by drug-resistant bacteria. In addition, taking into account that the extraction step is crucial for the successful development of new bioactive compounds, the use of novel solvents that increase the phytochemical effect, such as natural deep eutectic solvents (NADES), as well as the recent applications of these solvents as antimicrobials are discussed.

An opportunity for global antimicrobial stewardship research: Refugee populations.

Antimicrobial resistance is a well-known global health threat that has higher prevalence in the refugee population. Although guidance has been provided by the World Health Organization and Centers for Disease Control and Prevention on implementing antimicrobial stewardship in lower- and middle-income countries, as well as by the United Nations Refugee Agency on other infection prevention and control efforts, no specific guidance exists for implementation of stewardship in this population. We highlight challenges specific to this population, review recent studies of interest within this space, and propose a research agenda to help move stewardship forward in the refugee population. We advocate for the importance of this issue, particularly given recent current events of geopolitical volatility that render this population more vulnerable, in the setting of its already well-known numerous health challenges.

Genotypic characterization of multidrug resistant Escherichia coli isolates reveals co-existence of ESBL- and carbapenemase- encoding genes linked to ISCR1.

Antimicrobial resistance in food-producing animals has not yet judiciously been reported from Pakistan. Here, we report on the isolation rate of poultry-associated multidrug resistant extended spectrum β-lactamase (ESBL) -producing Escherichia coli in Peshawar, Pakistan. A total of 200 samples, 50 from retail-poultry meat, 50 from sick birds, 50 from the boiler farm-environment, and 50 from human beings working on or exposed to poultry were analyzed for isolation of ESBL -producing E. coli, ESBL -encoding genes and antimicrobial susceptibility. A total of 81 E. coli isolates [(50.0% Phylogroup-A, 33.3% D and 16.7% phylogroup B2)], were recovered, 36 (44.4%) of them were found to be ESBL -producers. PCR revealed that blaCTXM was the most prevalent (14/36 = 38.9%) ESBL -encoding gene followed by blaSHV2 (9/36 = 25%). Strikingly, co-occurrence of multiple ESBL - and/or carbapenemase-encoding genes in a single isolate was observed, and combination of blaCTXM + blaSHV2 was the most predominant (19.4%) followed by blaCTXM + blaNDM1 + blaOXA-48 (11.1%) and blaCTXM + blaOXA-48 (8.8%). All these ESBL producers were found to be multidrug resistant (MDR) and were carrying either integron 1 (48.5%) or 2 (51.5%). Finally, 14 of the 36 isolates were also found positive for variable region and insertion sequence common region 1, which was found linked to ESBL/carbapenemase encoding genes in 5/14 isolates suggesting its role in dissemination.

Antimicrobial Resistance in Pneumococcal Carriage Isolates from Children under 2 Years of Age in Rural Pakistan.

ABSTRACTAntimicrobial resistance is an emerging public health concern. Ten-valent pneumococcal vaccine (PCV10) was introduced in Pakistan’s Expanded Program on Immunization (EPI) in 2012 as a 3 + 0 schedule without catchup. From 2014 to 2018, children <2 years were randomly selected in two rural union councils of Matiari, Pakistan. Nasopharyngeal swabs were collected using standard WHO guidelines by trained staff and processed at Infectious Disease Research Laboratory at The Aga Khan University, Karachi using culture on sheep blood agar and Multiplex PCR methods described by CDC, USA. Pneumococcal isolates were identified by optochin sensitivity and bile solubility tests. Isolates were then tested for antimicrobial susceptibility by standard Kirby-Bauer disk-diffusion method on Mueller-Hinton Agar (MHA) with 5% sheep blood agar as per Clinical & Laboratory Standards Institute (CLSI) recommendations. Of 3140 children enrolled, pneumococcal isolates were detected in 2370 (75%). Vaccine coverage improved from 41% to 68.4%. Out of the 2370 isolates, 88.4%, 37.6% and 25% were resistant to cotrimoxazole, tetracycline and erythromycin, respectively. There was no resistance to penicillin, ceftriaxone, and vancomycin. For erythromycin, resistance increased from 20% in 2014/15 to 30.8% in 2017/18 and for tetracycline it increased from 34.9% to 41.8% both of which were explained by an increase in prevalence of serotype 19A. Pneumococcal isolates were susceptible to penicillin, ceftriaxone, and vancomycin. They were largely resistant to cotrimoxazole and tetracycline. There was an increase in erythromycin and tetracycline resistance attributed to increasing prevalence of serotype 19A. Pneumococcal isolates from carriage and invasive disease should be closely monitored for antimicrobial susceptibility.IMPORTANCE Antimicrobial resistance is an emerging public health concern particularly in low- and middle-income countries where there is poor regulation and easy availability of antibiotics. This is the first study from Pakistan to report antimicrobial resistance patterns of pneumococcus after vaccine introduction in the community. Pakistan was the first South-Asian country to introduce PCV10 in its Expanded Program on Immunization (EPI) in 2012 as a 3 + 0 schedule without catchup. In this study, we describe the PCV10 impact on antimicrobial resistance patterns of pneumococcal nasopharyngeal carriage in children younger than 2 years of age in a rural district in Pakistan after the introduction of the vaccine.

Antimicrobial resistance in commensal opportunistic pathogens isolated from non-sterile sites can be an effective proxy for surveillance in bloodstream infections

Antimicrobial resistance (AMR) surveillance in bloodstream infections (BSIs) is challenging in low/middle-income countries (LMICs) given limited laboratory capacity. Other specimens are easier to collect and process and are more likely to be culture-positive. In 8102 E. coli BSIs, 322,087 E. coli urinary tract infections, 6952 S. aureus BSIs and 112,074 S. aureus non-sterile site cultures from Oxfordshire (1998–2018), and other (55,296 isolates) rarer commensal opportunistic pathogens, antibiotic resistance trends over time in blood were strongly associated with those in other specimens (maximum cross-correlation per drug 0.51–0.99). Resistance prevalence was congruent across drug-years for each species (276/312 (88%) species-drug-years with prevalence within ± 10% between blood/other isolates). Results were similar across multiple countries in high/middle/low income-settings in the independent ATLAS dataset (103,559 isolates, 2004–2017) and three further LMIC hospitals/programmes (6154 isolates, 2008–2019). AMR in commensal opportunistic pathogens cultured from BSIs is strongly associated with AMR in commensal opportunistic pathogens cultured from non-sterile sites over calendar time, suggesting the latter could be used as an effective proxy for AMR surveillance in BSIs.

Integrating Scalable Genome Sequencing Into Microbiology Laboratories for Routine Antimicrobial Resistance Surveillance.

Antimicrobial resistance (AMR) is considered a global threat, and novel drug discovery needs to be complemented with systematic and standardized epidemiological surveillance. Surveillance data are currently generated using phenotypic characterization. However, due to poor scalability, this approach does little for true epidemiological investigations. There is a strong case for whole-genome sequencing (WGS) to enhance the phenotypic data. To establish global AMR surveillance using WGS, we developed a laboratory implementation approach that we applied within the NIHR Global Health Research Unit (GHRU) on Genomic Surveillance of Antimicrobial Resistance. In this paper, we outline the laboratory implementation at 4 units: Colombia, India, Nigeria, and the Philippines. The journey to embedding WGS capacity was split into 4 phases: Assessment, Assembly, Optimization, and Reassessment. We show that on-boarding WGS capabilities can greatly enhance the real-time processing power within regional and national AMR surveillance initiatives, despite the high initial investment in laboratory infrastructure and maintenance. Countries looking to introduce WGS as a surveillance tool could begin by sequencing select Global Antimicrobial Resistance Surveillance System (GLASS) priority pathogens that can demonstrate the standardization and impact genome sequencing has in tackling AMR.

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.