Gram-negative ESKAPE Pathogens Research Articles

Critical role of growth medium for detecting drug interactions in Gram-negative bacteria that model in vivo responses.

Drug-resistant bacterial infections are a growing concern and have only continued to increase during the SARS-CoV-2 pandemic. Though not routinely used for Gram-negative bacteria, drug combinations are sometimes used for serious infections and may become more widely used as the prevalence of extremely drug-resistant organisms increases. To date, reliable methods are not available for identifying beneficial drug combinations for a particular infection. Our study shows variability across strains in how drug interactions are impacted by growth conditions. It also demonstrates that testing drug combinations in tissue-relevant growth conditions for some strains better models what happens during infection and may better inform combination therapy selection.

PlyKp104, a Novel Phage Lysin for the Treatment of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Other Gram-Negative ESKAPE Pathogens.

Klebsiella pneumoniae and Pseudomonas aeruginosa are two leading causes of burn and wound infections, pneumonia, urinary tract infections, and more severe invasive diseases, which are often multidrug resistant (MDR) or extensively drug resistant. Due to this, it is critical to discover alternative antimicrobials, such as bacteriophage lysins, against these pathogens. Unfortunately, most lysins that target Gram-negative bacteria require additional modifications or outer membrane permeabilizing agents to be bactericidal. We identified four putative lysins through bioinformatic analysis of Pseudomonas and Klebsiella phage genomes in the NCBI database and then expressed and tested their intrinsic lytic activity in vitro. The most active lysin, PlyKp104, exhibited >5-log killing against K. pneumoniae, P. aeruginosa, and other Gram-negative representatives of the multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumonia, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species) without further modification. PlyKp104 displayed rapid killing and high activity over a wide pH range and in high concentrations of salt and urea. Additionally, pulmonary surfactants and low concentrations of human serum did not inhibit PlyKp104 activity in vitro. PlyKp104 also significantly reduced drug-resistant K. pneumoniae >2 logs in a murine skin infection model after one treatment of the wound, suggesting that this lysin could be used as a topical antimicrobial against K. pneumoniae and other MDR Gram-negative infections.

Phage-based therapy against biofilm producers in gram-negative ESKAPE pathogens

Persistent antibiotic use results in the rise of antimicrobial resistance with limited or no choice for multidrug-resistant (MDR) and extensively drug resistant (XDR) bacteria. This necessitates a need for alternative therapy to effectively combat clinical pathogens that are resistant to last resort antibiotics. The study investigates hospital sewage as a potential source of bacteriophages to control resistant bacterial pathogens. Eighty-one samples were screened for phages against selected clinical pathogens. Totally, 10 phages were isolated against A. baumannii, 5 phages against K. pneumoniae, and 16 phages were obtained against P. aeruginosa. The novel phages were observed to be strain-specific with complete bacterial growth inhibition of up to 6 h as monotherapy without antibiotics. Phage plus colistin combinations reduced the minimum-biofilm eradication concentration of colistin up to 16 folds. Notably, a cocktail of phages exhibited maximum efficacy with complete killing at 0.5–1 μg/ml colistin concentrations. Thus, phages specific to clinical strains have a higher edge in treating nosocomial pathogens with their proven anti-biofilm efficacy. In addition, analysis of phage genomes revealed close phylogenetic relations with phages reported from Europe, China, and other neighbouring countries. This study serves as a reference and can be extended to other antibiotics and phage types to assess optimum synergistic combinations to combat various drug resistant pathogens in the ongoing AMR crisis.

New understanding of multidrug efflux and permeation in antibiotic resistance, persistence, and heteroresistance.

Antibiotics effective against Gram-negative ESKAPE pathogens are a critical area of unmet need. Infections caused by these pathogens are not only difficult to treat but finding new therapies to overcome Gram-negative resistance is also a challenge. There are not enough antibiotics in development that target the most dangerous pathogens and there are not enough novel drugs in the pipeline. The major obstacle in the antibiotic discovery pipeline is the lack of understanding of how to breach antibiotic permeability barriers of Gram-negative pathogens. These barriers are created by active efflux pumps acting across both the inner and the outer membranes. Overproduction of efflux pumps alone or together with either modification of the outer membrane or antibiotic-inactivating enzymes and target mutations contribute to clinical levels of antibiotics resistance. Recent efforts have generated significant advances in the rationalization of compound efflux and permeation across the cell envelopes of Gram-negative pathogens. Combined with earlier studies and novel mathematical models, these efforts have led to a multilevel understanding of how antibiotics permeate these barriers and how multidrug efflux and permeation contribute to the development of antibiotic resistance and heteroresistance. Here, we discuss the new developments in this area.

An efflux-susceptible antibiotic-adjuvant with systemic efficacy against mouse infections

Scarcity of effective treatments against sepsis is daunting, especially under the contemporary standpoints on antibiotics resistance, entailing the development of alternative treatment strategies. Here, we describe the design and antibiotic adjuvant properties of a new lipopeptide-like pentamer, decanoyl-bis.diaminobutyrate-aminododecanoyl-diaminobutyrate-amide (C10BBc12B), whose sub-maximal tolerated doses combinations with inefficient antibiotics demonstrated systemic efficacies in murine models of peritonitis-sepsis and urinary-tract infections. Attempts to shed light into the mechanism of action using membrane-active fluorescent probes, suggest outer-membrane interactions to dominate the pentamer’s adjuvant properties, which were not associated with typical inner-membrane damages or with delayed bacterial growth. Yet, checkerboard titrations with low micromolar concentrations of C10BBc12B exhibited unprecedented capacities in potentiation of hydrophobic antibiotics towards Gram-negative ESKAPE pathogens, with an apparent low propensity for prompting resistance to the antibiotics. Assessment of the pentamer’s potentiating activities upon efflux inhibition incites submission of a hitherto unreported, probable action mechanism implicating the pentamer’s de-facto capacity to hijack bacterial efflux pumps for boosting its adjuvant activity through repetitive steps including outer-membrane adhesion, translocation and subsequent expulsion.

Structure-Uptake Relationship Studies of Oxazolidinones in Gram-Negative ESKAPE Pathogens.

The clinical success of linezolid for treating Gram-positive infections paired with the high conservation of bacterial ribosomes predicts that if oxazolidinones were engineered to accumulate in Gram-negative bacteria, then this pharmacological class would find broad utility in eradicating infections. Here, we report an investigative study of a strategically designed library of oxazolidinones to determine the effects of molecular structure on accumulation and biological activity. Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa strains with varying degrees of compromise (in efflux and outer membrane) were used to identify motifs that hinder permeation across the outer membrane and/or enhance efflux susceptibility broadly and specifically between species. The results illustrate that small changes in molecular structure are enough to overcome the efflux and/or permeation issues of this scaffold. Three oxazolidinone analogues (3e, 8d, and 8o) were identified that exhibit activity against all three pathogens assessed, a biological profile not observed for linezolid.

Designing Anti-Microbial Peptides Against Major β-Lactamase Enzymes in Clinically Important Gram-Negative Bacterial Pathogens: An In-Silico Study.

Anti-microbial resistance (AMR) creating healthcare concerns worldwide requires ardent exploration of therapeutic alternatives. Although anti-microbial peptides (AMP) are popular for broad-spectrum activity, recent evidence of increasing resistance to membrane-acting AMPs by ESKAPE pathogens has compelled us to design novel AMPs as therapeutic candidates. A library of 60 AMPs comprising natural AMPs and their mutants was constructed through in-silico methods. After physico-chemical property evaluations, each peptide in the library was subjected to flexible molecular docking against four major β-lactamases in Gram-negative ESKAPE pathogens. Among the potent AMP mutants, a Lactoferricin B-Mutant (M4) possessed uniformly high affinity with SHV1, OXA48, NDM1, and AmpC having energies -842.0Kcal/mol, -774.8Kcal/mol, -1103.3Kcal/mol, and -858.8Kcal/mol respectively. Coarse-grained clustering and flexibility analysis further accounted for the residue-level stable configurations of the protein-peptide complexes with high affinity. Highest affinity of Lactoferricin B_M4 was found with NDM1 due to H-bonds, salt-bridges, and hydrophobic interactions with the metallo-β-lactamase domain including crucial active-site residue Asp124. Molecular dynamics simulation further confirmed the stability of Lactoferricin B_M4-NDM1 complex having low residue-level root-mean square deviations (RMSD), atomic-level fluctuations, and radius of gyration (Rg). The study encourages experimental validations and similar methods to identify potential AMPs against drug-resistant pathogens.

Benzalkonium chloride antagonises aminoglycoside antibiotics and promotes evolution of resistance.

BackgroundBiocide disinfectants are essential tools in infection control, but their use can inadvertently contribute to emergence of antibiotic-resistant bacteria. In this study we systematically examine the effect of the biocide benzalkonium chloride, which is primarily used for surface disinfection but is also present as a preservative in many consumer products, on the activity of aminoglycoside antibiotics in Acinetobacter baumannii.MethodsThe effect of subinhibitory BAC on aminoglycoside treatment of A. baumannii ATCC17978 was investigated using time-to-kill assays, MIC determination, directed evolution experiments, fluctuation tests and labelled gentamicin accumulation assays. Further MIC determinations and directed evolution experiments were performed with additional Gram-negative ESKAPE pathogens.FindingsIn A. baumannii ATCC17978, BAC prevents gentamicin killing and drastically increases the frequency at which resistant mutants emerge, through reducing intracellular antibiotic accumulation. BAC also increases the MIC of multiple aminoglycoside antibiotics (kanamycin, tobramycin, streptomycin, gentamicin and amikacin). BAC promotes the emergence of mutants with reduced gentamicin susceptibility in other Gram-negative ESKAPE pathogens but does not always alter the MIC. These effects occur at BAC concentrations which are similar to residual levels in high-use environments, and just below the concentration range for BAC when used as a preservative in eye drops and ear drops.InterpretationOur results suggest that subinhibitory BAC has the potential to antagonise aminoglycoside activity and promote the emergence of bacterial mutants with reduced susceptibility. We suggest that the extremely widespread use of BAC in clinical and home settings and its long half-life mean there is potential for these interactions to occur in the environment, or in patients who use BAC-containing products while taking aminoglycosides to treat skin, eye or ear infections, although such co-exposure is likely to be rare. We suggest that biocide stewardship is needed to prevent the types of exposure that can contribute to antibiotic resistance.FundingThis work was funded by the National Health and Medical Research Council of Australia. The funders had no role in study design, interpretation or decision to publish.

Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including β-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of β-lactamases, the primary resistance mechanism associated with β-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPEpathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.

Facilitating Compound Entry as a Means to Discover Antibiotics for Gram-Negative Bacteria.

It has been over half a century since the last class of antibiotics active against the most problematic Gram-negative bacteria was approved by the Food and Drug Administration (FDA). The major challenge with developing antibiotics to treat these infections is not drug-target engagement but rather the inability of most small molecules to traverse the Gram-negative membranes, be retained, and accumulate within the cell. Despite an abundance of lead compounds, limited understanding of the physicochemical properties needed for compound accumulation (or avoidance of efflux) in Gram-negative bacteria has precluded a generalizable approach for developing Gram-negative antibiotics. Indeed, in many instances, despite years of intensive derivatization efforts and the synthesis of hundreds of compounds aimed at building in Gram-negative activity, little or no progress has been made in expanding the spectrum of activity for many Gram-positive-only antibiotics. In this Account, we describe the discovery and successful applications of a promising strategy for enhancing the accumulation of Gram-positive-only antibiotics as a means of imbuing compounds with broad-spectrum activity.Utilizing a prospective approach examining the accumulation in Escherichia coli for more than 180 diverse compounds, we found that small molecules have an increased likelihood to accumulate in E. coli when they contain an ionizable Nitrogen, have low Three-dimensionality, and are Rigid. Implementing these guidelines, codified as the "eNTRy rules" and assisted by web application www.entry-way.org, we have facilitated compound entry and systematically built Gram-negative activity into Gram-positive-only antibiotics. Though each antibiotic will have case-specific considerations, we describe a set of important criteria to consider when selecting candidate Gram-positive-only antibiotics for conversion to Gram-negative-active versions via the eNTRy rules. As detailed herein, using this blueprint the spectrum of activity was expanded for three antibiotic classes that engage three different biological targets: DNA gyrase inhibitor 6DNM, FabI inhibitor Debio-1452, and FMN riboswitch inhibitor Ribocil C. In each scenario, the eNTRy rules guided the synthesis of key analogues predisposed to accumulate in Gram-negative bacteria leading to compounds that display antibiotic activity (minimum inhibitory concentrations (MIC) ≤8 μg mL-1) against E. coli and other Gram-negative ESKAPE pathogens. While the eNTRy rules will continue to be refined and enhanced as more accumulation data is gathered, on the basis of these collective results and on other examples not covered herein it is clear that the eNTRy rules are actionable for the development of novel broad-spectrum antibiotics from Gram-positive-only compounds. By enabling the prediction of compound accumulation, the eNTRy rules should facilitate the process of discovering and developing novel antibiotics active against Gram-negative bacteria.

A FabG inhibitor targeting an allosteric binding site inhibits several orthologs from Gram-negative ESKAPE pathogens

The spread of antibiotic resistance within the ESKAPE group of human pathogenic bacteria poses severe challenges in the treatment of infections and maintenance of safe hospital environments. This motivates efforts to validate novel target proteins within these species that could be pursued as potential targets for antibiotic development. Genetic data suggest that the enzyme FabG, which is part of the bacterial fatty acid biosynthetic system FAS-II, is essential in several ESKAPE pathogens. FabG catalyzes the NADPH dependent reduction of 3-keto-acyl-ACP during fatty acid elongation, thus enabling lipid supply for production and maintenance of the cell envelope. Here we report on small-molecule screening on the FabG enzymes from A. baumannii and S. typhimurium to identify a set of µM inhibitors, with the most potent representative (1) demonstrating activity against six FabG-orthologues. A co-crystal structure with FabG from A. baumannii (PDB:6T65) confirms inhibitor binding at an allosteric site located in the subunit interface, as previously demonstrated for other sub-µM inhibitors of FabG from P. aeruginosa. We show that inhibitor binding distorts the oligomerization interface in the FabG tetramer and displaces crucial residues involved in the interaction with the co-substrate NADPH. These observations suggest a conserved allosteric site across the FabG family, which can be potentially targeted for interference with fatty acid biosynthesis in clinically relevant ESKAPE pathogens.

Antimicrobial resistance to rescue substances in Gram-negative ESKAPE pathogens – State-wide surveillance from the Saxony antibiotic network project

Antimicrobial resistance to rescue substances in Gram-negative ESKAPE pathogens – State-wide surveillance from the Saxony antibiotic network project

Dynamics of an LPS translocon induced by substrate and an antimicrobial peptide.

Lipopolysaccharide (LPS) transport to the outer membrane (OM) is a crucial step in the biogenesis of microbial surface defenses. Although many features of the translocation mechanism have been elucidated, molecular details of LPS insertion via the LPS transport (Lpt) OM protein LptDE remain elusive. Here, we integrate native MS with hydrogen-deuterium exchange MS and molecular dynamics simulations to investigate the influence of substrate and peptide binding on the conformational dynamics of LptDE. Our data reveal that LPS induces opening of the LptD β-taco domain, coupled with conformational changes on β-strands adjacent to the putative lateral exit gate. Conversely, an antimicrobial peptide, thanatin, stabilizes the β-taco, thereby preventing LPS transport. Our results illustrate that LPS insertion into the OM relies on concerted opening movements of both the β-barrel and β-taco domains of LptD, and suggest a means for developing antimicrobial therapeutics targeting this essential process in Gram-negative ESKAPE pathogens.

Repurposing Potential of Diminazene Aceturate as an Inhibitor of the E. coli DNA Gyrase B

Drug-resistant Escherichia coli (E. coli) has overburdened the healthcare facilities in recent years and is getting hard to combat, mandating search for novel therapeutics with a broad antibacterial spectrum and high chemotherapeutic index. The 24 kDa domain of DNA gyrase B that is involved in the ATPase activity has been reported to be a promising target for inhibitors. A PDB structure (1KZN) of the 24kD domain of gyrase B with the co-crystallized ligand clorobiocin was used for the docking studies to explore a library of 2924 FDA approved drugs from www.zinc.docking.org. FlexX docking module from Biosolve IT was used for receptor preparation and in silico docking experiments. Docking studies on the pocket created around the reference ligand clorobiocin revealed the best score with diminazene aceturate and it also demonstrated interactions with the crucial amino acids present within the pocket. Diminazene aceturate has been conventionally been used as an antiparasitic molecule in animals and it has also been demonstrated to exhibit repurposing potential in the treatment of disorders triggered due to overproduction of inflammatory cytokines, pulmonary hypertension, ischemia-induced cardiac pathophysiology, etc. among others. Findings from this study indicate the possibility of repurposing the age-old molecule diminazene aceturate into a DNA gyrase B antagonist to combat not just the drug-resistant E. coli but also other gram-negative ESKAPE pathogens. It may also aid in alleviating the inflammatory response induced in the body of the patients suffering from septicemia caused by a variety of Gram-negative bacterial pathogens.

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The "last-line" antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

Synthetic molecular evolution of host cell-compatible, antimicrobial peptides effective against drug-resistant, biofilm-forming bacteria

Novel classes of antibiotics and new strategies to prevent and treat infections are urgently needed because the rapid rise in drug-resistant bacterial infections in recent decades has been accompanied by a parallel decline in development of new antibiotics. Membrane permeabilizing antimicrobial peptides (AMPs) have long been considered a potentially promising, novel class of antibiotic, especially for wound protection and treatment to prevent the development of serious infections. Yet, despite thousands of known examples, AMPs have only infrequently proceeded as far as clinical trials, especially the chemically simple, linear examples. In part, this is due to impediments that often limit their applications in vivo. These can include low solubility, residual toxicity, susceptibility to proteolysis, and loss of activity due to host cell, tissue, and protein binding. Here we show how synthetic molecular evolution can be used to evolve potentially advantageous antimicrobial peptides that lack these impediments from parent peptides that have at least some of them. As an example of how the antibiotic discovery pipeline can be populated with more promising candidates, we evolved and optimized one family of linear AMPs into a new generation with high solubility, low cytotoxicity, potent broad-spectrum sterilizing activity against a panel of gram-positive and gram-negative ESKAPE pathogens, and antibiofilm activity against gram-positive and gram-negative biofilms. The evolved peptides have these activities in vitro even in the presence of concentrated host cells and also in vivo in the complex, cell- and protein-rich environment of a purulent animal wound model infected with drug-resistant bacteria.

Repurposing the scorpion venom peptide VmCT1 into an active peptide against Gram-negative ESKAPE pathogens

VmCT1 is a cationic antimicrobial peptide (AMP) from the venom of the scorpion Vaejovis mexicanus. VmCT1 and analogs were designed with single substitutions for verifying the influence of changes in physicochemical features described as important for AMPs antimicrobial and hemolytic activities, as well as their effect on VmCT1 analogs resistance against proteases action. The increase of the net positive charge by the introduction of an arginine residue in positions of the hydrophilic face of the helical structure affected directly the antimicrobial activity. Arg-substituted analogs presented activity against Gram-negative bacteria from the ESKAPE list of pathogens that were not observed for VmCT1. Additionally, peptides with higher net positive charge presented increased antimicrobial activity with values ranging from 0.39 to 12.5 μmol L−1 against Gram-positive and Gram-negative bacteria and fungi. The phenylalanine substitution by glycine (position 1), and the valine substitution by a proline residue (position 8) led to analogs with lower hemolytic activity (at concentrations 50 and 100 μmol L−1, respectively). These results revealed that it is possible to modulate the biological activities of VmCT1 derivatives by designing single substituted-analogs as prospective therapeutics against bacteria and fungi.

Broad Bactericidal Activity of the Myoviridae Bacteriophage Lysins LysAm24, LysECD7, and LysSi3 against Gram-Negative ESKAPE Pathogens.

The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting bacterial cell wall peptidoglycans to promote their osmotic lysis. However, relatively little is known regarding the spectrum of lysin bactericidal activity against Gram-negative bacteria. In this study, we present the results of in vitro activity assays of three putative and newly cloned Myoviridae bacteriophage endolysins (LysAm24, LysECD7, and LysSi3). The chosen proteins represent lysins with diverse domain organization (single-domain vs. two-domain) and different predicted mechanisms of action (lysozyme vs. peptidase). The enzymes were purified, and their properties were characterized. The enzymes were tested against a panel of Gram-negative clinical bacterial isolates comprising all Gram-negative representatives of the ESKAPE group. Despite exhibiting different structural organizations, all of the assayed lysins were shown to be capable of lysing Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Salmonella typhi strains. Less than 50 μg/mL was enough to eradicate growing cells over more than five orders of magnitude. Thus, LysAm24, LysECD7, and LysSi3 represent promising therapeutic agents for drug development.

Norfloxacin salts of carboxylic acids curtail planktonic and biofilm mode of growth in ESKAPE pathogens.

To enhance the antimicrobial and antibiofilm activity of norfloxacin against the planktonic and biofilm mode of growth in ESKAPE pathogens using chemically modified norfloxacin salts. Antimicrobial testing, synergy testing and time-kill curve analysis were performed to evaluate antibacterial effect of norfloxacin carboxylic acid salts against ESKAPE pathogens. In vivo efficacy to reduce bacterial bioburden was evaluated in zebrafish infection model. Crystal violet assay and live-dead staining were performed to discern antibiofilm effect. Membrane permeability, integrity and molecular docking studies were carried out to ascertain the mechanism of action. The carboxylic acid salts, relative to parent molecule norfloxacin, displayed two- to fourfold reduction in minimum inhibitory concentration against Staphylococcus aureus and Pseudomonas aeruginosa, in addition to displaying potent bacteriostatic effect against certain members of ESKAPE pathogens. In vivo treatments revealed that norfloxacin tartrate (SRIN2) reduced MRSA bioburden by greater than 1 log fold relative to parent molecule in the muscle tissue. In silico docking with gyrA of S. aureus showed increased affinity of SRIN2 towards DNA gyrase. The enhanced antibacterial effect of norfloxacin salts could be partially accounted by altered membrane permeability in S. aureus and perturbed membrane integrity in P. aeruginosa. Antibiofilm studies revealed that SRIN2 (norfloxacin tartrate) and SRIN3 (norfloxacin benzoate) exerted potent antibiofilm effect particularly against Gram-negative ESKAPE pathogens. The impaired colonization of both S. aureus and P. aeruginosa due to improved norfloxacin salts was further supported by live-dead imaging. Norfloxacin carboxylic acid salts can act as potential alternatives in terms of drug resensitization and reuse. Our study shows that carboxylic acid salts of norfloxacin could be effectively employed to treat both planktonic- and biofilm-based infections caused by select members of ESKAPE pathogens.

Structural Modification of Lipopolysaccharide Conferred by mcr-1 in Gram-Negative ESKAPE Pathogens.

mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceaemcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.