Hybrid Antibiotics Research Articles

An organometallic hybrid antibiotic of metronidazole with a Gold(I) N-Heterocyclic Carbene overcomes metronidazole resistance in Clostridioides difficile

Antimicrobial resistance (AMR) has been emerging as a major global health threat and calls for the development of novel drug candidates. Metal complexes have been demonstrating high efficiency as antibacterial agents that differ substantially from the established types of antibiotics in their chemical structures and their mechanism of action. One strategy to exploit this potential is the design of metal-based hybrid organometallics that consist of an established antibiotic and a metal-based warhead that contributes an additional mechanism of action different from that of the parent antibiotic. In this communication, we describe the organometallic hybrid antibiotic 2c, in which the drug metronidazole is connected to a gold(I) N-heterocyclic carbene warhead that inhibits bacterial thioredoxin reductase (TrxR). Metronidazole can be used for the treatment with the obligatory anaerobic pathogen Clostridioides difficile (C. difficile), however, resistance to the drug hampers its clinical success. The gold organometallic conjugate 2c was an efficient inhibitor of TrxR and it was inactive or showed only minor effects against eucaryotic cells and bacteria grown under aerobic conditions. In contrast, a strong antibacterial effect was observed against both metronidazole-sensitive and -resistant strains of C. difficile. This report presents a proof-of-concept that the design of metal-based hybrid antibiotics can be a viable approach to efficiently tackle AMR.Graphical abstractA metronidazole-gold hybrid metalloantibiotic with high efficacy against resistant C. difficile

Sol-Gel Synthesis of Silica-Poly (Vinylpyrrolidone) Hybrids with Prooxidant Activity and Antibacterial Properties.

The present work deals with the sol-gel synthesis of silica-poly (vinylpyrrolidone) hybrid materials. The nanohybrids (Si-PVP) have been prepared using an acidic catalyst at ambient temperature. Tetramethyl ortosilane (TMOS) was used as a silica precursor. Poly (vinylpyrrolidone) (PVP) was introduced into the reaction mixture as a solution in ethanol with a concentration of 20%. The XRD established that the as-prepared material is amorphous. The IR and 29Si MAS NMR spectra proved the formation of a polymerized silica network as well as the hydrogen bonding interactions between the silica matrix and OH hydrogens of the silanol groups. The TEM showed spherical particle formation along with increased agglomeration tendency. The efficacy of SiO2/PVP nanoparticles as a potential antimicrobial agent against a wide range of bacteria was evaluated as bacteriostatic, using agar diffusion and spot tests. Combined effects of hybrid nanomaterial and antibiotics could significantly reduce the bactericidal concentrations of both the antibiotic and the particles, and they could also eliminate the antibiotic resistance of the pathogen. The registered prooxidant activity of the newly synthesized material was confirmative and explicatory for the antibacterial properties of the tested substance and its synergetic combination with antibiotics. The effect of new hybrid material on Crustacea Daphnia magna was also estimated as harmless under concentration of 0.1 mg/mL.

Hybrid Molecules of Azithromycin with Chloramphenicol and Metronidazole: Synthesis and Study of Antibacterial Properties.

The sustained rise of antimicrobial resistance (AMR) causes a strong need to develop new antibacterial agents. One of the methods for addressing the problem of antibiotic resistance is through the design of hybrid antibiotics. In this work, we proposed a synthetic route for the conjugation of an azithromycin derivative with chloramphenicol and metronidazole hemisuccinates and synthesized two series of new hybrid molecules 4a-g and 5a-g. While a conjugation did not result in tangible synergy for wild-type bacterial strains, new compounds were able to overcome AMR associated with the inducible expression of the ermC gene on a model E. coli strain resistant to macrolide antibiotics. The newly developed hybrids demonstrated a tendency to induce premature ribosome stalling, which might be crucial since they will not induce a macrolide-resistant phenotype in a number of pathogenic bacterial strains. In summary, the designed structures are considered as a promising direction for the further development of hybrid molecules that can effectively circumvent AMR mechanisms to macrolide antibiotics.

The Hybrid Antibiotic Thiomarinol A Overcomes Intrinsic Resistance in Escherichia coli Using a Privileged Dithiolopyrrolone Moiety.

An impermeable outer membrane and multidrug efflux pumps work in concert to provide Gram-negative bacteria with intrinsic resistance against many antibiotics. These resistance mechanisms reduce the intracellular concentrations of antibiotics and render them ineffective. The natural product thiomarinol A combines holothin, a dithiolopyrrolone antibiotic, with marinolic acid A, a close analogue of mupirocin. The hybridity of thiomarinol A converts the mupirocin scaffold from inhibiting Gram-positive bacteria to inhibiting both Gram-positive and -negative bacteria. We found that thiomarinol A accumulates significantly more than mupirocin within the Gram-negative bacterium Escherichia coli, likely contributing to its broad-spectrum activity. Antibiotic susceptibility testing of E. coli mutants reveals that thiomarinol A overcomes the intrinsic resistance mechanisms that render mupirocin inactive. Structure-activity relationship studies suggest that the dithiolopyrrolone is a privileged moiety for improving the accumulation and antibiotic activity of the mupirocin scaffold without compromising binding to isoleucyl-tRNA synthetase. These studies also highlight that accumulation is required but not sufficient for antibiotic activity. Our work reveals a role of the dithiolopyrrolone moiety in overcoming intrinsic mupirocin resistance in E. coli and provides a starting point for designing dual-acting and high-accumulating hybrid antibiotics.

Antibacterial Resistance. The Challenge of Modernity

Antimicrobial resistance is considered by WHO as one of the most important threats to public health in the twenty-first century. According to forecasts, by 2025, many first-line antimicrobials will lose their effectiveness and the «post-antibiotic era» will begin. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and representatives of the genus Mycobacterium are classified by the American Society of Infectious Diseases as microorganisms that play a predominant role in the development of infections associated with medical care and leading to fatal consequences. The review highlights the mechanisms of antibiotic resistance and many variants of microbial resistance to antibiotics. Knowledge of the molecular mechanisms of the formation of resistance of microorganisms allows us to develop strategic directions for overcoming it. The search for new ways to prevent and overcome the formation of resistance of pathogens to antibiotics is an extremely important task of modern medical science. The effectiveness of hybrid antibiotics associated with chemical compounds with various specific effects is presented. The use of the main active factor of the bacterial virus, endolysin, both in its pure form and as part of homodimers, for example, lysoprotein, which is a complex of endolysin with human immunoglobulins, is considered promising. Phage therapy of the future is a personalized phage therapy that requires the creation of a library or bank of phages.

Unravelling patient pathways in the context of antibacterial resistance in East Africa

BackgroundA key factor driving the development and maintenance of antibacterial resistance (ABR) is individuals’ use of antibiotics (ABs) to treat illness. To better understand motivations and context for antibiotic use we use the concept of a patient treatment-seeking pathway: a treatment journey encompassing where patients go when they are unwell, what motivates their choices, and how they obtain antibiotics. This paper investigates patterns and determinants of patient treatment-seeking pathways, and how they intersect with AB use in East Africa, a region where ABR-attributable deaths are exceptionally high.MethodsThe Holistic Approach to Unravelling Antibacterial Resistance (HATUA) Consortium collected quantitative data from 6,827 adult outpatients presenting with urinary tract infection (UTI) symptoms in Kenya, Tanzania, and Uganda between February 2019- September 2020, and conducted qualitative in-depth patient interviews with a subset (n = 116). We described patterns of treatment-seeking visually using Sankey plots and explored explanations and motivations using mixed-methods. Using Bayesian hierarchical regression modelling, we investigated the associations between socio-demographic, economic, healthcare, and attitudinal factors and three factors related to ABR: self-treatment as a first step, having a multi-step treatment pathway, and consuming ABs.ResultsAlthough most patients (86%) sought help from medical facilities in the first instance, many (56%) described multi-step, repetitive treatment-seeking pathways, which further increased the likelihood of consuming ABs. Higher socio-economic status patients were more likely to consume ABs and have multi-step pathways. Reasons for choosing providers (e.g., cost, location, time) were conditioned by wider structural factors such as hybrid healthcare systems and AB availability.ConclusionThere is likely to be a reinforcing cycle between complex, repetitive treatment pathways, AB consumption and ABR. A focus on individual antibiotic use as the key intervention point in this cycle ignores the contextual challenges patients face when treatment seeking, which include inadequate access to diagnostics, perceived inefficient public healthcare and ease of purchasing antibiotics without prescription. Pluralistic healthcare landscapes may promote more complex treatment seeking and therefore inappropriate AB use. We recommend further attention to healthcare system factors, focussing on medical facilities (e.g., accessible diagnostics, patient-doctor interactions, information flows), and community AB access points (e.g., drug sellers).

Bifunctional antibiotic hybrids: A review of clinical candidates.

Antibiotic resistance is a top threat to human health and a priority across the globe. This problematic issue is accompanied by the decline of new antibiotics in the pipeline over the past 30years. In this context, an urgent need to develop new strategies to combat antimicrobial resistance is in great demand. Lately, among the possible approaches used to deal with antimicrobial resistance is the covalent ligation of two antibiotic pharmacophores that target the bacterial cells through a dissimilar mode of action into a single hybrid molecule, namely hybrid antibiotics. This strategy exhibits several advantages, including better antibacterial activity, overcoming the existing resistance towards individual antibiotics, and may ultimately delay the onset of bacterial resistance. This review sheds light on the latest development of the dual antibiotic hybrids pipeline, their potential mechanisms of action, and challenges in their use.

Laccases and Tyrosinases in Organic Synthesis.

Laccases (Lac) and tyrosinases (TYR) are mild oxidants with a great potential in research and industry. In this work, we review recent advances in their use in organic synthesis. We summarize recent examples of Lac-catalyzed oxidation, homocoupling and heterocoupling, and TYR-catalyzed ortho-hydroxylation of phenols. We highlight the combination of Lac and TYR with other enzymes or chemical catalysts. We also point out the biological and pharmaceutical potential of the products, such as dimers of piceid, lignols, isorhamnetin, rutin, caffeic acid, 4-hydroxychalcones, thiols, hybrid antibiotics, benzimidazoles, benzothiazoles, pyrimidine derivatives, hydroxytyrosols, alkylcatechols, halocatechols, or dihydrocaffeoyl esters, etc. These products include radical scavengers; antibacterial, antiviral, and antitumor compounds; and building blocks for bioactive compounds and drugs. We summarize the available enzyme sources and discuss the scalability of their use in organic synthesis. In conclusion, we assume that the intensive use of laccases and tyrosinases in organic synthesis will yield new bioactive compounds and, in the long-term, reduce the environmental impact of industrial organic chemistry.

Overcoming the rising incidence and evolving mechanisms of antibiotic resistance by novel drug delivery approaches - An overview.

Antimicrobial resistance is a normal evolutionary process for microorganisms. Antibiotics exerted accelerated selective pressure that hasten bacterial resistance through mutation, and acquisition external genes. These genes often carry multiple antibiotic resistant determinants allowing the recipient microbe an instant "super-bug" status. The extent of Antimicrobial Resistance (AMR) has reached a level of global crisis, existing antimicrobials are no long effective in treating infections caused by AMR pathogens. The great majority of clinically available antimicrobial agents are administered through oral and intra-venous routes. Overcoming antibacterial resistance by novel drug delivery approach offered new hopes, particularly in the treatment of AMR pathogens in sites less assessible through systemic circulation such as the lung and skin. In the current review, we will revisit the mechanism and incidence of important AMR pathogens. Finally, we will discuss novel drug delivery approaches including novel local antibiotic delivery systems, hybrid antibiotics, and nanoparticle-based antibiotic delivery systems.

Synthesis and evaluation of dual-action kanglemycin-fluoroquinolone hybrid antibiotics

Bacterial resistance threatens the utility of currently available antibiotics. Rifampicin, a cornerstone in the treatment of persistent Gram-positive infections, is prone to the development of resistance resulting from single point mutations in the antibiotic’s target, RNA polymerase. One strategy to circumvent resistance is the use of ‘hybrid’ antibiotics consisting of two covalently linked antibiotic entities. These compounds generally have two distinct cellular targets, reducing the probability of resistance development and potentially providing simplified pharmacological properties compared to combination therapies using the individual antibiotics. Here we evaluate a series of semi-synthetic hybrid antibiotics formed by linking kanglemycin A (Kang A), a rifampicin analog, and a collection of fluoroquinolones. Kang A is a natural product antibiotic which contains a novel dimethyl succinic acid moiety that offers a new attachment point for the synthesis of hybrid antibiotics. We compare the activity of the Kang A hybrids generated via the acid attachment point to a series of hybrids linked at the compound’s naphthoquinone ring system. Several hybrids exhibit activity against bacteria resistant to Kang A via the action of the partnered antibiotic, suggesting that the Kang scaffold may provide new avenues for generating antibiotics effective against drug-resistant infections.

Vancomyxins: Vancomycin-Polymyxin Nonapeptide Conjugates That Retain Anti-Gram-Positive Activity with Enhanced Potency against Gram-Negative Strains.

Vancomycin functions by binding to lipid II, the penultimate bacterial cell wall building block used by both Gram-positive and Gram-negative species. However, vancomycin is generally only able to exert its antimicrobial effect against Gram-positive strains as it cannot pass the outer membrane (OM) of Gram-negative bacteria. To address this challenge, we here describe efforts to conjugate vancomycin to the OM disrupting polymyxin E nonapeptide (PMEN) to yield the hybrid “vancomyxins”. In designing these hybrid antibiotics, different spacers and conjugation sites were explored for connecting vancomycin and PMEN. The vancomyxins show improved activity against Gram-negative strains compared with the activity of vancomycin or vancomycin supplemented with PMEN separately. In addition, the vancomyxins maintain the antimicrobial effect of vancomycin against Gram-positive strains and, in some cases, show enhanced activity against vancomycin-resistant strains. The hybrid antibiotics described here have reduced nephrotoxicity when compared with clinically used polymyxin antibiotics. This study demonstrates that covalent conjugation to an OM disruptor contributes to sensitizing Gram-negative strains to vancomycin while retaining anti-Gram-positive activity.

Inhibition of Isoleucyl-tRNA Synthetase by the Hybrid Antibiotic Thiomarinol.

Hybrid antibiotics are an emerging antimicrobial strategy to overcome antibiotic resistance. The natural product thiomarinol A is a hybrid of two antibiotics: holothin, a dithiolopyrrolone (DTP), and marinolic acid, a close analogue of the drug mupirocin that is used to treat methicillin-resistant Staphylococcus aureus (MRSA). DTPs disrupt metal homeostasis by chelating metal ions in cells, whereas mupirocin targets the essential enzyme isoleucyl-tRNA synthetase (IleRS). Thiomarinol A is over 100-fold more potent than mupirocin against mupirocin-sensitive MRSA; however, its mode of action has been unknown. We show that thiomarinol A targets IleRS. A knockdown of the IleRS-encoding gene, ileS, exhibited sensitivity to a synthetic analogue of thiomarinol A in a chemical genomics screen. Thiomarinol A inhibits MRSA IleRS with a picomolar Ki and binds to IleRS with low femtomolar affinity, 1600 times more tightly than mupirocin. We find that thiomarinol A remains effective against high-level mupirocin-resistant MRSA and provide evidence to support a dual mode of action for thiomarinol A that may include both IleRS inhibition and metal chelation. We demonstrate that MRSA develops resistance to thiomarinol A to a substantially lesser degree than mupirocin and the potent activity of thiomarinol A requires hybridity between DTP and mupirocin. Our findings identify a mode of action of a natural hybrid antibiotic and demonstrate the potential of hybrid antibiotics to combat antibiotic resistance.

Caerulomycin and collismycin antibiotics share a trans-acting flavoprotein-dependent assembly line for 2,2\u2019-bipyridine formation

Linear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) template the modular biosynthesis of numerous nonribosomal peptides, polyketides and their hybrids through assembly line chemistry. This chemistry can be complex and highly varied, and thus challenges our understanding in NRPS and PKS-programmed, diverse biosynthetic processes using amino acid and carboxylate building blocks. Here, we report that caerulomycin and collismycin peptide-polyketide hybrid antibiotics share an assembly line that involves unusual NRPS activity to engage a trans-acting flavoprotein in C-C bond formation and heterocyclization during 2,2’-bipyridine formation. Simultaneously, this assembly line provides dethiolated and thiolated 2,2’-bipyridine intermediates through differential treatment of the sulfhydryl group arising from l-cysteine incorporation. Subsequent l-leucine extension, which does not contribute any atoms to either caerulomycins or collismycins, plays a key role in sulfur fate determination by selectively advancing one of the two 2,2’-bipyridine intermediates down a path to the final products with or without sulfur decoration. These findings further the appreciation of assembly line chemistry and will facilitate the development of related molecules using synthetic biology approaches.

Next-Generation Antibiotics, Bacteriophage Endolysins, and Nanomaterials for Combating Pathogens.

This review presents various strategies to fight causative agents of infectious diseases. Species-specific programmable RNA-containing antibiotics open up new possibilities for creating next-generation of personalized drugs based on microbiome editing and can serve as a new tool for selective elimination of pathogenic bacterial species while keeping intact the rest of microbiota. Another promising approach in combating bacterial infections is genome editing using the CRISPR-Cas systems. Expanding knowledge on the molecular mechanisms of innate immunity has been actively used for developing new antimicrobials. However, obvious risks of using antibiotic adjuvants aimed at activation of the host immune system include development of the autoimmune response with subsequent organ damage. To avoid these risks, it is essential to elucidate action mechanisms of the specific ligands and signal molecules used as components of the hybrid antibiotics. Bacteriophage endolysins are also considered as effective antimicrobials against antibiotic-resistant bacteria, metabolically inactive persisters, and microbial biofilms. Despite significant advances in the design of implants with antibacterial properties, the problem of postoperative infections still remains. Different nanomodifications of the implant surface have been designed to reduce bacterial contamination. Here, we review bactericidal, fungicidal, and immunomodulating properties of compounds used for the implant surface nanomodifications, such as silver, boron nitride nanomaterials, nanofibers, and nanogalvanic materials.

Semisynthetic Analogs of the Antibiotic Fidaxomicin-Design, Synthesis, and Biological Evaluation.

The glycoslated macrocyclic antibiotic fidaxomicin (1, tiacumicin B, lipiarmycin A3) displays good to excellent activity against Gram-positive bacteria and was approved for the treatment of Clostridium difficile infections (CDI). Among the main limitations for this compound, its low water solubility impacts further clinical uses. We report on the synthesis of new fidaxomicin derivatives based on structural design and utilizing an operationally simple one-step protecting group-free preparative approach from the natural product. An increase in solubility of up to 25-fold with largely retained activity was observed. Furthermore, hybrid antibiotics were prepared that show improved antibiotic activities.

Engineered Streptomyces lividans Strains for Optimal Identification and Expression of Cryptic Biosynthetic Gene Clusters.

Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover “cryptic” secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.

Synthesis and evaluation of biological activity for dual-acting antibiotics on the basis of azithromycin and glycopeptides

One of the promising directions of the combined approach is the design of dual-acting antibiotics – heterodimeric structures on the basis of antimicrobial agents of different classes. In this study a novel series of azithromycin-glycopeptide conjugates were designed and synthesized. The structures of the obtained compounds were confirmed using NMR spectroscopy and mass spectrometry data including MS/MS analysis. All novel hybrid antibiotics were found to be either as active as azithromycin and vancomycin against Gram-positive bacterial strains or have superior activity in comparison with their parent antibiotics. One compound, eremomycin-azithromycin conjugate 16, demonstrated moderate activity against Enterococcus faecium and Enterococcus faecalis strains resistant to vancomycin, and equal to vancomycin’s activity for the treatment of mice with Staphylococcus aureus sepsis.

Covalently linked kanamycin – Ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance

To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics were synthesized, and their activity was determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The hybrids were antagonistic relative to the ciprofloxacin, but were substantially more potent than the parent kanamycin against Gram-negative bacteria, and overcame most dominant resistance mechanisms to aminoglycosides. Selected hybrids were 42–640 fold poorer inhibitors of bacterial protein synthesis than the parent kanamycin, while they displayed similar inhibitory activity to that of ciprofloxacin against DNA gyrase and topoisomerase IV enzymes. The hybrids showed significant delay of resistance development in both E. coli and B. subtilis in comparison to that of component drugs alone or their 1:1 mixture. More generally, the data suggest that an antagonistic combination of aminoglycoside-fluoroquinolone hybrids can lead to new compounds that slowdown/prevent the emergence of resistance.

Synthesis and Antimicrobial Evaluation of Novel Hybrid Antibiotics

Hybrid antibiotics are not found in nature as such. It is synthesized by combining the two or more antibiotics. Hybrid antibiotics can act through different modes of action combined in a single molecule. Molecules linked to one another by ester, amide, covalently bonded or by carbon chain linker. The expected benefits include activity against drug-resistant bacteria, an increased spectrum of activity and reduced chances for generating bacterial resistance. The formation of hybrid antibiotics having a synergistic effect to reduce the problem of antibacterial resistance. The main aim is to develop the hybrid molecules to increases the potency or synergistic effect as compared to giving them alone.

Design, synthesis and microbiological evaluation of ampicillin-tetramic acid hybrid antibiotics.

Exploiting iron-uptake pathways by conjugating β-lactam antibiotics with iron-chelators such as catechol and hydroxamic acid is a proven strategy to overcome permeability-related resistance in Gram-negative bacteria. Since naturally occurring iron chelating tetramic acids have not been previously examined for this purpose, an exploratory series of novel ampicillin-tetramic acid hybrids that structurally resemble ureidopenicillins was designed and synthesized. The new analogs were evaluated for the ability to chelate iron and their MIC activities determined against a representative panel of clinically significant bacterial pathogens. The tetramic acid β-lactam hybrids demonstrated a high affinity to iron in the order of 10−30 M3. The hybrids were less active against Gram-positive bacteria. However, against Gram-negative bacteria, their activity was species dependent with several hybrids displaying improved activity over ampicillin against wild-type Pseudomonas aeruginosa. The anti-Gram-negative activities of the hybrids improved in the presence of clavulanic acid revealing that the tetramic acid moiety did not provide added protection against β-lactamases. Additionally, the hybrids were found to be efflux pump substrates as their activities markedly improved against pump-inactivated strains. Unlike the catechol and hydroxamic acid siderophore β-lactam conjugates, the activities of the hybrids did not improve under iron-deficient conditions. These results suggest that the tetramic acid hybrids gain permeability via different membrane receptors, or they are out competed by native bacterial siderophores with stronger affinities for iron. This study provides a foundation for the further exploitation of the tetramic acid moiety to achieve novel β-lactam anti-Gram-negative agents, providing that efflux and β-lactamase mediated resistance is addressed.