Antimicrobial Inactivation Research Articles

Coupled environmental and demographic fluctuations shape the evolution of cooperative antimicrobial resistance.

There is a pressing need to better understand how microbial populations respond to antimicrobial drugs, and to find mechanisms to possibly eradicate antimicrobial-resistant cells. The inactivation of antimicrobials by resistant microbes can often be viewed as a cooperative behaviour leading to the coexistence of resistant and sensitive cells in large populations and static environments. This picture is, however, greatly altered by the fluctuations arising in volatile environments, in which microbial communities commonly evolve. Here, we study the eco-evolutionary dynamics of a population consisting of an antimicrobial-resistant strain and microbes sensitive to antimicrobial drugs in a time-fluctuating environment, modelled by a carrying capacity randomly switching between states of abundance and scarcity. We assume that antimicrobial resistance (AMR) is a shared public good when the number of resistant cells exceeds a certain threshold. Eco-evolutionary dynamics is thus characterised by demographic noise (birth and death events) coupled to environmental fluctuations which can cause population bottlenecks. By combining analytical and computational means, we determine the environmental conditions for the long-lived coexistence and fixation of both strains, and characterise a fluctuation-driven AMR eradication mechanism, where resistant microbes experience bottlenecks leading to extinction. We also discuss the possible applications of our findings to laboratory-controlled experiments.

Cationic Conjugated Microporous Polymers Coating for Dual‐Modal Antimicrobial Inactivation with Self‐Sterilization and Reusability Functions

AbstractCOVID‐19 pandemic outbreak poses a great threat to human health. Face masks have been considered as important personal protective equipment to prevent the COVID‐19 transmission. However, pathogens can survive up to several days on the fabrics of commercial masks, which increases the risk of direct/indirect microbial transmission. Herein, new cationic conjugated microporous polymers (CCMPs)‐based coating is developed, which possesses extended π‐conjugated skeletons and massive quaternary ammonium salt (QAS) groups, exhibiting dual‐modal antimicrobial inactivation, including sunlight‐driven photodynamic sterilization through the generation of reactive oxygen species and contact sterilization through QAS groups. As a result, the CCMPs coatings can rapidly and efficiently eradicate 99% of model microbes, such as Escherichia coli and Staphylococcus aureus under solar illumination, and also ensure the great antimicrobial effect in the absence of light. More importantly, the CCMPs coatings exhibit excellent durability, reusability as well as antimicrobial stability in humid environment. Contributing to the outstanding processability and formability, CCMPs can be in situ synthesized and coated over fibers through a simple spray procedure. Taken together, the design provides a promising strategy for developing reusable and self‐sterilizing antimicrobial fabrics, particularly for the application of face masks to tackle infectious pathogen and viruses in daily protection and medical applications.

Apatite-coated Ag/AgBr/TiO2 nanocomposites: Insights into the antimicrobial mechanism in the dark and under visible-light irradiation

For the first time, titania-based photocatalysts with different crystalline compositions (anatase/rutile) and morphology (irregular and faceted nanoparticles) were modified with Ag/AgBr and apatite (HA). The antimicrobial activity was tested for Escherichia coli (E. coli) bacteria and Candida albicans (C. albicans) fungi under visible-light illumination and in the dark for HA-covered and uncovered nanocomposites. It has been found that antimicrobial inactivation mechanism follows two different pathways, depending on the conditions (dark vs. irradiation), i.e., HA loading on the photocatalyst surface affects only the microorganism nourishment from the surrounding media (growth inhibition) in the dark, but significantly induces outer cell wall destruction (leading even to the mineralization) in the presence of light. Furthermore, 2-propanol oxidative decomposition was also investigated. The obtained results suggest that the improved photocatalytic activity of HA-covered samples originates from phosphate anions in HA, hindering the charge carriers’ recombination. In bacteria case, titania's properties are key factor for high bactericidal effects, such as anatase form (electrostatic attraction between microorganisms and photocatalyst surface) and fine crystallites, as opposed to yeast inactivation resembling 2-propanol oxidation, i.e., photocatalytic mechanism, based on reactive oxygen species. Concluding, HA/Ag/AgBr/TiO2 samples are promising “green” materials for decomposition of both microbiological and chemical pollutants.

Multidrug-Resistant Biofilms (MDR): Main Mechanisms of Tolerance and Resistance in the Food Supply Chain.

Biofilms are mono- or multispecies microbial communities enclosed in an extracellular matrix (EPS). They have high potential for dissemination and are difficult to remove. In addition, biofilms formed by multidrug-resistant strains (MDRs) are even more aggravated if we consider antimicrobial resistance (AMR) as an important public health issue. Quorum sensing (QS) and horizontal gene transfer (HGT) are mechanisms that significantly contribute to the recalcitrance (resistance and tolerance) of biofilms, making them more robust and resistant to conventional sanitation methods. These mechanisms coordinate different strategies involved in AMR, such as activation of a quiescent state of the cells, moderate increase in the expression of the efflux pump, decrease in the membrane potential, antimicrobial inactivation, and modification of the antimicrobial target and the architecture of the EPS matrix itself. There are few studies investigating the impact of the use of inhibitors on the mechanisms of recalcitrance and its impact on the microbiome. Therefore, more studies to elucidate the effect and applications of these methods in the food production chain and the possible combination with antimicrobials to establish new strategies to control MDR biofilms are needed.

Enhanced sunlight-absorption of Fe2O3 covered by PANI for the photodegradation of organic pollutants and antimicrobial inactivation

• Fabrication of core–shell S-scheme heterojunction Fe 2 O 3 -PANI nanocomposite via simple solution method. • Booted photodegradation of diverse organic pollutants under sunlight. • The enhanced antimicrobial characteristics against various bacterial strains. • The improved charge carrier’s kinetics via S-scheme with PANI. • A promising material against bacterial disinfection and an excellent stable photocatalyst. The current study focused on preparing S-scheme heterojunction Fe 2 O 3 -PANI nanocomposite and pristine Fe 2 O 3 using surfactant-assisted sol–gel and polymerization routes. The structural conformation was assessed through XRD diffractograms that confirmed the Fe 2 O 3 phase in both samples. FTIR data traces the metal–oxygen and carbon-related bonds. PL results showed that the recombination rate is retarded in nanocomposite. TEM images confirmed the covering of PANI over Fe 2 O 3 . EDX mapping showed that Fe, C, N, and O were present in the grown nanocomposite. XPS study revealed the binding energies of constituent atoms. The optical energy bandgap 2.8 and 2.7 eV for Fe 2 O 3 and Fe 2 O 3 -PANI was observed through UV–Vis analysis. The photocatalytic test against methyl blue (MB), eosin yellow (EY), and methyl red (MR) dyes were executed for 50 min under direct sunlight. The Fe 2 O 3 -PANI showed 99.8, 98.5, and 99.6% degradation efficiency against MB, EY, and MR dyes, respectively, which was higher than Fe 2 O 3 . The recyclability assessment confirmed the reusability upto four cycles. The antimicrobial property against E. coli, P-aeruginosa, K-pneumoniae, S. aureus, and P. vulgaris bacterial strains revealed a higher response for Fe 2 O 3 -PANI with the zone of inhibition 30, 31, 31, 30, and 30 nm, respectively. The enhanced performance of Fe 2 O 3 -PANI is being credited to S-scheme heterostructure supported with PANI, which can hinder the recombination process and support electron/hole separation. The results indicated that the as-grown nanocomposite could be a promising candidate against bacterial disinfection and an excellent stable photocatalyst.

Inactivation of Bacteria and Residual Antimicrobials in Hospital Wastewater by Ozone Treatment.

The emergence and spread of antimicrobial resistance (AMR) has become a persistent problem globally. In this study, an ozone treatment facility was established for an advanced hospital wastewater treatment in a core hospital facility in an urban area in Japan to evaluate the inactivation of antimicrobial-resistant bacteria and antimicrobials. Metagenomic DNA-seq analysis and the isolation of potential extended-spectrum β-lactamase (ESBL)-producing bacteria suggested that ozone exposure for at least 20 min is required for the adequate inactivation of DNA and ESBL-producing bacteria. Escherichia coli and Klebsiella species were markedly susceptible to 20-min ozone exposure, whereas Raoultella ornithinolytica and Pseudomonas putida were isolated even after an 80-min exposure. These ozone-resistant bacteria might play a pivotal role as AMR reservoirs in the environment. Nine antimicrobials (ampicillin, cefdinir, cefpodoxime, ciprofloxacin, levofloxacin, clarithromycin, chlortetracycline, minocycline, and vancomycin) were detected at 373 ng/L to 27 μg/L in the hospital wastewater, and these were removed (96–100% removal) after a 40-min treatment. These results facilitate a comprehensive understanding of the AMR risk posed by hospital wastewater and provides insights for devising strategies to eliminate or mitigate the burden of antimicrobial-resistant bacteria and the flow of antimicrobials into the environment. To the best of our knowledge, this is the first report on the implementation of a batch-type, plant-scale ozone treatment system in a hospital facility to execute and evaluate the inactivation of drug-resistant bacteria and antimicrobials.

Photodynamic and Contact Killing Polymeric Fabric Coating for Bacteria and SARS-CoV-2.

The development of low-cost, non-toxic, scalable antimicrobial textiles is needed to address the spread of deadly pathogens. Here, we report a polysiloxane textile coating that possesses two modes of antimicrobial inactivation, passive contact inactivation through amine/imine functionalities and active photodynamic inactivation through the generation of reactive oxygen species (ROS). This material can be coated and cross-linked onto natural and synthetic textiles through a simple soak procedure, followed by UV cure to afford materials exhibiting no aqueous leaching and only minimal leaching in organic solvents. This coating minimally impacts the mechanical properties of the fabric while also imparting hydrophobicity. Passive inactivation of Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) is achieved with >98% inactivation after 24 h, with a 23× and 3× inactivation rate increase against E. coli and MRSA, respectively, when green light is used to generate ROS. Up to 90% decrease in the infectivity of SARS-CoV-2 after 2 h of irradiated incubation with the material is demonstrated. These results show that modifying textiles with dual-functional polymers results in robust and highly antimicrobial materials that are expected to find widespread use in combating the spread of deadly pathogens.

Inert cationic iridium(iii) complexes with phenanthroline-based ligands: application in antimicrobial inactivation of multidrug-resistant bacterial strains

New iridium complexes with phenanthroline-based ligands show great potential as antimicrobials, being even more effective than the broad-spectrum antibiotic norfloxacin in Gram positive bacteria.

Enhancement in carrier separation of ZnO-Ho2O3-Sm2O3 hetrostuctured nanocomposite with rGO and PANI supported direct dual Z-scheme for antimicrobial inactivation and sunlight driven photocatalysis

In this work, a direct dual Z-scheme ZnO-Ho2O3-Sm2O3 (NC), polyaniline PANI-ZnO-Ho2O3-Sm2O3 (NCP), and reduced graphene oxide rGO-ZnO-Ho2O3-Sm2O3 (NCR) heterostructure nanocomposites were fabricated successfully. The XRD diffractograms confirmed that ZnO has hexagonal, while Ho2O3, and Sm2O3 have cubic phases in all grown composites. FTIR data exhibited the existence of the PANI, rGO, and metal–oxygen vibration. Raman spectra revealed the presence of optical phonon modes of individual oxides in nanocomposites. PL results disclose the lower charge carrier recombination rate in NCR. SEM images confirmed the anchoring of roughly spherical nanoparticles of NC with PANI and rGO. The UV–Vis analysis showed that the optical energy bandgap was 2.95 eV, 2.88 eV, and 2.81 eV for NC, NCP, and NCR, respectively. The EIS results exhibited a lower charge transfer resistance of NCR than others. The photocatalytic performance against MB, MR, and P-Nitro dyes under sunlight exhibited NCR > NCP > NC efficiency for all dyes in 60 min. The recyclability tests have confirmed the reusability upto six cycles. The antibacterial activity against E. coli, S. aureus, and P. Vulgarisbacteria displayed a higher activity for NCR. The boosted photocatalytic and antibacterial performance of NCR have ascribed the existence of Z-scheme supported with rGO, promote electron/hole separation and hinder the charge carrier’s recombination. The results clearly demonstrate that the as-grown nanocomposites can be used as an effective material for bacterial disinfection and wastewater treatment.

Whole Genome Analysis of Extensively Drug-Resistant Acinetobacter baumannii Clinical Isolates in Thailand.

Acinetobacter baumannii is recognized as a majority opportunistic nosocomial pathogen causing hospital-acquired infection worldwide. The increasing prevalence of extensively drug-resistant Acinetobacter baumannii (XDRAB) has become a rising concern in healthcare facilities and has impeded public health due to limitation of therapeutic options and are associated with high morbidity and mortality as well as longer hospitalization. Whole-genome sequencing of highly multidrug resistant A. baumannii will increase the understanding of resistant mechanisms, the emergence of novel resistance, genetic relationships among the isolates, source tracking, and treatment decisions in selected patients. This study revealed the genomic analysis to explore blaOXA-23 harboring XDRAB isolates in Thailand. Whole-genome sequencing of the two XDRAB isolates was carried out on a HiSeq2000 Illumina platform and susceptibility on antimicrobials was conducted. Both isolates revealed sequence types of international, clone II-carrying, multiple antimicrobial- resistant genes-ST195 and ST451. They were resistant to antimicrobial agents in all drug classes tested for Acinetobacter spp. They carried 18 antimicrobial-resistant genes comprising of 4 β-lactamase genes (blaOXA-23, blaOXA-66, blaTEM-1D, blaADC-25), 4 aminoglycoside-resistant genes (armA, aph(3')-Ia, aph(3")-Ib, aph(6)-Id), 3 macrolide-resistant genes (amvA, mphE, msrE), 1 sulfonamide- resistant gene (sul-2), 2 tetracycline-resistant genes (tetB, tetR), 1 resistant-nodulation-cell division (RND) antibiotic efflux pump gene cluster, 2 major facilitator superfamily (MFS) antibiotic efflux pump genes (abaF, abaQ), and 1 small multidrug-resistant (SMR) antibiotic efflux pump gene (abeS). Mutation of gyrA (S81L) occurred in both isolates. Whole-genome sequencing revealed both blaOXA-23 harboring XDRAB isolates were clustered under international clone II with different STs and carrying multiple antimicrobial-resistant genes conferred their resistance to antimicrobial agents. Inactivation of antimicrobials and target modification by enzymes and pumping antibiotics by efflux pump are mainly resistance mechanism of the XDRAB in this study.

Effects of different antioxidants on quality of meat patties treated with in-package cold plasma

Effects of antioxidants butylated hydroxytoluene (BHT, 0.02%), carnosine (1%), and plant extracts rosemary (RE, 1%), pine bark (PBE, 1%) and pomegranate (PGE, 1%) were investigated on total plate counts, pH, color, and lipid oxidation (LPO) in chicken breast meat patties packed in modified atmosphere and treated with dielectric-barrier-discharge (DBD) cold plasma (CP). The effect on mesophilic inactivation (> 50% reduction) by CP in stored patties varied with antioxidants. No effect was noted for BHT and PBE. RE and PGE enhanced it by >65%, but carnosine suppressed it completely. Antioxidants did not affect CP-induced pH reduction. Plant extracts significantly influenced CIEL*a*b* measurements and prevented CP effects on them during storage. BHT, carnosine, RE, and PGE reduced LPO by more than 18% in CP-treated, stored samples. PBE completely suppressed LPO in CP-treated, and PGE in non-CP-treated, stored patties. Natural plant extracts could improve microbial inactivation and inhibit LPO in CP-processed meat. Industrial relevanceAdding antioxidants in food matrix affects antimicrobial inactivation and prevents oxidative damages by in-package cold plasma (IPCP). The effectiveness varies with the antioxidant. Natural antioxidants rosemary, pomegranate, and pine bark extracts, can be used to enhance antimicrobial inactivation, suppress lipid oxidation, and maintain appearances of IPCP-processed meat products.

Copolymer-nanocapsules of zinc phenyl-thio-phthalocyanine and amphotericin-B in association with antimicrobial photodynamic therapy (A-PDT) applications against Candida albicans yeasts

Antimicrobial Photodynamic Therapy (A-PDT) is a modern and non-invasive therapeutic modality. Nanostructures like the polymeric nanocapsules (NC) has proved to be a system that has enormous potential to improve current antimicrobial therapeutic practice. NC of Zinc phenyl-thio-phthalocyanine and Amphotericin B association (NC/ZnS4Pc + AMB) built with poly(lactide-co-glycolide) (PLGA) 50:50 using the preformed polymer interfacial deposition method were developed at a 0.05 mg mL− 1 theoretical concentration to improve antifungal activity with two actives association and assistance from PDTa. It showed an average particle diameter of 253.8 ± 17.3, an average polydispersity index of 0.36 ± 0.01, and a negative Zeta potential average of -31.03 ± 5.54 for 158 days. UV–vis absorption and emission spectroscopy analyses did not show changes in photophysical properties in the steady-state of NC/ZnS4Pc + AMB counterparts free ZnS4Pc. The encapsulation percentage of actives was 89.24 % and 7.40 % for ZnS4Pc and AMB, respectively. Cell viability assay using NIH/3T3 ATCC® CRL-1658 ™ cells line showed no cytotoxicity for the concentrations tested. The photodynamic activity assay using NC/ZnS4Pc + AMB diluted showed fungal toxicity against Candida albicans yeast with energetic fluences of 12 J.cm-2 and 25 J.cm-2 by a decrease in cell viability. The MFC assay demonstrated a fungistatic activity for the conditions employed in the PDTa assay. The results show that NC/ZnS4Pc + AMB is a promising nanomaterial for antimicrobial inactivation using PDT.

Exploiting a Neutral BODIPY Copolymer as an Effective Agent for Photodynamic Antimicrobial Inactivation.

We report the synthesis and photophysical properties of a neutral BODIPY photosensitizing copolymer (poly-8-(4-hydroxymethylphenyl)-4,4-difluoro-2,6-diethynyl-4-bora-3a,4a-diaza-s-indacene) containing ethynylbenzene links between the BODIPY units. The copolymer absorbs further towards the red in the UV-vis spectrum compared to the BODIPY precursor. Photolysis of the polymer produces a singlet excited state which crosses to the triplet surface in less than 300 ps. This triplet state was used to form singlet oxygen with a quantum yield of 0.34. The steps leading to population of the triplet state were studied using time-resolved spectroscopic techniques spanning the pico- to nanosecond timescales. The ability of the BODIPY polymer to generate a biocidal species for bactericidal activity in both solution- and coating-based studies was assessed. When the BODIPY copolymer was dropcast onto a surface, 4 log and 6 log reductions in colony forming units/ml representative of Gram-positive and Gram-negative bacteria, respectively, under illumination at 525 nm were observed. The potent broad-spectrum antimicrobial activity of a neutral metal-free copolymer when exposed to visible light conditions may have potential clinical applications in infection management.

Inactivation of Clinically Frequently Used Antimicrobial Agents By BacT/ALERT FAN Plus and BACTEC Aerobic and Anaerobic Culture Media in Simulated Blood Cultures: First Comparative Evaluation in China Mainland.

PurposeTo evaluate the antimicrobial inactivation capabilities of BacT/ALERT (FA Plus and FN Plus) and BACTEC (Plus Aerobic/F and Lytic/10 Anaerobic/F) media.Patients and MethodsThe inactivation capabilities of the commercial blood culture media were compared using 21 microorganism-antimicrobial combinations in simulated adult blood cultures.ResultsBacT/ALERT culture media demonstrated higher detection rates than the BACTEC culture media. The recovery rates of the aerobic bottles were 74/115 (64.3%) for FA Plus bottles and 64/115 (55.7%) for BACTEC Aerobic Plus bottles. The BacT/ALERT FAN Plus culture media exhibited a shorter time to detection (TTD). The TTD of FA Plus media was 14.7 h, 4.85 h shorter than the BACTEC Aerobic media (19.55 h), while the TTDs of FN Plus media and BACTEC Anaerobic media were 16.8 h and 18.4 h, respectively.ConclusionBacT/ALERT (FA Plus and FN Plus) media showed relative, but not absolute, advantages, as it had higher detection rates and shorter TTD and thus can be selectively applied to patients with prior use of antimicrobial agents before blood culture samples are taken.

Nanotechnology-based antimicrobials and delivery systems for biofilm-infection control.

Bacterial-infections are mostly due to bacteria in an adhering, biofilm-mode of growth and not due to planktonically growing, suspended-bacteria. Biofilm-bacteria are much more recalcitrant to conventional antimicrobials than planktonic-bacteria due to (1) emergence of new properties of biofilm-bacteria that cannot be predicted on the basis of planktonic properties, (2) low penetration and accumulation of antimicrobials in a biofilm, (3) disabling of antimicrobials due to acidic and anaerobic conditions prevailing in a biofilm, and (4) enzymatic modification or inactivation of antimicrobials by biofilm inhabitants. In recent years, new nanotechnology-based antimicrobials have been designed to kill planktonic, antibiotic-resistant bacteria, but additional requirements rather than the mere killing of suspended bacteria must be met to combat biofilm-infections. The requirements and merits of nanotechnology-based antimicrobials for the control of biofilm-infection form the focus of this Tutorial Review.

Photooxidation Properties of Photosensitizer/Direct Dye Patterned Polyester/Cotton Fabrics

Towards the goal of developing anti-infective textiles based on a photodynamic inactivation mechanism, here we present the design, dyeing procedure, characterization, substrate photooxidation studies, and antibacterial efficacy of methylene blue-dyed polyester fabrics, termed MB-polyester. Dye-uptake and apparent K/S (absorption and scattering coefficient) values as a function of MB concentration % (o.w.f) were determined, and were found to correlate. Photooxidation studies employing the model substrate 1,5-dihydroxynaphthalene (1,5-DHN) revealed that the MB-polyester fabrics were able to generate singlet oxygen in an illumination time-dependent manner. Antibacterial efficacy was evaluated against Staphylococcus aureus (ATCC-29213), with our best results achieving a 99.89 % (~3 log units) reduction in Colony-Forming Units (CFU)/mL after only 30 min illumination (Xenon lamp, 3500 mW/cm2, 420–780 nm). On the basis of these results with MB-polyester, we subsequently designed patterned dual-dyed polyester/cotton fabrics, wherein an alternating pattern of MBdyed polyester was combined with direct dyes-dyed cotton, and showed that their ability to sensitize singlet oxygen (1O2) in the photooxidation reaction of 1,5-DHN was maintained. Taken together, these findings suggest that MB is a suitable photosensitizer (PS) against S. aureus for the practical development of low-cost polyester-based antimicrobial textiles, and can potentially be used in the production of diverse form-patterned textiles that possess a photodynamic antimicrobial inactivation mechanism.

Antimicrobial activity inactivation and toxic immune reactions induce Epilepsy in humans

Antimicrobial activity inactivation and toxic immune reactions induce Epilepsy in humans

Preparation and characterization of bioadhesive system containing hypericin for local photodynamic therapy

Hypericin (Hyp) is a natural photoactive pigment utilized in the treatment of different types of cancer and antimicrobial inactivation using photodynamic therapy (PDT). Hyp is poorly soluble in water leading to problems of administration, getting close contact with the site, and bio-availability. Therefore, this study aimed to develop bioadhesive thermoresponsive system containing Hyp for local PDT. Carbomer 934P, poloxamer 407, and Hyp were used to prepare the thermoresponsive bioadhesive formulations. They were characterized for sol-gel transition temperature, mechanical, mucoadhesive, rheological (continuous flow and oscillatory) and dielectric properties, syringeability, in vitro Hyp release kinetics, ex vivo permeability, and photodynamic activity. The formulations displayed suitable gelation temperature and rheological characteristics. The compressional, mechanical and mucoadhesive properties, as well the syringeability showed the easiness of administration and the permanence of the system adhered to the mucosa or skin. The dielectric analysis helped to understand the Hyp availability, and its release presented an anomalous behavior. The system did not permeate the pig skin nor rat intestine and showed good biological photodynamic activity. Therefore, data obtained from the bioadhesive system indicate a potentially useful role as a platform for local hypericin delivery in PDT, suggesting it is worthy of in vivo evaluation.

Cationic Amphiphiles Increase Activity of Aminoglycoside Antibiotic Tobramycin in the Presence of Airway Polyelectrolytes

It is empirically known that anionic polyelectrolytes present in cystic fibrosis (CF) airways due to bacterial infection significantly decrease the activity of cationic antimicrobials via electrostatic binding. In this work, we use synchrotron small-angle X-ray scattering to investigate the interaction between tobramycin, an aminoglycoside antibiotic commonly administered to CF patients via inhalation, with DNA, which is found in high concentrations in the CF airway. We find that interactions between DNA and tobramycin are significantly modified by the presence of mixtures of amphiphilic molecules. We measure a hierarchy of self-assembled structures formed between tobramycin, DNA, and the amphiphile mixtures and show how interactions between these components can be controlled. Results indicate that mixtures of cationic and negative curvature amphiphiles optimized for DNA binding via charge matching and curvature matching can competitively displace bound tobramycin from DNA and thereby drastically suppress tobramycin-DNA binding and resultant antimicrobial inactivation. Growth inhibition assays confirm the increased activity of tobramycin in the presence of DNA with the addition of the amphiphiles. These results suggest that optimized cationic amphiphile solutions have the potential to enhance antimicrobial function in highly infected environments that contain increased concentrations of anionic inflammatory polymers.

The Key Residue for Substrate Transport (Glu14) in the EmrE Dimer Is Asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.