Summary
Background
Antimicrobial resistance (AMR) poses a critical threat to public health and disproportionately affects the health and well-being of persons in low-income and middle-income countries. Our aim was to identify synthetic antimicrobials termed conjugated oligoelectrolytes (COEs) that effectively treated AMR infections and whose structures could be readily modified to address current and anticipated patient needs.
Methods
Fifteen chemical variants were synthesized that contain specific alterations to the COE modular structure, and each variant was evaluated for broad-spectrum antibacterial activity and for in vitro cytotoxicity in cultured mammalian cells. Antibiotic efficacy was analyzed in murine models of sepsis; in vivo toxicity was evaluated via a blinded study of mouse clinical signs as an outcome of drug treatment.
Findings
We identified a compound, COE2-2hexyl, that displayed broad-spectrum antibacterial activity. This compound cured mice infected with clinical bacterial isolates derived from patients with refractory bacteremia and did not evoke bacterial resistance. COE2-2hexyl has specific effects on multiple membrane-associated functions (e.g., septation, motility, ATP synthesis, respiration, membrane permeability to small molecules) that may act together to negate bacterial cell viability and the evolution of drug-resistance. Disruption of these bacterial properties may occur through alteration of critical protein–protein or protein-lipid membrane interfaces—a mechanism of action distinct from many membrane disrupting antimicrobials or detergents that destabilize membranes to induce bacterial cell lysis.
Interpretation
The ease of molecular design, synthesis and modular nature of COEs offer many advantages over conventional antimicrobials, making synthesis simple, scalable and affordable. These COE features enable the construction of a spectrum of compounds with the potential for development as a new versatile therapy for an imminent global health crisis.
Funding
U.S. Army Research Office, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.
Keywords
Evidence before this study
Antimicrobial resistance (AMR) poses a critical threat to public health, disease management and global healthcare practices, with the highest burden in low-resource settings. Of particular concern are multidrug-resistant pathogens that are resistant to all, or nearly all, available antibiotics. Despite the scale and urgency, few promising drug candidates are currently in the clinical pipeline to address current and anticipated patient needs. Although multiple new compounds that function via traditional mechanisms show promise against AMR bacteria, they often are vulnerable to the same resistance mechanisms; e.g., drug efflux for synthetic fluoroquinolone derivatives. Recent discoveries have defined a promising path towards the development of antimicrobials that are not prone to antimicrobial resistance, including the natural product, teixobactin; and Irresistin, identified by small molecule library screening of unique compounds. Despite these significant advancements, considerable hurdles remain including the complexity of lead-candidate synthesis, modification, scalability and toxicity.
Added value of this study
Conjugated oligoelectrolytes (COEs) are a class of small synthetic molecules that were designed to insert into bacterial membranes and function as electron transporters, but some were found to inhibit bacterial growth in culture. By screening a diverse array of COEs for antibacterial activity, we identified a specific COE, COE2-2hexyl, that had broad-spectrum activity. Notably, this compound cured mice infected with pathogens derived from patients with refractory bacteremia, and did not evoke bacterial resistance. COE2-2hexyl had specific effects on multiple membrane-associated functions that may act together to disrupt bacterial cell viability and the evolution of drug-resistance through a mechanism of action distinct from most membrane disrupting antimicrobials or detergents which destabilize membranes to induce cell lysis.
Implications of all the available evidence
The diversity and ease of COE design and chemical synthesis have the potential to establish a new standard for drug design and personalized antibiotic treatment. These COE features enable the construction of a spectrum of compounds with the potential as a new versatile therapy for the emergence and rapid global spread of pathogens that are resistant to all, or nearly all, existing antimicrobial medicines.
Introduction
The World Health Organization (WHO) identified antimicrobial resistance among the major threats to global health, food security and economic stability,
World Health Organization
Antimicrobial resistance.
attributing to 1.27 million deaths annually with the highest burden in low- and middle-income countries.
- Murray C.
- Ikuta K.
- Sharara F.
- et al.
Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis.
This public health crisis is predicted to worsen due to climate change—a force-multiplier for the spread of infectious disease and antimicrobial resistance.
- Burnham J.
Climate change and antibiotic resistance: a deadly combination.
Of particular concern are CDC urgent/WHO critical priority pathogens that are resistant to all—or nearly all—available antibiotics.
Centers for Disease Control and Prevention
Antibiotic resistance threats in the United States, 2019.
,
World Health Organization
WHO publishes list of bacteria for which new antibiotics are urgently needed.
These include carbapenem-resistant Acinetobacter, Enterobacterales, and Pseudomonas aeruginosa (CRAB, CRE, CRPA); Clostridioides difficile; drug-resistant Neisseria gonorrhoeae; and extended spectrum β-lactamase producing Enterobacteriaceae (ESBL). Despite the imminent public health threat, few promising drug candidates are currently in the clinical pipeline
Pew Charitable Trusts
Tracking the global pipeline of antibiotics in development, 2021.
,
- Butler M.
- Gigante V.
- Sati H.
- et al.
Analysis of the clinical pipeline of treatments for drug resistant bacterial infections: despite progress, more action is needed.
due to the high costs of drug development and the risk that a new antibiotic becomes ineffective due to bacterial resistance or is reserved as a drug of last resort.
- Lewis K.
The science of antibiotic discovery.
,
- Theuretzbacher U.
- Outterson K.
- Engel A.
- Karlén A.
The global preclinical antibacterial pipeline.
Other factors include the diminished incentives for pharmaceutical research and development for diseases that require relatively short courses of treatment (infectious diseases) compared to blockbuster drugs for pervasive diseases (cancer, cardiovascular diseases, hyperlipidemia and immune disorders).
- Plackett B.
Why big pharma has abandoned antibiotics.
,
- Urquhart L.
Top companies and drugs by sales in 2020.
Product profiles of new antimicrobials that do not evoke bacterial resistance are critical to both clinical and marketplace needs.
- Brown E.
- Wright G.
Antibacterial drug discovery in the resistance era.
Recent discoveries have defined a promising path towards developing antibiotics that do not evoke bacterial resistance, including the natural product teixobactin that specifically targets Gram-positive bacteria, and Irresistin, a broad-spectrum antibiotic identified by small molecule library screening of unique compounds.
- Ling L.
- Schneider T.
- Peoples A.
- et al.
A new antibiotic kills pathogens without detectable resistance.
,
- Martin II, J.
- Sheehan J.
- Bratton B.
- et al.
A dual-mechanism antibiotic kills gram-negative bacteria and avoids drug resistance.
However, significant hurdles remain including complexity of synthesis, modification, scalability and toxicity of lead candidates.
Conjugated oligoelectrolytes (COEs) are a class of small amphiphilic molecules that share a modular structure that can spontaneously interact with lipid bilayers (Fig. 1a).
- Garner L.
- Park J.
- Dyar S.
- Chworos A.
- Sumner J.
- Bazan G.
Modification of the optoelectronic properties of membranes via insertion of amphiphilic phenylenevinylene oligoelectrolytes.
,
- Yan H.
- Catania C.
- Bazan G.
Membrane-intercalating conjugated oligoelectrolytes: impact on bioelectrochemical systems.
The ease of molecular design and synthesis allows the construction of a spectrum of bacterial interfacing synthetic compounds that can be readily modified to alter membrane affinity and other properties (solubility, charge, stability).
- Wang B.
- Fronk S.
- Rengert Z.
- Limwongyut J.
- Bazan G.
Conjugated oligoelectrolytes: materials for acceleration of whole cell biocatalysis.
,
- Yan H.
- Rengert Z.
- Thomas A.
- Rehermann C.
- Hinks J.
- Bazan G.
Influence of molecular structure on the antimicrobial function of phenylenevinylene conjugated oligoelectrolytes.
COE intercalation into phospholipid bilayers is driven by the hydrophobic centre and the terminal ionic functionalities, consisting of the conjugated aromatic core and hydrocarbon pendants. These pendant moieties resemble the fatty acid centre of the bilayer, while the cationic end groups and terminal acyl chains interact via coulombic and hydrophobic interactions with the membrane surface functionalities (Fig. 1b and c). Although COEs were initially designed to insert into bacterial membranes and function as electron transporters, some were found to inhibit bacterial growth in culture for a limited number of pathogens.
- Limwongyut J.
- Nie C.
- Moreland A.
- Bazan G.
Molecular design of antimicrobial conjugated oligoelectrolytes with enhanced selectivity toward bacterial cells.
,
- Zhou C.
- Chia G.
- Ho J.
- et al.
Informed molecular design of conjugated oligoelectrolytes to increase cell affinity and antimicrobial activity.
Such findings launched an effort to synthesize and screen a diverse array of COE chemical variants (altered length of the conjugated aromatic backbone, distribution of ionic groups, and hydrophobic substitutions) for antibacterial activity against 17 clinically relevant Gram-negative and Gram-positive pathogens. Here we report on a specific COE, COE2-2hexyl, that exhibited broad-spectrum activity, effectively treated mice infected with multidrug-resistant (MDR) bacteria and was not prone to bacterial resistance.
Fig. 1Conjugated oligoelectrolytes (COEs) and comparative bactericidal activity and mammalian cell cytotoxicity. a, COEs share a modular structure that spontaneously integrates into the bacterial membrane. b and c, COE structural modules are depicted by coloured boxes. The intercalation into phospholipid bilayers is driven by the hydrophobic centre and the terminal ionic functionalities, consisting of the conjugated aromatic core (gold module) and hydrocarbon pendants (blue module), which resemble the fatty acid centre of the bilayer. Additionally, the cationic end groups (red module) and terminal acyl chains (pink module) interact via coulombic and hydrophobic interactions with the membrane surface functionalities; specific example: COE2-2hexyl. Bactericidal activity. Exponential-phase cultures (∼108 cells) of either d, S. Typhimurium 14028 or e, CA-MRSA USA300 were incubated with 10 × MIC of either COE2-2hexyl (20 μg/mL; 10 μg/mL, respectively) or ciprofloxacin (0.156 μg/mL; 5 μg/mL, respectively) for 4 h, and viability was enumerated by direct colony count (n ≥ 3, SEM). Mammalian cell cytotoxicity. COEs at the designated concentration (1–20 μg/mL) were incubated for 18 h with f, murine macrophage (RAW 264.7) and g, human epithelial cell lines (HEp-2). Mammalian cell cytotoxicity was determined by the trypan blue vital stain exclusion method,
- Kamiloglu S.
- Sari G.
- Ozdal T.
- Capanoglu E.
Guidelines for cell viability assays.
and the unstained viable cells were counted using a hemocytometer (n = 6, SD).
Methods
Synthesis and characterization of conjugated oligoelectrolytes
COE syntheses and product characterizations were as previously described (detailed experimental methods, synthetic schemes, yields and NMR spectra).
- Yan H.
- Rengert Z.
- Thomas A.
- Rehermann C.
- Hinks J.
- Bazan G.
Influence of molecular structure on the antimicrobial function of phenylenevinylene conjugated oligoelectrolytes.
,
- Limwongyut J.
- Nie C.
- Moreland A.
- Bazan G.
Molecular design of antimicrobial conjugated oligoelectrolytes with enhanced selectivity toward bacterial cells.
,
- Zhou C.
- Chia G.
- Ho J.
- et al.
Informed molecular design of conjugated oligoelectrolytes to increase cell affinity and antimicrobial activity.
,
- Tiihonen A.
- Cox-Vazquez S.
- Liang Q.
- et al.
Predicting antimicrobial activity of conjugated oligoelectrolyte molecules via machine learning.
,
- Bazan G.
- Zhang Z.
- Moreland A.
- et al.
Short conjugated oligoelectrolytes and uses thereof.
Briefly, the alkylation steps were performed by Williamson ether synthesis with a carbonate base. The COE conjugated backbones were derived via Horner−Wadsworth−Emmons or McMurry coupling reactions. The specific COEs were obtained after quaternization of the terminal alkyl halide groups with excess trimethylamine or other amines. Intermediates and COE products were purified using multiple strategies (i.e., liquid−liquid extraction, column chromatography, precipitation, solvent removal under vacuum, etc.) and subsequently characterized by NMR or mass spectroscopy. The maximum solubility of COE2-2hexyl is 400 μg/mL in sterile H2O. COE2-2hexyl MIC testing was performed on at least three independent batch syntheses.
Bacterial strains and culture conditions
Gram-negative bacterial isolates included: A. baumannii ATCC 19606; A. baumanii ATCC 17978; E. coli DH5α, E. coli ATCC 25922, E. coli MG1655, E. coli BW25113, E. coli BW25113 ΔmutL::kan, K. pneumoniae ATCC 13883; CRE K. pneumoniae (MT3325), derived from a urinary/sepsis patient obtained from Santa Barbara Cottage Hospital (2017); N. gonorrhoeae ATCC 700825; N. gonorrhoeae ATCC 49226; S. flexneri ATCC 29903; P. aeruginosa ATCC 10145; S. enterica serovar Typhimurium ATCC 14028; Y. pseudotuberculosis (YPIII). Gram-positive clinical isolates included methicillin-resistant (MRSA) and -sensitive (MSSA) S. aureus: CA-MRSA USA300, MSSA Newman and 3 isolates derived from sepsis patients obtained from Santa Barbara Cottage Hospital (2016) termed MRSA Blood (MT3302); MRSA Wound (MT3315); MSSA Blood (MT3305).
- Ersoy S.
- Heithoff D.
- Barnes L.
- et al.
Correcting a fundamental flaw in the paradigm for antimicrobial susceptibility testing.
S. pneumoniae clinical isolates included D39 (ser. 2) and Daw 1 (ser. 6).
Bacterial strains were grown as previously described.
- Yang W.
- Heithoff D.
- Aziz P.
- et al.
Accelerated aging and clearance of host anti-inflammatory enzymes by discrete pathogens fuels sepsis.
Briefly, Gram-negative bacteria were isolated after overnight growth on Luria–Bertani (LB) agar and incubated at 37 °C in ambient air or, for Yersinia, 48 h at room temperature. N. gonorrhoeae were isolated on Chocolate agar (Becton Dickinson) for 48 h at 37 °C in a 5% CO2 incubator. Gram-positive S. aureus strains were isolated on Tryptic Soy Broth (TSB) agar and incubated at 37 °C in ambient air. S. pneumoniae strains were grown overnight on Columbia CNA agar with 5% sheep blood (Becton Dickinson) and grown in Todd-Hewitt Broth (THB) supplemented with 2% yeast extract incubated at 37 °C in a 5% CO2 incubator.
MIC assays
COE structural variants were evaluated for MIC against a collection of nine clinical bacterial isolates via broth microdilution (n ≥ 9).
Clinical and Laboratory Standards Institute
Performance standards for antimicrobial resistance testing; twenty-fourth informational supplement.
,
Clinical and Laboratory Standards Institute
Performance standards for antimicrobial susceptibility testing.
,
European Committee on Antimicrobial Susceptibility Testing
Breakpoint tables for interpretation of MICs and zone diameters.
Gram-negative bacteria: E. coli ATCC 25922 (MT3277); K. pneumoniae ATCC 13883 (MT1947); P. aeruginosa ATCC 10145 (MT1945); and S. Typhimurium 14028; Gram-positive bacteria: MSSA Newman; MSSA blood isolate (MT3305); CA-MRSA USA300; MRSA blood isolate (MT3302); and MRSA wound isolate (MT3315). Standard AST medium is Mueller-Hinton Broth (MHB) supplemented with CaCl2 and MgCl2 to make cation-adjusted MHB (Ca-MHB).
Clinical and Laboratory Standards Institute
Performance standards for antimicrobial resistance testing; twenty-fourth informational supplement.
Unless otherwise specified, bacteria were grown overnight at 37 °C in Ca-MHB broth in ambient air. Yersinia was cultured at 28 °C. N. gonorrhea was grown in modified Chocolate broth
- Shapiro M.
- Heifetz C.
- Sesnie J.
Comparison of microdilution and agar dilution procedures for testing antibiotic susceptibility of Neisseria gonorrhoeae.
incubated at 37 °C for 20 h in a 5% CO2 incubator. S. aureus MIC assays were done by direct inoculation: five to seven S. aureus colonies from TSB agar were used to inoculate 1 mL Ca-MHB. S. pneumoniae was grown overnight on Columbia CNA agar with 5% sheep blood, and five colonies were inoculated into 0.5 mL Ca-MHB supplemented with 5% lysed horse blood (Lampire Biological Laboratories), and incubated 4 h at 37 °C in a 5% CO2 incubator.
Cell culture
The murine macrophage RAW 264.7 (ATCC TIB-71) and the human epithelial HEp-2 (ATCC CCL-23) cell lines were obtained from the American Type Culture Collection, Rockville, MD, and maintained in minimum essential medium (MEM) supplemented with l-glutamine and 10% heat-inactivated bovine growth-supplemented calf serum (HyClone Laboratories, Logan, UT). Cells were grown in a humidified atmosphere of 5% carbon dioxide and 95% air at 37 °C in 75-cm2 plastic flasks (Corning Glass Works, Corning, NY). Cultured cells were harvested by scraping with a rubber policeman and plated at a density of 5 × 104 to 1 × 105 cells/mL in 1 mL of supplemented MEM in 24-well dishes (Corning) and grown for 24 h to approximately 80–90% confluence (1 × 105–2 × 105 cells/well) (adapted from
- Fleckenstein J.
- Kopecko D.
- Warren R.
- Elsinghorst E.
Molecular characterization of the tia invasion locus from enterotoxigenic Escherichia coli.
).
COE cytotoxicity in cultured mammalian cells
In vitro cytotoxicity was determined by the trypan blue vital stain exclusion method.
- Kamiloglu S.
- Sari G.
- Ozdal T.
- Capanoglu E.
Guidelines for cell viability assays.
COEs or antibiotics were added to cultured murine macrophage (RAW 264.7) or human epithelial (HEp-2) 80–90% confluent monolayers in 24-well cell culture plates (Corning) at 0, 1, 4, 10 or 20 μg/mL in 1 mL cell culture media. The culture was incubated for 18 h at 37 °C in a 5% CO2 incubator, washed once with 1 mL PBS, and cells were harvested by scraping into 0.1 mL PBS. Cells were diluted 1:5 in PBS, and 10 μL of the diluted cells were added to an equal volume of 0.4% trypan blue, and the unstained viable and total cell number were counted using a hemocytometer. Standard deviation (SD) was determined from 6 biological replicates for each condition.
Bactericidal activity assay
S. Typhimurium 14028 or CA-MRSA USA300 were grown overnight in LB or TSB respectively, diluted 1:100, and incubated at 37 °C with shaking to obtain exponential-phase cultures. ∼108 cells of S. Typhimurium 14028 or CA-MRSA USA300 were incubated with either 10 × MIC of COE2-2hexyl (20 μg/mL; 10 μg/mL, respectively) or ciprofloxacin (0.156 μg/mL; 5 μg/mL, respectively) 4 h, and viability was enumerated by direct colony count. Standard error of the mean (SEM) was determined from ≥3 biological replicates for each condition.
In vivo toxicity assay
Toxicity of COE2-2hexyl was assessed in mice relative to that of low- and high-dose treatment with polymyxin B (PMB). The proportion of mice developing abnormal attitude scores was compared between groups using Fisher’s exact test with Bonferroni adjustment for multiple comparisons. A linear mixed model was also fitted with attitude score as the outcome variable, treatment group was evaluated as a factor and mouse as a random effect. Differences in attitude scores were determined for PMB treatment among low- and high- dose groups [i.p., 30 mg/kg/day (B.I.D) vs. 45 mg/kg/day (T.I.D.)]
- Kubicek-Sutherland J.
- Heithoff D.
- Ersoy S.
- et al.
Host-dependent induction of transient antibiotic resistance: a prelude to treatment failure.
and mock-treated BALB/c mice (n = 10); and between the COE2-2hexyl treated (i.v., 2 mg/kg/day) (S.I.D.) and mock-treated C57BL/6 mice (n = 10). Attitude score scale: 0 = normal appearance/activity/clinical signs; 1 = slight abnormal hair coat; normal activity/clinical signs; 2 = abnormal hair coat; normal activity/clinical signs; 3 = abnormal appearance/activity/clinical signs, euthanize.
Bacterial infections and antibiotic treatment
Bacterial infections were performed as previously described.
- Yang W.
- Heithoff D.
- Aziz P.
- et al.
Accelerated aging and clearance of host anti-inflammatory enzymes by discrete pathogens fuels sepsis.
,
- Heithoff D.
- Pimienta G.
- Mahan S.
- et al.
Coagulation factor protein abundance in the pre-septic state predicts coagulopathic activities that arise during late-stage murine sepsis.
Mice were i.v. infected with either MRSA (MT3302) or CRE K. pneumoniae (MT3325) at a dose of 1 × 108 cfu (20 × LD50) and treated for 3 days (beginning 2 h post-infection) with a single-daily dose of COE2-2hexyl (i.v., 2 mg/kg/day; maximum soluble dose) (n = 10). Additionally, another CRE K. pneumoniae cohort was treated with a twice-daily dose of colistin (CST) (i.p. 30 mg/kg/day)
- Ersoy S.
- Heithoff D.
- Barnes L.
- et al.
Correcting a fundamental flaw in the paradigm for antimicrobial susceptibility testing.
(n = 10). Survival was scored up to day 5 and compared to infected, mock-treated animals (MRSA, n = 10; K. pneumoniae, n = 20). The COE2-2hexyl dose is equivalent to 28 μg/mL plasma concentration at the time of administration (28 × MIC for MRSA; 7 × MIC for K. pneumoniae). Unless otherwise specified, all animal experiments were carried out with 8–12-week-old C57BL/6J mice and used equal numbers of males and females. All mice in the study were provided sterile pellet food and water ad libitum.
DiBAC4 and PI staining
Overnight cultures of E. coli MG1655 cells were diluted 100-fold in LB media, grown to log phase with shaking at 37 °C, diluted in LB to an OD600 = 0.2 and aliquoted (2 mL each) into polypropylene tubes. COE2-2hexyl was added at the indicated final concentrations and cells were incubated at 37 °C with shaking for the times indicated. At the end of the incubation period, cells were harvested by centrifugation (15,000 × g, 1 min, 23 °C), resuspended in an equal volume of phosphate-buffered saline, pH 7.2 (PBS), and DiBAC4(3) and/or PI were added at final concentrations of 0.01 μg/mL and 6.68 μM respectively, and incubated for 30 min at 37 °C. Samples were then analysed using an Accuri C6 flow cytometer (Becton Dickinson) using FL1 (533/30 nm, DiBAC4(3)) and FL3 (670 nm, PI) fluorophore filters. SD was determined from 3 biological replicates.
ATP analysis
Exponentially growing E. coli MG1655 cells were diluted in LB medium to OD600 = 0.2, COE2-2hexyl was added to 2 mL cultures of these cells in polypropylene tubes and incubated with shaking for the indicated times at 37 °C. At the end of the incubation time, 100 μL samples were dispensed into 3 wells (triplicate) of an opaque black microtiter plate and 100 μL of BacTiter-Glo reagent (Promega) was added. After incubation with gentle shaking for 5 min at 23 °C, luminescence was measured using a Perkin–Elmer Wallac 1420 multilabel counter. Cultures similarly grown and processed without the addition of COE2-2hexyl served as control. In parallel, serially-diluted pure ATP preparations were subjected to the same assay and the luminescence output measured and plotted to calculate ATP levels in samples. SD was determined from 3 biological replicates.
Oxygen consumption and protein quantification
Overnight cultures of E. coli MG1655 cells were diluted 1:100 with LB media and grown with shaking at 37 °C to OD600 = 0.6. Cultures were then diluted in LB medium to OD600 = 0.2, 5 mL aliquots were taken in polypropylene tubes, and COE2-2hexyl was added and incubated at 37 °C with shaking for the indicated times. Aerobic respiration was measured at 23 °C in a sealed stirred cuvette with a Clarke oxygen electrode as recommended by the manufacturer (Qubit Systems). Calibration was carried out after air was bubbled into the oxygen electrode sample chamber (100% O2 saturation) and after addition of a few grains of sodium sulphite (0% saturation). E. coli samples (3 mL) were added to the calibrated cuvette equipped with a stir bar; the top plunger was lowered, and air bubbles were expelled through the top port. Readings were taken every second and the slope was calculated by least-squares analysis.
- Aoki S.
- Webb J.
- Braaten B.
- Low D.
Contact-dependent growth inhibition causes reversible metabolic downregulation in Escherichia coli.
Protein quantification was carried out using the DC protein assay (Bio-Rad). Briefly, bacterial cultures (1 mL) were harvested by centrifugation (15,000 × g, 1 min, 23 °C), and cell pellets were washed 1× with 200 μL PBS. The pellets were resuspended in 100 μL 1× Laemmli buffer, heated at 100 °C for 5 min, centrifuged at 15,000 × g for 5 min and the supernatant solutions collected. Samples were analysed according to the Bio-Rad protocol measuring absorbance at 750 nm, measuring dilutions of a bovine serum albumin control solution in parallel to quantify total protein content. SD was determined from 3 biological replicates.
Cloxacillin sensitivity
The effect of COE2-2hexyl on cloxacillin sensitivity was measured using E. coli DL5916 (MG1655 acrB::EZ-Tn ΔaraBAD::spcR) and DL5850 (acrB+ΔaraBAD::spcR). Serial dilutions of COE2-2hexyl were made in LB (1 mL, polypropylene tubes), 10 μL of E. coli (OD600 = 0.2) was added to each tube and tubes incubated overnight with shaking to estimate the MIC for each E. coli strain (in triplicate, experiment repeated 3 times). Similarly, the MIC of cloxacillin was determined for both strains, both in the absence of COE2-2hexyl and in the presence of 0.5 × MIC COE2-2hexyl (1 μg/mL). SD was determined from 4 biological replicates.
Isolation of COER mutants by serial dilution
Three independent 10 mL cultures of bacteria were grown overnight, serially diluted 1:10 in the presence of either 0.5 × MIC or 1 × MIC COE2-2hexyl in polypropylene flasks, and incubated 20 h at 37 °C with shaking (MIC: S. Typhimurium 14028, 2 μg/mL; MRSA CA–USA300, 1 μg/mL). If growth was scored in the presence of 1 × MIC, the serial dilution procedure was repeated at the initial- and double-the drug concentration (1 and 2 × MIC). If no growth was scored at 1 × MIC, the 0.5 × MIC culture was used to repeat the serial dilution procedure (0.5 and 1 × MIC). If growth then occurred at 1 × MIC, the serial dilution procedure was repeated at the initial- and double-the drug concentration (1 and 2 × MIC). This procedure was repeated for 21 consecutive days.
Isolation of COER mutants by morbidostat-based experimental evolution
The morbidostat measures the growth rates of evolving microbial populations and automatically adjusts drug concentrations to maintain a constant drug-induced inhibition.
- Leyn S.
- Zlamal J.
- Kurnasov O.
- et al.
Experimental evolution in morbidostat reveals converging genomic trajectories on the path to triclosan resistance.
,
- Zlamal J.
- Leyn S.
- Iyer M.
- et al.
Shared and unique evolutionary trajectories to ciprofloxacin resistance in gram-negative bacterial pathogens.
The hardware, software components and experimental methods and work-flow were as described.
- Leyn S.
- Zlamal J.
- Kurnasov O.
- et al.
Experimental evolution in morbidostat reveals converging genomic trajectories on the path to triclosan resistance.
Briefly, bacterial populations were placed under increasing selective drug pressure in parallel reactors. Total genomic DNA from bacterial cell populations from each reactor were sampled and sequenced on a daily basis; and mutations and possible mechanisms of resistance were determined by variant calling bioinformatics. Experimental validation was performed by verification of identified mutations and measurement of the MIC change of representative clones.
Ethics statement
Human subjects approval was obtained from the Institutional Human Subjects Use Committee of the University of California, Santa Barbara and the Institutional Review Board of Santa Barbara Cottage Hospital. All animal experimentation was conducted following the National Institutes of Health guidelines for housing and care of laboratory animals and performed in accordance with Institutional regulations after pertinent review and approval by the Institutional Animal Care and Use Committee at the University of California, Santa Barbara.
Statistical analyses
Log-rank (Mantel–Cox) test was used to compare differences in survival between groups for Kaplan–Meier survival curves; significance was determined using GraphPad Prism version 9.0. P values of less than 0.05 were considered significant. The exact value of n, representing the number of mice, was indicated in the figure legends. Fisher’s exact test and Restricted Maximum Likelihood (REML) were used to compare differences of in vivo toxicity utilizing RStudio
RStudio Team
Integrated development environment for R.
and the fmsb (version 0.7.3)
- Nakazawa M.
fmsb: functions for medical statistics book with some demographic data, R package version 0.7.32022.
and lme 4 (version 1.1–29)
- Bates D.
- Mächler M.
- Bolker B.
- Walker S.
Fitting linear mixed-effects models using lme 4.
packages.
Role of the funding source
The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.
Results
In vitro COE screen for antibacterial activity and cytotoxicity
Fifteen chemical variants were synthesized that contain specific alterations to the COE modular structure, including length of conjugated aromatic backbone, linkage type, length of hydrocarbon pendants, and distribution of cationic groups and terminal acyl chains (Supplementary Table S1; see Methods). Each structural variant was evaluated for antibacterial activity against a collection of nine bacterial clinical isolates via antibiotic susceptibility testing (AST)—defining the minimal inhibitory concentration (MIC) of each compound
Clinical and Laboratory Standards Institute
Performance standards for antimicrobial resistance testing; twenty-fourth informational supplement.
,
Clinical and Laboratory Standards Institute
Performance standards for antimicrobial susceptibility testing.
,
European Committee on Antimicrobial Susceptibility Testing
Breakpoint tables for interpretation of MICs and zone diameters.
—and for in vitro cytotoxicity relative to ciprofloxacin (CIP), a broad-spectrum fluoroquinolone antibiotic (Supplementary Table S1). Following incubation of a murine macrophage cell line (RAW 264.7) with these compounds, membrane integrity as a measure of cytotoxicity was determined by the trypan blu
00026-9&id=gr1.jpg){kind=link}