Activity Of Aminoglycosides Research Articles

Over-the-counter antibiotics compromising aminoglycoside activity.

Antimicrobial resistance (AMR) is a global issue that needs addressing. While antibiotic stewardship has improved often by restricting antibiotic use, some antibiotics that are still sold legally over the counter (OTC), notably in sore throat medications. Recent findings suggest OTC antibiotics could trigger cross-resistance to antibiotics used in clinical treatments, whether systemic or topical. Here we investigated the impact of three antibiotics contained in OTC sore throat medicines on emerging AMR in vitro. Bacterial pathogens were exposed to a bactericidal concentration of an aminoglycoside in the presence or absence of a during-use concentration of bacitracin, gramicidin or tyrothricin in a time-kill assay. Damage to the bacterial membrane was also investigated by measuring potassium leakage and membrane potential alteration post-OTC antibiotic exposure. Gramicidin (15 µg/mL) significantly decreased the bactericidal activity of amikacin, tobramycin or gentamicin in Acinetobacter baumannii. It also decreased gentamicin bactericidal activity in Enterobacter cloacae, Escherichia coli and Klebsiella pneumoniae, while tyrothricin decreased the aminoglycoside efficacy in E. cloacae and E. coli. Gramicidin significantly decreased bacterial membrane potential and caused significant potassium leakage. Gramicidin and to some extent tyrothricin impacted aminoglycoside efficacy by affecting membrane potential, which is essential for aminoglycosides uptake. Thus, some OTC antibiotics can interfere with aminoglycoside activity, which could in turn affect treatment efficacy. Although the likelihood of OTC antibiotics and aminoglycosides being used at the same time might not be common, this research highlights one potential reason for OTC antibiotics' usage to result in treatment failure and their contribution to AMR development.

Resistance against aminoglycoside antibiotics via drug or target modification enables community-wide antiphage defense.

The ongoing arms race between bacteria and phages has forced bacteria to evolve a sophisticated set of antiphage defense mechanisms that constitute the bacterial immune system. In our previous study, we highlighted the antiphage properties of aminoglycoside antibiotics, which are naturally secreted by Streptomyces. Successful inhibition of phage infection was achieved by addition of pure compounds and supernatants from a natural producer strain emphasizing the potential for community-wide antiphage defense. However, given the dual functionality of these compounds, neighboring bacterial cells require resistance to the antibacterial activity of aminoglycosides to benefit from the protection they confer against phages. In this study, we tested a variety of different aminoglycoside resistance mechanisms acting via drug or target (16S rRNA) modification and demonstrated that they do not interfere with the antiphage properties of the molecules. Furthermore, we confirmed the antiphage impact of aminoglycosides in a community context by coculturing phage-susceptible, apramycin-resistant Streptomyces venezuelae with the apramycin-producing strain Streptoalloteichus tenebrarius. Given the prevalence of aminoglycoside resistance among natural bacterial isolates, this study highlights the ecological relevance of chemical defense via aminoglycosides at the community level.

The two-component regulatory systems GraRS and SrrAB mediate Staphylococcus aureus susceptibility to Pep5 produced by clinical isolate of Staphylococcus epidermidis.

Staphylococcus aureus is a common bacterium on the skin and in the nose that sometimes causes severe illness. Bacteriocins, antimicrobial peptides, or proteins produced by bacteria are candidates for the treatment of S. aureus infection. In this study, we found that a clinical Staphylococcus epidermidis strain, KSE112, produced the lantibiotic Pep5, which showed anti-S. aureus activity. The complete nucleotide sequence of the Pep5-encoding plasmid was determined. Several S. aureus two-component regulatory systems (TCSs) are known to be involved in bacteriocin susceptibility. Therefore, susceptibility tests were performed using TCS-inactivated S. aureus mutants to determine which TCS is responsible for Pep5 susceptibility; the ΔgraRS mutant exhibited increased susceptibility to Pep5, while the ΔsrrAB mutant exhibited decreased susceptibility. GraRS is known to regulate dltABCD and mprF in concert with vraFG, and Pep5 susceptibility was significantly increased in the ΔdltABCD, ΔmprF, and ΔvraFG mutants. Regarding the ΔsrrAB mutant, cross-resistance to aminoglycosides was observed. As aminoglycoside activity is known to be affected by aerobic respiration, we focused on qoxABCD and cydAB, which are quinol oxidase genes that are necessary for aerobic respiration and have downregulated the expression in the ΔsrrAB mutant. We constructed ΔqoxABCD and ΔcydAB mutants and found that qoxABCD inactivation decreased susceptibility to Pep5 and aminoglycosides. These results indicate that reduced aerobic respiration due to the reduced qoxABCD expression in the ΔsrrAB mutant decreased Pep5 activity.IMPORTANCEThe emergence of drug-resistant bacteria, including MRSA, is a severe health problem worldwide. Thus, the development of novel antimicrobial agents, including bacteriocins, is needed. In this report, we found a Pep5-producing strain with anti-S. aureus activity. We determined the complete sequence of the Pep5-encoding plasmid for the first time. However, in S. aureus, GraRS and its effectors conferred decreased susceptibility to Pep5. We also revealed that another TCS, SrrAB, affects susceptibility Pep5 and other lantibiotics by controlling aerobic respiration. In our study, we investigated the efficacy of Pep5 against S. aureus and other Gram-positive bacteria and revealed that respiratory constancy regulated by TCS is required for the antimicrobial activity of nisin, nukacin, and Pep5. These findings provide important information for the clinical application of bacteriocins and suggest that they have different properties among similar pore-forming lantibiotics.

In vitro activity of plazomicin and other aminoglycosides against Klebsiella pneumoniae multidrug-resistant strains.

Plazomicin is a new aminoglycoside with broad-spectrum activity against multidrug-resistant strains. The aim of this study was to assess the susceptibility of the K. pneumoniae strains to plazomicin and other aminoglycosides. The activity of plazomicin in combination with ceftazidim-avibactam or meropenem with selected strains was evaluated. The study involved 60 ESβL-positive K. pneumoniae isolates and 50 carbapenemase-positive. The susceptibility to aminoglycosides was tested using the gradient strip. The in vitro activities of plazomicin and ceftazidim-avibactam or meropenem were evaluated using the MTSTM cross synergy method. Plazomicin exhibited high activity against K. pneumoniae with MICs ranging from 0.19 to 4 µg ml-1 for ESβL-positive strains and from 0.25 to 256 µg ml-1 for carbapenemase-positive strains. No antagonism was identified with any combinations. Plazomicin demonstrated excellent in vitro activity against analyzed strains, suggesting that this antibiotic may be an effective therapeutic option in the treatment of infections caused by MDR K. pneumoniae strains.

Enhancing bactericidal activities of ciprofloxacin by targeting the trans-translation system that is involved in stress responses in Klebsiella pneumoniae.

Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1M NaCl), hydrogen peroxide (40mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

A Pharmacodynamic Study of Aminoglycosides against Pathogenic E. coli through Monte Carlo Simulation.

This research focuses on combating the increasing problem of antimicrobial resistance, especially in Escherichia coli (E. coli), by assessing the efficacy of aminoglycosides. The study specifically addresses the challenge of developing new therapeutic approaches by integrating experimental data with mathematical modeling to better understand the action of aminoglycosides. It involves testing various antibiotics like streptomycin (SMN), kanamycin (KMN), gentamicin (GMN), tobramycin (TMN), and amikacin (AKN) against the O157:H7 strain of E. coli. The study employs a pharmacodynamics (PD) model to analyze how different antibiotic concentrations affect bacterial growth, utilizing minimum inhibitory concentration (MIC) to gauge the effective bactericidal levels of the antibiotics. The study's approach involved transforming bacterial growth rates, as obtained from time-kill curve data, into logarithmic values. A model was then developed to correlate these log-transformed values with their respective responses. To generate additional data points, each value was systematically increased by an increment of 0.1. To simulate real-world variability and randomness in the data, a Gaussian scatter model, characterized by parameters like κ and EC50, was employed. The mathematical modeling was pivotal in uncovering the bactericidal properties of these antibiotics, indicating different PD MIC (zMIC) values for each (SMN: 1.22; KMN: 0.89; GMN: 0.21; TMN: 0.32; AKN: 0.13), which aligned with MIC values obtained through microdilution methods. This innovative blend of experimental and mathematical approaches in the study marks a significant advancement in formulating strategies to combat the growing threat of antimicrobial-resistant E. coli, offering a novel pathway to understand and tackle antimicrobial resistance more effectively.

Synthesis, antibacterial and antimonooxidase activity of 4-(2-methyl-1,3-oxazol-5-yl)benzenesulfonamide

A new method for obtaining 4-(2-methyl-1,3-oxazol-5-yl)benzenesulfonamide an antiglaucomatous drug candidate oxazopt was developed using a diazotization reaction. A combined potentiated effect of the antimicrobial action of carbapenems (meropenem) and aminoglycosides (gentamicin) against antibiotic-resistant gram-positive bacteria Enterococcus faecium, Enterococcus faecalis and gram-negative bacteria Escherichia coli and antimonooxidase effect in vitro was revealed for this compound.

A novel ruthenium-silver based antimicrobial potentiates aminoglycoside activity against Pseudomonas aeruginosa.

The rapid dissemination of antibiotic resistance combined with the decline in the discovery of novel antibiotics represents a major challenge for infectious disease control that can only be mitigated by investments in novel treatment strategies. Alternative antimicrobials, including silver, have regained interest due to their diverse mechanisms of inhibiting microbial growth. One such example is AGXX, a broad-spectrum antimicrobial that produces highly cytotoxic reactive oxygen species (ROS) to inflict extensive macromolecular damage. Due to the connections identified between ROS production and antibiotic lethality, we hypothesized that AGXX could potentially increase the activity of conventional antibiotics. Using the gram-negative pathogen Pseudomonas aeruginosa, we screened possible synergistic effects of AGXX on several antibiotic classes. We found that the combination of AGXX and aminoglycosides tested at sublethal concentrations led to a rapid exponential decrease in bacterial survival and restored the sensitivity of a kanamycin-resistant strain. ROS production contributes significantly to the bactericidal effects of AGXX/aminoglycoside treatments, which is dependent on oxygen availability and can be reduced by the addition of ROS scavengers. Additionally, P. aeruginosa strains deficient in ROS detoxifying/repair genes were more susceptible to AGXX/aminoglycoside treatment. We further demonstrate that this synergistic interaction was associated with a significant increase in outer and inner membrane permeability, resulting in increased antibiotic influx. Our study also revealed that AGXX/aminoglycoside-mediated killing requires an active proton motive force across the bacterial membrane. Overall, our findings provide an understanding of cellular targets that could be inhibited to increase the activity of conventional antimicrobials. IMPORTANCE The emergence of drug-resistant bacteria coupled with the decline in antibiotic development highlights the need for novel alternatives. Thus, new strategies aimed at repurposing conventional antibiotics have gained significant interest. The necessity of these interventions is evident especially in gram-negative pathogens as they are particularly difficult to treat due to their outer membrane. This study highlights the effectiveness of the antimicrobial AGXX in potentiating aminoglycoside activities against P. aeruginosa. The combination of AGXX and aminoglycosides not only reduces bacterial survival rapidly but also significantly re-sensitizes aminoglycoside-resistant P. aeruginosa strains. In combination with gentamicin, AGXX induces increased endogenous oxidative stress, membrane damage, and iron-sulfur cluster disruption. These findings emphasize AGXX's potential as a route of antibiotic adjuvant development and shed light on potential targets to enhance aminoglycoside activity.

Contribution of different mechanisms to aminoglycoside resistance in clinical isolates of Acinetobacter baumannii

The antibiotics overuse for infection treatment was the sparkle in the spreading of multi-drug resistance Acinetobacter baumannii in hospitals. In our study, we evaluated the contribution of the aminoglycoside resistance mechanisms of A. baumannii to the resistance surge in some selected Egyptian hospitals with a checkerboard assay application to retrieve the aminoglycoside activity. The resistance profile analysis of collected 200 A. baumannii isolates revealed a multidrug-resistant pattern with limited susceptibilities to aminoglycosides. Analysis of the prevalence of aminoglycoside-modifying enzyme (AMEs) genes revealed the presence of the six AMEs genes either singly or in combination in selected isolates and aph (3)-VIa gene was the predominant one. At the same time, four efflux pump genes of AdeABC and AdeKJL family showed significant (P < 0.001) up-regulation levels. Moreover, the implementation of combination strategy showed fourteen synergistic activities against two high-level aminoglycoside-resistance (HLAR) A. baumannii isolates. The findings highlighted the alarming levels of aminoglycoside resistance in A. baumannii isolates, which proved that a common enzymatic modification mechanism acts synergistically with decreased antibiotic accumulation in acquiring aminoglycoside resistance. Additionally, the study provides useful information for the promising synergistic combination therapy that reduces the therapeutic dose of aminoglycosides used and subsequently increases their clinical application.

Genomics and Antimicrobial Susceptibility of Clinical Pseudomonas aeruginosa Isolates from Hospitals in Brazil.

Pseudomonas aeruginosa, an opportunistic pathogen causing infections in immunocompromised patients, usually shows pronounced antimicrobial resistance. In recent years, the frequency of carbapenemases in P. aeruginosa has decreased, which allows use of new beta-lactams/combinations in antimicrobial therapy. Therefore, the in vitro evaluation of these drugs in contemporary isolates is warranted. We evaluated the antimicrobial susceptibility and genomic aspects of 119 clinical P. aeruginosa isolates from 24 different hospitals in Brazil in 2021-2022. Identification was performed via MALDI-TOF-MS, and antimicrobial susceptibility was identified through broth microdilution, gradient tests, or disk diffusion. Whole-genome sequencing was carried out using NextSeq equipment. The most active drug was cefiderocol (100%), followed by ceftazidime-avibactam (94.1%), ceftolozane-tazobactam (92.4%), and imipenem-relebactam (81.5%). Imipenem susceptibility was detected in 59 isolates (49.6%), and the most active aminoglycoside was tobramycin, to which 99 (83.2%) isolates were susceptible. Seventy-one different sequence types (STs) were detected, including twelve new STs described herein. The acquired resistance genes blaCTX-M-2 and blaKPC-2 were identified in ten (8.4%) and two (1.7%) isolates, respectively. Several virulence genes (exoSTUY, toxA, aprA, lasA/B, plcH) were also identified. We found that new antimicrobials are effective against the diverse P. aeruginosa population that has been circulating in Brazilian hospitals in recent years.

Enhancement of β-Lactam-Mediated Killing of Gram-Negative Bacteria by Lysine Hydrochloride.

Widespread bacterial resistance among Gram-negative bacteria is rapidly depleting our antimicrobial arsenal. Adjuvants that enhance the bactericidal activity of existing antibiotics provide a way to alleviate the resistance crisis, as new antimicrobials are becoming increasingly difficult to develop. The present work with Escherichia coli revealed that neutralized lysine (lysine hydrochloride) enhances the bactericidal activity of β-lactams in addition to increasing bacteriostatic activity. When combined, lysine hydrochloride and β-lactam increased expression of genes involved in the tricarboxylic acid (TCA) cycle and raised reactive oxygen species (ROS) levels; as expected, agents known to mitigate bactericidal effects of ROS reduced lethality from the combination treatment. Lysine hydrochloride had no enhancing effect on the lethal action of fluoroquinolones or aminoglycosides. Characterization of a tolerant mutant indicated involvement of the FtsH/HflkC membrane-embedded protease complex in lethality enhancement. The tolerant mutant, which carried a V86F substitution in FtsH, exhibited decreased lipopolysaccharide levels, reduced expression of TCA cycle genes, and reduced levels of ROS. Lethality enhancement by lysine hydrochloride was abolished by treating cultures with Ca2+ or Mg2+, cations known to stabilize the outer membrane. These data, plus damage observed by scanning electron microscopy, indicate that lysine stimulates β-lactam lethality by disrupting the outer membrane. Lethality enhancement of β-lactams by lysine hydrochloride was also observed with Acinetobacter baumannii and Pseudomonas aeruginosa, thereby suggesting that the phenomenon is common among Gram-negative bacteria. Arginine hydrochloride behaved in a similar way. Overall, the combination of lysine or arginine hydrochloride and β-lactam offers a new way to increase β-lactam lethality with Gram-negative pathogens. IMPORTANCE Antibiotic resistance among Gram-negative pathogens is a serious medical problem. The present work describes a new study in which a nontoxic nutrient increases the lethal action of clinically important β-lactams. Elevated lethality is expected to reduce the emergence of resistant mutants. The effects were observed with significant pathogens (Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa), indicating widespread applicability. Examination of tolerant mutants and biochemical measurements revealed involvement of endogenous reactive oxygen species in response to outer membrane perturbation. These lysine hydrochloride-β-lactam data support the hypothesis that lethal stressors can stimulate the accumulation of ROS. Genetic and biochemical work also revealed how an alteration in a membrane protease, FtsH, abolishes lysine stimulation of β-lactam lethality. Overall, the work presents a method for antimicrobial enhancement that should be safe, easy to administer, and likely to apply to other nutrients, such as arginine.

Comparative Activities of Novel Therapeutic Agents against Molecularly Characterized Clinical Carbapenem-Resistant Enterobacterales Isolates.

Limited treatment options exist for the treatment of carbapenem-resistant Enterobacterales (CRE) bacteria. Fortunately, there are several recently approved antibiotics indicated for CRE infections. Here, we examine the in vitro activity of various novel agents (eravacycline, plazomicin, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam) and comparators (tigecycline, amikacin, levofloxacin, fosfomycin, polymyxin B) against 365 well-characterized CRE clinical isolates with various genotypes. Nonduplicate isolates collected from the largest public health hospital in Singapore between 2007 and 2020 were subjected to antimicrobial susceptibility testing (broth microdilution or antibiotic gradient test strips). Susceptibilities were defined using Clinical and Laboratory Standards Institute (CLSI) or Food and Drug Administration (FDA) interpretative criteria. Sequence types and resistance mechanisms were characterized using short-read whole-genome sequencing. Overall, tigecycline and plazomicin exhibited the highest susceptibility rates (89.6% and 80.8%, respectively). However, the tigecycline susceptibility breakpoint utilized here may be outdated in view of prevailing pharmacokinetic-pharmacodynamic (PK/PD) data. Susceptibility varied by carbapenemase genotype; the β-lactam/β-lactamase inhibitor combinations were equally active (92.3 to 99.2% susceptible) against KPC producers, but only ceftazidime-avibactam retained high susceptibility (98.7%) against OXA-48-like producers. Against metallo-β-lactamase producers, only plazomicin exhibited moderate activity (77.0% susceptible). Aminoglycoside activity was also influenced by carbapenemase genotypes. This work provides an insight into the comparative activity and presumptive utility of novel agents in this geographic region. IMPORTANCE This study determined the susceptibilities of carbapenem-resistant Enterobacterales isolates to various novel antimicrobial agents (ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, eravacycline, and plazomicin). Whole-genome sequencing was performed for all strains. Our study findings provide insights into the comparative activities of novel agents in this geographic region. Plazomicin and ceftazidime-avibactam exhibited the lowest nonsusceptibility rates and may be considered promising agents in the management of carbapenem-resistant Enterobacterales infections. We note also that antibiotic activity is influenced by genotypes and that understanding the geographic region's molecular epidemiology could aid in the definition of the presumptive utility of novel agents and contribute to antibiotic decision-making.

Heat shock potentiates aminoglycosides against gram-negative bacteria by enhancing antibiotic uptake, protein aggregation, and ROS

The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli. This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae. Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.

Antimicrobial Activity of Zinc Oxide, titanium Dioxide and Silver Nanoparticles Against Mithicillin-Resistant Staphylococcus aureus Isolates

The different investigation has been carried out on the biological activities of silver, zinc oxide and titanium dioxide nanoparticles. But no study till now has identified the activity of particles' size or type, nor a study has proved their best synergism activity with antibiotics in one experiment against the same isolates.&#x0D; In this study different sizes of the three types of nanoparticles (NPs) was used to detect the antibacterial activity against methicillin resistant Staphylococcus aureus, which was isolated from patients (with burns and wound) at Erbil hospitals. Different antibiotics also, was evaluated against the same isolates. The Mithicillin-Resistant S. aureus (MRSA) isolates showed multidrug-resistant to most using antibiotics like; ampicillin, amoxicillin, gentamicin, tetracycline clindamycin and erythromycin. The minimum inhibitory concentration (MIC) of these antibiotics at then was conjugated with the nanoparticles' MIC to show the synergism effect by well diffusion method.&#x0D; The results showed that the phenotypic inhibition zones had been increased minimum 2 mm to maximum 18 mm, that confirmed the synergism activity when the antibiotics conjugated with NPs. These results signify that these nanoparticle potentate the antimicrobial action of (beta lactums, aminoglycosides, tetracycline, macrolides and lincosamides) a possible utilization of nano compound in combination effect against MRSA .

Antibiofilm Activity and Synergistic Effects of Thymol-Loaded Poly (Lactic-Co-Glycolic Acid) Nanoparticles with Amikacin against Four Salmonella enterica Serovars.

Salmonella species are frequently linked to biofilm-associated infections. Biofilm formation intensively reduces the efficacy of antibiotics and the host immune system. Therefore, new therapeutic strategies are needed. Thymol, the main monoterpene phenol found in Thymus vulgaris, has been shown to possess potent antibiofilm activity. Our previous findings showed that thymol enhanced the antibiofilm activity of aminoglycosides against Salmonella enterica serovars. However, the clinical potential of thymol has not yet been realized due to its low aqueous solubility and high volatility. Nano-based drug delivery systems have emerged as a novel strategy to resolve these problems. This study aimed to investigate the antibiofilm activity of thymol-loaded poly (lactic-co-glycolic acid) nanoparticles (TH-NPs) and their synergism when used in combination with amikacin antibiotics. The antibacterial activity of TH-NPs was evaluated using the broth microdilution method. Biofilm formation and antibiofilm assays were performed by the miniaturized microtiter plate method. Interaction studies between TH-NPs and amikacin against biofilm were determined using the checkerboard method. TH-NPs exhibited antibacterial activity against planktonic cells of S. enterica serovars that were more efficient (8 to 32 times) than free thymol alone. S. Typhimurium and S. Choleraesuis isolates were considered strong biofilm producers. The combination of TH-NPs with amikacin showed synergistic activity in the inhibition and eradication of S. enterica serovar biofilm. The minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) of amikacin were reduced by 32 to 128-fold when used in combination with TH-NPs. Time-kill kinetic studies showed that the combination of TH-NPs with amikacin possesses bactericidal action. This study suggests that the combination of TH-NPs with amikacin can be an alternative to overcome biofilm-associatedSalmonella diseases and therefore should be further explored as a model to search for new antibiofilm drugs.

Chenodeoxycholic Acid-Amikacin Combination Enhances Eradication of Staphylococcus aureus.

The rise of antibiotic resistance and dearth of novel antibiotics have posed a serious health crisis worldwide. In this study, we screened a combination of antibiotics and nonantibiotics providing a viable strategy to solve this issue by broadening the antimicrobial spectrum. We found that chenodeoxycholic acid (CDCA), a cholic acid derivative of the traditional Chinese medicine (TCM) Tanreqing (TRQ), synergizes with amikacin against Staphylococcus aureus in vitro, and this synergistic killing was effective against diverse methicillin-resistant S. aureus (MRSA) variants, including small-colony variants (SCVs), biofilm strains, and persisters. The CDCA-amikacin combination protects a mouse model from S. aureus infections. Mechanistically, CDCA increases the uptake of aminoglycosides in a proton motive force-dependent manner by dissipating the chemical potential and potentiates reactive oxygen species (ROS) generation by inhibiting superoxide dismutase activity. This work highlights the potential use of TCM components in treating S. aureus-associated infections and extend the use of aminoglycosides in eradicating Gram-positive pathogens. IMPORTANCE Multidrug resistance (MDR) is spreading globally with increasing speed. The search for new antibiotics is one of the key strategies in the fight against MDR. Antibiotic resistance breakers that may or may not have direct antibacterial action and can either be coadministered or conjugated with other antibiotics are being studied. To better expand the antibacterial spectrum of certain antibiotics, we identified one component from a traditional Chinese medicine, Tanreqing (TRQ), that increased the activity of aminoglycosides. We found that this so-called agent, chenodeoxycholic acid (CDCA), sensitizes Staphylococcus aureus to aminoglycoside killing and protects a mouse model from S. aureus infections. CDCA increases the uptake of aminoglycosides in a proton motive force-dependent manner by dissipating the chemical potential and potentiates ROS generation by inhibiting superoxide dismutase activity in S. aureus. Our work highlights the potential use of TCM or its effective components, such as CDCA, in treating antibiotic resistance-associated infections.

Mortality risks associated with empirical antibiotic activity in Escherichia coli bacteraemia: an analysis of electronic health records.

BackgroundReported bacteraemia outcomes following inactive empirical antibiotics (based on in vitro testing) are conflicting, potentially reflecting heterogeneity in causative species, MIC breakpoints defining resistance/susceptibility, and times to rescue therapy.MethodsWe investigated adult inpatients with Escherichia coli bacteraemia at Oxford University Hospitals, UK, from 4 February 2014 to 30 June 2021 who were receiving empirical amoxicillin/clavulanate with/without other antibiotics. We used Cox regression to analyse 30 day all-cause mortality by in vitro amoxicillin/clavulanate susceptibility (activity) using the EUCAST resistance breakpoint (>8/2 mg/L), categorical MIC, and a higher resistance breakpoint (>32/2 mg/L), adjusting for other antibiotic activity and confounders including comorbidities, vital signs and blood tests.ResultsA total of 1720 E. coli bacteraemias (1626 patients) were treated with empirical amoxicillin/clavulanate. Thirty-day mortality was 193/1400 (14%) for any active baseline therapy and 52/320 (16%) for inactive baseline therapy (P = 0.17). With EUCAST breakpoints, there was no evidence that mortality differed for inactive versus active amoxicillin/clavulanate [adjusted HR (aHR) = 1.27 (95% CI 0.83–1.93); P = 0.28], nor of an association with active aminoglycoside (P = 0.93) or other active antibiotics (P = 0.18). Considering categorical amoxicillin/clavulanate MIC, MICs > 32/2 mg/L were associated with mortality [aHR = 1.85 versus MIC = 2/2 mg/L (95% CI 0.99–3.73); P = 0.054]. A higher resistance breakpoint (>32/2 mg/L) was independently associated with higher mortality [aHR = 1.82 (95% CI 1.07–3.10); P = 0.027], as were MICs > 32/2 mg/L with active empirical aminoglycosides [aHR = 2.34 (95% CI 1.40–3.89); P = 0.001], but not MICs > 32/2 mg/L with active non-aminoglycoside antibiotic(s) [aHR = 0.87 (95% CI 0.40–1.89); P = 0.72].ConclusionsWe found no evidence that EUCAST-defined amoxicillin/clavulanate resistance was associated with increased mortality, but a higher resistance breakpoint (MIC > 32/2 mg/L) was. Additional active baseline non-aminoglycoside antibiotics attenuated amoxicillin/clavulanate resistance-associated mortality, but aminoglycosides did not. Granular phenotyping and comparison with clinical outcomes may improve AMR breakpoints.

Discovery of first-in-class nanomolar inhibitors of heptosyltransferase I reveals a new aminoglycoside target and potential alternative mechanism of action

A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell surfaces. While many labs have discovered or designed novel small molecule inhibitors, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo2-lipid A suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a Ki of 600 ± 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes’ protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. Our research suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI, which directly leads to modification of LPS production in vivo. This finding could change our understanding of how aminoglycoside antibiotics function, with interruption of LPS biosynthesis being an additional and important mechanism of aminoglycoside action. Further research to discern the microbiological impact of aminoglycosides on cells is warranted, as inhibition of the ribosome may not be the sole and primary mechanism of action. The inhibition of HepI by aminoglycosides may dramatically alter strategies to modify the structure of aminoglycosides to improve the efficacy in fighting bacterial infections.

N-Butanol Potentiates Subinhibitory Aminoglycosides against Bacterial Persisters and Multidrug-Resistant MRSA by Rapidly Enhancing Antibiotic Uptake.

Potentiation of traditional antibiotics is of significance for combating antibiotic-resistant bacteria that have become a severe threat to human and animal health. Here, we report that 1 min co-treatment with n-butanol greatly and specifically enhances the bactericidal action of aminoglycosides by 5 orders of magnitude against stationary-phase Staphylococcus aureus cells, with n-propanol and isobutanol showing less potency. This combined treatment also rapidly kills various S. aureus persisters, methicillin-resistant S. aureus (MRSA) cells, and numerous Gram-positive and -negative pathogens including some clinically isolated multidrug-resistant pathogens (e.g., S. aureus, Staphylococcus epidermidis, and Enterococcus faecalis) in vitro, as well as S. aureus in mice. Mechanistically, the potentiation results from the actions of aminoglycosides on their conventional target ribosome rather than the antiseptic effect of n-butanol and is achieved by rapidly enhancing the bacterial uptake of aminoglycosides, while salts and inhibitors of proton motive force (e.g., CCCP) can diminish this uptake. Importantly, such n-butanol-enhanced antibiotic uptake even enables subinhibitory concentrations of aminoglycosides to rapidly kill both MRSA and conventional S. aureus cells. Given n-butanol is a non-metabolite in the pathogens we tested, our work may open avenues to develop a metabolite-independent strategy for aminoglycoside potentiation to rapidly eliminate antibiotic-resistant/tolerant pathogens, as well as for reducing the toxicity associated with aminoglycoside use.

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.