Gyrase and Topoisomerase IV: Recycling Old Targets for New Antibacterials to Combat Fluoroquinolone Resistance.

Beyond their requisite functions in many critical DNA processes, the bacterial type II topoisomerases, gyrase and topoisomerase IV, are the targets of fluoroquinolone antibacterials. These drugs act by stabilizing gyrase/topoisomerase IV-generated DNA strand breaks and by robbing the cell of the catalytic activities of these essential enzymes. Since their clinical approval in the mid-1980s, fluoroquinolones have been used to treat a broad spectrum of infectious diseases and are listed among the five "highest priority" critically important antimicrobial classes by the World Health Organization. Unfortunately, the widespread use of fluoroquinolones has been accompanied by a rise in target-mediated resistance caused by specific mutations in gyrase and topoisomerase IV, which has curtailed the medical efficacy of this drug class. As a result, efforts are underway to identify novel antibacterials that target the bacterial type II topoisomerases. Several new classes of gyrase/topoisomerase IV-targeted antibacterials have emerged, including novel bacterial topoisomerase inhibitors, Mycobacterium tuberculosis gyrase inhibitors, triazaacenaphthylenes, spiropyrimidinetriones, and thiophenes. Phase III clinical trials that utilized two members of these classes, gepotidacin (triazaacenaphthylene) and zoliflodacin (spiropyrimidinetrione), have been completed with positive outcomes, underscoring the potential of these compounds to become the first new classes of antibacterials introduced into the clinic in decades. Because gyrase and topoisomerase IV are validated targets for established and emerging antibacterials, this review will describe the catalytic mechanism and cellular activities of the bacterial type II topoisomerases, their interactions with fluoroquinolones, the mechanism of target-mediated fluoroquinolone resistance, and the actions of novel antibacterials against wild-type and fluoroquinolone-resistant gyrase and topoisomerase IV.

Fate and removal of fluoroquinolone antibiotics in mesocosmic wetlands: Impact on wetland performance, resistance genes and microbial communities.

The fate of fluoroquinolone antibiotics norfloxacin and ofloxacin were investigated in mesocosmic wetlands, along with their effects on nutrients removal, antibiotic resistance genes (ARGs) and epiphytic microbial communities on Hydrilla verticillate using bionic plants as control groups. Approximately 99% of norfloxacin and ofloxacin were removed from overlaying water, and H. verticillate inhibited fluoroquinolones accumulation in surface sediments compared to bionic plants. Partial least squares path modeling showed that antibiotics significantly inhibited the nutrient removal capacity (0.55) but had no direct effect on plant physiology. Ofloxacin impaired wetland performance more strongly than norfloxacin and more impacted the primary microbial phyla, whereas substrates played the most decisive role on microbial diversities. High antibiotics concentration shifted the most dominant phyla from Proteobacteria to Bacteroidetes and inhibited the Xenobiotics biodegradation function, contributing to the aggravation in wetland performance. Dechloromonas and Pseudomonas were regarded as the key microorganisms for antibiotics degradation. Co-occurrence network analysis excavated that microorganisms degrade antibiotics mainly through co-metabolism, and more complexity and facilitation/reciprocity between microbes attached to submerged plants compared to bionic plants. Furthermore, environmental factors influenced ARGs mainly by altering the community dynamics of differential bacteria. This study offers new insights into antibiotic removal and regulation of ARGs accumulation in wetlands with submerged macrophyte.

Physicochemical properties as a function of lomefloxacin biological activity.

OBJECTIVE: Aim: The goal is to discover QSAR of Lomefloxacin as antibacterial activity. PATIENTS AND METHODS: Materials and Methods: A number of lomefloxacins analogs activities were studied by program Windows Chem SW. The analogues were obtained and energy minimization was carried out through Molecular Modeling Program, the calculations were performed using General Atomic and Molecular Electronic Structure System (GAMESS) software. RESULTS: Results: There were six descriptions (N-quinoline more (-) ev charge, Kinetic Energy, Potential Energy, Log p, Log S, F6 charge) results have highly compatible of physicochemical properties with lomefloxacin analogs activities. It can be used to estimate the activities depending on QSAR equation of lomefloxacin analogs. CONCLUSION: Conclusions: The parameters used for calculation were depending on the quantum chemical was employed in deriving from computational study of properties and can used to predict the activities of certain analogs of Lomefloxacins as antibacterial compounds.

Expression of norA, norB and norC efflux pump genes mediating fluoroquinolones resistance in MRSA isolates.

INTRODUCTION: Although fluoroquinolones are used to treat methicillin-resistant Staphylococcus aureus (MRSA)-induced infections, acquisition of antibiotic resistance by bacteria has impaired their clinical relevance. We aimed to evaluate the frequency of norA, norB, and norC efflux pump genes-mediating fluoroquinolones resistance and measure their expression levels in MRSA isolates. METHODOLOGY: 126 S. aureus isolates were collected from different clinical samples of adult hospitalized patients and identified by conventional microbiological methods. MRSA was diagnosed by cefoxitin disc diffusion method and minimum inhibitory concentration (MIC) of ciprofloxacin by broth microdilution method. The expression levels of efflux pump genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: 80 (63.5%) MRSA isolates were identified and showed high level of resistance to erythromycin (80%), gentamicin (75%), clindamycin (65%) and ciprofloxacin (60 %). norA, norB and norC were detected in 75%, 35% and 55% of the MRSA isolates respectively. norC was the most commonly overexpressed gene measured by qRT-PCR, occurring in 40% of MRSA isolates, followed by norA (35%) and norB (30%). The expression of these genes was significantly higher in ciprofloxacin-resistant than quantitative real-time PCR ciprofloxacin-sensitive MRSA isolates. CONCLUSIONS: This study showed high prevalence and overexpression of efflux pump genes among MRSA isolates which indicates the significant role of these genes in the development of multidrug resistance against antibiotics including fluoroquinolones.

Patterns and profiles of drug resistance-conferring mutations in Mycobacterium tuberculosis genotypes isolated from tuberculosis-suspected attendees of spiritual holy water sites in Northwest Ethiopia.

Purpose: This study examined the patterns and frequency of genetic changes responsible for resistance to first-line (rifampicin and isoniazid), fluoroquinolones, and second-line injectable drugs in drug-resistant Mycobacterium tuberculosis (MTB) isolated from culture-positive pulmonary tuberculosis (PTB) symptomatic attendees of spiritual holy water sites (HWSs) in the Amhara region. Patients and methods: From June 2019 to March 2020, a cross-sectional study was carried out. A total of 122 culture-positive MTB isolates from PTB-suspected attendees of HWSs in the Amhara region were evaluated for their drug resistance profiles, and characterized gene mutations conferring resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolones (FLQs), and second-line injectable drugs (SLIDs) using GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0. Drug-resistant MTB isolates were Spoligotyped following the manufacturer's protocol. Results: Genetic changes (mutations) responsible for resistance to RIF, INH, and FLQs were identified in 15/122 (12.3%), 20/122 (16.4%), and 5/20 (25%) of MTB isolates, respectively. In RIF-resistant, rpoB/Ser531Lue (n = 12, 80%) was most frequent followed by His526Tyr (6.7%). Amongst INH-resistant isolates, katG/Ser315Thr1 (n = 19, 95%) was the most frequent. Of 15 MDR-TB, the majority (n = 12, 80%) isolates had mutations at both rpoB/Ser531Leu and katG/Ser315Thr1. All 20 INH and/or RIF-resistant isolates were tested with the MTBDRsl VER 2.0, yielding 5 FLQs-resistant isolates with gene mutations at rpoB/Ser531Lue, katG/Ser315Thr1, and gyrA/Asp94Ala genes. Of 20 Spoligotyped drug-resistant MTB isolates, the majority (n = 11, 55%) and 6 (30%) were SIT149/T3-ETH and SIT21/CAS1-Kili sublineages, respectively; and they were any INH-resistant (mono-hetero/multi-). Of 15 RIF-resistant (RR/MDR-TB) isolates, 7 were SIT149/T3-ETH, while 6 were SIT21/CAS1-Kili sublineages. FLQ resistance was detected in four SIT21/CAS1-Kili lineages. Conclusion: In the current study, the most common gene mutations responsible for resistance to INH, RIF, and FLQs were identified. SIT149/T3-ETH and SIT21/CAS1-Kili constitute the majority of drug-resistant TB (DR-TB) isolates. To further understand the complete spectrum of genetic changes/mutations and related genotypes, a sequencing technology is warranted.

Potential acetylcholinesterase inhibitors to treat Alzheimer's disease.

OBJECTIVE: Alzheimer's disease (AD) is identified by neuropathological symptoms, and there is now no effective treatment for the condition. A lack of the brain neurotransmitter acetylcholine has been related to the etiology of Alzheimer's disease. Acetylcholinesterase is an enzyme that breaks down acetylcholine to an inactive form and causes the death of cholinergic neurons. Conventional treatments were used but had less effectiveness. Therefore, there is a crucial need to identify alternative compounds with potential anti-cholinesterase agents and minimal undesirable effects. MATERIALS AND METHODS: Fluoroquinolones and benzimidazole-benzothiazole derivatives offer antimicrobial, anti-inflammatory, anti-oxidant, anti-diabetic, and anti-Alzheimer activities. To enhance the chemical portfolio of cholinesterase inhibitors, a variety of fluoroquinolones and benzimidazole-benzothiazole compounds were evaluated against acetylcholinesterase (AChE) butyrylcholinesterase (BChE) enzymes. For this purpose, molecular docking and adsorption, distribution, metabolism, excretion, and toxicology ADMET models were used for in-silico studies for both AChE and BChE enzymes to investigate possible binding mechanisms and drug-likeness of the compounds. The inhibitory effect of docked heterocyclic compounds was also verified in vitro against AChE and BChE enzymes. Fluoroquinolones (Z, Z3, Z4, Z6, Z8, Z12, Z15, and Z9) and benzimidazole-benzothiazole compounds (TBIS-16, TBAF-1 to 9) passed through the AChE inhibition assay and their IC50 values were calculated. RESULTS: The compound 1-ethyl-6-fluoro-7-(4-(2-(4-nitrophenylamino)-2-oxoethyl)piperazin-1-yl) -4-oxo-1,4 di-hydroquinoline-3-carboxylic acid and 2-((1H-benzo[d]imidazol-2-yl)methyl)-N'-(3-bromobenzyl)-4-hydroxy-2H-thiochromene-3-carbohydrazide 1,1-dioxide (Z-9 and TBAF-6) showed the lowest IC50 values against AChE/BChE (0.37±0.02/2.93±0.03 µM and 0.638±0.001/1.31±0.01 µM, respectively) than the standard drug, donepezil (3.9±0.01/4.9±0.05 µM). During the in-vivo investigation, behavioral trials were performed to analyze the neuroprotective impact of Z-9 and TBAF-6 compounds on AD mouse models. The groups treated with Z-9 and TBAF-6 compounds had better cognitive behavior than the standard drug. CONCLUSIONS: This study found that Z-9 (Fluoroquinolones) and TBAF-6 (benzimidazole-benzothiazole) compounds improve behavioral and biochemical parameters, thus treating neurodegenerative disorders effectively.

Harnessing Graphene-Modified Electrode Sensitivity for Enhanced Ciprofloxacin Detection.

Increased evidence has documented a direct association between Ciprofloxacin (CFX) intake and significant disruption to the normal functions of connective tissues, leading to severe health conditions (such as tendonitis, tendon rupture and retinal detachment). Additionally, CFX is recognized as a potential emerging pollutant, as it seems to impact both animal and human food chains, resulting in severe health implications. Consequently, there is a compelling need for the precise, swift and selective detection of this fluoroquinolone-class antibiotic. Herein, we present a novel graphene-based electrochemical sensor designed for Ciprofloxacin (CFX) detection and discuss its practical utility. The graphene material was synthesized using a relatively straightforward and cost-effective approach involving the electrochemical exfoliation of graphite, through a pulsing current, in 0.05 M sodium sulphate (Na2SO4), 0.05 M boric acid (H3BO3) and 0.05 M sodium chloride (NaCl) solution. The resulting material underwent systematic characterization using scanning electron microscopy/energy dispersive X-ray analysis, X-ray powder diffraction and Raman spectroscopy. Subsequently, it was employed in the fabrication of modified glassy carbon surfaces (EGr/GC). Linear Sweep Voltammetry studies revealed that CFX experiences an irreversible oxidation process on the sensor surface at approximately 1.05 V. Under optimal conditions, the limit of quantification was found to be 0.33 × 10-8 M, with a corresponding limit of detection of 0.1 × 10-8 M. Additionally, the developed sensor's practical suitability was assessed using commercially available pharmaceutical products.

Target-Mediated Fluoroquinolone Resistance in Neisseria gonorrhoeae: Actions of Ciprofloxacin against Gyrase and Topoisomerase IV.

Fluoroquinolones make up a critically important class of antibacterials administered worldwide to treat human infections. However, their clinical utility has been curtailed by target-mediated resistance, which is caused by mutations in the fluoroquinolone targets, gyrase and topoisomerase IV. An important pathogen that has been affected by this resistance is Neisseria gonorrhoeae, the causative agent of gonorrhea. Over 82 million new cases of this sexually transmitted infection were reported globally in 2020. Despite the impact of fluoroquinolone resistance on gonorrhea treatment, little is known about the interactions of this drug class with its targets in this bacterium. Therefore, we investigated the effects of the fluoroquinolone ciprofloxacin on the catalytic and DNA cleavage activities of wild-type gyrase and topoisomerase IV and the corresponding enzymes that harbor mutations associated with cellular and clinical resistance to fluoroquinolones. Results indicate that ciprofloxacin interacts with both gyrase (its primary target) and topoisomerase IV (its secondary target) through a water-metal ion bridge that has been described in other species. Moreover, mutations in amino acid residues that anchor this bridge diminish the susceptibility of the enzymes for the drug, leading to fluoroquinolone resistance. Results further suggest that ciprofloxacin primarily induces its cytotoxic effects by enhancing gyrase-mediated DNA cleavage as opposed to inhibiting the DNA supercoiling activity of the enzyme. In conclusion, this work links the effects of ciprofloxacin on wild-type and resistant gyrase to results reported for cellular and clinical studies and provides a mechanistic explanation for the targeting and resistance of fluoroquinolones in N. gonorrhoeae.

Safety of fluoroquinolones / Seguridad de las fluoroquinolonas

Fluoroquinolones (FQs) are one of the most commonly prescribed classes of antibiotics. Although they were initially well tolerated in randomized clinical trials, subsequent epidemiological studies have reported an increased risk of threatening, severe, long-lasting, disabling and irreversible adverse effects (AEs), related to neurotoxicity and collagen degradation, such as tendonitis, Achilles tendon rupture, aortic aneurysm, and retinal detachment. This article reviews the main potentially threatening AEs, the alarms issued by regulatory agencies and therapeutic alternatives. (AU)

Las fluoroquinolonas son una de las clases de antibióticos más prescritas. Aunque inicialmente fueron bien toleradas en ensayos clínicos aleatorizados, estudios epidemiológicos posteriores han informado de un mayor riesgo de efectos adversos efectos adversos amenazantes, graves, duraderos, incapacitantes e irreversibles, relacionados con la neurotoxicidad y la degradación del colágeno, como tendinitis, rotura del tendón de Aquiles, aneurisma aórtico y desprendimiento de retina. Este artículo repasa los principales efectos adversos potencialmente amenazadores, las alarmas emitidas por las agencias reguladoras y las alternativas terapéuticas. (AU)

How Should We Activate Ferrate(VI)? Fe(IV) and Fe(V) Tell Different Stories about Fluoroquinolone Transformation and Toxicity Changes.

Many studies have investigated activation of ferrate (Fe(VI)) to produce reactive high-valent iron intermediates to enhance the oxidation of micropollutants. However, the differences in the risk of pollutant transformation caused by Fe(IV) and Fe(V) have not been taken seriously. In this study, Fe(VI)-alone, Fe3+/Fe(VI), and NaHCO3/Fe(VI) processes were used to oxidize fluoroquinolone antibiotics to explore the different effects of Fe(IV) and Fe(V) on product accumulation and toxicity changes. The contribution of Fe(IV) to levofloxacin degradation was 99.9% in the Fe3+/Fe(VI) process, and that of Fe(V) was 89.4% in the NaHCO3/Fe(VI) process. The cytotoxicity equivalents of levofloxacin decreased by 1.9 mg phenol/L in the Fe(IV)-dominant process while they significantly (p < 0.05) increased by 4.7 mg phenol/L in the Fe(V)-dominant process. The acute toxicity toward luminescent bacteria and the results for other fluoroquinolone antibiotics also showed that Fe(IV) reduced the toxicity and Fe(V) increased the toxicity. Density functional theory calculations showed that Fe(V) induced quinolone ring opening, which would increase the toxicity. Fe(IV) tended to oxidize the piperazine group, which reduced the toxicity. These results show the different-pollutant transformation caused by Fe(IV) and Fe(V). In future, the different risk outcomes during Fe(VI) activation should be taken seriously.

Mechanistic study of the electrochemical oxidation of fluoroquinolones: Ciprofloxacin, danofloxacin, enoxacin, levofloxacin and lomefloxacin.

The fluoroquinolones ciprofloxacin, danofloxacin, enoxacin, levofloxacin and lomefloxacin, occur in water bodies worldwide and therefore pose a threat to the aquatic environment. Advanced purification procedures, such as electrochemical oxidation, may act as a remedy since they contribute to eliminating contaminants and prevent micropollutants from entering open water bodies. By electrochemical treatment in a micro-flow reactor equipped with a boron-doped diamond (BDD) electrode, the fluoroquinolones were efficiently degraded. A total of 15 new products were identified using high-performance high-resolution chromatography coupled with high-resolution multifragmentation mass spectrometry. The ecotoxicity of the emerging transformation products was estimated through in silico quantitative structure activity relationship analysis. Almost all transformation products were predicted less ecotoxic than the initial compounds. The fluoroquinolone degradation followed three major mechanisms depending on the voltage during the electrochemical oxidation. At approximately 1 V, the reactions started with the elimination of molecular hydrogen from the piperazine moiety. At approx. 1.25 V, methyl and methylene groups were eliminated. At 1.5 V, hydroxyl radicals, generated at the BDD electrode, led to substitution at the piperazine ring. This novel finding of the three reactions depending on voltage contributes to the mechanistic understanding of electrochemical oxidation as potential remedy against fluoroquinolones in the aquatic environment.

Hydroxyl-containing triazine-based conjugated microporous polymers for solid phase extraction of fluoroquinolone antibiotics in the environment and food samples.

Fluoroquinolones (FQs) are a category of broadly used antibiotics. Development of an effective and sensitive approach for determination of trace FQs in environmental and food samples is still challenging. Herein, the hydroxyl-containing triazine-based conjugated microporous polymers (CMPs-OH) was constructed and served as SPE absorbent for the efficient enrichment of FQs. Based on DFT simulations, the excellent enrichment capacity between CMPs-OH and FQs was contributed by hydrogen bonding and π-π interactions. In combination with high-performance liquid chromatography-tandem mass spectrometry, the proposed approach exhibited a wide linear range (0.2-400 ng L-1), low detection limits (0.05-0.15 ng L-1), and good intraday and interday precisions under optimal conditions. In addition, the established method was effectively utilized for the determination of FQs in fourteen samples with recoveries between 82.6 % and 109.2 %. This work provided a feasible sample pretreatment method for monitoring FQs in environmental and food matrices.

Host-Guest Doping Modulated Afterglow Emission of Fluoroquinolones for Their Separation-Free Detection and Discrimination.

Detection and discrimination of fluoroquinolones (FQs) are crucial for food safety but remain a formidable challenge due to their minor differences in molecular structures and the serious interferences from food matrices. Herein, we propose an afterglow assay for the detection and discrimination of FQs through modulating their room-temperature phosphorescence (RTP) and thermally activated delayed fluorescence (TADF) properties by a host-guest doping strategy. FQs were doped into the boric acid host, forming boronic anhydride structures and hydrogen bonds, which prompted the RTP and TADF performance of FQs by stabilizing their excited states, preventing triplet exciton quenching, and reducing the energy gap between singlet and triplet states. The FQs can be quantitatively detected through monitoring the afterglow intensity of host-guest systems, as low as 0.25 µg/mL. The differences in the afterglow intensity and emission lifetime allowed accurate discrimination of 11 types of FQs through pattern recognition methods. Aided by the delayed signal detection model of afterglow emission, the background signal and the interferences from food matrices were effectively eliminated, which endow the detection and discrimination of mixed FQs in commercial meat samples, without multiple-step separation processes.

On-site extraction using a 3D printed device coated with Zn/Co-ZIF-derived carbon followed by an on-line SIA-HPLC-FL system for fluoroquinolones determination in wastewater.

A 3D printed device covered with Zn/Co-ZIF-derived carbon allows the on-site extraction of fluoroquinolones (FQs) from wastewater, avoiding the sample transportation to the laboratory, and the subsequent elution, separation and determination using an on-line flow system based on sequential injection analysis (SIA) coupled to HPLC-FL. Several parameters that affect the extraction efficiency and desorption were optimized including the sorption phase immobilization technique on the 3D device, extraction time, pH effect, sample volume as well as the type of eluent, eluent volume, and flow rate. Under optimum conditions, detection limits of 3-9 ng L-1 were achieved for norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin and difloxacin. The precision expressed as relative standard deviation (%RSD, n = 3), showed intraday and interday ranges of 1.5-5.3% and 2.8-5.7%, respectively, demonstrating a good precision of the proposed methodology. To assess matrix effects and accuracy of the proposed method in real samples, recovery studies were performed without and with FQs spiked at different concentrations (0.5-10 µg L-1) to wastewater samples, showing good recoveries in the range of 91-104%. The results allow to confirm the applicability of MOF-derived carbons as adsorbents for on-site extraction, and the satisfactory separation and quantification of FQs by a SIA-HPLC-FL on-line system after their desorption with small eluent volumes.

Longitudinal antimicrobial susceptibility trends of canine Staphylococcus pseudintermedius.

Antimicrobial resistance within Staphylococcus pseudintermedius poses a significant risk for the treatment of canine pyoderma and as a reservoir for resistance and potential zoonoses, but few studies examine long-term temporal trends of resistance. This study assesses the antimicrobial resistance prevalence and minimum inhibitory concentration (MIC) trends in S. pseudintermedius (n=1804) isolated from canine skin samples at the Cornell University Animal Health Diagnostic Center (AHDC) between 2007 and 2020. Not susceptible (NS) prevalence, Cochran-Armitage tests, logrank tests, MIC50 and MIC90 quantiles, and survival analysis models were used to evaluate resistance prevalence and temporal trends to 23 antimicrobials. We use splines as predictors in accelerated failure time (AFT) models to model non-linear temporal trends in MICs. Multidrug resistance was common among isolates (47%), and isolates had moderate to high NS prevalence to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, the macrolides/lincosamides, the tetracyclines, and trimethoprim-sulfamethoxazole. However, low levels of NS to amikacin, rifampin, and vancomycin were observed. Around one third of isolates (38%) were found to be methicillin resistant S. pseudintermedius (MRSP), and these isolates had a higher prevalence of NS to all tested antimicrobials than methicillin susceptible isolates. Amongst the MRSP isolates, one phenotypically vancomycin resistant isolate (MIC >16 µg/mL) was identified, but genomic sequence data was unavailable. AFT models showed increasing MICs across time to the beta-lactams, chloramphenicol, the fluoroquinolones, gentamicin, and the macrolides/lincosamides, and decreasing temporal resistance (decreasing MICs) to doxycycline was observed amongst isolates. Notably, ATF modeling showed changes in MIC distributions that were not identified using Cochran-Armitage tests on prevalence, MIC quantiles, and logrank tests. Increasing resistance amongst these S. pseudintermedius isolates highlights the need for rational, empirical prescribing practices and increased antimicrobial resistance (AMR) surveillance to maintain the efficacy of current therapeutic agents. AFT models with non-linear predictors may be a useful, breakpoint-independent, surveillance tool alongside other modeling methods and antibiograms.

The effects of antibiotic exposures on the gut resistome during hematopoietic cell transplantation in children.

Antibiotic resistance is a global threat driven primarily by antibiotic use. We evaluated the effects of antibiotic exposures on the gut microbiomes and resistomes of children at high risk of colonization by antibiotic-resistant bacteria. We performed shotgun metagenomic sequencing of 691 serially collected fecal samples from 80 children (<18 years) undergoing hematopoietic cell transplantation. We evaluated the effects of aerobic (cefepime, vancomycin, fluoroquinolones, aminoglycosides, macrolides, and trimethoprim-sulfamethoxazole) and anaerobic (piperacillin-tazobactam, carbapenems, metronidazole, and clindamycin) antibiotic exposures on the diversity and composition of the gut microbiome and resistome. We identified 372 unique antibiotic resistance genes (ARGs); the most frequent ARGs identified encode resistance to tetracyclines (n = 88), beta-lactams (n = 84), and fluoroquinolones (n = 79). Both aerobic and anaerobic antibiotic exposures were associated with a decrease in the number of bacterial species (aerobic, ß = 0.71, 95% CI: 0.64, 0.79; anaerobic, ß = 0.66, 95% CI: 0.53, 0.82) and the number of unique ARGs (aerobic, ß = 0.81, 95% CI: 0.74, 0.90; anaerobic, ß = 0.73, 95% CI: 0.61, 0.88) within the gut metagenome. However, only antibiotic regimens that included anaerobic activity were associated with an increase in acquisition of new ARGs (anaerobic, ß = 1.50; 95% CI: 1.12, 2.01) and an increase in the relative abundance of ARGs in the gut resistome (anaerobic, ß = 1.62; 95% CI: 1.15, 2.27). Specific antibiotic exposures were associated with distinct changes in the number and abundance of ARGs for individual antibiotic classes. Our findings detail the impact of antibiotics on the gut microbiome and resistome and demonstrate that anaerobic antibiotics are particularly likely to promote acquisition and expansion of antibiotic-resistant bacteria.

The intracellular concentrations of fluoroquinolones determined the antibiotic resistance response of Escherichia coli.

The extensive consumption of antibiotics has been reported to significantly promote the generation of antibiotic resistance (ABR), however, a quantitative causal relationship between antibiotic exposure and ABR response is absent. This study aimed to pinpoint the accurate regulatory concentration of fluoroquinolones (FQs) and to understand the biochemical mechanism of the mutual action between FQ exposure and FQ resistance response. Highly sensitive analytical methods were developed by using UPLC-MS/MS to determine the total residual, extracellular residual, total intracellular, intracellular residual and intracellular degraded concentration of three representative FQs, including ciprofloxacin (CIP), ofloxacin (OFL) and norfloxacin (NOR), with detection limits in the range of 0.002-0.057 µg/L, and recoveries in the range of 80-93%. The MICs of Escherichia coli (E. coli) were 7.0-31.4-fold of the respective MIC0 after 40-day FQ exposure, and significant negative associations were discovered between the intracellular (residual, degraded or the sum) FQ concentrations and FQ resistance. Transcriptional expression and whole-genome sequencing results indicated that reduced membrane permeability and enhanced multi-drug efflux pumps contributed to the decreasing intracellular concentration. These results unveiled the pivotal role of intracellular concentration in triggering FQ resistance, providing important information to understand the dose-response relationship between FQ exposure and FQ resistance response, and ascertain the target dose metric of FQs for eliminating FQ resistance crisis.

Two synonymous single-nucleotide polymorphisms promoting fluoroquinolone resistance of Escherichia coli in the environment.

Single-nucleotide polymorphism (SNP) is one of the core mechanisms that respond to antibiotic resistance of Escherichia coli (E. coli), which is a major issue in environmental pollution. A specific type of SNPs, synonymous SNPs, have been generally considered as the "silent" SNPs since they do not change the encoded amino acid. However, the impact of synonymous SNPs on mRNA splicing, nucleo-cytoplasmic export, stability, and translation was gradually discovered in the last decades. Figuring out the mechanism of synonymous SNPs in regulating antibiotic resistance is critical to improve antimicrobial therapy strategies in clinics and biological treatment strategies of antibiotic-resistant E. coli-polluted materials. With our newly designed antibiotic resistant SNPs prediction algorithm, Multilocus Sequence Type based Identification for Phenotype-single nucleotide polymorphism Analysis (MIPHA), and in vivo validation, we identified 2 important synonymous SNPs 522 G>A and 972 C>T, located at hisD gene, which was previously predicted as a fluoroquinolone resistance-related gene without a detailed mechanism in the E. coli samples with environmental backgrounds. We first discovered that hisD causes gyrA mutation via the upregulation of sbmC and its downstream gene umuD. Moreover, those 2 synonymous SNPs of hisD cause its own translational slowdown and further reduce the expression levels of sbmC and its downstream gene umuD, making the fluoroquinolone resistance determining region of gyrA remains unmutated, ultimately causing the bacteria to lose their ability to resist drugs. This study provided valuable insight into the role of synonymous SNPs in mediating antibiotic resistance of bacteria and a new perspective for the treatment of environmental pollution caused by drug-resistant bacteria.

Virtual clinical QT exposure-response studies - A translational computational approach.

BACKGROUND AND PURPOSE: A recent paradigm shift in proarrhythmic risk assessment suggests that the integration of clinical, non-clinical, and computational evidence can be used to reach a comprehensive understanding of the proarrhythmic potential of drug candidates. While current computational methodologies focus on predicting the incidence of proarrhythmic events after drug administration, the objective of this study is to predict concentration-response relationships of QTc as a clinical endpoint. EXPERIMENTAL APPROACH: Full heart computational models reproducing human cardiac populations were created to predict the concentration-response relationship of changes in the QT interval as recommended for clinical trials. The concentration-response relationship of the QT-interval prolongation obtained from the computational cardiac population was compared against the relationship from clinical trial data for a set of well-characterized compounds: moxifloxacin, dofetilide, verapamil, and ondansetron. KEY RESULTS: Computationally derived concentration-response relationships of QT interval changes for three of the four drugs had slopes within the confidence interval of clinical trials (dofetilide, moxifloxacin and verapamil) when compared to placebo-corrected concentration-ΔQT and concentration-ΔQT regressions. Moxifloxacin showed a higher intercept, outside the confidence interval of the clinical data, demonstrating that in this example, the standard linear regression does not appropriately capture the concentration-response results at very low concentrations. The concentrations corresponding to a mean QTc prolongation of 10 ms were consistently lower in the computational model than in clinical data. The critical concentration varied within an approximate ratio of 0.5 (moxifloxacin and ondansetron) and 1 times (dofetilide, verapamil) the critical concentration observed in human clinical trials. Notably, no other in silico methodology can approximate the human critical concentration values for a QT interval prolongation of 10 ms. CONCLUSION AND IMPLICATIONS: Computational concentration-response modelling of a virtual population of high-resolution, 3-dimensional cardiac models can provide comparable information to clinical data and could be used to complement pre-clinical and clinical safety packages. It provides access to an unlimited exposure range to support trial design and can improve the understanding of pre-clinical-clinical translation.