Comparative genomics of quinolone-resistant Escherichia coli from broilers and humans in Norway.

BACKGROUND: The usage of fluoroquinolones in Norwegian livestock production is very low, including in broiler production. Historically, quinolone-resistant Escherichia coli (QREC) isolated from Norwegian production animals rarely occur. However, with the introduction of a selective screening method for QREC in the Norwegian monitoring programme for antimicrobial resistance in the veterinary sector in 2014; 89.5% of broiler caecal samples and 70.7% of broiler meat samples were positive. This triggered the concern if there could be possible links between broiler and human reservoirs of QREC. We are addressing this by characterizing genomes of QREC from humans (healthy carriers and patients) and broiler isolates (meat and caecum). RESULTS: The most frequent mechanism for quinolone resistance in both broiler and human E. coli isolates were mutations in the chromosomally located gyrA and parC genes, although plasmid mediated quinolone resistance (PMQR) was also identified. There was some relatedness of the isolates within human and broiler groups, but little between these two groups. Further, some overlap was seen for isolates with the same sequence type isolated from broiler and humans, but overall, the SNP distance was high. CONCLUSION: Based on data from this study, QREC from broiler makes a limited contribution to the incidence of QREC in humans in Norway.

Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates.

BACKGROUND: The emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing. RESULTS: Out of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6')-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene. CONCLUSIONS: Our results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

Management of Neisseria gonorrhoeae infection: from drug resistance to drug repurposing.

INTRODUCTION: Neisseria gonorrhoeae is a common sexually transmitted disease connected with extensive drug resistance to many antibiotics. Presently, only expanded spectrum cephalosporins (ceftriaxone and cefixime) and azithromycin remain useful for its management. AREAS COVERED: New chemotypes for the classical antibiotic drug target gyrase/topoisomerase IV afforded inhibitors with potent binding to these enzymes, with an inhibition mechanism distinct from that of fluoroquinolones, and thus less prone to mutations. The α-carbonic anhydrase from the genome of this bacterium (NgCAα) was also validated as an antibacterial target. EXPERT OPINION: By exploiting different subunits from the gyrase/topoisomerase IV as well as new chemotypes, two new antibiotics reached Phase II/III clinical trials, zoliflodacin and gepotidacin. They possess a novel inhibition mechanism, binding in distinct parts of the enzyme compared to the fluoroquinolones. Other chemotypes with inhibitory activity in these enzymes were also reported. NgCAα inhibitors belonging to a variety of classes were obtained, with several sulfonamides showing MIC values in the range of 0.25-4 µg/mL and significant activity in animal models of this infection. Acetazolamide and similar CA inhibitors might thus be repurposed as antiinfectives. The scientific/patent literature has been searched for on PubMed, ScienceDirect, Espacenet, and PatentGuru, from 2016 to 2024.

Comparative genomic analysis of Brazilian Mesomycoplasma ovipneumoniae strains revealed genomic differences associated with the geographic origin and health status and mutations in the gyrA.

Sheep respiratory disease (SRD) is a multifactorial illness commonly affecting sheep. Mesomycoplasma (Mycoplasma) ovipneumoniae is one of the most important etiological agents of SRD and should be better understood, especially in countries where it was recently detected, such as Brazil. Also, the intensive use of quinolones in mycoplasmal infections increases the selective pressure for resistance to this drug class, and no data about antimicrobial resistance in Brazil is available. Therefore, this study aimed to perform a comparative genomic analysis of newly isolated Brazilian M. ovipneumoniae strains, identify point mutations in target genes that may be associated with antibiotic resistance, and perform a phylogenomic analysis of these strains with available genome representatives of M. ovipneumoniae. Glucose-fermenting fried egg-like colonies identified as M. ovipneumoniae were obtained after a culture of tracheobronchial lavage from infected sheep. The genomes were sequenced, de novo assembled and comparatively evaluated. Important putative virulence factors were detected in all isolates: the analysis of the average nucleotide homology of all these genes with the M. ovipneumoniae ATCC 29419 revealed associations between clpB, lgt, tuf, and dnaJ genes and geographic location. In addition, nucleotide substitutions in a few positions of the Quinolone-Resistant Determinant Region of the gyrA gene, including the Ser83Ala, were detected. The phylogenomic analysis showed that the Brazilian isolates belonged to two different clades corresponding to geographic location, and the isolates from São Paulo showed high similarity, which differs from isolates from Rio de Janeiro. This first genomic analysis of the Brazilian M. ovipneumoniae genomes demonstrates strain segregation according to location and health status, reinforcing the importance of continuous surveillance and diagnostics of this bacteria causing sheep respiratory disease in the Brazilian flocks.

Exploring the interaction of N-(benzothiazol-2-yl)pyrrolamide DNA gyrase inhibitors with the GyrB ATP-binding site lipophilic floor: A medicinal chemistry and QTAIM study.

N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.

Rapid, DNA-induced interface swapping by DNA gyrase.

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (-1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface 'swapping' (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed 'swivelling' mechanism for DNA gyrase (Gubaev et al., 2016).

Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

Estimation of passive gastrointestinal absorption of new dual DNA gyrase and topoisomerase IV inhibitors using PAMPA and biopartitioning micellar chromatography and quantitative structure-retention relationship analysis.

DNA gyrase and topoisomerase IV play significant role in maintaining the correct structure of DNA during replication and they have been identified as validated targets in antibacterial drug discovery. Inadequate pharmacokinetic properties are responsible for many failures during drug discovery and their estimation in the early phase of this process maximizes the chance of getting useful drug candidates. Passive gastrointestinal absorption of a selected group of thirteen dual DNA gyrase and topoisomerase IV inhibitors was estimated using two in vitro tests - parallel artificial membrane permeability assay (PAMPA) and biopartitioning micellar chromatography (BMC). Due to good correlation between obtained results, passive gastrointestinal absorption of remaining ten compounds was estimated using only BMC. With this experimental setup, it was possible to identify compounds with high values of retention factors (k) and highest expected passive gastrointestinal absorption, and compounds with low values of k for which low passive gastrointestinal absorption is predicted. Quantitative structure-retention relationship (QSRR) modelling was performed by creating multiple linear regression (MLR), partial least squares (PLS) and support vector machines (SVM) models. Descriptors with the highest influence on retention factor were identified and their interpretation can be used for the design of new compounds with improved passive gastrointestinal absorption.

The unusual mode of action of the polyketide glycoside antibiotic cervimycin C.

Cervimycins A-D are bis-glycosylated polyketide antibiotics produced by Streptomyces tendae HKI 0179 with bactericidal activity against Gram-positive bacteria. In this study, cervimycin C (CmC) treatment caused a spaghetti-like phenotype in Bacillus subtilis 168, with elongated curved cells, which stayed joined after cell division, and exhibited a chromosome segregation defect, resulting in ghost cells without DNA. Electron microscopy of CmC-treated Staphylococcus aureus (3 × MIC) revealed swollen cells, misshapen septa, cell wall thickening, and a rough cell wall surface. Incorporation tests in B. subtilis indicated an effect on DNA biosynthesis at high cervimycin concentrations. Indeed, artificial downregulation of the DNA gyrase subunit B gene (gyrB) increased the activity of cervimycin in agar diffusion tests, and, in high concentrations (starting at 62.5 × MIC), the antibiotic inhibited S. aureus DNA gyrase supercoiling activity in vitro. To obtain a more global view on the mode of action of CmC, transcriptomics and proteomics of cervimycin treated versus untreated S. aureus cells were performed. Interestingly, 3 × MIC of cervimycin did not induce characteristic responses, which would indicate disturbance of the DNA gyrase activity in vivo. Instead, cervimycin induced the expression of the CtsR/HrcA heat shock operon and the expression of autolysins, exhibiting similarity to the ribosome-targeting antibiotic gentamicin. In summary, we identified the DNA gyrase as a target, but at low concentrations, electron microscopy and omics data revealed a more complex mode of action of cervimycin, which comprised induction of the heat shock response, indicating protein stress in the cell.IMPORTANCEAntibiotic resistance of Gram-positive bacteria is an emerging problem in modern medicine, and new antibiotics with novel modes of action are urgently needed. Secondary metabolites from Streptomyces species are an important source of antibiotics, like the cervimycin complex produced by Streptomyces tendae HKI 0179. The phenotypic response of Bacillus subtilis and Staphylococcus aureus toward cervimycin C indicated a chromosome segregation and septum formation defect. This effect was at first attributed to an interaction between cervimycin C and the DNA gyrase. However, omics data of cervimycin treated versus untreated S. aureus cells indicated a different mode of action, because the stress response did not include the SOS response but resembled the response toward antibiotics that induce mistranslation or premature chain termination and cause protein stress. In summary, these results point toward a possibly novel mechanism that generates protein stress in the cells and subsequently leads to defects in cell and chromosome segregation.

Chemical Composition, Anti-bacterial Activity and Molecular Docking Studies of Essential Oil Isolated from Sa Sam Nam (Launaea sarmentosa).

Launaea sarmentosa, also known as Sa Sam Nam, is a widely used remedy in Vietnamese traditional medicine and cuisine. However, the chemical composition and bioactivity of its essential oil have not been elucidated yet. In this study, we identified 40 compounds (98.6% of total peak area) in the essential oil via GC-MS analysis at the first time. Among them, five main compounds including Thymohydroquinone dimethyl ether (52.4%), (E)-α-Atlantone (9.0%), Neryl isovalerate (6.6%), Davanol D2 (isomer 2) (3.9%), and trans-Sesquisabinene hydrate (3.9%) have accounted for 75.8% of total peak area. The anti-bacterial activity of the essential oil against 4 microorganisms including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa has also investigated via agar well diffusion assay. The results showed that the essential oil exhibited a strong antibacterial activity against Bacillus subtilis with the inhibition zones ranging from 8.2 to 18.7 mm. To elucidate the anti-bacterial effect mechanism of the essential oil, docking study of five main compounds of the essential oil (Thymohydroquinone dimethyl ether, (E)-α-Atlantone, Neryl isovalerate, Davanol D2 (isomer 2), and trans-Sesquisabinene hydrate) against some key proteins for bacterial growth such as DNA gyrase B, penicillin binding protein 2A, tyrosyl-tRNA synthetase, and dihydrofolate reductase were performed. The results showed that the main constituents of essential oil were highly bound with penicillin binding protein 2A with the free energies ranging -27.7 to -44.8 kcal/mol, which suggests the relationship between the antibacterial effect of essential oil and the affinity of main compounds with penicillin binding protein. In addition, the free energies of main compounds of the essential oil with human cyclooxygenase 1, cyclooxygenase 2, and phospholipase A2, the crucial proteins related with inflammatory response were less than diclofenac, a non-steroidal antiinflammatory drug. These findings propose the essential oil as a novel and promising anti-bacterial and anti-inflammatory medicine or cosmetic products.

Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

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.

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.

Construction and Docking Studies of Novel Pyrimido[4,5-b]quinolines as Antimicrobial Agents.

In order to develop novel antimicrobial agents, we prepared quinoline bearing pyrimidine analogues 2-7, 8 a-d and 9 a-d and their structures were elucidated by spectroscopic techniques. Furthermore, our second aim was to predict the interactions between the active compounds and enzymes (DNA gyrase and DHFR). In this work, fourteen pyrimido[4,5-b]quinoline derivatives were prepared and assessed for their antimicrobial potential by estimating zone of inhibition. All the screened candidates displayed antibacterial potential with zone of inhibition range of 9-24 mm compared with ampicillin (20-25 mm) as a reference drug. Moreover, the target derivatives 2 (ZI=16), 9 c (ZI=17 mm) and 9 d (ZI=16 mm) recorded higher antifungal activity against C. albicans to that exhibited by the antifungal drug amphotericin B (ZI=15 mm). Finally, the most potent pyrimidoquinoline compounds (2, 3, 8 c, 8 d, 9 c and 9 d) were docked inside DHFR and DNA gyrase active sites and they recorded excellent fitting within the active regions of DNA gyrase and DHFR. These outcomes revealed us that compounds (2, 3, 8 c, 8 d, 9 c and 9 d) could be lead compounds to discover novel antibacterial candidates.

Interactions between Gepotidacin and Escherichia coli Gyrase and Topoisomerase IV: Genetic and Biochemical Evidence for Well-Balanced Dual-Targeting.

Antimicrobial resistance is a global threat to human health. Therefore, efforts have been made to develop new antibacterial agents that address this critical medical issue. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial in clinical development. Recently, phase III clinical trials for gepotidacin treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, were stopped for demonstrated efficacy. Because of the clinical promise of gepotidacin, it is important to understand how the compound interacts with its cellular targets, gyrase and topoisomerase IV, from E. coli. Consequently, we determined how gyrase and topoisomerase IV mutations in amino acid residues that are involved in gepotidacin interactions affect the susceptibility of E. coli cells to the compound and characterized the effects of gepotidacin on the activities of purified wild-type and mutant gyrase and topoisomerase IV. Gepotidacin displayed well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli cells, which was reflected in a similar inhibition of the catalytic activities of these enzymes by the compound. Gepotidacin induced gyrase/topoisomerase IV-mediated single-stranded, but not double-stranded, DNA breaks. Mutations in GyrA and ParC amino acid residues that interact with gepotidacin altered the activity of the compound against the enzymes and, when present in both gyrase and topoisomerase IV, reduced the antibacterial activity of gepotidacin against this mutant strain. Our studies provide insights regarding the well-balanced dual-targeting of gyrase and topoisomerase IV by gepotidacin in E. coli.

Proposal of Helicobacter higonensis sp. nov. isolated from a human clinical specimen, and emended description of Helicobacter valdiviensis Collado, 2014.

We have previously isolated a gram-negative microaerophilic strain, PAGU2000T from a patient presenting with a fever in Kumamoto Prefecture, Japan. The present study aimed to comprehensively analyze the taxonomy of the isolated strain using a polyphasic approach. The 16S rRNA gene sequence analysis indicated that the strain was a member of enterohepatic Helicobacter. The strain PAGU2000T shared a 97.5% 16S rRNA gene nucleotide identity with Helicobacter valdiviensis, and this taxonomic position was confirmed by phylogenetic analysis of the GyrA amino acid sequences. The proposed strain PAGU2000T has a 1.482 Mbp chromosome with a DNA G + C content of 31.3 mol% and encodes 1520 predicted coding sequences. The average nucleotide identity between the strain PAGU2000T and type strain of H. valdiviensis was 70.3%, which was lower than the recommended threshold of 95% for species delineation. The strain PAGU2000T was a motile, non-spore-forming, and spiral-shaped bacterium, exhibiting catalase and oxidase activities but not urease and nitrate reduction. This study demonstrates that the isolate represents a novel species within enterohepatic Helicobacter, for which the name Helicobacter higonensis is proposed (type strain: PAGU2000T = GTC 16811T = LMG 33095T). In this study, we describe the phenotypic and morphological features of this strain and propose an emended description of some biochemical traits of H. valdiviensis.

Identification of novel resistance-associated mutations and discrimination within whole-genome sequences of fluoroquinolone-resistant Mycobacterium tuberculosis isolates.

This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and allele mutation frequencies in identifying FQ-resistant Mycobacterium tuberculosis (MTB) isolates. A random selection of isolates was extracted from an ongoing collection. Drug resistance was determined using the resazurin microtiter assay (REMA) as the gold standard. Mutation loci and the burden of mutations in the quinolone resistance-determining region (QRDR) were elucidated through whole-genome sequencing (WGS). Novel amino acid mutations, namely, G520D and G520T, were identified in the gyrB and associated with FQ resistance. In the context of distinguishing FQ-resistant isolates, the AUC for the QRDR mutation frequency burden (0.969) surpassed that of the mutation locus (0.929), and this difference was statistically significant (P = 0.03). Furthermore, using the resistance mutation locus as a reference, setting the QRDR mutation frequency burden threshold at 1.31% resulted in a 3.60% increase in the accuracy of classifying FQ-resistant isolates (NRI = 3.60%, P < 0.001). The QRDR mutation frequency burden appears to offer superior diagnostic efficacy in discriminating FQ-resistant isolates compared to qualitative detection of mutant loci.IMPORTANCEFluoroquinolone (FQ) drugs are recommended as second-line drugs for the treatment of multidrug-resistant tuberculosis. With the massive use of FQ drugs in the clinical treatment of tuberculosis (TB), there is an increasing rate of drug resistance to FQ drugs. In this study, we identified and demonstrated novel amino acid mutations associated with FQ resistance in Mycobacterium tuberculosis (MTB), and we quantified the mutation sites and identified the quinolone resistance-determining region (QRDR) mutation frequency burden as a novel diagnostic method for FQ resistance. We hope that the results of this study will provide data support and a theoretical basis for the rapid diagnosis of FQ-resistant MTB.

Evaluating the potency of zoliflodacin against Helicobacter pylori: In vitro activity and conserved GyrB target.

BACKGROUND: The current standard treatment for Helicobacter pylori infection, which involves a combination of two broad-spectrum antibiotics, faces significant challenges due to its detrimental impact on the gut microbiota and the emergence of drug-resistant strains. This underscores the urgent requirement for the development of novel anti-H. pylori drugs. Zoliflodacin, a novel bacterial gyrase inhibitor, is currently undergoing global phase III clinical trials for treating uncomplicated Neisseria gonorrhoeae. However, there is no available data regarding its activity against H. pylori. MATERIALS AND METHODS: We evaluated the in vitro activity of zoliflodacin against H. pylori clinical isolates (n = 123) with diverse multidrug resistance. We performed DNA gyrase supercoiling and microscale thermophoresis assays to identify the target of zoliflodacin in H. pylori. We analyzed 2262 H. pylori whole genome sequences to identify Asp424Asn and Lys445Asn mutations in DNA gyrase subunit B (GyrB) that are associated with zoliflodacin resistance. RESULTS: Zoliflodacin exhibits potent activity against all tested isolates, with minimal inhibitory concentration (MIC) values ranging from 0.008 to 1 µg/mL (MIC50: 0.125 µg/mL; MIC90: 0.25 µg/mL). Importantly, there was no evidence of cross-resistance to any of the four first-line antibiotics commonly used against H. pylori. We identified GyrB as the primary target of zoliflodacin, with Asp424Asn or Lys445Asn substitutions conferring resistance. Screening of 2262 available H. pylori genomes for the two mutations revealed only one clinical isolate carrying Asp424Asn substitution. CONCLUSION: These findings support the potential of zoliflodacin as a promising candidate for H. pylori treatment, warranting further development and evaluation.

Novel Thiazole Derivatives Containing Imidazole and Furan Scaffold: Design, Synthesis, Molecular Docking, Antibacterial, and Antioxidant Evaluation.

Carbothioamides 3a,b were generated in high yield by reacting furan imidazolyl ketone 1 with N-arylthiosemicarbazide in EtOH with a catalytic amount of conc. HCl. The reaction of carbothioamides 3a,b with hydrazonyl chlorides 4a-c in EtOH with triethylamine at reflux produced 1,3-thiazole derivatives 6a-f. In a different approach, the 1,3-thiazole derivatives 6b and 6e were produced by reacting 3a and 3b with chloroacetone to afford 8a and 8b, respectively, followed by diazotization with 4-methylbenzenediazonium chloride. The thiourea derivatives 3a and 3b then reacted with ethyl chloroacetate in ethanol with AcONa at reflux to give the thiazolidinone derivatives 10a and 10b. The produced compounds were tested for antioxidant and antibacterial properties. Using phosphomolybdate, promising thiazoles 3a and 6a showed the best antioxidant activities at 1962.48 and 2007.67 µgAAE/g dry samples, respectively. Thiazoles 3a and 8a had the highest antibacterial activity against S. aureus and E. coli with 28, 25 and 27, 28 mm, respectively. Thiazoles 3a and 6d had the best activity against C. albicans with 26 mm and 37 mm, respectively. Thiazole 6c had the highest activity against A. niger, surpassing cyclohexamide. Most compounds demonstrated lower MIC values than neomycin against E. coli, S. aureus and C. albicans. A molecular docking study examined how antimicrobial compounds interact with DNA gyrase B crystal structures. The study found that all of the compounds had good binding energy to the enzymes and reacted similarly to the native inhibitor with the target DNA gyrase B enzymes' key amino acids.

A platform for predicting mechanism of action based on bacterial transcriptional responses identifies an unusual DNA gyrase inhibitor.

In the search for much-needed new antibacterial chemical matter, a myriad of compounds have been reported in academic and pharmaceutical screening endeavors. Only a small fraction of these, however, are characterized with respect to mechanism of action (MOA). Here, we describe a pipeline that categorizes transcriptional responses to antibiotics and provides hypotheses for MOA. 3D-printed imaging hardware PFIboxes) profiles responses of Escherichia coli promoter-GFP fusions to more than 100 antibiotics. Notably, metergoline, a semi-synthetic ergot alkaloid, mimics a DNA replication inhibitor. In vitro supercoiling assays confirm this prediction, and a potent analog thereof (MLEB-1934) inhibits growth at 0.25 µg/mL and is highly active against quinolone-resistant strains of methicillin-resistant Staphylococcus aureus. Spontaneous suppressor mutants map to a seldom explored allosteric binding pocket, suggesting a mechanism distinct from DNA gyrase inhibitors used in the clinic. In all, the work highlights the potential of this platform to rapidly assess MOA of new antibacterial compounds.