Gyrase Subunit Research Articles

Greener and Highly Efficient Synthesis, In Silico ADMET and Molecular Docking Studies of Potent Antimicrobial Thiophene Clubbed Pyrazole‐1,2,3‐Triazole Hybrids

AbstractTo achieve environmentally benign synthesis and develop pharmacologically active compounds, a library of novel thiophene‐clubbed pyrazole‐1,2,3‐triazole hybrids was designed and successfully synthesized using L‐ascorbic acid as a green acid catalyst and ethanol as a green solvent. The reaction was carried out using microwave and ultrasonic irradiation methods to achieve a highly efficient reaction route. All title compounds were characterized and evaluated for their potential in vitro antimicrobial activity using Ciprofloxacin and Nystatin as standard drugs, in which compounds with chloro and naphthyl substitutions exhibited excellent antibacterial activity at concentrations of 25 μg/mL and compounds with methyl and methoxy substitutions exhibited excellent antifungal activity at concentrations of 100 μg/mL. Additionally, the molecular docking study showed that all compounds examined had excellent binding energies ranging from −9.3 to −10.3 kcal/mol, and naphthyl substitution displayed the highest binding energy (−10.3 kcal/mol) on the target protein of E. coli DNA gyrase subunit B. Furthermore, the final moieties were evaluated for in silico ADMET prediction to examine their drug‐likeness profile and toxic effects. The present study revealed that this structural information will be helpful in future antimicrobial drug design and development.

Phytochemical profiling, in vitro biological activities, and in silico (molecular docking and absorption, distribution, metabolism, excretion, toxicity) studies of Polygonum cognatum Meissn.

Polygonum cognatum Meissn, a perennial herbaceous belonging to the Polygonaceae family, is an aromatic plant. High-performance liquid chromatography/diode array detector method was developed and validated for the phytochemical analysis of the plant. Also, various methods were used to investigate the antioxidant, antimicrobial, and cytotoxic activities of the methanolic extracts. Antioxidant activities were researched by 2,2'-diphenyl-1-picrylhydrazyl and cupric reducing antioxidant capacity methods. Among the tested standard microbial strains, Candida albicans was found to be more sensitive with a 24.60±0.55mm inhibition zone according to the diffusion tests. In the microdilution tests, the minimum inhibitory concentration and minimum bactericidal/fungicidal concentration values were 4.75 and ≥ 4.75mg/mL, respectively, for all tested pathogens. Human colon carcinoma cells were used to investigate cytotoxicity by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromideanalysis (IC50 =2891μg/mL for Plant A, IC50 =3291μg/mL for Plant B). Molecular docking and absorption, distribution, metabolism, excretion, and toxicity analysis were used to explain inhibition mechanisms of major phenolic compounds of plants against Tankyrase 1, Tankyrase 2 enzymes, and deoxyribonucleic acid gyrase subunit B and found compatible with experimental results.

In silico Study on Structural Inhibition of Bacterial DNA Gyrase by Major Secondary Metabolites Found in Grape Seed Extract

In the early 20th century, during the era of investigating and identifying essential “vitamins”, scientific research focused on grape seed extracts and their bioactive components, particularly polyphenols. Extensive studies have demonstrated that grape seed extract, rich in proanthocyanidins, offers protection against a wide spectrum of diseases, encompassing inflammation, cardiac ailments, peptic ulcers, hypertension, diabetes, cancer, and microbial infections. To explore potential secondary metabolites within grape seed extract that could serve as structural inhibitors of bacterial DNA Gyrase, molecular docking studies were performed. The docking results revealed that two phytochemicals, namely (-)-catechin and Procyanidin-B2, exhibited the highest potency in inhibiting DNA gyrase subunit B. Subsequent in silico physicochemical and pharmacokinetic parameter predictions were conducted using specialized web servers for the examined phytochemicals. Notably, (-)-catechin displayed superior inhibitory and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) characteristics, suggesting its potential for utilization in synthesizing novel antibacterial compounds.

Synthesis, characterization and biological activity of a novel N1, N5-bis[2-hydroxyphenyl]-2-methyl-1,5-diaminopentane Schiff base: Effect of methylene spacers on biological activities

A novel ONNO donor tetradentate Schiff base ligand N,N'-bis[2- hydroxyphenyl]-2-methyl-1,5-diaminopentane (1) is synthesized and characterized by using 1H, 13C NMR spectroscopy and mass spectrometry. Biological studies on newly synthesized ligand are carried out and results are compared with the biological activities of known Schiff base ligands: N,N'-bis[{2- hydroxyphenyl }]-1,4-diaminobutane (2) and N,N'-bis[{2- hydroxyphenyl }]-1,3-diaminopropane (3). This comparative study highlights the effect of increasing methylene linkage on the biological activity of these ligands. The anti-microbial activities of all three ligands have been screened against two distinct gram-positive and gram-negative bacteria. In-vitro results of this comparative analysis are also supported by comprehensive molecular docking studies with DNA gyrase subunit B protein of gram-positive bacterium S. aureus and FabH–CoA complex of gram-negative bacterium E. coli. Experimental studies reveal that the spacer group plays a vital role in controlling the biological activities of the ligand. These findings are in good agreement with the theoretical findings. The study demonstrates that the biological activity of ligands is enhanced with increments in the number of spacers. Thus, the newly synthesized ligand (1) with the most extended spacer among the three showed the highest biological activity compared to other two ligands, similar to the popular drug Ciprofloxacin.

Bacillus velezensis BV01 Has Broad-Spectrum Biocontrol Potential and the Ability to Promote Plant Growth.

To evaluate the potential of a bacterial strain as a fungal disease control agent and plant growth promoter, its inhibitory effects on phytopathogens such as Bipolaris sorokiniana, Botrytis cinerea, Colletotrichum capsici, Fusarium graminearum, F. oxysporum, Neocosmospora rubicola, Rhizoctonia solani, and Verticillium dahliae were investigated. The results showed that the inhibitory rates in dual-culture and sterile filtrate assays against these eight phytopathogens ranged from 57% to 83% and from 36% to 92%. The strain was identified as Bacillus velezensis based on morphological and physiological characterization as well as phylogenetic analyses of 16S rRNA and the gyrase subunit A protein (gyrA) regions. The results demonstrated that B. velezensis was able to produce fungal cell-wall-degrading enzymes, namely, protease, cellulase, and β-1,3-glucanase, and the growth-promotion substances indole-3-acetic acid (IAA) and siderophore. Furthermore, B. velezensis BV01 had significant control effects on wheat root rot and pepper Fusarium wilt in a greenhouse. Potted growth-promotion experiments displayed that BV01 significantly increased the height, stem diameter, and aboveground fresh and dry weights of wheat and pepper. The results imply that B. velezensis BV01, a broad-spectrum biocontrol bacterium, is worth further investigation regarding its practical applications in agriculture.

A peptide nucleic acid probe-based multiplex qPCR assay for rapid and accurate detection and quantification of fish-pathogenic Edwardsiella species

Edwardsiellosis causes severe economic losses in the aquaculture industry worldwide. Accurate identification of the fish pathogenic Edwardsiella species is crucial for preventing disease spread and providing effective treatment strategies in the aquaculture industry. These species have high phenotypic and genetic similarities (> 99% in 16S rRNA sequences), which makes their differentiation challenging. In this study, we developed a multiplex real-time PCR diagnostic assay based on the single-copy DNA gyrase subunit B (gyrB) gene using a peptide nucleic acid (PNA) probe to discriminate among four fish pathogenic Edwardsiella species, E. tarda, E. piscicida, E. anguillarum, and E. ictaluri. Through phylogenetic analysis based on gyrB sequences (1462 bp), 57 Korean Edwardsiella isolates were affiliated to E. piscicida, E. anguillarum, and E. tarda, accounting for host and geographical origin specificity. The assay targeted a conserved region in gyrB, and designed four probes, each capable of distinguishing among the four Edwardsiella species, even in cases of 1–3 nucleotide mismatch, based on differences in melting temperature. The assay exhibited high specificity and sensitivity, with reaction efficiency in the range 85–100% and a reliable quantifiable limit of 102 copies per reaction for all fluorescence signals. In addition, it could detect and quantify the four species, even in mixed infections, allowing accurate identification within 2 h in a single reaction. This PNA probe-based multiplex qPCR assay could be a practical and reliable diagnostic tool in the field and provide correct taxonomic assignment for further research on the widened host range and niche, pathogenicity, and susceptibility of each Edwardsiella species, to help establish appropriate treatment and vaccine strategies.

Benzimidazole and piperidine containing novel 1,2,3-triazole hybrids as anti-infective agents: Design, synthesis, in silico and in vitro antimicrobial efficacy.

Cu alkyne-azide cycloaddition was used to easily synthesize a library of novel heterocycles containing benzimidazole and piperidine based 1,2,3-triazole(7a-7l) derivatives. The synthesized analogs were characterized by various spectroscopic techniques like FTIR, 1 H nuclear magnetic resonance (NMR), 13 C NMR, and mass spectrometry. All these novel bioactive compounds (7a-7l) were evaluated for in vitro antibacterial and antifungal efficacy. Compound 7k exhibited appreciable potent activity against Escherichia coli strain. Compounds 7a, 7b, 7f, and 7i showed excellent potent activity against all bacterial strains. Compound 7b, 7c, 7d, and 7g derivatives showed excellent effects when tested in vitro for antifungal activity against various fungal strains. Additionally, a molecular docking investigation revealed that compound 7k has the ability to bind to the active site of the E. coli DNA gyrase subunit protein and form hydrogen bonds with significant amino acid residues Asp73 and Asp49 in the active sites. In a 100 ns molecular dynamics simulation, the E. coli DNA gyrase protein's steady capacity to bind compound 7k was shown by the low measured root mean square deviation, which was an indication of the complex's conformational stability.

Molecular identification of Pseudomonas aeruginosa and antibiotic resistance testing

Pseudomonas aeruginosa is a Gram-negative bacillus considered an opportunistic pathogen, usually associated with nosocomial infections in immunocompromised patients. Among the infections caused, the most frequent are urinary tract infections, wound infections, pneumonia, and bacteremia. It has intrinsic and acquired resistance to antibiotics, due to the low permeability of its outer membrane, the overexpression of efflux pumps, and the production of enzymes that degrade the antibiotic. In this study, 61 samples of P. aeruginosa collected from the General Hospital “Dr. Aquiles Calles Ramírez" from the city of Tepic, which were phenotypically identified using the Vitek2 automated system. Subsequently, they were molecularly identified with the polymerase chain reaction technique, targeting the gyrB gene that codes for the B subunit of DNA gyrase. In 60 of the samples, amplification of the gyrB gene was obtained and only one, no amplification was observed. The results showed that infections caused by P. aeruginosa are more frequent in male patients, in age range of 41-60 years. Urine culture being the origin of the sample that most predominated. The sensitivity-resistance analysis to antibiotics showed that most of the strains are resistant to β-lactams, except for meropenem, which presented a high sensitivity in most of the strains, as well as in the case of ciprofloxacin, amikacin, and gentamicin.

DFT and In-silico Investigations, along with In-vitro Antitumor and Antimicrobial Assessments of Pharmacological Molecules.

Molecules bearing an active methylene bridge are one of the most fruitful and remarkable precursors that have been incorporated into the synthetic strategy of an assortment of bioactive compounds. The reactive methylene derivatives have been endowed with multiple reactions, which target biological and medicinal applications and result from their structural diversity and discrete reactivity. The present report endeavors to synthesize, characterize, and in-vitro evaluate several novel propanoic acids, coumarin, and pyrazole derivatives as antimicrobial and antiproliferative agents. The in-silico molecular docking, physicochemical, pharmacokinetic/ADMET, bioactivity, and drug-likeness predictions were conducted for all the synthesized compounds. The highest docking score is -9.9 and -8.3 kcal/mol, respectively, for compound 9 (azocoumarin) and 13 (acrylic acid derivative) with the target proteins E. coli topoisomerase II, DNA gyrase subunit B and PI3K p110α domain, respectively. Moreover, this study predicts the synthesized molecules that may inhibit the novel COVID-19, obtained through virtual screenings only, where compounds 9, 13, 14, 17, and 19 came to the limelight with good docking scores i.e., more than -8 Kcal/mol. Safety profiling of the most potent compound 9 was utilized against normal cell lines and the hemolytic effect on RBCs. The in-silico ADMET studies of the synthesized compounds revealed moderate to good -likeness, high gastro intestinal (GI) absorption, and inhibiting the Cytochrome CYP2C19 and CYP2C9 and all the derivatives possess non-cancerous nature. The in-vitro screening demonstrated that several of the novel molecules are promising drug candidates. The density functional theory (DFT) theoretical calculations were performed to calculate the energy levels of the FMOs and their energy gaps, dipolemoment, andmolecular electrostatic potential. Such parameters, along with the physicochemical parameters, could be a good tool to confirm biological activity.

Green Synthesis of Zinc Oxide Nanoparticles Using Cymbopogon citratus Extract and Its Antibacterial Activity.

Excessive use of antimicrobial medications including antibiotics has led to the emerging menace of antimicrobial resistance, which, as per the World Health Organization (WHO), is among the top ten public health threats facing humanity, globally. This necessitates that innovative technologies be sought that can aid in the elimination of pathogens and hamper the spread of infections. Zinc oxide (ZnO) has multifunctionality owing to its extraordinary physico-chemical properties and functionality in a range of applications. In this research, ZnO nanoparticles (NPs) were synthesized from zinc nitrate hexahydrate, by a green synthesis approach using Cymbopogon citratus extract followed by characterization of the NPs. The obtained X-ray diffraction peaks of ZnO NPs matched with the standard JCPDS card (no. 89-510). The particles had a size of 20-24 nm, a wurtzite structure with a high crystallinity, and hexagonal rod-like shape. UV-Vis spectroscopy revealed absorption peaks between 369 and 374 nm of ZnO NPs synthesized from C. citratus extract confirming the formation of ZnO. Fourier transform infrared confirmed the ZnO NPs as strong absorption bands were observed in the range of 381-403 cm-1 corresponding to Zn-O bond stretching. Negative values of the highest occupied molecular orbital-lowest unoccupied molecular orbital for ZnO NPs indicated the good potential to form a stable ligand-protein complex. Docking results indicated favorable binding interaction between ZnO and DNA gyrase subunit b with a binding energy of -2.93 kcal/mol. ZnO NPs at various concentrations inhibited the growth of Escherichia coli and Staphylococcus aureus. Minimum inhibitory concentration values of ZnO NPs against E. coli and S. aureus were found to be 92.07 ± 0.13 and 88.13 ± 0.35 μg/mL, respectively, at a concentration of 2 mg/mL. AO/EB staining and fluorescence microscopy revealed the ability of ZnO NPs to kill E. coli and S. aureus cells. Through the findings of this study, it has been shown that C. citratus extract can be used in a green synthesis approach to generate ZnO NPs, which can be employed as alternatives to antibiotics and a tool to eliminate drug-resistant microbes in the future.

Prevalence and mechanisms of ciprofloxacin resistance in Escherichia coli isolated from hospitalized patients, healthy carriers, and wastewaters in Iran

BackgroundThis study was aimed to evaluate the prevalence and molecular characteristics of ciprofloxacin resistance among 346 Escherichia coli isolates collected from clinical specimens (n = 82), healthy children (n = 176), municipal wastewater (n = 34), hospital wastewater (n = 33), poultry slaughterhouse wastewater (n = 12) and livestock (n = 9) slaughterhouse wastewater in Iran.MethodsCiprofloxacin minimum inhibitory concentration (MIC) was determined by agar dilution assay. Phylogroups and plasmid-mediated quinolone resistance (PMQR) genes were identified using PCR. Mutations in gyrA, gyrB, parC, and parE genes and amino acid alterations were screened through sequencing assay. The effect of efflux pump inhibitor (PAβN) on ciprofloxacin MICs in ciprofloxacin-resistant isolates was investigated using the microdilution method.ResultsIn total, 28.03% of E. coli isolates were phenotypically resistant to ciprofloxacin. Based on sources of isolation, 64.63%, 51.51%, 33.33%, 14.70%, 10.22% and 8.33% of isolates from clinical specimens, hospital wastewater, livestock wastewater, municipal wastewater, healthy children and poultry wastewater were ciprofloxacin-resistant, respectively. Eighty-one point eighty-one percent (Ser-83 → Leu + Asp-87 → Asn; 78.78% and Ser-83 → Leu only; 3.03% (of ciprofloxacin-resistant E. coli isolates showed missense mutation in GyrA subunit of DNA gyrase, while no amino-acid substitution was noted in the GyrB subunit. DNA sequence analyses of the ParC and ParE subunits of topoisomerase IV exhibited amino-acid changes in 30.30% (Ser-80 → Ile + Glu-84 → Val; 18.18%, Ser-80 → Ile only; 9.10% and Glu-84 → Val only; 3.03%0 (and 15.38% (Ser-458 → Ala) of ciprofloxacin-resistant E. coli isolates, respectively. The PMQR genes, aac(6')-Ib-cr, qnrS, qnrB, oqxA, oqxB, and qepA were detected in 43.29%, 74.22%, 9.27%, 14.43%, 30.92% and 1.03% of ciprofloxacin-resistant isolates, respectively. No isolate was found to be positive for qnrA and qnrD genes. In isolates harboring the OqxA/B efflux pump, the MIC of ciprofloxacin was reduced twofold in the presence of PAβN, as an efflux pump inhibitor. The phylogroups B2 (48.45%) and A (20.65%) were the most predominant groups identified in ciprofloxacin-resistant isolates.ConclusionsThis study proved the high incidence of ciprofloxacin-resistant E. coli isolates in both clinical and non-clinical settings in Iran. Chromosomal gene mutations and PMQR genes were identified in ciprofloxacin resistance among E. coli population.

Phenotypic and genetic insights into efflux pump mechanism in Mycoplasma anserisalpingitidis.

Mycoplasma anserisalpingitidis is one of the most important waterfowl-pathogenic mycoplasmas. Due to inadequate antibiotic treatment, many strains with high minimal inhibitory concentration (MIC) values for multiple drugs have been isolated lately. Decreased antibiotic susceptibility in several Mycoplasma species are known to be associated with mutations in topoisomerase and ribosomal genes, but other strategies such as active efflux pump mechanisms were also described. The scope of this study was the phenotypic and genetic characterization of the active efflux mechanism in M. anserisalpingitidis. We measured the MIC values in the presence and absence of different efflux pump inhibitors (EPIs), such as carbonyl cyanide m-chlorophenylhydrazine (CCCP), orthovanadate (OV), and reserpine (RSP). Moreover, bioinformatic tools were utilized to detect putative regulatory sequences of membrane transport proteins coding genes, while comparative genome analysis was performed to reveal potential markers of antibiotic resistance. Out of the three examined EPIs, CCCP decreased the MICs at least two-fold below the original MICs (in 23 cases out of 36 strains). In the presence of OV or RSP, MIC value differences could be seen only if modified dilution series (10% decrease steps were used instead of two-fold dilutions) were applied (in 24/36 cases with OV and 9/36 with RSP). During comparative genome analysis, non-synonymous single nucleotide polymorphisms (nsSNPs) were identified in genes encoding ABC membrane transport proteins, which were displayed in higher percentages in M. anserisalpingitidis strains with increased MICs. In terms of other genes, a nsSNP was identified in DNA gyrase subunit A (gyrA) gene which can be related to decreased susceptibility to enrofloxacin. The present study is the first to highlight the importance of efflux pump mechanisms in M. anserisalpingitidis. Considering the observed effects of the EPI CCCP against this bacterium, it can be assumed, that the use of EPIs would increase the efficiency of targeted antibiotic therapy in the future control of this pathogen. However, further research is required to obtain a more comprehensive understanding of efflux pump mechanism in this bacterium.

Bacillus velezensis FX-6 suppresses the infection of Botrytis cinerea and increases the biomass of tomato plants.

Botrytis cinerea causing tomato gray mold is a major cause of economic loss in tomato production. It is urgent and necessary to seek an effective and environmentally friendly control strategy to control tomato grey mold disease. In this study, Bacillus velezensis FX-6 isolated from the rhizosphere of plants displayed significant inhibitory ability against B. cinerea and could promote tomato plant growth. FX-6 could effectively inhibit the growth of Botrytis cinerea mycelium in vitro and in vivo, and the inhibitory rate in vitro could reach 78.63%. According to morphological observations and phylogenetic trees based on sequences of the 16S rDNA and gyrA (DNA gyrase subunit A) genes, the strain FX-6 was identified as Bacillus velezensis. In addition, B. velezensis FX-6 showed antagonistic activity against seven phytopathogens, this indicated that FX-6 had broad-spectrum biocontrol activity. We also found that FX-6 fermentation broth had the strongest antagonistic activity against B. cinerea when the culture time was 72 hours, and the inhibition rate was 76.27%. The growth promotion test revealed that strain FX-6 significantly promoted tomato seed germination and seedling growth. Further deeply study on growth-promoting mechanism indicated that the FX-6 produced IAA and siderophore, and had ACC deaminase activity. The trait of significant biological control activity and growth promoting effect on tomato imply that B. velezensis FX-6 has the potential to be used as a biocontrol agent for tomato gray mold management.

Molecular docking, synthesis, and antibacterial activity of the analogs of 1-allyl-3-benzoylthiourea.

The incidence of antibiotic resistance rapidly emerges over the globe. In the present study, the synthesis of thiourea derivatives as antibacterial agents and their biological evaluation are reported. Preliminary studies were done by molecular docking of four analogs of 1-allyl-3-benzoylthiourea, clorobiocin, and ciprofloxacin on the DNA gyrase subunit B receptor (PDB: 1KZN). The nucleophilic substitution reaction of benzoyl chloride analogs to the allylthiourea yielded four 1-allyl-3-benzoylthiourea analogs (Cpd 1-4). The reactions were done by a modified Schotten Baumann method. The in vitro antimicrobial activities were determined using the agar dilution method against methicillin-resistant Staphylococcus aureus (MRSA), Salmonella typhi, Escherichia coli, and Pseudomonas aeruginosa. The in-silico study showed that Cpd 1-4 possesses a good interaction on the DNA gyrase subunit B receptor compared to the ciprofloxacin. Cpd 3 had the best binding affinity with a rerank score of - 91.2304. Although the candidate compounds showed unsatisfactory antibacterial activity, they indicated an increasing trend of growth inhibition along with the increment of concentration. Cpd 1 and 4 exhibited in vitro antibacterial activities against MRSA with a minimum inhibitory concentration value of 1000 µg/mL, better compared to the other compounds. Despite lacking antibacterial activity, all the synthesized compounds showed an increased trend of growth inhibition along with the increment of concentration. Therefore, additional development should be implemented to the compounds of interest in which optimization of lipophilicity and steric properties are suggested.

Identification and pathogenicity of multidrug-resistant Elizabethkingia miricola isolated from farmed American bullfrogs Rana catesbeiana in China with in vitro screening of herbal antimicrobial agents.

In 2021, an outbreak of an infectious disease characterized by torticollis, cataracts, and neurological disorders caused massive mortality in farmed American bullfrogs Rana catesbeiana in Hubei province, China. We identified the causal agent in this outbreak, characterized its pathogenicity, and screened candidate antimicrobial agents for future disease control. Bacterium was isolated from the diseased American bullfrogs and identified based on biochemical tests, sequence analyses (16S ribosomal RNA; DNA gyrase subunit B), and experimental challenge. Furthermore, antibiotic sensitivity of the isolated strain was detected with Kirby-Bauer paper diffusion method, and the antibacterial activity of 60 traditional Chinese herbal extracts against the isolated strain was evaluated by agar disc diffusion and broth dilution assays. We identified Elizabathkingia miricola strain FB210601 as the causative agent of this disease. The isolated E. miricola strain FB210601 exhibited extensive antibiotic resistance to all tested quinolones, β-lactam antibiotics, and aminoglycosides. Eight herbal extracts exhibited excellent antimicrobial activity against E. miricola FB210601, especially Caesalpinia sappan and Rhus chinensis, with minimal inhibitory concentrations less than 0.2 mg/mL. Additionally, the combined effects of two-component herbal mixtures containing C. sappan or R. chinensis were greater than those of the individual extracts. Our results provide a reference for understanding the pathogenesis of Elizabethkingia infection in frogs. Furthermore, this study will aid in the application of herbal extracts for protection against infections caused by multidrug-resistant Elizabathkingia in the future.

Analysing the Impact of Bedaquiline Resistance in Fluoroquinolone-Resistant Clinical Isolates and Investigating the Molecular Interactions of BDQ with the atpE Gene

The progress of the tuberculosis eradication programme is hindered by the global health issue of drug-resistant tuberculosis, which is a significant threat to the population. Resistance-associated mutations vary geographically, so investigate the prevalence of resistance to fluoroquinolone (anti-TB) drugs and its association with resistance-related mutations in clinical isolates of Tamilnadu (north zone) and Puducherry was carried out. And the resultant fluoroquinolone drug-resistant tuberculosis strains will be subjected to the molecular analysis of bedaquiline resistance to study the mutation in the atpE gene. The study includes 430 specimens received at Intermediate Reference Laboratory, State TB training and Demonstration Centre in the Government Hospital for Chest Diseases for Molecular testing between January 2020 and December 2021. Samples were analysed by GenoType MTBDRslV.2 for the molecular detection of mutations in the gyrA and gyrB genes. FQ resistance was observed in 32 samples (7.4%), most of which were naïve and previous treatment failure cases. Twenty-five isolates had mutations in DNA gyrase subunit -A (gyrA), while mutations in gyrB were observed in only 7 isolates. Mutational analysis revealed that the mutations mainly alter codon 94 (replacing aspartic acid with glycine, D94G), and 90 (replacing alanine with valine, A90V). In Isoniazid Resistance, MDR, and treatment failure cases, the resistance to Fluoroquinolones was most commonly associated with the D94G mutation. A molecular stimulation study evaluates the effect of BDQ on atpE gene by docking, using AutoDock 4.2, showing hydrogen, hydrophobic and electrostatic interactions.

The potential target of bithionol against Staphylococcus aureus: design, synthesis and application of biotinylated probes Bio-A2.

This study aims to explore the potential targets of bithionol in Staphylococcus aureus.The four bithionol biotinylated probes Bio-A2-1, Bio-A2-2, Bio-A2-3, and Bio-A2-4 were synthesized, the minimal inhibitory concentrations (MICs) of these probes against S. aureus were determined. The bithionol binding proteins in S. aureus were identified through immunoprecipitation and LC-MS/MS with bithionol biotinylated probe. The biotinylated bithionol probes Bio-A2-1 and Bio-A2-3 displayed antibacterial activities against S. aureus. The Bio-A2-1 showed lower MICs than Bio-A2-3, and both with the MIC50/MIC90 at 12.5/12.5 μM against S. aureus clinical isolates. The inhibition rates of bithionol biotinylated probes Bio-A2-1 and Bio-A2-3 on the biofilm formation of S. aureus were comparable to that of bithionol, and were stronger than that of Bio-A2-2 and Bio-A2-4. The biofilm formation of 10 out of 12S. aureus clinical isolates could be inhibited by Bio-A2-1 (at 1/4×, or 1/2× MICs). There are three proteins identified in S. aureus through immunoprecipitation and LC-MS/MS with bithionol biotinylated probe Bio-A2-1: Protein translocase subunit SecA 1 (secA1), Alanine--tRNA ligase (alaS) and DNA gyrase subunit A (gyrA), and in which the SecA1 protein the highest coverage and the most unique peptides. The LYS112, GLN143, ASP213, GLY496 and ASP498 of SecA1 protein act as hydrogen acceptors to form 6 hydrogen bonds with bithionol biotinylated probe Bio-A2-1 by molecular docking analysis. In conclusion, the bithionol biotinylated probe Bio-A2-1 has antibacterial and anti-biofilm activities against S. aureus, and SecA1 was probably one of the potential targets of bithionol in S. aureus.

Neisseria gonorrhoeae diagnostic escape from a gyrA-based test for ciprofloxacin susceptibility and the effect on zoliflodacin resistance: a bacterial genetics and experimental evolution study.

The aetiological bacterial agent of gonorrhoea, Neisseria gonorrhoeae, has become resistant to each of the first-line antibiotics used to treat it, including ciprofloxacin. One diagnostic approach to identify ciprofloxacin-susceptible isolates is to determine codon 91 in the gene encoding the A subunit of DNA gyrase, gyrA, where coding for the wild-type serine (gyrA91S) is associated with ciprofloxacin susceptibility and phenylalanine (gyrA91F) with resistance. The aim of this study was to investigate the possibility of diagnostic escape from gyrA susceptibility testing. We used bacterial genetics to introduce pairwise substitutions in GyrA positions 91 (S or F) and 95 (D, G, or N), which is a second site in GyrA associated with ciprofloxacin resistance, into five clinical isolates of N gonorrhoeae. All five isolates encoded GyrA S91F, an additional substitution in GyrA at position 95, substitutions in ParC that are known to cause an increased minimum inhibitory concentration (MIC) to ciprofloxacin, and GyrB 429D, which is associated with susceptibility to zoliflodacin (a spiropyrimidinetrione-class antibiotic in phase 3 trials for treatment of gonorrhoea). We evolved these isolates to assess for the existence of pathways to ciprofloxacin resistance (MIC ≥1 μg/mL) and measured MICs for ciprofloxacin and zoliflodacin. In parallel, we searched metagenomic data for 11 355 N gonorrhoeae clinical isolates with reported ciprofloxacin MICs that were publicly available from the European Nucleotide Archive for strains that would be identified as susceptible by gyrA codon 91-based assays. Three clinical isolates of N gonorrhoeae with substitutions in GyrA position 95 associated with resistance (G or N) maintained intermediate ciprofloxacin MICs (0·125-0·5 μg/mL), which has been associated with treatment failure, despite reversion of GyrA position 91 from phenylalanine to serine. From an in-silico analysis of the 11 355 genomes from N gonorrhoeae clinical isolates, we identified 30 isolates with gyrA codon 91 encoding a serine and a ciprofloxacin resistance-associated mutation at codon 95. The reported MICs for these isolates varied from 0·023 μg/mL to 0·25 μg/mL, including four with intermediate ciprofloxacin MICs (associated with substantially increased risk of treatment failure). Finally, through experimental evolution, one clinical isolate of N gonorrhoeae bearing GyrA 91S acquired ciprofloxacin resistance through mutations in the gene encoding for the B subunit of DNA gyrase (gyrB) that also conferred reduced susceptibility to zoliflodacin (ie, MIC ≥2 μg/mL). Diagnostic escape from gyrA codon 91 diagnostics could occur through either reversion of the gyrA allele or expansion of circulating lineages. N gonorrhoeae genomic surveillance efforts might benefit from including gyrB, given its potential for contributing to ciprofloxacin and zoliflodacin resistance, and diagnostic strategies that reduce the likelihood of escape, such as the incorporation of multiple target sites, should be investigated. Diagnostics that guide antibiotic therapy can have unintended consequences, including novel resistance determinants and antibiotic cross-resistance. US National Institutes of Health National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, and the Smith Family Foundation.

Taxonomic Positions and Secondary Metabolite-Biosynthetic Gene Clusters of Akazaoxime- and Levantilide-Producers

Micromonospora sp. AKA109 is a producer of akazaoxime and A-76356, whereas Micromonospora sp. AKA38 is that of levantilide C. We aimed to clarify their taxonomic positions and identify biosynthetic gene clusters (BGCs) of these compounds. In 16S rRNA gene and DNA gyrase subunit B gene (gyrB) sequence analyses, strains AKA109 and AKA38 were the most closely related to Micromonospora humidisoli MMS20-R2-29T and Micromonospora schwarzwaldensis HKI0641T, respectively. Although Micromonospora sp. AKA109 was identified as M. humidisoli by the gyrB sequence similarity and DNA-DNA relatedness based on whole genome sequences, Micromonospora sp. AKA38 was classified to a new genomospecies. M. humidisoli AKA109 harbored six type-I polyketide synthase (PKS), one type-II PKS, one type-III PKS, three non-ribosomal peptide synthetase (NRPS) and three hybrid PKS/NRPS gene clusters, among which the BGC of akazaoxime and A-76356 was identified. These gene clusters are conserved in M. humidisoli MMS20-R2-29T. Micromonospora sp. AKA38 harbored two type-I PKS, one of which was responsible for levantilide C, one type-II PKS, one type-III PKS, two NRPS and five hybrid PKS/NRPS gene clusters. We predicted products derived from these gene clusters through bioinformatic analyses. Consequently, these two strains are revealed to be promising sources for diverse non-ribosomal peptide and polyketide compounds.

Isolation and evaluation of Bacillus subtilis RSS-1 as a potential biocontrol agent against Sclerotinia sclerotiorum on oilseed rape

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape in the world. Because of the absence of resistant varieties and the disadvantages of chemical control, application of antifungal microbes has become an eco-friendly and effective measure to control this disease. In this study, Bacillus subtilis strain RSS-1, isolated from soil samples, was identified based on morphological, physiological and biochemical tests, and DNA gyrase subunit A (gyrA), gyrB, DNA-directed RNA polymerase subunit beta (rpoB) and rpoC gene sequence analysis. It significantly inhibited mycelial growth and sclerotial production of S. sclerotiorum in vitro. In greenhouse experiments, all three tested concentrations (106, 107, 108 cfu mL−1) of cell fermentation broth and culture filtrate significantly reduced the severity of sclerotinia stem rot on oilseed rape (P < 0.05). RSS-1 was more effective at reducing disease severity when applied 24 h before inoculation with S. sclerotiorum than at 24 h post inoculation, suggesting that RSS-1 should be applied as a prophylactic rather than a curative biological agent. Colonization tests indicated that the population density of RSS-1 on rapeseed leaves significantly decreased (P < 0.05) over 6 days. However, RSS-1 could stably colonize in rhizospheric soil of rapeseed over 30 days. Challenge inoculation tests showed that RSS-1 significantly inhibited the activities of polygalacturonase and cellulase and accumulation of oxalic acid during the S. sclerotiorum infection. These results suggest that RSS-1 was a potential biological agent for controlling sclerotinia stem rot caused by S. sclerotiorum on oilseed rape.