In this work, we describe an improved series of N-phenylpyrrolamide inhibitors that exhibit potent activity against DNA gyrase and are highly effective against high-priority gram-positive bacteria. The most potent compounds show low nanomolar IC50 values against Escherichia coli DNA gyrase, and in addition, compound 7c also inhibits E. coli topoisomerase IV in the nanomolar concentration range, making it a promising candidate for the development of potent dual inhibitors for these enzymes. All tested compounds show high selectivity towards the human isoform DNA topoisomerase IIα. Compounds 6a, 6d, 6e and 6f show MIC values between 0.031 and 0.0625 μg/mL against vancomycin-intermediate S. aureus (VISA) and Enterococcus faecalis strains. Compound 6g shows an inhibitory effect against the methicillin-resistant S. aureus strain (MRSA) with a MIC of 0.0625 μg/mL and against the E. faecalis strain with a MIC of 0.125 μg/mL. In a time-kill assay, compound 6d showed a dose-dependent bactericidal effect on the MRSA strain and achieved bactericidal activity at 8 × MIC after 8 h. The duration of the post-antibiotic effect (PAE) on the MRSA strain for compound 6d was 2 h, which corresponds to the PAE duration for ciprofloxacin. The compounds were not cytotoxic at effective concentrations, as determined in an MTS assay on the MCF-7 breast cancer cell line.

Clostridia are abundant in the human gut and comprise families associated with host health such as Oscillospiraceae, which has been correlated with leanness. However, culturing bacteria within this family is challenging, leading to their detection primarily through 16S rRNA amplicon sequencing, which has a limited ability to unravel diversity at low taxonomic levels, or by shotgun metagenomics, which is hindered by its high costs and complexity. In this cross-sectional study involving 114 Colombian adults, we used an amplicon-based sequencing strategy with alternative markers-gyrase subunit B (gyrB) and DNA K chaperone heat protein 70 (dnaK)-that evolve faster than the 16S rRNA gene. Comparing the diversity and abundance observed with the three markers in our cohort, we found a reduction in the diversity of Clostridia, particularly within Lachnospiraceae and Oscillospiraceae among obese individuals [as measured by the body mass index (BMI)]. Within Lachnospiraceae, the diversity of Ruminococcus_A negatively correlated with BMI. Within Oscillospiraceae, the genera CAG-170 and Vescimonas also exhibited this negative correlation. In addition, the abundance of Vescimonas was negatively correlated with BMI. Leveraging shotgun metagenomic data, we conducted a phylogenetic and genomic characterization of 120 metagenome-assembled genomes from Vescimonas obtained from a larger sample of the same cohort. We identified 17 of the 72 reported species. The functional annotation of these genomes showed the presence of multiple carbohydrate-active enzymes, particularly glycosyl transferases and glycoside hydrolases, suggesting potential beneficial roles in fiber degradation, carbohydrate metabolism, and butyrate production.
OBJECTIVE: The gut microbiota is diverse across various taxonomic levels. At the intra-species level, it comprises multiple strains, some of which may be host-specific. However, our understanding of fine-grained diversity has been hindered by the use of the conserved 16S rRNA gene. While shotgun metagenomics offers higher resolution, it remains costly, may fail to identify specific microbes in complex samples, and requires extensive computational resources and expertise. To address this, we employed a simple and cost-effective analysis of alternative genetic markers to explore diversity within Clostridia, a crucial group within the human gut microbiota whose diversity may be underestimated. We found high intra-species diversity for certain groups and associations with obesity. Notably, we identified Vescimonas, an understudied group. Making use of metagenomic data, we inferred functionality, uncovering potential beneficial roles in dietary fiber and carbohydrate degradation, as well as in short-chain fatty acid production.

With the rapid emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), various levels of resistance against existing anti-tuberculosis (TB) drugs have developed. Consequently, the identification of new anti-TB targets and drugs is critically urgent. DNA gyrase subunit B (GyrB) has been identified as a potential anti-TB target, with novobiocin and SPR719 proposed as inhibitors targeting GyrB. Therefore, elucidating the molecular interactions between GyrB and its inhibitors is crucial for the discovery and design of efficient GyrB inhibitors for combating multidrug-resistant TB. In this study, we revealed the detailed binding mechanisms and dissociation processes of the representative inhibitors, novobiocin and SPR719, with GyrB using classical molecular dynamics (MD) simulations, tau-random acceleration molecular dynamics (τ-RAMD) simulations, and steered molecular dynamics (SMD) simulations. Our simulation results demonstrate that both electrostatic and van der Waals interactions contribute favorably to the inhibitors\' binding to GyrB, with Asn52, Asp79, Arg82, Lys108, Tyr114, and Arg141 being key residues for the inhibitors\' attachment to GyrB. The τ-RAMD simulations indicate that the inhibitors primarily dissociate from the ATP channel. The SMD simulation results reveal that both inhibitors follow a similar dissociation mechanism, requiring the overcoming of hydrophobic interactions and hydrogen bonding interactions formed with the ATP active site. The binding and dissociation mechanisms of GyrB with inhibitors novobiocin and SPR719 obtained in our work will provide new insights for the development of promising GyrB inhibitors.

DNA gyrase is essential for the successful replication of circular chromosomes, such as those found in most bacterial species, by relieving topological stressors associated with unwinding the double-stranded genetic material. This critical central role makes gyrase a valued target for antibacterial approaches, as exemplified by the highly successful fluoroquinolone class of antibiotics. It is reasonable that the activity of gyrase could be intrinsically regulated within cells, thereby helping to coordinate DNA replication with doubling times. Numerous proteins have been identified to exert inhibitory effects on DNA gyrase, although at lower doses, it can appear readily reversible and therefore may have regulatory value. Some of these, such as the small protein toxins found in plasmid-borne addiction modules, can promote cell death by inducing damage to DNA, resulting in an analogous outcome as quinolone antibiotics. Others, however, appear to transiently impact gyrase in a readily reversible and non-damaging mechanism, such as the plasmid-derived Qnr family of DNA-mimetic proteins. The current review examines the origins and known activities of protein inhibitors of gyrase and highlights opportunities to further exert control over bacterial growth by targeting this validated antibacterial target with novel molecular mechanisms. Furthermore, we are gaining new insights into fundamental regulatory strategies of gyrase that may prove important for understanding diverse growth strategies among different bacteria.

Tuberculosis (TB), the second leading infectious killer, causes serious public health problems worldwide. To develop novel anti-TB agents, many biochemical studies have targeted the subunit B of DNA gyrase (GyrB), which captures a second DNA segment and responses for ATP hydrolysis. Here, we investigated specific interactions between GyrB residues and existing pyrrolamide derivatives at an electronic level using ab initio fragment molecular orbital (FMO) calculations and designed potent inhibitors against GyrB. The evaluated binding affinities between GyrB and pyrrolamides were confirmed to be consistent with the IC50 values obtained from previous experiments. Thus, we employed the most potent pyrrolamide (compound 1) as a lead compound and proposed novel pyrrolamide derivatives. The specific interactions between GyrB and these derivatives were investigated using molecular mechanic optimizations and FMO calculations. The results revealed that our proposed derivatives had strong hydrogen bonds with Asp79 and Arg141 and exhibited electrostatic interactions with Glu56 and Ile84 of GyrB. In addition, the binding affinity between GyrB and compound 1 was enhanced significantly by the replacement at the R3 site of compound 1. The present results may provide structural concepts for the rational design of potent GyrB inhibitors as anti-TB agents.Communicated by Ramaswamy H. Sarma.

Vibrio vulnificus can cause disease in aquatic animals and humans, therefore, rapid and simple field detection of pathogenic V. vulnificus is important for early disease prevention. In this study, a novel recombinase-aided amplification (RAA) combined test strip with double T-lines (RAA-TS-DTL) was developed for the rapid detection of V. vulnificus in aquatic products. Pathogenic V. vulnificus was detected using the virulence vvhA gene and the housekeeping gene gyrB gene as the dual target of the test strip. The RAA-TS-DTL method showed 100% specificity for V. vulnificus, and no cross-reaction was observed with Vibrio spp. or other bacteria (n = 14). Furthermore, sensitive detection of V. vulnificus in oysters was achieved. The LODs of the gyrB and vvhA genes were 6 CFU/mL and 23 CFU/mL, respectively, which was about five times higher than that of the commercial test strip. The method was validated with spiked samples (n = 60) of fish, shrimp and oyster. The consistency between RAA-TS-DTL and the traditional culture method was 97.9%. In addition, the entire process of detection, including preparation of the sample, could be completed within 50 min. Our results indicated that the developed RAA-TS-DTL was a reliable and useful tool for rapid screening or on-site detection of pathogenic V. vulnificus in aquatic products and aquaculture water.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic-protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

UNASSIGNED: TB lymphadenitis is still a problem that needs serious treatment. In Indonesia, it was reported that 53% of TB cases were extrapulmonary tuberculosis, with the most cases being Lymphadenitis TB, 11.6%. In children, 43% of extrapulmonary tuberculosis cases are TB lymphadenitis. Diagnosis is quite difficult; a method of determining the diagnosis and appropriate comprehensive treatment is required in managing TB Lymphadenitis.
UNASSIGNED: In this study, 15 fine needle aspiration biopsy aspirate samples were subjected to molecular examination using the gyrB-polymerase chain reaction method and histopathological observations using the smear method with hematoxylin-eosin staining. Observation of preparations using a microscope with a magnification of 200x.
UNASSIGNED: The histopathological characteristics of the fine needle aspiration biopsy aspirate showed positive results in 4 out of 15 samples, with epithelioid cells arranged in a characteristic granuloma structure, necrotic debris was visible, and cells joined together to form multinucleated giant cells as an inflammatory response to Mycobacterium tuberculosis complex infection. In this study, 6 out of 15 (40%) were detected to be positive in the diagnosis based on molecular detection using a specific target gene gyrB - polymerase chain reaction .
UNASSIGNED: Characteristic features on histopathological examination associated with gyrB - positive polymerase chain reaction on lymphadenitis fine needle aspiration biopsy aspirate samples can be used as a determinant diagnosis of tuberculous lymphadenitis.

Bacterial blight of pomegranate caused by Xanthomonas auxonopodis pv.punicae (Xap) threaten the existence of a group of farmers for the past few decades who rely on pomegranate cultivation for their livelihood since it will cause huge yield loss. The primary focus of this study was to conduct a thorough analysis of the characterization of this blight incitant Xap. Physiological, biochemical, and molecular characteristics of six phytopathogenic strains of Xap, designated as PBF1 (PBF: Pomegranate Blight Fruit), PBF2, PBF3, PBF4, PBF5, and PBF6, isolated from the infected fruits were examined. Bacterial colonies were featured as gram-negative, yellow-pigmented circular with a glistening appearance. An attempt to determine the best culture medium, favouring bacterial proliferation was successfully done with four distinct medium, Nutrient Glucose Agar (NGA), Nutrient sucrose Agar (NSA), Yeast Dextrose Calcium Carbonate Agar (YDCA) and Yeast Glucose Calcium Carbonate Agar (YGCA) and comparatively, significant growth was found in NGA (66.66%) followed by YDCA (33%). According to the antibiotic susceptibility results, both ampicillin and streptomycin were determined as potentially effective drugs in preventing the proliferation of Xap (P 0.05). The reactive oxygen species-mediated plant immune response during host-pathogen interaction was confirmed by accessing the presence of H2O2 accumulation in infected leaves via 3,3 - diaminobenzidine (DAB) -staining technique. Bacterial isolates from this study were confirmed by two universal constitutive genes such as gyrB and 16S rRNA. From the BLAST analysis, the isolates were identified as Xap with base pair lengths of 1408bp, 1180bp, and 1159bp, which correspond to PBF1, PBF2, and PBF3, respectively. A neighbor-joining phylogenetic tree study explaining a strong phylogenetic relationship between the query sequence and closely related bacterial species.

BACKGROUND: Multi-drug-resistant tuberculosis (MDR-TB) has become a major public health concern globally. Mutations in first- and second-line drug targets such as katG, inhA, rpoB, rrs, eis, gyrA, and gyrB have been associated with drug resistance. Monitoring predominant mutations in the MDR-TB patient population is essential to monitor and devise future therapeutic regimes. The present study is aimed to characterize genetic mutations in MDR isolates of Mycobacterium tuberculosis (MTB) bacilli conferring resistance to a second-line anti-tuberculosis drug in the Eastern Indian population.
METHODS: This cross-sectional study was conducted in the Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, and in the Tuberculosis Demonstration & Training Centre, Agamkuan, Patna. A total of 3270 patients suspected to have MDR-TB were recruited in the study. Two sputum samples, one on the spot, and the other in the morning were collected from each patient and the diagnosis of rifampicin-sensitive (RS)/rifampicin-resistant (RR/MDR) TB was done by Gene-Xpert test. One hundred fifty RS-TB samples and 150 RR/MDR-TB samples were considered for line probe assay (LPA). RS samples were subjected to first-line LPA using Genotype® MTBDR Plus ver 2.0 and RR/MDR samples were considered for second-line LPA using Genotype® MTBDRsl ver 2.0. All sputum samples were subjected to sputum smear microscopy using the Ziehl-Neelsen staining method. Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 26.0 (IBM Corp. Armonk, NY) and R (version 4.1; R Core Team 2021).
RESULTS: In the present study, out of 3270 patients, we detected RR/MDR-TB in 235 patients (7.19%), RS-TB in 812 patients (24.83%), the rest of the patients negative for MTB (2223, 67.98%). Out of 150 RR/MDR-TB sputum samples tested, resistance to fluoroquinolone (FQ) was observed in 41 samples. The selected patients had predominantly FQ resistance due to the gyrA gene mutations (97.56%, n=40) compared to the gyrB gene mutations (2.44%, n=1). We observed >60% of the mutations in the gyrA gene in codon 94 (MUT3C (D94G), MUT3A (D94A), and MUT3D (D94H). In addition, we found the mutations MUT1 (A90V) and MUT2 (S91P) in the codons 90 and 91 of the gyrA gene in the considered MTB patient population.
CONCLUSIONS: The identified genes can be further validated to be considered as therapeutic targets, but more therapeutics and advanced strategies should be applied in the management of MTB.