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Background Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. Objectives To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. Methods Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. Results By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1–100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. Conclusions These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.

Although the presence of catenanes (i.e., intermolecular tangles) in chromosomal DNA stabilizes interactions between daughter chromosomes, a lack of resolution can have serious consequences for genomic stability. In all species, from bacteria to humans, type II topoisomerases are the enzymes primarily responsible for catenating/decatenating DNA. DNA topology has a profound influence on the rate at which these enzymes alter the superhelical state of the double helix. Therefore, the effect of supercoil handedness on the ability of human topoisomerase IIα and topoisomerase IIβ and bacterial topoisomerase IV to catenate DNA was examined. Topoisomerase IIα preferentially catenated negatively supercoiled over positively supercoiled substrates. This is opposite to its preference for relaxing (i.e., removing supercoils from) DNA and may prevent the enzyme from tangling the double helix ahead of replication forks and transcription complexes. The ability of topoisomerase IIα to recognize DNA supercoil handedness during catenation resides in its C-terminal domain. In contrast to topoisomerase IIα, topoisomerase IIβ displayed little ability to distinguish DNA geometry during catenation. Topoisomerase IV from three bacterial species preferentially catenated positively supercoiled substrates. This may not be an issue, as these enzymes work primarily behind replication forks. Finally, topoisomerase IIα and topoisomerase IV maintain lower levels of covalent enzyme-cleaved DNA intermediates with catenated over monomeric DNA. This allows these enzymes to perform their cellular functions in a safer manner, as catenated daughter chromosomes may be subject to stress generated by the mitotic spindle that could lead to irreversible DNA cleavage.

The emergence of resistance to antibacterial drugs remains an important global threat that necessitates an urgent need for the discovery of alternative drugs. This study was undertaken to synthesize some novel nitroaryl/heteroaryl hydrazone derivatives as potential antibacterial agents. After synthesizing by a simple reaction between quinoline/quinazoline hydrazine and nitroaryl/heteroaryl aldehydes, all the compounds were screened for their antibacterial activities, cytotoxicity and in silico investigations. The compound, 2-(4-nitrobenzylidene)-1-(quinazolin-4-yl)hydrazine (1b), displayed significant antimicrobial activity against several susceptible and resistant bacteria without any cytotoxicity. Moreover, scanning electron microscopy (SEM) revealed the complete destruction of Staphylococcus aureus and Escherichia coli following exposure to this compound after 2 h exposure. The in silico studies confirmed the better binding energy of these compounds in comparison with the reference drugs in complex with topoisomerase IV and bacterial ribosomal receptor. Compound 1b can be considered a promising lead compound for the development of broad-spectrum antibacterial medications after further studies.

Introduction The mechanisms of fluoroquinolone-resistance of Mycoplasma genitalium were analysed by a new method. Methods M. genitalium strains from urinary sediments of patients with urethritis were isolated and examined antimicrobial susceptibilities and the mutations in ParC, GyrA and 23S rRNA. Docking models between gyrase and topoisomerase IV with sitafloxacin showed that two binding modes in which the amine moiety at the C-7 position rotated could be constructed. Results Among 18 strains, 13 strains had mutations with amino-acid changes at Serine 83 in ParC. The MICs of moxifloxacin or sitafloxacin for three strains with only S83I in ParC were 2, 1 and 8 mg/L (moxifloxacin) or 0.13, 0.13 and 1 mg/L (sitafloxacin), respectively. In contrast, the MICs of moxifloxacin or sitafloxacin for 3 strain with S83N in ParC were 0.25, 0.13 and 0.25 mg/L (moxifloxacin) or 0.06, 0.03, and 0,03 mg/L (sitafloxacin), respectively, not significantly different from wild-type isolates. The docking model of sitafloxacin and topoisomerase IV showed that the oxygen atom at the gamma position of Serine 83 of ParC interacted with the sitafloxacin carboxylate moiety. When the S83I substitution occurs, the isoleucine side chain is lipophilic and the residue hydropathy changes from hydrophilicity to hydrophobicity and important H-bond interactions between serine and the carboxylate moiety are lost. When the serine 83 to asparagine substitution (S83N) occurred, the asparagine side chain is hydrophilic and the residue hydropathy does not change. Conclusion The docking model suggests that Ser83 replacements causes attenuation or loss of activity of fluoroquinolones such as sitafloxacin.

Introduction: Resistance to fluoroquinolones is mainly due to point mutations that gave rise to amino acid substitutions in the quinolone resistance-determining regions of either gyrA or parC genes, which may be augmented by plasmid mediated resistance. Accordingly, the main aim of the study was to investigate the mutations in gyrA and parC genes as well as the qnrA and qnrB genes acquisition. Methodology: 193 Klebsiella pneumoniae and Escherichia coli isolates were collected, identified and MICs for ciprofloxacin, levofloxacin and moxifloxacin were determined. Polymerase Chain Reaction to investigate qnrA, qnrB, gyrA and parC genes followed by DNA sequencing analysis to identify mutations in gyrA and parC genes. Results: The most prominent mutation in gyrA gene was ser83leu, followed by asp87asn, and lys154arg. Regarding parC mutations, ser80ile was the most detected. Other mutations val141ala and glu84ala were also noted. In addition to a substitution mutation at codon 157 of leucine to tyrosin. To the best of our knowledge this mutation was not previously reported. qnrB was the most detected gene, as 64.7% Klebsiella pneumoniae and 57.1% Escherichia coli were positive. qnrA gene was detected in 11% Klebsiella pneumoniae and 4% of Escherichia coli isolates tested. Conclusions: This study suggests that the indiscriminate use of fluoroquinolones resulted in the increase of development of resistance either through mutations in the quinolone resistance-determining regions of either gyrA or parC genes augmented by plasmid mediated resistance. The irrational use of new fluoroquinolones such as moxifloxacin has created selective pressure for the appearance of new mutation.

Pandemics are large-scale outbreaks of infectious disease that can greatly increase morbidity and mortality all the globe. Since past 1990 till twentieth century, these infectious diseases have been major threat all over the globe associated with poor hygiene and sanitation. In light of these epidemics, researches have gained enormous rise in the developing the potential therapeutic treatment. Thus, revolutionized antibiotics have led to the near eradication of such ailments. Around 50 million prescription of antibiotics written in US per year according to center for disease control and prevention (CDC) report. There is a wide range of antibiotics available which differ in their usage and their mechanism of action. Among these quinoline and quinolone class of antibiotics get attention as they show tremendous potential in fighting the epidemics. Quinoline and quinolone comprise of two rings along with substitutions at different positions which is synthetically obtained by structural modifications of quinine. Quinoline and quinolone antibiotics exhibit extensive activities approved by FDA in the treatment of the several ailments such as gastrointestinal infections, urinary tract infections, prostate inflammation, malaria, gonorrhea, skin infection, colorectal cancer, respiratory tract infections. These are active against both gram-negative and gram-positive bacteria. This basic core of quinoline and quinolone is vital due to its capability of targeting the pathogen causing disease and beneficial in treating the infectious disease. They inhibit the synthesis of nucleic acid of bacteria which results in the rupture of bacterial chromosome due to the interruption of enzymes such as DNA gyrase and topoisomerase IV. There are various quinoline and quinolone compounds that are synthetically derived by applying different synthesis approaches which show a wide range of pharmacological activities in several diseases. The most commonly used are fluoro, chloro, and hydroxychloro derivatives of quinoline and quinolone. These compounds are helpful in the treatment of numerous epidemics as a chief and combination therapy. These quinoline and quinolone pharmacophore fascinate the interest of researchers as they inhibit the entry of virus in host cell and cease its replication by blocking the host receptor glycosylation and proteolytic processing. They act as immune modulator by inhibiting autophagy and reduction of both lysosomal activity and production of cytokine. Therefore, quinoline and quinolone derivatives attain significance in area of research and treatment of various life-threatening epidemics such as SARS, Zika virus, Ebola virus, dengue, and COVID-19 (currently). In this chapter, the research and advancements of quinoline- and quinolone-based antibiotics in epidemic management are briefly discussed.

The bacterial condensin MukB and the cellular chromosomal decatenase, topoisomerase IV interact and this interaction is required for proper condensation and topological ordering of the chromosome. Here, we show that Topo IV stimulates MukB DNA condensation by stabilizing loops in DNA: MukB alone can condense nicked plasmid DNA into a protein–DNA complex that has greater electrophoretic mobility than that of the DNA alone, but both MukB and Topo IV are required for a similar condensation of a linear DNA representing long stretches of the chromosome. Remarkably, we show that rather than MukB stimulating the decatenase activity of Topo IV, as has been argued previously, in stoichiometric complexes of the two enzymes each inhibits the activity of the other: the ParC subunit of Topo IV inhibits the MukF-stimulated ATPase activity of MukB and MukB inhibits both DNA crossover trapping and DNA cleavage by Topo IV. These observations suggest that when in complex on the DNA, Topo IV inhibits the motor function of MukB and the two proteins provide a stable scaffold for chromosomal DNA condensation.

Background & Objective: A facile and efficient method for the synthesis of novel derivatives of FQ citrate conjugates with 1,2,4-triazoles and 1,3,4-oxadiazole scaffolds 8-11 using conventional, as well as microwave irradiation methods, was reported. Based on these original building blocks, the new derivatives of 3, 7-disubstituted fluoroquinolones bearing the oxadiazolyl-triazole groups were obtained. These invaluable derivatives are of great interest in medicinal and pharmaceutical studies because of their important biological properties. Methods: All the reactions were examined under conventional as well as microwave mediated conditions. The structures of obtained compounds were confirmed by 1H NMR, 13C NMR, IR HRMS spectroscopy, and elemental analysis. The antibacterial and antifungal activities of these compounds were screened against Gram-positive, Gram-negative bacteria, and fungal stains by the agar well diffusion method. Cytotoxic assay of the title compounds was evaluated against cervical carcinoma cell line (HeLa) by using the MTT assay. The crystal structure of the Quinolone-DNA cleavage complex of type IV topoisomerase from S. pneumoniae (PDB ID: 3RAE) complex was obtained from the Protein Database (PDB, http:// www.rcsb.org). Molecular properties prediction-drug likeness was studied by Molinspiration and Molsoft software, while lipophilicity and solubility parameters were studied using the Osiris program. Results: A novel approach for the synthesis of benzylthio-1,2,4-triazole and 1,3,4-oxadiazoles core with regioisomeric norfloxacin citrate conjugates was developed. Among the title compounds, 11b, 10a reveal pronounced activity against S. pneumoniae with minimum inhibitory concentrations of 0.89, 0.96 mg/mL and MBCs of 2.95, 2.80 mg/mL, respectively. Minimum Fungicidal Concentration (MFC) has been determined for each compound against two fungal strains. Compound 11b showed maximum anti-cancer activity against HeLa cell line with IC50 value 11.3 ± 0.41 comparable to standard drug DXN. For binding mode, active site residues and docking energies (ΔG =-7.9 Kcal/mol) for ligand 9b exhibited the highest hydrogen bonding (3.59274 A˚), Pi– Alkyl (5.14468 A˚) interactions with amino acid LEU479 of 3RAE protein. The compounds following the Lipinski ‘Rule of five’ were synthesized for antimicrobial and anti-cancer screening as oral bioavailable drugs/leads. Maximum drug likeness model score 1.52, 1.41 was found for compounds 10d, 11b. Conclusion: The present work, through simple synthetic approaches, led to the development of novel hybrids of fluoroquinolone containing citrate-triazole-oxadiazole pharmacophores that exhibited remarkable biological activities against different microorganisms and cell lines. The compounds showed suitable druglike properties and are expected to present good bioavailability profile. An efficient combination of molecular modeling and biological activity provided an insight into QSAR guidelines that could aid in further development and optimization of the norfloxacin derivatives.

Copper complexes with 1,3-disubstituted thiourea derivatives, all containing 3-(trifluoromethyl)phenyl tail and 1-alkyl/halogen-phenyl substituent, were synthesized. The experimental spectroscopic studies and theoretical calculation revealed that two ligands coordinate to Cu(II) in a bidentate fashion via thiocarbonyl S and deprotonated N atoms of thiourea moiety. Such monomers are characteristic of alkylphenylthiourea complexes, whereas the formation of a sandwich-type dimer is observed for halogeno derivatives. For the first time, the structural identifications of CuN2S2-based complexes using experimental and theoretical X-ray absorption near edge structure are demonstrated. The dimeric halogeno derivatives showed higher antimicrobial activity in comparison with alkylphenylthiourea complexes. The Cu(II) complex of 1-(4-chloro-3-nitrophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea was active against 19 strains of methicillin-resistant Staphylococci (MIC = 2 µg/mL). This derivative acted as a dual inhibitor of DNA gyrase and topoisomerase IV isolated from Staphylococcus aureus. Additionally, complexes of halogenphenylthiourea strongly inhibited the growth of mycobacteria isolated from tuberculosis patients, even fourfold stronger than the reference isoniazid. The complexes exerted weak to moderate antitumor activity (towards SW480, SW620, and PC3) being non-toxic towards normal HaCaT cells.

Nitroimidazoles such as metronidazole are used as anti-infective drugs against anaerobic bacteria. Upon

Aim This study aimed to investigate the occurrence of antibiotic resistance phenotype and simultaneously understand its genetic basis in Escherichia coli isolated from the cloacal swabs of commercial chickens from north India. Methods and results Escherichia coli isolates were assessed for susceptibility to 14 different antibiotics using the disc-diffusion technique and were screened for the presence of 22 antibiotic resistance genes (ARGs) by employing PCR. Isolates were found to be highly resistant to fluoroquinolones (nalidixic acid 91%, norfloxacin 73% and ciprofloxacin 66%), tetracycline (71%), beta-lactams (ampicillin 49% and amoxicillin/clavulanic acid 37%), co-trimoxazole (48%), streptomycin (31%) and chloramphenicol (28%); and comparatively less resistant to cefazolin (13%), amikacin (10%), aztreonam (4%), gentamicin (4%) and ceftriaxone (3%). Sixty-three percent of isolates were resistant to more than four different drugs. Abundance of plasmid-borne ARGs like tetA (83%), sul3 (44%), aadA1 (44%), strA (43%), strB (41%), qnrS (38%), sul2 (28%) and aac(6)-Ib-cr (15%) was observed among the isolates. Forty-five percent of isolates possessed more than five different ARGs. Quinolone resistance-determining region (QRDR) mutations within gyrA and parC genes were found to be the major determiners of quinolone resistance. QRDR mutations included leu83, asn87 and gly87 within gyrase-A polypeptide and ile80 and lys84 within topoisomerase IV (encoded by parC). Conclusions Our findings suggest the abuse of antibiotics as feed additives and prophylactic drugs in Indian poultry sector. It also projects this industry as an active hotspot for the replication and selection of ARGs. Significance and impact of the study Our findings would provide evidence to the authorities for formulating effective strategies for restricting antibiotic usage as non-therapeutic agents in food animals. Occurrence of both plasmid-borne and chromosome-borne resistance towards quinolones can drive movement of resistance phenotype across bacterial species and vertical movement of resistance along the bacterial generations, respectively, which can pose mitigation challenges.

Fluoroquinolones are potentially effective against Elizabethkingia anophelis. We investigated the MIC, mutant prevention concentration (MPC), and target gene mutations of fluoroquinolones in E. anophelis. Eighty-five E. anophelis isolates were collected from five hospitals in Taiwan. The MIC and MPC of ciprofloxacin and levofloxacin were examined for all E. anophelis except 17 isolates, in which ciprofloxacin MPC could not be determined due to drug precipitation caused by overly high drug concentration. Mutations in the quinolone resistance-determining regions of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) in the clinical isolates and fluoroquinolone-selected mutants were examined. Overall, 23.5% and 71.8% of the isolates tested were susceptible to ciprofloxacin and levofloxacin, respectively. The MPC(50) of ciprofloxacin was 128 mg/L, and the MPC(50) of levofloxacin was 51.2 mg/L. The MPC(50)/MIC(50) ratio for ciprofloxacin was 64, whereas that for levofloxacin was 25.6. The coefficient of determination between the MPC and MIC for ciprofloxacin and levofloxacin was 0.72 and 0.56, respectively, in the linear regression analysis. Preexisting mutations in GyrA (S83I, S83R, and D87Y) were identified in 18 clinical isolates, all of which were resistant to both ciprofloxacin and levofloxacin. Additional amino acid substitutions in GyrA were identified in all ciprofloxacin- and levofloxacin-selected mutants. Furthermore, GyrB alterations (D431N or D431H) were found in nine levofloxacin-treated isolates. Given that maintaining the serum concentrations of fluoroquinolones above MPCs is impossible under presently recommended doses, the selection of mutant E. anophelis strains seems inevitable.

Objectives Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global threat and novel treatment alternatives are imperative. Herein, susceptibility to the novel antimicrobial zoliflodacin, currently in a global Phase 3 randomized controlled clinical trial for gonorrhoea treatment, was investigated by screening for zoliflodacin GyrB target mutations in publicly available gonococcal genomes and, where feasible, determination of the associated zoliflodacin MIC. Methods The European Nucleotide Archive was queried using the search term ‘Taxon: 485’. DNA sequences from 27 151 gonococcal isolates were analysed and gyrB, gyrA, parC and parE alleles characterized. Results GyrB amino acid alterations were rare (97.0% of isolates had a wild-type GyrB sequence). GyrB V470L (2.7% of isolates) was the most prevalent alteration, followed by S467N (0.12%), N. meningitidis GyrB (0.092%), V470I (0.059%), Q468R/P (0.015%), A466T (0.0074%), L425I + L465I (0.0037%), L465I (0.0037%), G482S (0.0037%) and D429V (0.0037%). Only one isolate (0.0037%) carried a substitution in a resistance-associated GyrB codon (D429V), resulting in a zoliflodacin MIC of 8 mg/L. None of the other detected gyrB, gyrA, parC or parE mutations caused a zoliflodacin MIC outside the wild-type MIC distribution. Conclusions The zoliflodacin target GyrB was highly conserved among 27 151 global gonococcal isolates cultured in 1928–2021. The single zoliflodacin-resistant clinical isolate (0.0037%) was cultured from a male patient in Japan in 2000. Evidently, this strain has not clonally expanded nor has the gyrB zoliflodacin-resistance mutation disseminated through horizontal gene transfer to other strains. Phenotypic and genomic surveillance, including gyrB mutations, of zoliflodacin susceptibility are imperative.

Background Infection of Salmonella enterica subsp. enterica serovar Typhi is the primary etiology of typhoid fever globally and is common in many developing countries, especially those with dense populations and poor environmental sanitation. Antibiotic fluoroquinolones were used for the treatment in the 1980s due to the resistance to the first-line antibiotics. However, many cases of treatment failure of fluoroquinolones in typhoidal patients have been reported from numerous countries in Asia, Europe, Africa, and America. Mutations in quinolone resistance determining regions (QRDR) genes, gyrA, gyrB, parC, and parE, are found in fluoroquinolone-resistant Salmonella Typhi. Contrast reports came from the S. Typhi isolates in Indonesia, mainly Jakarta and the surroundings, obtained from patients with typhoid fever, with good sensitivity to the fluoroquinolones, i.e., nalidixic acid, ciprofloxacin, moxifloxacin, and levofloxacin. The present study, therefore, aimed to identify the hotspot sequences of gyrA, gyrB, parC, and parE genes of the local S. Typhi strains based on their susceptibility to fluoroquinolones from patients with typhoid fever in Jakarta and its satellite cities. Results A total of 28 isolates were identified as S. Typhi. All isolates were susceptible to nalidixic acid, levofloxacin, and moxifloxacin. Twenty-seven isolates (96.4%) were susceptible to ciprofloxacin, with one isolate (3.6%) being intermediate. The hotspot sequences of gyrA, gyrB, parC, and parE genes from all isolates were identical to the fluoroquinolone-sensitive reference sequence Salmonella enterica subsp. enterica serovar Typhi Ty2 (NCBI GenBank AE014613.1), including the isolate with intermediate susceptibility. The mutation was not found, and amino acid deduced from all hotspots in susceptible and intermediate isolates showed no replacement in all reported codons. Conclusions This study showed that the local S. Typhi strains from Jakarta and surroundings were susceptible to fluoroquinolones (nalidixic acid, ciprofloxacin, levofloxacin, and moxifloxacin), and the hotspot sequences of the gyrA, gyrB, parC, and parE genes were all identical to the reference sequence. Thus, the hotspot sequences of the gyrA, gyrB, parC, and parE genes seemingly were conserved in Jakarta’s local S. Typhi strains and could be considered wild type. The phenotypic susceptibility was consistent with the genotypic characteristic without non-synonymous mutations associated with drug resistance.

Developing novel antimicrobial agents has become a necessitate due to the increasing rate of microbial resistance to antibiotics. All the newly adamantane derivatives were evaluated for their antimicrobial activities against six MDR clinical pathogenic isolates. The results exhibited that 13 compounds have from potent to good activity. Among those, five derivatives (6, 7, 9, 14a, and 14b) displayed the potent activities against the different isolates tested (MIC 0.25 µg/ml with bacteria and8 µg/ml with fungi) compared with Ciprofloxacin (CIP) and Fluconazole (FCA). Additionally, the potent adamantanes showed bactericidal and fungicidal effects based on (MBCs and MFCs) and the time-kill assay. The most active adamantane derivatives 7 and 14b exhibited a synergistic effect of ΣFIC ≤ 0.5 with CIP and FCA against the bacterial and fungal isolates. Moreover, no antagonistic effect appeared for the tested derivatives. Additionally, the interaction of DNA gyrase and topoisomerase IV enzymes with the compounds 6, 7, 9, 14a, and 14b exhibited potent antimicrobial activity using in vitro biochemical assays and gel-based DNA-supercoiling inhibition method. The activity of DNA gyrase and topoisomerase IV enzymes showed inhibitory activity (IC

The emergence of drug-resistant bacterial strains continues to be one of the major challenges of medicine. For this reason, the importance of searching for novel structures of antibacterial drugs chemically different from the currently known antibiotics is still of great importance. In this study, we synthesized the thiosemicarbazide and 1,3,4-thiadiazole derivatives and tested them for antibacterial activity. In in vitro tests, we examined the activity of the synthesized substances against Gram-positive and Gram-negative bacteria strains. While all 1,3,4-thiadiazoles tested lacked significant activity, the antimicrobial response of the thiosemicarbazides was moderate and it was also dependent on the type and position of the substituent on the phenyl ring. The highest activity towards all Gram-positive bacteria strains was shown by all three linear compounds containing the trifluoromethylphenyl group in the structure. The MIC (minimum inhibitory concentration) values were in the range of 3.9–250 µg/mL. Additionally, we try to explain the mechanism of the antibacterial activity of the tested compounds using the molecular docking to DNA gyrase and topoisomerase IV, following previous reports on the molecular basis of the activity of thiosemicarbazides. Docking simulations allow the purposing dual mechanism of the antibacterial activity of the synthesized compounds through inhibition of topoisomerase IV DNA gyrase with the moderate prevalence of the topoisomerase pathway.

The increased use of polyphenols nowadays poses the need for identification of their new pharmacological targets. Recently, structure similarity-based virtual screening of DrugBank outlined pseudopurpurin, a hydroxyanthraquinone from

Acinetobacter baumannii has emerged as an important nosocomial pathogen due to its ability to survive in hospital settings and its antimicrobial resistance. It is one of the key pathogens in ventilator-associated pneumonia (VAP). The aim of this study was to characterize the mechanisms of quinolone resistance among A. baumannii isolates causing VAP and to investigate the presence of the novel abaQ gene among them. Quinolone-resistant A. baumannii isolates causing VAP were collected over a period of 4 months. Mutations within gyrA and parC were analyzed and the presence of qnrA, qnrB, qnrS, and abaQ was investigated genotypically. Twenty-one A. baumannii isolates were collected, most of them (76.2%) were extensively drug-resistant (XDR) and only one isolate (4.8%) was pandrug-resistant (PDR). All isolates showed high level of resistance to ciprofloxacin, while qnrA, qnrB and qnrS were absent among our isolates. This is the first report of A. baumannii isolates co-harboring Ser81Leu in gyrA and Ser84Leu in parC together with the novel abaQ gene. Interestingly, a new mutation in gyrA quinolone resistance-determining region Arg89Cys was detected among two of our isolates. The emergence of XDR and PDR isolates among A. baumannii causing VAP is an alarming threat.

We designed and synthesized a series of inhibitors of the bacterial enzymes DNA gyrase and DNA topoisomerase IV, based on our recently published benzothiazole-based inhibitor bearing an oxalyl moiety. To improve the antibacterial activity and retain potent enzymatic activity, we systematically explored the chemical space. Several strategies of modification were followed: varying substituents on the pyrrole carboxamide moiety, alteration of the central scaffold, including variation of substitution position and, most importantly, modification of the oxalyl moiety. Compounds with acidic, basic, and neutral properties were synthesized. To understand the mechanism of action and binding mode, we have obtained a crystal structure of compound 16a, bearing a primary amino group, in complex with the N-terminal domain of E. coli gyrase B (24 kDa) (PDB: 6YD9). Compound 15a, with a low molecular weight of 383 Da, potent inhibitory activity on E. coli gyrase (IC50 = 9.5 nM), potent antibacterial activity on E. faecalis (MIC = 3.13 [micro]M), and efflux impaired E. coli strain (MIC = 0.78 [micro]M), is an important contribution for the development of novel gyrase and topoisomerase IV inhibitors in Gram-negative bacteria. Abstract. Bibliografija: str. 2439-2440. EU H2020 ITN-ETN Project INTEGRATE EU FP7 Integrated Project MAREX Academy of Finland ARRS Biotechnology and Biosciences Research Council (UK) Institute Strategic Programme Grant B Wellcome Trust - (Investigator Award)

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA. Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC, respectively, contributed to the horizontal transfer of resistance in H. influenzae. Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 μg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 μg/mL).

Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of β-lactamases were detected by phenotypic methods, while presence of β-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different β-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajima’s D test and Fu’s Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, β-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them had integrase I gene and had high level of mutations in QRDR regions in gyrA, parC and parE genes. All these factors may play an important role in the invasiveness of these strains and make them difficult to treat. Isolation of these strains from different medical centers, indicate the need for a strict application of infection control measures in Medical centers in the North West Bank-Palestine that aim to reduce expense and damage caused by P. aeruginosa infections. Molecular analyses showed that Palestinian fluoroquinolone resistant P. aeruginosa haplotypes are not genetically differentiated; however, more mutations may exist in these strains.

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) is an important causative agent of nosocomial infections. Mutations in the quinolone resistance-determining regions (QRDRs) of the gyr and par genes have been described. This study aimed to characterise phenotypic and genotypic fluoroquinolone resistance in 69 MRSA isolates of different clonal lineages from hospitals in Rio de Janeiro, Brazil. Methods QRDR mutations in the gyrA, gyrB, parC and parE genes were detected by DNA sequencing. Minimum inhibitory concentrations (MICs) for ciprofloxacin and moxifloxacin were determined by broth microdilution. The occurrence of associations between mutations and MICs among the different clonal lineages of MRSA isolates was then verified. Results Most isolates from the USA400/ST1/SCCmec IV lineage, but mainly USA100/ST5/SCCmec II isolates, which have been more recently found in Rio de Janeiro hospitals, showed different patterns of mutations, including double mutation in the QRDR of parC (Ser-80⿿⿿⿿Tyr and Glu-84⿿⿿⿿Lys/Gly) and/or gyrA (Ser-84⿿⿿⿿Leu and/or Glu-88⿿⿿⿿Lys) associated with higher moxifloxacin and ciprofloxacin MICs (MIC90, ⿥8⿿mg/L and 256⿿mg/L, respectively). On the other hand, all USA800/ST5/SCCmec IV and the BEC/ST239/SCCmec III isolates, which have disappeared from hospitals, showed single mutations in parC (Ser-80⿿⿿⿿Phe) and gyrA (Ser-84⿿⿿⿿Leu or Glu-88⿿⿿⿿Gly) and lower fluoroquinolones MICs (MIC90, ⿥2⿿mg/L and ⿥16⿿mg/L). Conclusion This study highlights an increase in the number and types of mutations in the QRDRs ofgyrA and parC associated with high fluoroquinolones MICs that may be related to changes in the epidemiological profile of MRSA isolates from hospitals in Rio de Janeiro.

This work did a new exploration towards aminothiazolquinolone oximes as potentially multi-targeting antimicrobial agents. A class of novel hybrids of quinolone, aminothiazole, piperazine and oxime fragments were designed for the first time, conveniently synthesized as well as characterized by 1H NMR, 13C NMR and HRMS spectra. Biological activity showed that some of the synthesized compounds exhibited good antimicrobial activities in comparison with the reference drugs. Especially, O-methyl oxime derivative 10b displayed excellent inhibitory efficacy against MRSA and S. aureus 25923 with MIC values of 0.009 and 0.017 mM, respectively. Further studies indicated that the highly active compound 10b showed low toxicity toward BEAS-2B and A549 cell lines and no obvious propensity to trigger the development of bacterial resistance. Quantum chemical studies have also been conducted and rationally explained the structural features essential for activity. The preliminarily mechanism exploration revealed that compound 10b could not only exert efficient membrane permeability by interfering with the integrity of cells, bind with topoisomerase IV–DNA complex through hydrogen bonds and π-π stacking, but also form a steady biosupramolecular complex by intercalating into DNA to exert the efficient antibacterial activity. The supramolecular interaction between compound 10b and human serum albumin (HSA) was a static quenching, and the binding process was spontaneous, where hydrogen bonds and van der Waals force played vital roles in the supramolecular transportation of the active compound 10b by HSA.

A series of novel fluoroquinolone-Safirinium dye hybrids was synthesized by means of tandem Mannich-electrophilic amination reactions from profluorophoric isoxazolones and antibiotics bearing a secondary amino group at position 7 of the quinoline ring. The obtained fluorescent spiro fused conjugates incorporating quaternary nitrogen atoms were characterized by H-1 NMR, IR, MS, and elemental analysis. All the synthetic analogues (3a-h and 4a-h) were evaluated for their in vitro antimicrobial, bactericidal, and antibiofilm activities against a panel of Gram positive and Gram-negative pathogenic bacteria. The most active Safirinium Q derivatives of lomefloxacin (4d) and ciprofloxacin (4e) exhibited molar-based antibacterial activities comparable to the unmodified drugs and displayed considerable inhibitory potencies in E. coli DNA gyrase supercoiling assays with IC50 values in the low micromolar range. Zwiterionic hybrids were noticeably less lipophilic than the parent quinolones in micellar electrokinetic chromatography (MECK) experiments. The tests performed in the presence of phenylalanine-arginine-beta-naphthylamide (PA beta N) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) revealed that the conjugates are to some extent subject to bacterial efflux and cellular accumulation, respectively. Moreover, the hybrids did not exhibit notable cytotoxicity towards the HEK 293 control cell line and demonstrated low propensity for resistance development, as exemplified for compounds 3g and 4b. Finally, molecular docking experiments revealed that the synthesized compounds were able to bind in the fluoroquinolone-binding mode at S. aureus DNA gyrase and S. pneumoniae topoisomerase IV active sites. (C) 2019 Elsevier Masson SAS. All rights reserved.

Novel antibacterial agents needed constantly to counter the ever emergent resistance development to commercially available drugs; one of the effective synthetic antibacterial classes is fluoroquinolone (FQ). This study includes structure activity relationship based design and synthesis of novel fluoroquinolone molecules active against resistant pathogens bearing mutations of DNA gyrase and/or topoisomerase IV which also express efflux pumps. Here, series of compounds were prepared by treating 1-cyclopropyl-6,7-difluoro-8-methoxy-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid as a core with various 4-substituted-3,3-dialkyl piperidines as side chains, through conventional synthetic approaches. Subsequently, antibacterial activities of these fluoroquinolones were examined against Streptococcus pneumoniae, SPN 5844 (Moxi resistant DNA gyrase and topo IV mutant) and SPN 706 (FQ efflux positive). The current manuscript covers50 examples of fluoroquinolone NCEs, amongst 20 NCEs have shown MIC in the range of (0.4 to6.25 μg/ml) for SPN 5844 and (0.1-12.5 μg/ml) for SPN 706 strains. During the course of this study; WCK 919, comprising two chiral isomers; WCK 1152 and WCK 1153 were emerged as lead among the different series synthesized. Advance studies suggested either WCK 1152 or WCK 1153 are the worthy candidates for further clinical developments for respiratory infections caused by resistant pneumococci and staphylococci. However, on the basis of in house preclinical work, WCK 1152 had been selected for phase-1 domestic clinical trials.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections. Although being potent, susceptibility to CNS side effects limits their use. It was observed that improvements in absorption, activity and side effects were achieved via modifications at the N atom of the C7 of the side chain. To meet the increasing demand for development of new antibacterial agents, nineteen novel ciprofloxacin-sulfonamide hybrid molecules were designed, synthesized and characterized by IR, 1H NMR and 13C NMR as potential antibacterial agents with dual DNA gyrase/topoisomerase IV inhibitory activity. Most of the synthesized compounds showed significant antibacterial activity that was revealed by testing their inhibitory activity against DNA gyrase, DNA topoisomerase IV as well as their minimum inhibitory concentration against Staphylococcus aureus. Six ciprofloxacin-sulfonamide hybrids (3f, 5d, 7a, 7d, 7e and 9b) showed potent inhibitory activity against DNA topoisomerase IV, compared to ciprofloxacin (IC50: 0.55 μM), with IC50 range: 0.23–0.44 μM. DNA gyrase was also efficiently inhibited by five ciprofloxacin-sulfonamide hybrids (3f, 5d, 5e, 7a and 7d) with IC50 range: 0.43–1.1 μM (IC50 of ciprofloxacin: 0.83 μM). Compounds 3a and 3b showed a marked improvement in the antibacterial activity over ciprofloxacin against both Gram-positive and Gram-negative pathogens, namely, Staphylococcus aureus Newman and Escherichia coli ATCC8739, with MIC = 0.324 and 0.422 μM, respectively, that is 4.2-fold and 3.2-fold lower than ciprofloxacin (MIC = 1.359 μM) against the Gram-positive Staphylococcus aureus, and MIC = 0.025 and 0.013 μM, respectively, that is 10.2-fold and 19.6-fold lower than ciprofloxacin (MIC = 0.255 μM) against the Gram-negative Escherichia coli ATCC8739. Also, the most active compounds showed lower CNS and convulsive side effects compared to ciprofloxacin with a concomitant decrease in GABA expression.

The human gut houses a complex group of bacterial genera, including both opportunistic pathogens and commensal micro-organisms. These are regularly exposed to antibiotics, and their subinhibitory concentrations play a pivotal role in shaping the microbial responses. This study was aimed to investigate the effects exerted by sub-MICs of nalidixic acid (NA) on the growth rate, bacterial motility, biofilm formation and expression of outer membrane proteins (OMPs) in a commensal strain of E. coli. The NA-sensitive strain was sequentially passaged under sub-MICs of NA. E-test was used to determine the MIC values of NA. Results indicated significant changes in the growth profile of commensal E. coli upon exposure to NA at sub-MICs. Differential expression of OMPs was observed in cells treated with sub-MICs of NA. Bacterial motility was reduced under 1/2 MIC of NA. Interestingly, successive passaging under 1/2 MIC of NA led to the emergence of resistant E. coli with an increased MIC value of 64 µg ml-1 in just 24 days. The NA-resistant variant was confirmed by comparing its 16S rRNA sequence to that of the sensitive commensal strain. Mutations in the Quinolone Resistance-Determining Regions (QRDRs) of chromosomal gyrA, and Topoisomerase IV-encoding parC genes were detected in NA-resistant E. coli. Our results demonstrate how antibiotics play an important role as signalling molecules or elicitors in driving the pathogenicity of commensal bacteria in vitro.

Ciprofloxacin is one of the most widely used antibiotics for treating Pseudomonas aeruginosa infections. However P. aeruginosa acquires mutations that confer ciprofloxacin resistance, making treatment more difficult. Resistance is multifactorial, with mutations in multiple genes influencing the resistance phenotype. However the contributions of individual mutations and mutation combinations to the amounts of ciprofloxacin that P. aeruginosa can tolerate are not well understood. Engineering P. aeruginosa strain PAO1 to contain mutations in any one of the resistance-associated genes gyrA, nfxB, rnfC, parC and parE showed that only gyrA mutations increased the Minimum Inhibitory Concentration (MIC) for ciprofloxacin. Mutations in parC and parE increased the MIC of a gyrA mutant, making the bacteria ciprofloxacin resistant. Mutations in nfxB and rnfC increased the MIC, conferring resistance, only if both were mutated in a gyrA background. Mutations in all of gyrA, nfxB, rnfC and parC/E further increased the MIC. These findings reveal an epistatic network of gene-gene interactions in ciprofloxacin resistance. We used this information to predict ciprofloxacin resistance/susceptibility for 274 isolates of P. aeruginosa from their genome sequences. Antibiotic susceptibility profiles were predicted correctly for 84% of the isolates. The majority of isolates for which prediction was unsuccessful were ciprofloxacin resistant, demonstrating the involvement of additional as yet unidentified genes and mutations in resistance. Our data show that gene-gene interactions can play an important role in antibiotic resistance and can be successfully incorporated into models predicting resistance phenotype.

A series of ciprofloxacin/thiazolidine-2,4-dione hybrids 3a-m were prepared and identified by IR, 1HNMR, 13CNMR and elemental analysis. The antibacterial activity results of the designed hybrids revealed a shift of spectrum toward Gram-positive bacteria. They exhibited excellent activity against S. aureus ATCC 6538, with the most potent compounds being 3a, 3e, 3g, 3i, 3k, 3l and 3m possessing MICs of 0.02, 2.03, 0.64, 0.35, 1.04, 0.22 and 0.36 µM, respectively, compared to their parent compound ciprofloxacin (MIC: 5.49 µM). They also showed interesting activity against MRSA AUMC 261 with 3a, 3e and 3l showing MIC values of 5 nM. Reduced activity was observed against Gram-negative bacteria with compound 3l exhibiting a slightly higher activity against K. pneumoniae ATCC10031 with a MIC value of 0. 08 µM. Mechanistically, the incorporation of thiazolidine-2,4-dione ring into ciprofloxacin retained its ability to inhibit DNA synthesis via inhibiting both topoisomerase IV and DNA gyrase of S. aureus. Compounds 3a, 3l and 3m were more potent than ciprofloxacin for topoisomerase IV (IC50 = 0.3–1.9 μM) and gyrase (IC50 = 0.22–0.31 µM) inhibition, which coincide with their antibacterial activity against S. aureus ATCC 6538. Docking against DNA gyrase active site confirmed the ability of the tested compounds to form stable complexes with the enzyme; like that of ciprofloxacin, 3a, 3i, 3k, 3m and 3l reconsidered promising broad-spectrum antibacterial agents targeting topoisomerase IV and gyrase enzymes and have good activity against MRSA.

The worldwide distribution of qnr genes found on plasmids and their presence on the chromosomes of aquatic bacteria, such as Vibrio vulnificus, one of the suspected sources, suggests an origin before the development of synthetic quinolones. However, their native function remains unknown. Previous work indicated that expression of qnrVv in V. vulnificus was induced by cold shock. To investigate its role further, we constructed single in-frame deletion mutants in qnrVv and cspA (the gene for cold shock protein) and a double mutant in qnrVv and cspA in V. vulnificus ATCC 17562 to evaluate the response to different environmental conditions and stresses and to exposure to various DNA-damaging agents. We found that qnrVv is involved in resistance to ciprofloxacin, levofloxacin, and mitomycin C and in the cold shock response in V. vulnificus. Moreover, ΔqnrVv and ΔcspA mutants showed slower growth when they were treated with bile salts at 37°C and then shifted to 15°C (cold shock) without bile salts in the medium, with the effect being stronger in the double mutant. This transition may mimic what happens when V. vulnificus is ingested into the gastrointestinal tract and released in its natural environment. Cold shock and bile salts induced the expression of cspA and DNA gyrase and topoisomerase IV genes. However, no induction was found in the ΔqnrVv mutant, suggesting that the qnrVv gene is involved in the response to DNA damage and nucleic acid secondary structure.

Background: Methicillin-resistant S. aureus (MRSA) has already tormented humanity and the environment for a long time and is responsible for many difficult-to-treat infections. Unfortunately, there are limited therapeutic options, and MRSA isolates with complete resistance to vancomycin, the first-line drug for the treatment of MRSA infections, have already emerged in recent years. Moxifloxacin retained activity against mutant bacterial strains with various levels of fluoroquinolones resistance and had a lower potential to select for resistant mutants. Isatin is a versatile structure, and its derivatives are potent inhibitors of many enzymes and receptors. The fluoroquinolone- isatin derivatives demonstrated excellent antibacterial activity against both drug-sensitive and drug-resistant organisms. The structure-activity relationship elucidated that incorporation of 1,2,3-triazole moiety into the C-7 position of fluoroquinolone skeleton was favorable to the antibacterial activity. Accordingly, fluoroquinolone derivatives with isatin and 1,2,3-triazole fragments at the side chain on the C-7 position are promising candidates to fight against drug-resistant bacteria. Objective: To explore more active moxifloxacin derivatives to fight against MRSA and enrich the structure-activity relationships. Methods: The synthesized moxifloxacin derivatives 7a-i and 14a-f were evaluated for their antibacterial activity against a panel of MRSA strains by means of standard two-fold serial dilution method. Results: The majority of the synthesized moxifloxacin derivatives were active against most of the tested MRSA strains with MIC values in a range of 1 to 64 μg/mL. The mechanistic investigations revealed that topoisomerase IV was one of the targets for antibacterial activity. Conclusion: These derivatives are useful scaffolds for the development of novel topoisomerase IV inhibitors.

Purpose Haemophilus influenzae strains with low susceptibility to quinolones have recently emerged in the paediatric field in Japan. These strains are judged as 'susceptible' in routine susceptibility tests, although they may survive after quinolone treatment. Therefore, we aimed to construct a simple and cost-effective identification method for low-susceptibility strains using disc diffusion assays. Methodology A total of 33 H. influenzae clinical isolates and a control strain were used. For the disc diffusion assay, levofloxacin, norfloxacin, nalidixic acid and pipemidic acid were employed. Correlations between the inhibition zone diameter and amino acid substitutions were evaluated. Results All of the tested strains formed clear inhibition zones on both levofloxacin and norfloxacin discs. By contrast, none of the low-susceptibility strains showed inhibition zones against nalidixic acid, while the low-susceptibility strains with amino acid substitutions in both GyrA and ParC did not show inhibition zones against pipemidic acid discs, indicating that low-susceptibility strains can be detected with high sensitivity and specificity by the presence or absence of inhibition zones for earlier quinolones. Conclusion A disc diffusion test combining results from nalidixic acid and pipemidic acid can detect low-susceptibility strains harbouring amino acid substitutions without the need for genetic analysis. This test can help reduce inappropriate and unnecessary fluoroquinolone use.

Global growth in antibiotic resistance is a major social problem. A high level of resistance to fluoroquinolones requires the concurrent presence of at least 3 mutations in the target proteins—2 in DNA gyrase (GyrA) and 1 in topoisomerase IV (ParC), which occur in a stepwise manner. In the Escherichia coli chromosome, the gyrA and parC loci are positioned about 1 Mb away from each other. Here we show that the 3 fluoroquinolone resistance mutations are tightly associated genetically in naturally occurring strains. In the latest pandemic uropathogenic and multidrug-resistant E. coli clonal group ST1193, the mutant variants of gyrA and parC were acquired not by a typical gradual, stepwise evolution but all at once. This happened as part of 11 simultaneous homologous recombination events involving 2 phylogenetically distant strains of E. coli, from an uropathogenic clonal complex ST14 and fluoroquinolone-resistant ST10. The gene exchanges swapped regions between 0.5 and 139 Kb in length (183 Kb total) spread along 976 Kb of chromosomal DNA around and between gyrA and parC loci. As a result, all 3 fluoroquinolone resistance mutations in GyrA and ParC have simultaneously appeared in ST1193. Based on molecular clock estimates, this potentially happened as recently as

Objectives This study assessed genetic alterations in gyrA, gyrB, parC and parE and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes among Escherichia coli and Klebsiella pneumoniae isolates from urinary tract infections (UTIs) in Azerbaijan, Iran. Methods A total of 205 clinical isolates of E. coli (n = 177) and K. pneumoniae (n = 28) were obtained from UTIs. Antimicrobial susceptibility was determined by disk diffusion and agar dilution assays. The presence of PMQR genes was determined by PCR, and sequencing of the gyrA, gyrB, parC and parE was performed. Results The rate of fluoroquinolone (FQ) resistance among the isolates was 77.1%. The Ser83Leu mutation in gyrA was observed in all 60 FQ-resistant isolates selected for direct sequencing. The second most common mutation in gyrA was Asp87Asn. Frequent mutations in parC were Ser80Ile and Glu84Val. Ser359Ala + Ser367Thr and Gly385Cys mutations in gyrB were identified in one isolate each of K. pneumoniae and E. coli, respectively. The parE gene had mutations at Ile529Leu, Ser458Ala and Leu416Phe. Overall, PMQR determinants were identified in 90% of E. coli and 100% of K. pneumoniae. The prevalence of PMQR genes was as follows: aac(6′)-Ib-cr, 71.7%; oqxB, 51.7%; oqxA, 36.7%; qnrB, 28.3%; qnrS, 21.7%; qnrD, 16.7%; qepA, 5.0%; qnrA, 1.7%; and qnrC, 1.7%. Conclusions FQ resistance rates were high. Mutations in DNA gyrase and topoisomerase IV and the prevalence of PMQR genes in E. coli and K. pneumoniae isolates were alarming. Moreover, the combination of these resistance mechanisms plays an important role in high-level FQ resistance.

Background: An increasing pattern of fluoroquinolone resistance (FQR) among bacterial pathogens has been described worldwide. In this study, we compared the patterns of genetic mechanisms that conf...

DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases. peerReviewed

Purpose. Mycoplasma hominis is considered among the causes of urogenital infections and shows increasing resistance to fluoroquinolones. However, data regarding the fluoroquinolone resistance mechanism of M. hominis in Southwest China are limited. This study aimed to investigate gene mutations of quinolone resistance-determining regions (QRDRs) of M. hominis isolated from clinical urogenital samples in a Chinese hospital. Methodology. Strains of M. hominis were identified by 16S rRNA gene sequencing. The minimal inhibitory concentrations (MICs) of fluoroquinolones were determined by the broth microdilution method, following CLSI guidelines. PCR was used to amplify the QRDRs of the genes gyrA, gyrB, parC and parE. Positive products were sequenced, and gene mutations and amino acid substitutions were analysed by DNAMAN software and BLAST. Results. The resistance rates of M. hominis to ciprofloxacin (CIP), levofloxacin (LVX), moxifloxacin (MXF) and gatifloxacin (GAT) were 90.5, 85.7, 73.8 and 71.4 %, respectively. A total of 57 isolates of M. hominis were screened, among which 52 strains demonstrated different resistant phenotypes to fluoroquinolones, 41 harboured amino acid substitutions of GyrA S153L, 51 harboured ParC S91I and 22 harboured ParC K144R. ParE A463S and ParC A154T were recorded for the first time and no amino acid change was detected in GyrB. Conclusion. The resistance of M. hominis to fluoroquinolones in Southwest China is mainly related to mutations in QRDRs of either gyrA or parC. High-level resistance is associated with mutations in both DNA gyrase and topoisomerase IV.

The potential of fluoroquinolones as remarkable antibacterial agents evolved from their ability to generate 'poison' complexes between type IIA topoisomerases [topo2As (DNA gyrases and topoisomerases IV)] and DNA. However, the overuse of fluoroquinolones coupled with chromosomal mutations in topo2As has increased incidence of resistance and consequently undermined the application of the currently available fluoroquinolones in clinical practice. In this study, the molecular mechanism of interaction between the secondary metabolites of Crescentia cujete (an underutilized plant with proven anti-bacterial activity) and topo2As was investigated using computational methods. Through molecular docking, the top five compounds with the best affinity for each topo2A were identified and subjected to molecular dynamics simulation over a period of 100 ns. The results revealed that the identified compounds had higher binding energy values than the reference standards against the topo2As except for topoisomerase IV ParC, and this was consistent with the results of the structural stability and compactness of the resulting complexes. Specifically, cistanoside D (-49.18 kcal/mol), chlorogenic acid (-55.55 kcal/mol), xylocaine (-33.08 kcal/mol), and naringenin (-35.48 kcal/mol) had the best affinity for DNA gyrase A, DNA gyrase B, topoisomerase IV ParC, and topoisomerase IV ParE, respectively. Of the constituents of C. cujete evaluated, only apigenin and luteolin had affinity for all the four targets. These observations are indicative of the identified compounds as potential inhibitors of topo2As as evidenced from the molecular interactions including hydrogen bonds established with the active site amino acids of the respective targets. This is the first in silico report on the antibacterial effect of C. cujete and the findings would guide structural modification of the identified compounds as novel inhibitors of topo2As for further in vitro and in vivo assessments.

MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis-dependent DNA loop-extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity. Other structural determinants and interactions that regulate the ATPase activity of MukBEF are not clear. Here, we have investigated the MukBEF ATPase activity, identifying intersubunit and intrasubunit interactions by protein-protein crosslinking and site-specific mutagenesis. We show that interactions between the hinge of MukB and its neck region are essential for the ATPase activity, that the ParC subunit of topoisomerase IV inhibits the MukB ATPase by preventing this interaction, that MukE interaction with DNA is likely essential for viability, and that interactions between MukF and the MukB neck region are necessary for ATPase activity and viability.

A series of hybridized pyrrolidine compounds with a 1,2,4-oxadiazole moiety were synthesized to develop effective molecules against the enzymes DNA gyrase and topoisomerase IV (Topo IV). Compounds 8-20 were developed based on a previously disclosed series of compounds from our lab, but with small structural modifications in the hopes of increasing the compounds' biological activity. In comparison to novobiocin, with IC

Objectives The aim of this study was to determine mutations associated with a quinolone-resistant (QR) phenotype of Flavobacterium columnare isolates. Methods The susceptibility of 53 F. columnare isolates to 11 antimicrobials, including 2 quinolones, was investigated by the disk diffusion method. Oxolinic acid (OXO) was subsequently chosen for minimum inhibitory concentration (MIC) assay. Sequence analysis of four genes within the quinolone resistance-determining regions (QRDRs) of OXO-resistant F. columnare compared with susceptible isolates was subsequently performed. Results The disk diffusion assay revealed that the majority of isolates were susceptible to all tested antimicrobials. However, 14 and 8 isolates were resistant to the quinolone antibiotics OXO and nalidixic acid, respectively. No multidrug resistance was observed. The MIC assay revealed five additional isolates that were resistant to OXO (≥4 μg/mL), making a total of 19 OXO-resistant isolates observed in this study. DNA sequencing identified missense mutations both in parC and gyrA but not in gyrB or parE in QR F. columnare isolates. Mutation in parC resulted in the change His87 → Tyr. For gyrA, 15 isolates of Thai origin exhibited a change at residue Ser83 to either Phe, Tyr or Ala, whereas 3 Vietnamese isolates contained two mutation sites (Ser83 → Phe and Asp87 → Tyr). Conclusion This study is the first to reveal that QR phenotype F. columnare isolates harboured missense mutations both in parC and gyrA but not in gyrB or parE of the QRDRs.

This study aimed to investigate the antimicrobial resistance profile to quinolones, the presence of quinolone-resistant determinants and the plasmid replicon typing in environmental Achromobacter sp. isolated from Brazil. Soil and water samples were used for bacterial isolation. The antimicrobial susceptibility testing was performed by minimum inhibitory concentration method. The detection of mutations in the quinolone resistance-determining regions (QRDR) genes, the presence of plasmid-mediated quinolone resistance (PMQR) genes, and plasmid replicons were performed by PCR. A total of 16 isolates was obtained from different cultures, cities, and states of Brazil. All isolates were non-susceptible to ciprofloxacin, norfloxacin, and levofloxacin. Some mutations in QRDR genes were found, including Gln-83-Leu and Asp-87-Asn in the gyrA and Gln-80-Ile and Asp-84-Ala in the parC. Different PMQR genes were detected, such as qnrA, qnrB, qnrS, oqxA, and oqxB. Three different plasmid families were detected, being most presented the ColE-like, followed by IncFIB and IncA/C. The presence of different PMQR genes and plasmids in the isolates of the present study shows that environmental bacteria can act as reservoir of important genes of resistance to fluoroquinolones, which is of great concern, due to the potential of horizontal dissemination of these genes. Besides that, there are no studies reporting these results in Achromobacter sp. isolates.

Introduction Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium . Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated. Aim To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H). Methods Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated. Results From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3–100) and specificity 99.3% (95% CI:96.3–100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94–1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4–65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7–99.0), suggesting that other SNPs are contributing to resistance. Conclusion MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.

7 p.-4 fig., The topology of DNA duplexes changes during replication and also after deproteinization in vitro. Here we describe these changes and then discuss for the first time how the distribution of superhelical stress affects the DNA topology of replication intermediates,taking into account the progression of replication forks. The high processivity of Topo IV to relax the left-handed (+) supercoiling that transiently accumulates ahead of the forks is not essential, since DNA gyrase and swiveling of the forks cooperate with Topo IV to accomplish this task in vivo. We conclude that despite Topo IV has a lower processivity to unlink the right-handed (+) crossings of pre-catenanes and fully replicated catenanes, this is indeed itsmain role in vivo. This would explain why in the absence of Topo IV replication goes-on, but fully replicated sister duplexes remain heavily catenated., This work was sustained by grant PINV15-573 from the Paraguayan CONACYT-PROCIENCIA program to M.J.F.N. and BFU2014-56835 from the Spanish Ministerio de Economía y Competitividad to J.B.S.

Significance Since conjugative plasmids are usually large and may carry genes encoding functions that are detrimental to the bacterial host, minimizing plasmid copy number is critical for reducing the host burden. Toxin–antitoxin (TA) systems are one of the conserved modules on conjugative plasmids. Here, we demonstrate the functional significance of a large group of antitoxins on conjugative plasmids: the antitoxin acts as an unexpected player in the negative control of plasmid replication. For the plasmid-encoded PrpT/PrpA TA system, the antitoxin can control toxin production by binding to PrpT and by reducing plasmid copy number. This work shows that the antitoxin can directly regulate plasmid replication, expanding our understanding of the physiological role of TA systems., Toxin–antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin–antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

The susceptibility ofA total of 10 isolates showed result resistance to levofloxacin and gyrA gene mutation in the amino acid changes. Nucleotide sequence analysis revealed a point mutation in QRDR (quinolone resistance determining region) of gyrA Ser83→Leu, Asp87→Asn. The silent mutation was also found at codon Val85, Arg91, Ser111, Thr123.Mutation in the

Bacterial resistance to the main widespread antibiotics, such as those based on quinolones, is a concern of the scientific community, leading to the search for new classes of molecules that can be used as an alternative. Here, we investigate the crystalline and chemical characteristics of a thioxopyrimide to understand its demonstrated biological activity and to identify which portion of the molecule can be used as a framework to develop new antibiotics. For this purpose, structural studies of ethyl 4-methyl-2-phenyl-6-thioxo-1,6-dihydro-5-pyrimidinecarboxylate were carried out aided by Hirshfeld surface analysis, as well as theoretical calculations on frontier molecular orbitals, molecular electrostatic potential, and conformational stability, in addition to docking studies targeting topoisomerase IV. The docking results show a reasonable accommodation of the molecule at the topoisomerase IV binding site and interact mainly by hydrogen bonds between the thioxopyrimidine portion with Glu198, Thr292, and Gly225, aided by hydrophobic interactions involving the rest of the molecule. These results suggest a relationship between the antibacterial activity shown mainly with the 4-thioxopyrimidine portion, leading to the investigation of new compounds that use this scaffold.