Novel berberine derivatives: Design, synthesis, antimicrobial effects, and molecular docking studies / 中国天然药物

A series of berberine derivatives were synthesized by introducing substituted benzyl groups at C-9. All these synthesized compounds (4a-4m) were screened for their in vitro antibacterial activity against four Gram-positive bacteria and four Gram-negative bacteria and evaluated for their antifungal activity against three pathogenic fungal strains. All these compounds displayed good antibacterial and antifungal activities, compared to reference drugs including Ciprofloxacin and Fluconazole; Compounds 4f, 4g, and 4l showed the highest antibacterial and antifungal activities. Moreover, all the synthesized compounds were docked into topoisomerase II-DNA complex, which is a crucial drug target for the treatment of microbial infections. Docking results showed that H-bond, π-π stacked, π-cationic, and π-anionic interactions were responsible for the strong binding of the compounds with the target protein-DNA complex.

Novel berberine derivatives: Design, synthesis, antimicrobial effects, and molecular docking studies / 中国天然药物

A series of berberine derivatives were synthesized by introducing substituted benzyl groups at C-9. All these synthesized compounds (4a-4m) were screened for their in vitro antibacterial activity against four Gram-positive bacteria and four Gram-negative bacteria and evaluated for their antifungal activity against three pathogenic fungal strains. All these compounds displayed good antibacterial and antifungal activities, compared to reference drugs including Ciprofloxacin and Fluconazole; Compounds 4f, 4g, and 4l showed the highest antibacterial and antifungal activities. Moreover, all the synthesized compounds were docked into topoisomerase II-DNA complex, which is a crucial drug target for the treatment of microbial infections. Docking results showed that H-bond, π-π stacked, π-cationic, and π-anionic interactions were responsible for the strong binding of the compounds with the target protein-DNA complex.

Resistance mechanism of Mycoplasma hominis to quinolones in Chengdu area / 中华检验医学杂志

Objective To detect the resistance of Mycoplasma hominis to quinolones in Chengdu area,explore resistance mechanism of topoisomerase gene gyrA, gyrB, parC and parE mutations associated with drug resistance and provide epidemiological data.Methods Mycoplasma hominis was identified by 16SrRNA gene sequencing technique and antibiotic susceptibility test was carried out by broth microdilution method.Resistance genes were amplified by PCR,whereas sequence alignment was analyzed by DNAMAN software and BLAST.Results Resistance rates of Mycoplasma hominis to ciprofloxacin, levofloxacin, moxifloxacin and gatifloxacin were 92.4%(61/66),87.9%(58/66),71.2%(47/66)and 66.7%(44/66),respectively.Totally 45 strains with different susceptibility to quinolones were screened for amplification and sequencing of topoisomerase genes, of which, 31 strains resistant to moxifloxacin and gatifloxacin harbored GyrA S153L amino acid mutation, 68.9%(31/45), 41 strains resistant to ciprofloxacin and levofloxacin harbored ParC S91I amino acid mutation, 91.1%(41/45).In addition, a new amino acid substitution of ParE A463S was found in 2 high-level resistant strains.No amino acid change was found in GyrB.Conclusions Resistance of Mycoplasma hominis to quinolones is closely associated with amino acid changes caused by mutations in gyrA and parC genes.Different quinolones have different targeting roles and high level resistance is associated with multiple gene mutations.

Discovery of a New DNA Gyrase A Inhibitor, 4-(1-methyl-6-nitroquinolin-1-ium-4-yl)amino-N-4-(1-methylpyridin-1-ium-4-yl)aminophenylbenzamide

Escherichia coli (E. coli) is a clinically important causative organism that can lead to urinary tract infections. Quinolone antibiotics are among the first-line treatments for urinary tract infections. However, the frequency of resistance to quinolone in E. coli has been increasing. Therefore, new antimicrobial agents that can be used for treatment in lieu of quinolone antibiotics are needed. In this study, thirty-six compounds with higher scores in a virtual screening based on the three-dimensional structure of E. coli DNA gyrase were selected for in vitro antimicrobial activity testing. An in vitro test confirmed the antimicrobial activity of 4-[(1-methyl-6-nitroquinolin-1-ium-4-yl)amino]-N-[4-[(1-methylpyridin-1-ium-4-yl)amino]phenyl]benzamide (ZINC18057104) against E. coli among the 36 compounds. The minimum inhibitory concentration (MIC) of ZINC18057104 against E. coli ATCC® 25922™ was 2 μg/ml, and the MIC₅₀ and MIC₉₀ for the 72 quinolone-resistant E. coli clinical isolates were 4 and 64 μg/ml, respectively. ZINC18057104, which has a quinoline structure which is similar to the quinolone antibiotics, is predicted to exhibit antimicrobial activity in quinolone-resistant E. coli because it has different molecular interactions with the DNA gyrase than that of existing quinolone antibiotics.

Quinolonas: Perspectivas actuales y mecanismos de resistencia / Quinolones: Nowadays perspectives and mechanisms of resistance

Quinolones are a family of synthetic broad-spectrum antimicrobial drugs whose target is the synthesis of DNA. They directly inhibit DNA replication by interacting with two enzymes; DNA gyrase and topoisomerase IV. They have been widely used for the treatment of several community and hospital acquired infections, in the food processing industry and in the agricultural field, making the increasing incidence of quinolone resistance a frequent problem associated with constant exposition to diverse microorganisms. Resistance may be achieved by three non-exclusive mechanisms; through chromosomic mutations in the Quinolone Resistance-Determining Regions of DNA gyrase and topoisomerase IV, by reducing the intracytoplasmic concentrations of quinolones actively or passively and by Plasmid-Mediated Quinolones-Resistance genes, [Qnr determinant genes of resistance to quinolones, variant gene of the aminoglycoside acetyltransferase (AAC(6')-Ib-c)] and encoding genes of efflux pumps (qepA and oqxAB)]. The future of quinolones is uncertain, however, meanwhile they continue to be used in an irrational way, increasing resistance to quinolones should remain as an area of primary priority for research.

Las quinolonas son un grupo de antimicrobianos sintéticos de amplio espectro, cuyo objetivo es la síntesis del ADN. Inhiben directamente su replicación al interactuar con dos enzimas; ADN girasa y topoisomerasa IV. Se han utilizado ampliamente para el tratamiento de infecciones intra y extra-hospitalarias, en el campo de la agricultura y en el procesamiento de alimentos, lo que hace que el incremento de resistencia a quinolonas sea un problema cada vez más frecuente, asociado a la constante exposición de diversos microorganismos. La resistencia puede alcanzarse mediante tres mecanismos no excluyentes entre sí; a través de mutaciones cromosómicas en genes codificantes que afectan las regiones determinantes de resistencia a quinolonas de ADN girasa y topoisomerasa IV, al reducir las concentraciones intracitoplásmicas de quinolonas de manera activa o pasiva y por genes de resistencia a quinolonas mediados por plásmidos [genes de resistencia a quinolonas determinates de qnr, gen variante de la aminoglucósido acetil transferasa (AAC(6’)-lb-cr) y genes codificadores de bombas de eflujo (qepAy oqxAB)]. El futuro de las quinolonas es incierto; sin embargo, mientras continúen empleándose para el manejo de infecciones en el ser humano, el incremento de resistencia a quinolonas debe permanecer como un área de importancia primaria para la investigación.

Characterization of antimicrobial resistance and application of RFLP for epidemiological monitoring of thermophilic Campylobacter spp. isolated from dogs and humans in Korea / 대한수의학회지

An antimicrobial susceptibility test was conducted to compare the resistance rates among Campylobacter spp. isolates from dogs (n = 50) raised under diverse conditions and humans (n = 50). More than 60% of Campylobacter (C.) jejuni from dogs and humans showed resistance to nalidixic acid, enrofloxacin and ciprofloxacin. C. jejuni isolates from humans showed higher resistance to tetracycline (83.3%) and ampicillin (91.3%) than those from dogs. None of the C. jejuni or Campylobacter coli isolates from humans or dogs were resistant to erythromycin. Overall, 85% of Campylobacter spp. isolates showed a multidrug resistant phenotype. Nucleotide sequencing analysis of the gryA gene showed that 100% of NA(R)/CIP(R) C. jejuni isolates from dogs and humans had the Thr-86th-Ile mutation, which is associated with fluoroquinolone resistance. flaA PCR restriction fragment length polymorphism (RFLP) typing to differentiate the isolates below the species level revealed 12 different clusters out of 73 strains. The human isolates belonged to eight different RFLP clusters, while five clusters contained dog and human isolates.

Study on the resistance-related genes of Shigella sonnei with decreased susceptibility to fluoroquinolones / 中华传染病杂志

Objective To investigate the resistance-related genes of Shigella sonnei with decreased susceptibility to fluoroquinolones.MethodsA total of 131 strains of Shigella sonnei were analyzed for their antimicrobial susceptibility.Mutations within the quinolone resistance determining regions (QRDR) of gyrA and parC were detected by polymerase chain reaction (PCR) and PCR products were then sequenced. Meanwhile, the plasmid-mediated quinolone resistance (PMQR) genes,qnr and aac(6')-Ib-cr were screened by PCR.ResultsResistance rates of 131 Shigella sonnei isolates to nalidixic acid,tetracycline,ampicillin and trimethoprim-sulfamethoxazole were 100.0％,93.9％,93.2％ and 92.8％,respectively.All strains were susceptible to norfloxacin,ciprofloxacin,levofloxacin,while 94％ nalidixic acid-resistant Shigella sonnei strains showed reduced susceptibilities to fluoroquinolones.All of nalidixic acid-resistant Shigella sonnei strains presented a single mutation at codon 83 (Ser→Leu) of gyrA genes,but no mutations were detected in parC gene.And PMQR genes qnr and aac (6’)-Ib-cr were not detected.Conclusions The nalidixic acid-resistant Shigella sonnei strains with reduced susceptibility to fluoroquinolones are common in the clinical practice,which may mainly due to a single mutation at codon 83 (Ser→Leu) of gyrA genes.

Reduced Levofloxacin Susceptibility in Clinical Respiratory Isolates of Haemophilus Influenzae Is Not yet Associated with Mutations in the DNA Gyrase and Topoisomerase II Genes in Korea

Among 155 clinical respiratory isolates of Haemophilus influenzae in Korea, 6 (3.9%) isolates had reduced levofloxacin susceptibility (MICs > or = 0.5 microg/mL). These six isolates had no significant quinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, or parE. This phenomenon suggests that neither evolution nor spread of any significant QRDRs mutations in clinical isolates occurred in Korea. Therefore, continued surveillance is necessary to observe the evolution of antibiotic-resistance and take measures to avoid the spread of drug-resistant clones.

Reduced Levofloxacin Susceptibility in Clinical Respiratory Isolates of Haemophilus Influenzae Is Not yet Associated with Mutations in the DNA Gyrase and Topoisomerase II Genes in Korea

Among 155 clinical respiratory isolates of Haemophilus influenzae in Korea, 6 (3.9%) isolates had reduced levofloxacin susceptibility (MICs > or = 0.5 microg/mL). These six isolates had no significant quinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, or parE. This phenomenon suggests that neither evolution nor spread of any significant QRDRs mutations in clinical isolates occurred in Korea. Therefore, continued surveillance is necessary to observe the evolution of antibiotic-resistance and take measures to avoid the spread of drug-resistant clones.

The study on levofloxacin resistance and gyrA gene mutation of Helicobacter pylori in Jinhua Zhejiang province / 中华消化杂志

Objective To analyze levofloxacin resistance in Helicobacter pylori (Hp) and the sequence difference of gyrA gene in levofloxacin resistance and sensitive Hp strains. To explore the function of gyrA gene mutation in the development of levofloxacin resistance Hp strain.Methods From July 2007 to December 2008 in Department of Gastroenterology, Jinhua People hospital of Zhejiang Province, the gastric mucosa from gastroscopy biopsy of chronic gastritis and peptic ulcer patients were cultured in Hp selective medium under microaerobic condition at 37 degrees for three to five days. The Hp strains were isolated and identified by oxidase test, catalase test,urease test and UreA gene detecting. The levofloxacin susceptibility was determined by E-test and then resistance and sensitive strains were screened. The genomic DNA of Hp strains was isolated. The gyrA gene was amplified by PCR and the sequences were analyzed. Results 38 clinical isolated Hp strains were passed the levofloxacin susceptibility E-test, among those the minimum inhibitory concentration (MIC) of 12 strains was over 1.0 μg/ml, the percentage of resistant strain was 31.58%, while the sensitive stains was 68.42%. The gyrA gene sequence result indicated 10 resistant strain with 261, 271 and 272 10 site mutation, 2 strains with C261A mutation, one strain with C261G mutation, two strains with G271A mutation, 2 strains with A272G mutation, 2 strains with C261A,G271T andA272G mutation, 1 strain with C261G and A272G mutation. However, no mutation sites were found in 26 sensitive strains. Conclusion The rate of levofloxacir-resistance in isolated clinical Hp strain was high. The drug-resistance was associated with 261,271 and 272. site mutation of gyrA gene.

Mutaciones en genes gyrA y gyrB en cepas de bacilos Gram negativos aisladas en hospitales chilenos y su relación con la resistencia a fluoroquinolonas / Mutations in gyrA and gyrB genes among strains of Gram-negative bacilli isolated from Chilean hospitals and their relation with resistance to fluoroquinolones

Background: A progressive frequency of resistance to fluorquinolones is observed among Gram-negative bacilli. Aim: To investigate the mechanism of resistance to fluoroquinolones mediated by mutations affecting gyrA and gyrB genes in strains of Gram negative bacüli isolated from CMean hospitals. Material and method: Minimal inhibitory concentration of fluoroquinolones was determined in 91 randomly selected nalidixic acid-resistant strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, isolated from hospitals of 12 Chilean cities. Quinolone resistance determining region (QRDR) was amplified by PCR and mutations were determined by restriction fragment length polymorphism (RFLP) and DNA sequencing. Results: Strains with mutation in codon 83 of gyrA showed decreased susceptibility to ciprofloxacin with MICs ranging from 0.25 to 1024 fig/ml. The sequencing of PCR products for gyrA indicated amino acid changes in the QRDR region. One strain ofE. coli presented a double mutation, in codon 83 Ser to Leu as well as in codon 87 Asp to Asn. In strains ofK. pneumoniae, however, the change of codon 83 was Ser to Tyr, in A. baumannii was Ser to Leu and in P. aeruginosa was Thr to He. No strains with mutations affecting gyrB were found. Conclusions: Mutations in codon 83 of gyrA is a frequent genetic event involved in the mechanism leading to decreased susceptibility to fluoroquinolone in strains of Gram-negative bacilli.

Comparison of QRDR Mutations in Fluoroquinolone-resistant Klebsiella pneumoniae Between Fluoroquinolone Inducing Strains in vitro and Clinical Isolates / 中华医院感染学杂志

OBJECTIVE To study the differences of quinolones-resistant determining region(QRDR) in DNA gyrase A(gyrA) subunit between drug-resistant Klebsiella pneumoniae induced by fluoroquinolone in vitro and strains isolated from clinical isolates.Furthermore,to reveal the relation between different QRDR gene mutations and drug-resistance. METHODS Ten sensitive K.pneumoniae strains were selected and induced into drug-resistant strains by ciprofloxacin.The QRDR in gyrA by polymerase chain reaction(PCR) was amplified and compared their DNA sequences with clinically isolated quinolone-resistant K.pneumoniae. RESULTS We had found several gene mutations of QRDR in fluoroquinolone-resistant K.pneumoniae induced by ciprofloxacin,including Ser 83(TCC)→Phe(TTC) and Ile(ATC);and Gln106(CAG)→Leu(CTG).And the alteration of Gln106(CAG)→Leu(CTG) was a new discovery. CONCLUSIONS There are gene mutations of QRDR in fluoroquinolone-resistant K.pneumoniae induced by ciprofloxacin.The mutation of Ser 83(TCC) and Gln106(CAG) is probably the molecular basis of inducing drug-resistant strains.

Mutations in the gyrA and parC genes of quinolone-resistant Shigella flexneri clinical isolates / 中华传染病杂志

Objective To identify the mutations of DNA gyrase gyrA and Topoisomerase Ⅳ parC genes in quinolone-resistant Shigella flexneri clinical isolates and evaluate the relevance of amino acid changes in GyrA and ParC to quinolone resistance. Methods Based on the antimicrobial susceptibility testing, 47 S.flexneri clinical isolates with different quinolone susceptibility were selected and the fragments including the quinolone resistance-determining region (QRDR) in gyrA and parC were PCR amplified and sequenced. SAS (V 8.2) software was used to analyze the correlation between quinolone resistance and changes in GyrA and ParC. Results Sense mutation(s) in gyrA and parC were commonly observed in all of 44 quinolone-resistant isolates, whereas no sense mutation was found in the 3 quinolone-susceptible ones. The most frequent mutation is at codon 83(TCG→TTG) of gyrA, which was observed in 43 quinolone-resistant isolates. The mixed model analysis indicated that the alterations in amino acid 83 of GyrA and amino acid 80 of ParC are close related to nalidixic acid resistance (P

A mutation in QRDR in the ParC subunit of topoisomerase IV was responsible for fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae

Forty-one strains of Streptococcus pneumoniae were isolated at Seoul National University Children's Hospital from 1991 to 1997. Isolates were divided into six groups based on MICs of three quinolones, ciprofloxacin, ofloxacin and norfloxacin. Sequencing showed that the isolates which were intermediately resistant to three quinolones or resistant to at least one kind of quinolone had one missense mutation, Lys137-->Asn(AAG-->AAT) substitution in the ParC subunit of topoisomerase IV without additional mutation in QRDR of the GyrA subunit of DNA gyrase. In conclusion, the ParC subunit of DNA topoisomerase IV is the primary target site for fluoroquinolone in S. pneumoniae and Lys137-->Asn substitution renders the quinolone resistance in S. pneumoniae.

Alignment and phylogenetic analysis of type II DNA topoisomerases.

DNA topoisomerases have been evolved to solve the topological problems of DNA during replication, transcription, recombination and segregation. Discovery of several new enzymes and their characterization has necessitated this compilation. This analysis shows the distinct evolutionary relatedness of type II DNA topoisomerases. A striking feature is the absence of a contiguous stretch of about 160 amino acids in one of the subunits of prokaryotic type II enzymes, which might have important implications to their structure and function.

Rapid Detection of Mutations of Neisseria gonorrhoeae gyrA Gene Using DNA Chip / 中华皮肤科杂志

Objective To develop a new method-DNA chip to be used for rapid detection of mutations of Neisseria gonorrhoeae gyrA gene. Methods Probes were designed according to the sequence of Neisseria gonorrhoeae gyrA genes, and DNA chip was fabricated accordingly. DNA fragment which contains gyrA gene mutation was amplified using PCR technique, labeled with Cy5 fluorescence, and then hybridized with DNA chip. Results of DNA sequencing were used as the control. Results All of the 50 urogenital swab specimens were detected using DNA chip. The consistency between the DNA chip and drug sensitivity test, and between the DNA chip and DNA sequencing were 100% and 98%, respectively. Conclusions DNA chip is a rapid technique with high sensitivity and specificity for the detection of mutations of Neisseria gonorrhoeae gyrA gene. This method can be used for the detection of drug resistance in clinical practice.