Insight into DNA Gyrase inhibition using quantitative structure-Activity relationships and structure-based drug design approaches

The emergence of certain bacterial strains resistant to antibiotics has become a major public health problem, hence the need to develop new antibiotic molecules. Bacterial DNA gyrase, a type II DNA topoisomerase found in all bacteria is a proven target for antibacterial chemotherapy. Our objective is designing novel DNA Gyrase inhibitors using Quantitative StructureActivity Relationships and Structure-Based Drug Design Approaches. We used bioinformatics tools, biological databases like PDB (Protein DataBank), Binding Databases and software's like, MarvinView, MarvinSketch, PyMOL, AutoDockTools-1.5.6. The 3D crystal structure of DNA Gyrase was extracted from PDB (code: 4DHU) and we characterized the active site. Using 83 compounds with different Ki were extracted from Binding Databases, we built and validated a QSAR Model (PLS regression) and we confirmed the interesting correlation between predicted and experimental Ki (R2=0,843). Four molecules were chosen to be docked into DNA Gyrase active site using AutoDockTools. The compound which has the low Ki (Benzimidazole urea analogue 5) shows more binding affinity with score value of ΔG= -8,6 kcal/mol than the others compounds. So, it would be very interesting to synthesis this promising compound and to test in vitro its antibacterial properties.

In Silico Screening of DNA Gyrase B Potent Flavonoids for the Treatment of Clostridium difficile Infection from PhytoHub Database

Abstract Clostridium difficile infection (CDI) is the most common hospital acquired diarrheal disease with its increasing incidence and mortality rate globally. DNA Gyrase B (GyrB) is a key component of DNA replication process across all bacterial genera; thus, this offers a potential target for the treatment of CDI. In the present study, several virtual screening approaches were employed to identify a novel C. difficile GyrB inhibitor. The 139 known metabolites were screened out from the 480 flavonoids in PhytoHub database. Molinspiration and PROTOX II servers were used to calculate the ADME properties and oral toxicity of the metabolites, whereas mutagenicity, tumorigenicity, irritant, and reproductive effect were predicted using DataWarrior program. The binding mode and the binding efficiency of the screened flavonoids against the GyrB were studied using FlexX docking program. From virtual screening of 139 metabolites, we found 25 flavonoids with no mutagenicity, tumorigenicity, irritant, and reproductive effect. Docking study suggested that flavonoids 1030 ((-)-epicatechin 3'-O-sulfate), 1032 ((-)-epicatechin 4'-O-sulfate), 1049 (3'-O-methyl-(-)-epicatechin 4-O-sulfate), 1051 (3'-O-methyl-(-)-epicatechin 7-O-sulfate), 1055 (4'-O-methyl-(-)-epicatechin 7-O-sulfate) and 1317 (quercetin sulfate) have significantly higher binding affinity than the known GyrB inhibitor novobiocin. The results from molecular dynamics simulation and free energy calculations based on solvated interaction energy suggested that (-)-epicatechin 3'-O-sulfate could be a potential drug candidate in the management of CDI.

A Novel Mismatched PCR-Restriction Fragment Length Polymorphism Assay for Rapid Detection of gyrA and parC Mutations Associated With Fluoroquinolone Resistance in Acinetobacter baumannii

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.

Antimicrobial resistance profiles of vancomycin-resistant Enterococcus species isolated from laboratory mice

Isolates of 24 enterococci, 5 Enterococcus casseliflavus and 19 Enterococcus gallinarum, possessing vanC genes and showing low-level resistance to vancomycin were obtained from mice from commercial mouse breeding companies. Since some of these isolates showed resistance to other antibiotics, the purpose of this study was to clarify the resistant profiles of these isolates. One E. casseliflavus isolate showed resistance to erythromycin with a minimal inhibitory concentration (MIC) of 8 μg/mL and also showed apparent resistance to fluoroquinolones with an MIC of 32 μg/mL for ciprofloxacin. The MICs of 2 other fluoroquinolone-resistant E. casseliflavus and E. gallinarum isolates were 3 and 6 μg/mL, respectively. These 3 resistant isolates showed an absence of macrolide- and fluoroquinolone-resistant genes, including amino acid substitutions in the quinolone resistance determining regions of DNA gyrase and topoisomerase IV. Resistance to tetracycline was detected in 2 E. gallinarum isolates that were highly resistant, exhibiting MICs of 48 and 64 μg/mL and possessing tet(O) genes. The results indicate that antibiotic-resistant enterococci are being maintained in some laboratory mouse strains that have never been treated with an antibiotic.

Antibacterial and Pharmacological Evaluation of Fluoroquinolones: A Chemoinformatics Approach

Fluoroquinolone (FQ) antibiotics are an important class of synthetic antibacterial agents. These are the most extensively used drugs for treating bacterial infections in the field of both human and veterinary medicine. Herein, the antibacterial and pharmacological properties of four fluoroquinolones: lomefloxacin, norfloxacin, ciprofloxacin, and ofloxacin have been studied. The objective of this study was to analyze the antibacterial characteristics of the different fluoroquinolones. Also, the pharmacological properties of the compounds including the Lipinski rule of five, absorption, distribution, metabolism, and excretion, LD50, drug likeliness, and toxicity were evaluated. We found that among all four FQ molecules, ofloxacin showed the highest antibacterial activity through in silico assays with a strong interaction (−38.52 kJ/mol) with the antibacterial target protein (topoisomerase-II DNA gyrase enzyme). The pharmacological and pharmacokinetic analysis also showed that the compounds ciprofloxacin, ofloxacin, lomefloxacin and norfloxacin have good pharmacological properties. Notably, ofloxacin was found to possess an IGC50 (concentration needed to inhibit 50% growth) value of 0.286 μg/L against the Tetrahymena pyriformis protozoa. It also tested negative for the Ames toxicity test, showing its non-carcinogenic character.

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment

Abstract Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n = 47) and fecal samples (n = 94) from cows, and samples from water (n = 23) and milk lines (n = 19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

[Alterations of gyrA in Enterobacteriaceae isolated of urinary tract infection resistance to quinolones antibiotics]

Background: Urinary tract infection is one of the most common infections, which if not treated, it can cause serious problems in patients. One of the ways to treat of this infection is antibiotic therapy. Nowaday, antibiotic resistance in microorganisms is a main problem for physicians and patients in the world. The aim of this study was to evaluate the genetic resistance to quinolones antibiotics in Enterobacteriaceae isolated in urine samples

Materials and methods: 100 bacteria of Enterobacteriaceae family were isolated from suspected samples of urinary infection. Antibiotic susceptibility of isolated bacteria to quinolone antibiotics, including ciprofloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin and enrofloaxin, was performed by disc diffusion method according to standard guidelines [CLSI 2014]. PCR was performed by specific primers of gyrA gene

Results: Hundred bacteria were isolated of clinical urine sample including 60 E.coli, 32 Klebsiella, 3 Enterobacter, and 5 Proteus. Antibiotic resistance to ciprofloxacin were 36%, nalidixic acid 45%, norfloxacin 38%, ofloxacin 38%, levofloxacin 35% and enrofloaxin 39%. Totally, 36 bacteria were resist to all antibiotics, which 29 bacteria [80.55%] revealed mutation in gyrA gene. Conclusion: This study revealed that Ecoli isolates carry a mutation in gyrA genes. This mutation has an important role in antibiotic resistance to quinolons

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.

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran

Abstract The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. Of the total 100 isolates, 64 were resistant to ciprofloxacin. No amino acid alterations were detected in gyrA or parC genes of the ciprofloxacin susceptible or ciprofloxacin intermediate isolates. Thr-83 → Ile substitution in gyrA was found in all 64 ciprofloxacin resistant isolates. Forty-four (68.75%) of them had additional substitution in parC. A correlation was found between the number of the amino acid alterations in the QRDR of gyrA and parC and the level of ciprofloxacin and levofloxacin resistance of the P. aeruginosa isolates. Ala-88 → Pro alteration in parC was generally found in high level ciprofloxacin resistant isolates, which were suggested to be responsible for fluoroquinolone resistance. These findings showed that in P. aeruginosa, gyrA was the primary target for fluoroquinolone and additional mutation in parC led to highly resistant isolates.

Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α.

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.

Quinolone resistance and ornithine decarboxylation activity in lactose-negative Escherichia coli

Quinolones and fluoroquinolones are widely used to treat uropathogenic Escherichia coli infections. Bacterial resistance to these antimicrobials primarily involves mutations in gyrA and parC genes. To date, no studies have examined the potential relationship between biochemical characteristics and quinolone resistance in uropathogenic E. coli strains. The present work analyzed the quinolone sensitivity and biochemical activities of fifty-eight lactose-negative uropathogenic E. coli strains. A high percentage of the isolates (48.3%) was found to be resistant to at least one of the tested quinolones, and DNA sequencing revealed quinolone resistant determining region gyrA and parC mutations in the multi-resistant isolates. Statistical analyses suggested that the lack of ornithine decarboxylase (ODC) activity is correlated with quinolone resistance. Despite the low number of isolates examined, this is the first study correlating these characteristics in lactose-negative E. coli isolates.

Characterization of mannitol-fermenting methicillin-resistant staphylococci isolated from pigs in Nigeria

This study was conducted to determine the species distribution, antimicrobial resistance pheno- and genotypes and virulence traits of mannitol-positive methicillin-resistant staphylococci (MRS) isolated from pigs in Nsukka agricultural zone, Nigeria. Twenty mannitol-positive methicillin-resistant coagulase-negative staphylococcal (MRCoNS) strains harboring the mecA gene were detected among the 64 Staphylococcus isolates from 291 pigs. A total of 4 species were identified among the MRCoNS isolates, namely, Staphylococcus sciuri (10 strains), Staphylococcus lentus (6 strains), Staphylococcus cohnii (3 strains) and Staphylococcus haemolyticus (one strain). All MRCoNS isolates were multidrug-resistant. In addition to β-lactams, the strains were resistant to fusidic acid (85%), tetracycline (75%), streptomycin (65%), ciprofloxacin (65%), and trimethoprim/sulphamethoxazole (60%). In addition to the mecA and blaZ genes, other antimicrobial resistance genes detected were tet(K), tet(M), tet(L), erm(B), erm(C), aacA-aphD, aphA3, str, dfrK, dfrG, cat_pC221, and cat_pC223. Thirteen isolates were found to be ciprofloxacin-resistant, and all harbored a Ser84Leu mutation within the QRDR of the GyrA protein, with 3 isolates showing 2 extra substitutions, Ser98Ile and Arg100Lys (one strain) and Glu88Asp and Asp96Thr (2 strains). A phylogenetic tree of the QRDR nucleotide sequences in the gyrA gene revealed a high nucleotide diversity, with several major clusters not associated with the bacterial species. Our study highlights the possibility of transfer of mecA ...

Pesquisa de genes de resistência a quinolonas em bacilos Gram negativos de origem clínica e ambiental / Research for genes of quinolone resistance in Gram negative bacilli from clinical and environmental origin

Introdução. Quinolonas são antimicrobianos sintéticos que inibem as enzimas DNA-girase e topoisomerase IV resultando na morte bacteriana. São altamente eficazes no tratamento de infecções bacterianas, especialmente causadas por bactérias Gram negativas, e portanto amplamente utilizados na medicina humana e veterinária, na qual também são empregados como profiláticos. Porém, o uso indiscriminado e inadequado levou ao aumento de bactérias resistentes a estes compostos. Esta resistência pode ocorrer devido a mutações nas enzimas DNA-girase e topoisomerase IV, e também por genes contidos em plasmídeos. Estes últimos são os principais responsáveis pela disseminação e circulação da resistência entre o meio ambiente e o ambiente hospitalar. Objetivos. Pesquisar genes de resistência a antimicrobianos do grupo das quinolonas em bactérias Gram negativas de origem clínica e ambiental que apresentam resistência fenotípica a este grupo. Material e Métodos. 73 cepas de Enterobacteriaceae e Aeromonas sp. de origem clínica e ambiental foram selecionadas para o estudo, e avaliadas quanto à sensibilidade aos antimicrobianos do grupo das quinolonas e à pesquisa de genes de resistência a este mesmo grupo e mutações no gene que codifica a enzima DNA-girase por meio de PCR e sequenciamento. Resultados. Das 73 cepas previamente selecionadas para compor o estudo, 65 foram utilizadas, devido à exclusão de perfis clonais similares. Nestas, foram observados os genes, qnrS1 (1,5 por cento ), qnrS2 (26,2 por cento ), qnrB1 (3,1 por cento ), qnrB19 (12,3 por cento ), qnrD1 (1,5 por cento ), aac(6)-Ib-cr (10,8 por cento ), oqxA (43,1 por cento ) e oqxB (41,5 por cento ), e duas variantes determinadas qnrB-like (3,1 por cento ) e qnrB69-like (1,5 por cento ). Os genes qnrA, qnrC e qepA não foram identificados. Mutações na enzima DNA-girase foram observadas em 97,9 por cento das cepas positivas para algum dos genes pesquisados...

Introduction. Quinolones are synthetic antimicrobial agents that inhibit DNA gyrase and topoisomerase IV enzymes resulting in bacterial death. They are highly effective in the treatment of bacterial infections, especially the ones caused by Gram negative bacteria, as well as for prophylaxy. Therefore they are widely used in human and veterinary medicine. However, indiscriminate and improper use led to an increase of bacteria resistance to these compounds. This resistance can be due to mutations in DNA gyrase and topoisomerase IV enzymes and also by genes contained in plasmids, which are mainly responsible for the spread and transmission of resistance between the environment and the hospital set. Objectives. To search for genes of resistance to quinolone antimicrobial agents in Gram-negative bacteria from clinical and environmental strains that present phenotypic resistance to this group. Material and Methods. 73 strains of Enterobacteriaceae and Aeromonas spp., from clinical and environmental origin, were selected for this study, and evaluated for antimicrobial susceptibility of quinolone and search of resistance genes in this same group and also for mutations in the gene encoding the enzyme DNA gyrase by PCR and sequencing. Results. Of the 73 strains previously selected to compose this study, 65 were used, due to the exclusion of similar clonal profiles. In these, genes qnrS1 (1.5 per cent ), qnrS2 (26.2 per cent ) qnrB1 (3.1 per cent ), qnrB19 (12.3 per cent ) qnrD1 (1.5 per cent ) aac(6')-Ib-cr (10.8 per cent ) oqxA (43.1 per cent ) and oqxB (41.5 per cent ) were observed, and two variants were named as qnrB-like (3.1 per cent ) and qnrB69-like (1.5 per cent ). The qnrA, qnrC and qepA genes were not identified. Mutations in DNA gyrase enzyme were observed in 97.9 per cent of the positive strains for at least one of the genes studied. It was possible to establish the association of aac(6')-Ib-cr with class 1 integron gene in four strains...

Molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis clinical isolates

To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.

The Drug Resistance Profile of Mycobacterium abscessus Group Strains from Korea

BACKGROUND: Bacteria of the Mycobacterium abscessus group are the second most common pathogens responsible for lung disease caused by nontuberculous mycobacteria in Korea. There is still a lack of studies investigating the genetic mechanisms involved in M. abscessus resistance to antibiotics other than clarithromycin. This study investigated the characteristics of drug resistance exhibited by M. abscessus clinical isolates from Korea. METHODS: We performed drug susceptibility testing for a total of 404 M. abscessus clinical strains. Subspecies were differentiated by molecular biological methods and examined for mutations in drug resistance-related genes. RESULTS: Of the 404 strains examined, 202 (50.00%), 199 (49.26%), and 3 (0.74%) strains were identified as M. abscessus, M. massiliense, and M. bolletii, respectively. Of the 152 clarithromycin-resistant strains, 6 possessed rrl mutations, while 4 of the 30 amikacin-resistant strains contained rrs mutations, and 5 of the 114 quinolone-resistant strains had gyr mutations. All mutant strains had high minimal inhibitory concentration values for the antibiotics. CONCLUSIONS: Our results showed the distribution of the strains with mutations in drug resistance-related genes was low in the M. abscessus group. Furthermore, we performed drug susceptibility testing and sequence analyses to determine the characteristics of these genes in the M. abscessus group.

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.

Induction in vitro and stability of Mycobacterium tuberculosis resistance to ofloxacin / 中华预防医学杂志

<p><b>OBJECTIVE</b>To induce Mycobacterium tuberculosis (MTB) resistance with ofloxacin (Ofx) of stepwise increasing concentration in vitro, investigate stability to fluoroquinolone (FQs) antibiotic of MTB, and analyze the molecular mechanism and mutation specialty of drug resistance preliminarily.</p><p><b>METHODS</b>MTB Standard strain H37RV and 24 clinical isolates susceptible to Ofx were selected and experimentally serially subcultured in liquid culture medium containing increasing concentration of Ofx and induced the drug resistance to Ofx. Variety of Minimal Inhibitory Concentrations (MICs) to FQs drugs were detected by microwell-MIC-test method. Mutations of quinolone resistance determining region (QRDR) of gyrA gene were sequenced and identified. Relationship of different mutation sites and drug resistant degree were analyzed. A total of 6 MTB clinical isolates resistant to Ofx and induced drug resistant isolates in vitro were serially subcultured in liquid culture medium without drug. Variety of drug resistant stability, including MIC and mutation of gyrA gene were detected.</p><p><b>RESULTS</b>MIC values of 21 Ofx susceptible isolates after induction were eight times higher than before, which were induced to drug resistant strains successfully and also resistant to Lfx and Mfx. Hot mutations of QRDR of gyrA gene were detected by sequencing, except one strain. Mutation of codon 94 occurred in 60% (12/20) of the strains with mutations and corresponding value of 50% Minimal Inhibitory Concentrations(MIC50) was ≥ 8 µg/ml. In all, 4 of 6 MTB clinical isolates resistant to Ofx harbored mutation of codon 90 (67%) , but the corresponding value of MIC50 was 2 µg/ml. After 21 serially subcultured in liquid culture medium without drug, MIC values of 6 clinical isolates resistant to Ofx were not changed obviously and mutations were also not changed. After 11 times serially subcultured in culture medium without drug, MIC values of induced drug resistant strains were also not changed obviously, but new mutations were detected in QRDR of 3 isolates.</p><p><b>CONCLUSION</b>MTB strains resistant to three kinds of FQs antibiotic were obtained by induction in vitro with Ofx. Codons 88, 94 mutations of QRDR of gyrA gene were related to the high level FQs drug resistance of MTB. Drug resistant stability of MTB to FQs was strong, and it is difficult for MTB to resume susceptibility.</p>

Contribution of different mechanisms to the resistance to fluoroquinolones in clinical isolates of Salmonella enterica

OBJECTIVES: To study the potential factors include gene mutation, efflux pump and alteration of permeability associated with quinolone-resistance of Salmonella enterica strains isolated from patients with acute gastroenteritis and to evaluate the degree of synergistic activity of efflux pump inhibitors when combined with ciprofloxacin against resistant isolates. METHODS: Antimicrobial resistance patterns of fifty-eight Salmonella isolates were tested. Five isolates were selected to study the mechanism of resistance associated with quinolone group, including mutation in topoisomerase-encoding gene, altered cell permeability, and expression of an active efflux system. In addition, the combination between antibiotics and efflux pump inhibitors to overcome the microbial resistance was evaluated. RESULTS: Five Salmonella isolates totally resistant to all quinolones were studied. All isolates showed alterations in outer membrane proteins including disappearance of some or all of these proteins (Omp-A, Omp-C, Omp-D and Omp-F). Minimum inhibitory concentration values of ciprofloxacin were determined in the presence/absence of the efflux pump inhibitors: carbonyl cyanide m-chlorophenylhydrazone, norepinephrin and trimethoprim. Minimum inhibitory concentration values for two of the isolates were 2-4 fold lower with the addition of efflux pump inhibitors. All five Salmonella isolates were amplified for gyrA and parC genes and only two isolates were sequenced. S. Enteritidis 22 had double mutations at codon 83 and 87 in addition to three mutations at parC at codons 67, 76 and 80 whereas S. Typhimurium 57 had three mutations at codons 83, 87 and 119, but no mutations at parC. CONCLUSIONS: Efflux pump inhibitors may inhibit the major AcrAB-TolC in Salmonella efflux systems which are the major efflux pumps responsible for multidrug resistance in Gramnegative clinical isolates.

Inappropriateness of Quinolone in Scrub Typhus Treatment Due to gyrA Mutation in Orientia tsutsugamushi Boryong Strain

The use of quinolone for treatment of rickettsial diseases remains controversial. Recent clinical studies suggest that quinolone is not as effective as others in patients with rickettsial diseases including scrub typhus, although the mechanism is not well understood. In this study, we evaluated the mutation in gyrA associated with quinolone resistance. We prospectively enrolled scrub typhus patients, collected blood samples and clinical data from October, 2010 to November, 2011. Among the 21 patients enrolled, one initially received ciprofloxacin for 3 days but was switched to doxycycline due to clinical deterioration. We obtained the gyrA gene of Orientia tsutsugamushi from 21 samples (20 Boryong strain, 1 Kato strain) and sequenced the quinolone resistance-determining region. All of 21 samples had the Ser83Leu mutation in the gyrA gene, which is known to be associated with quinolone resistance. This suggests that quinolones may be avoided for the treatment of serious scrub typhus.