Identification of ciprofloxacin resistance by SimpleProbe™, High Resolution Melt and Pyrosequencing™ nucleic acid analysis in biothreat agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis

Loveless, Bonnie M.; Yermakova, Anastasiya; Christensen, Deanna R.; Kondig, John P.; Heine, Henry S.; Wasieloski, Leonard P.; Kulesh, David A.

doi:10.1016/j.mcp.2010.01.003

Cited by 40 publications

(50 citation statements)

References 25 publications

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“…The length, GC content, and sequence complementarities of the DNA fragments contribute to the melting characteristics of double-stranded DNA (29). This technique has successfully been applied in mutation scanning, single nucleotide polymorphism (SNP) genotyping, and identification of many bacterial species, including screening for RIF and INH resistance in M. tuberculosis (6,19,(23)(24)(25). With its simple and fast work flow, HRMA is easy to perform and can be used for a large number of samples in a short time (35).…”

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confidence: 99%

Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis

et al. 2011

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A high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosis by targeting resistance-associated mutations in the katG, mabA-inhA promoter, rpoB, and gyrA genes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosis isolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhA promoter, rpoB, and gyrA genes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katG and/or mabA-inhA promoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoB mutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrA mutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosis which could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.One-third of the world's population is reportedly infected with Mycobacterium tuberculosis, causing high mortality and morbidity. Eight million to nine million new tuberculosis (TB) cases and about 2 million deaths are reported each year (9, 18, 28). Multidrug-resistant TB (MDR-TB) is defined as resistance of the isolate to at least isoniazid (INH) and rifampin (RIF); extensively drug-resistant TB (XDR-TB) is MDR-TB in which the isolate has additional resistance to any of fluoroquinolones and at least one of three injectable second-line antituberculosis drugs used in TB treatment, namely, capreomycin, kanamycin, and amikacin (14, 21). MDR-TB and XDR-TB are major obstacles to the control of TB worldwide. More than 2.5 million patients were diagnosed with active TB in 116 countries and regions in 2006; of them, 4.8% had MDR-TB (3). About one in six (15%) patients with MDR-TB had previously received treatment for TB for at least 1 month (18), and at least 8.0% had XDR-TB, according to incomplete data collected from 37 countries between 2002 and 2007 (41).Drug susceptibility testing using molecular techniques can enhance the identification of drug-resistant M. tuberculosis. Several scanning methods are available for detection of drug resistance mutations, including denaturing gradient gel electrophoresis, conformation-sensitive gel electrophoresis, temperature gradient capillary electrophoresis, denaturing high-performance liquid chromatography (dHPLC), and high-density oligonucleotide arrays (15,20,22,30,31,33). These methods vary in sensitivity and...

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Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis

et al. 2011

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“…holarctica strain (URFtCIPR isolate) (9). This mutation was also observed in an F. tularensis SchuS4 strain isolated after in vitro exposure to increasing amounts of ciprofloxacin and was accompanied by the G259T (D87Y) GyrA QRDR substitution (10).…”

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confidence: 74%

“…These drugs inhibit DNA replication through interaction with complexes composed of DNA and either of the two target enzymes, DNA gyrase or topoisomerase IV, which belong to the type IIA topoisomerases (8). As in most Gramnegative bacteria, the primary target for FQs in Francisella strains is thought to be the DNA gyrase (9)(10)(11), which functions as an A 2 B 2 heterotetrameric complex able to catalyze negative supercoiling of the bacterial circular chromosome (12). Resistance to FQs can result from single point mutations in GyrA and GyrB, leading to conformational changes of the whole complex, which in turn impair antibiotic-target interactions.…”

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Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

Caspar

Siebert

Sutera

et al. 2017

Antimicrob Agents Chemother

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Fluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC 50 ) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella.

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“…[14] Resistance to ciprofloxacin is caused by the changes in the amino acids sequences around the enzyme active site resulting in reduced drug affinity thereby allowing for the continued bacteria cell growth.…”

Section: World Journal Of Pharmacy and Pharmaceutical Sciencesmentioning

confidence: 99%

Antibacterial Activity of Methanolic Extract of Mangifera Indica (Bark) and Osyris Lanceolata (Leaves) From Western Region of Nepal

Bhandari¹

2017

WJPPS

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Nature has been a source of medicinal agents since times immemorial.The importance of herbs in the management of human ailments cannot be over emphasized. Different extracts from traditional medicinal plants have been tested to identify the source of the therapeutic effects. needs to be purified through antibacterial activity guided fractionation to isolate and identify the compounds responsible for the antibacterial activity. So, this study provides the platform to carry out further researches to give light upon specific compounds which are responsible for their antibacterial activities.

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Identification of ciprofloxacin resistance by SimpleProbe™, High Resolution Melt and Pyrosequencing™ nucleic acid analysis in biothreat agents: Bacillus anthracis, Yersinia pestis and Francisella tularensis

Cited by 40 publications

References 25 publications

Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis

Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

Antibacterial Activity of Methanolic Extract of Mangifera Indica (Bark) and Osyris Lanceolata (Leaves) From Western Region of Nepal

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