Molecular Basis of High-Level Ciprofloxacin Resistance in
            <i>Neisseria gonorrhoeae</i>
            Strains Isolated in Denmark from 1995 to 1998

Su, Xiaohong; Lind, Inga

doi:10.1128/aac.45.1.117-123.2001

Cited by 47 publications

(34 citation statements)

References 30 publications

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“…All the mutation sites selected for detection in the gyrA and parC genes in this study have been reported previously (1,2,7,8,16,17,23). The sequences of the gyrA and parC genes from different N. gonorrhoeae isolates were aligned by use of Lasergene software (DNAStar, Madison, Wis.).…”

Section: Methodsmentioning

confidence: 99%

“…In the Far East and Western Pacific region, including the People's Republic of China, resistant strains may account for at least 10% of all N. gonorrhoeae isolates (18,27). Several mechanisms of ciprofloxacin resistance have been identified and characterized in N. gonorrhoeae strains that have decreased susceptibilities to ciprofloxacin (2,6,7,8,16,21,23,25). Genotyping studies of genetic mutations resulting in ciprofloxacin resistance in N. gonorrhoeae isolates have shown that amino acid substitutions within the quinolone (i.e., ciprofloxacin) resistance-determining regions (QRDRs) in the gyrA and parC genes play crucial roles in resistance to these agents.…”

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confidence: 99%

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Detection of gyrA and parC Mutations Associated with Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of Oligonucleotide Biochip Technology

Zhou

Cao

et al. 2004

J Clin Microbiol

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An oligonucleotide biochip that specifically detects point mutations in the gyrA and parC genes of Neisseria gonorrhoeae was designed and subsequently evaluated with 87 untreated clinical specimens. The susceptibilities of the N. gonorrhoeae strains were tested to determine the prevalence of ciprofloxacin-resistant strains in Anhui Province, People's Republic of China. Conventional DNA sequencing was also performed to identify mutations in gyrA and parC and to confirm the biochip data. The study demonstrates that all of the point mutations in the gyrA and parC genes of N. gonorrhoeae were easily discriminated by use of the oligonucleotide biochip. Fifteen different alteration patterns involved in the formation of ciprofloxacin resistance were identified by the biochip assay. Double mutations in both Ser91 and Asp95 of the GyrA protein were seen in all nonsensitive isolates. Double mutations in Ser91 and Asp95 of GyrA plus mutation of Glu91 or Ser87 of the ParC protein lead to significant high-level resistance to ciprofloxacin in N. gonorrhoeae isolates. The results obtained by use of the oligonucleotide biochip were identical to those obtained by use of DNA sequencing. In conclusion, the oligonucleotide biochip technology has potential utility for the rapid and reliable identification of point mutations in the drug resistance genes of N. gonorrhoeae.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Detection of gyrA and parC Mutations Associated with Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of Oligonucleotide Biochip Technology

Zhou

Cao

et al. 2004

J Clin Microbiol

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“…PFGE DNA preparation followed standard procedure, and PFGE analyses were performed following the interpretative procedures described previously (10)(11)(12). The PFGE patterns were analyzed using GelCompar II software program (Applied Maths, Belgium).…”

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confidence: 99%

Prosthetic Valve Endocarditis Due to Neisseria skkuensis, a Novel Neisseria Species

et al. 2012

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eWe describe the first reported case of endocarditis due to Neisseria skkuensis. The organism from the blood cultures taken on admission day was identified initially as unidentified Gram-negative cocci by Vitek2. Finally, it was identified as Neisseria skkuensis by 16 rRNA gene sequence analysis. CASE REPORTA 41-year-old man was admitted to our hospital with a 1-week history of febrile sense, chills, sweating, aggravation of dyspnea, and hypotension during hemodialysis. He had a complicated history, including liver cirrhosis caused by chronic hepatitis B infection and chronic kidney disease due to glomerulonephritis. He had received entecavir since 2009 and hemodialysis since 2000. In addition to these, he had undergone a mechanical mitral valve replacement due to infective endocarditis caused by methicillinresistant Staphylococcus aureus more than 1 year ago. He denied having had dental treatment or drug abuse since mitral valve replacement.On arrival at the emergency department, the patient's vital signs were as follows: blood pressure, 86/52 mm Hg; respiratory rate, 22 breaths per min; and temperature, 37°C. Physical examination revealed metallic heart sounds without murmur and no abdominal tenderness with positive shifting dullness. A chest radiography showed cardiomegaly and pulmonary edema. Laboratory investigations revealed a C-reactive protein concentration of 10.0 mg/dl (normal [N], Ͻ0.3 mg/dl), an erythrocyte sedimentation rate of 37 mm/h (N, Ͻ22 mm/h), and a procalcitonin concentration of 152.2 ng/ml. The white blood cell (WBC) count was 9,850/mm 3 with dominant segmented neutrophils (85%), hemoglobin (Hb) at 7.5 g/dl, platelet count of 57,000/mm 3 , blood urea nitrogen at 49.5 mg/dl, creatinine at 6.55 mg/dl (N, Ͻ1.3 mg/dl), and total bilirubin at 1.2 mg/dl (N, Ͻ1.5 mg/dl). He was coagulopathic with a prothrombin time of 23

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“…Another limitation is the possibility of detecting synonymous mutations, which translate as "false mutants," although the frequency of synonymous mutations at these loci appears to be very low. Outlier mutations have been observed at loci not targeted by our probes (6,19,27,28), but these occur infrequently and have only been observed in the presence of at least one mutation detectable with our probes. We intended to develop an assay to screen for clinically important mutations (i.e., those associated with a ciprofloxacin MIC of Ն4 g/ml, requiring a change in therapy) and not for definitive genetic analysis.…”

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confidence: 99%

Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

et al. 2004

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Mutations in quinolone resistance-determining regions (QRDRs) have been associated with quinoloneresistant Neisseria gonorrhoeae (QRNG). Since diagnostic nucleic acid amplification tests for gonococci are now in frequent use, molecular detection of QRNG could facilitate surveillance in the absence of culture. Here we describe a real-time molecular assay for detecting QRDR mutations in gonococci.Quinolone-resistant Neisseria gonorrhoeae (QRNG) strains are rapidly emerging. Well-characterized quinolone resistancedetermining region (QRDR) mutations correlate with decreased gonococcal antimicrobial susceptibility to fluoroquinolones (MIC, Ն1 g/ml) (2-6, 8-10, 13, 19, 21, 24, 26-28).Using well-characterized isolates, we developed a method for detecting QRDR mutations utilizing Taqman chemistry and the ABI 7900HT Prism sequence detector (Applied Biosystems, Foster City, Calif.).N. gonorrhoeae strains. We evaluated 80 isolates collected in 2000 to 2001 in Israel that were characterized previously (9, 29).DNA isolation, QRDR amplification, and direct sequencing. Genomic DNA was purified (Promega Wizard, Promega Corp., Madison, Wis.), and QRDRs were amplified (11, 12). The forward primers used were GyrA Forward (NG-GYRA-Z; 5Ј-CAAATTCGCCCTCGAAACCCT-3Ј; nucleotides [nt] 30 to 50 of the gyrA gene, 368-bp product) and ParC Forward (NG-PARC-Z; 5Ј-GCCCGTGCAGCGGCGCAT-3Ј; nt 138 to 155 of the parC gene, 219-bp product). Reaction mixtures had a 50-l total volume: 25 l of PCR Master mix, 22 l of sterile water, 1 l each of forward and reverse primers, and 1 l of DNA template. PCR products were sequenced with forward primers, and data were aligned with QRDR sequences for amino acids (aa) 91 to 95 of gyrA (GenBank accession no. U08817) and aa 86 to 92 of parC (GenBank accession no. U08907). The three mutation patterns identified are shown in Table 1.ABI primer and probe design. Primers and probes (Table 2) were developed by using Primer Express 2.0 software (Applied Biosystems, Foster City, Calif.). Probes encompassed aa 91 and 95 of gyrA and aa 86, 87, and 88 of parC, the loci most often associated with resistance (2-6, 8-10, 13, 19, 21, 24, 26-28). Using National Center for Biotechnology Information BLAST analysis, primers and probes were designed to match only gonococcal target sequences.ABI real-time PCR. Diplex PCRs were performed in 96-well plates with the following per well: 25 l of Promega PCR Master mix, 21 l of sterile water, 0.2 l of each set of forward and reverse primers (0.2 M final concentration of each), 0.5 l of each probe (0.2 M final concentration of each), and 1 l of DNA template, for a total reaction volume of 50 l. Cycles included one 2-min hold (50°C); a 10-min denaturation (95°C); and 40 cycles of denaturation (95°C for 30s), annealing (60°C for 30s), and extension (72°C for 30s). Control wells included blanks, wild-type (WT) strains, strains with one mutation, and strains with multiple mutations, as determined by sequencing. The lower-level detection limit was established at five genome copies by using publi...

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Molecular Basis of High-Level Ciprofloxacin Resistance in Neisseria gonorrhoeae Strains Isolated in Denmark from 1995 to 1998

Cited by 47 publications

References 30 publications

Detection of gyrA and parC Mutations Associated with Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of Oligonucleotide Biochip Technology

Detection of gyrA and parC Mutations Associated with Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of Oligonucleotide Biochip Technology

Prosthetic Valve Endocarditis Due to Neisseria skkuensis, a Novel Neisseria Species

Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

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