Molecular Characterization of Markers Associated With Antimicrobial Resistance in Neisseria gonorrhoeae Identified From Residual Clinical Samples

Melendez, Johan H.; Hardick, Justin; Barnes, Mathilda; Barnes, Perry; Geddes, Christopher D.; Gaydos, Charlotte A.

doi:10.1097/olq.0000000000000755

Cited by 11 publications

(16 citation statements)

References 30 publications

(51 reference statements)

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“…Two-hundred microliters of each bacterial suspension were extracted for DNA using the automated MagNA Pure LC instrument (Roche Diagnostics, Indianapolis, IN, USA). Following DNA extraction, the isolates were confirmed as NG using a previously described PCR assay targeting the OPA gene [ 11 , 12 ].…”

Section: Methodsmentioning

confidence: 99%

“…In order to elucidate the molecular mechanism associated with resistance to fluoroquinolones and penicillin, all isolates were tested with two previously described real-time PCR assays. A PCR assay targeting GyrA wild-type sequences was used to detect mutations associated with ciprofloxacin resistance [ 12 , 19 ]. The presence of a plasmid mediating resistance to penicillin was ascertained using a real-time PCR assay [ 20 ] as previously described [ 12 ].…”

Section: Methodsmentioning

confidence: 99%

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Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates in Baltimore, Maryland, 2016: The Importance of Sentinel Surveillance in the Era of Multi-Drug-Resistant Gonorrhea

et al. 2018

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The increasing rates of gonorrhea infections and the global emergence and spread of multi-drug-resistant Neisseria gonorrhoeae (NG) threaten the successful management of gonorrhea. In the era of nucleic acid amplification tests (NAATs), surveillance projects are urgently needed to monitor prevalence and trends in the antimicrobial susceptibility of NG. In this study, we retrospectively determined the susceptibility profile of NG isolates to previously and currently prescribed antimicrobials. NG isolates collected in Baltimore, Maryland between January and October 2016 were evaluated by the E-test method and/or molecular methods for susceptibility to ceftriaxone, azithromycin, ciprofloxacin, tetracycline, gentamicin, and penicillin. One-hundred and forty-three NG isolates from African-American males (98.6%), primarily heterosexual (88.8%), ranging in age from 15 to 69 years of age were included in the study. Ciprofloxacin resistance was observed in 44.1% of isolates. Plasmid-mediated resistance to penicillin and tetracycline resistance was detected in 22.4% and 10.1% of isolates, respectively. Three isolates (2.1%) displayed high-level resistance to azithromycin (minimum inhibitory concentration (MIC) > 256 μg/mL). Forty-three percent of isolates were resistant or had decreased susceptibility to three antimicrobials (ciprofloxacin, tetracycline, and penicillin). All isolates were susceptible to ceftriaxone and gentamicin. Overall, the epidemiology of antimicrobial resistant NG in Baltimore continues to evolve, and the emergence of azithromycin resistance in this population emphasizes the need for continued sentinel surveillance programs to monitor susceptibility trends and aid in treatment recommendations.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates in Baltimore, Maryland, 2016: The Importance of Sentinel Surveillance in the Era of Multi-Drug-Resistant Gonorrhea

et al. 2018

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“…Two-hundred microliters of each bacterial suspension was extracted for DNA using the automated MagNA Pure LC instrument (Roche Diagnostics, Indianapolis, IN, USA). Following DNA extraction, the isolates were confirmed as NG using a previously described PCR assay targeting the opa gene [18,19]. For non-viable isolates, 200 µL of the isolate-containing TSB media was extracted for total DNA as described above.…”

Section: Ng Isolatesmentioning

confidence: 99%

“…In order to determine ciprofloxacin susceptibility at the molecular level, all isolates (viable and non-viable) were analyzed by real-time PCR for the presence/absence of mutation(s) in the gyrA gene using a previously described assay [19,20]. The PCR assay targets wildtype gyrA sequences, which are highly predictive of ciprofloxacin susceptibility [9].…”

Section: Molecular Characterization Of Ciprofloxacin Susceptibilitymentioning

confidence: 99%

Can Ciprofloxacin be Used for Precision Treatment of Gonorrhea in Public STD Clinics? Assessment of Ciprofloxacin Susceptibility and an Opportunity for Point-of-Care Testing

et al. 2019

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Background: Given the lack of new antimicrobials to treat Neisseria gonorrhoeae (NG) infections, reusing previously recommended antimicrobials has been proposed as a strategy to control the spread of multi-drug-resistant NG. We assessed ciprofloxacin susceptibility in a large sample set of NG isolates and identified correlates associated with ciprofloxacin-resistant NG infections. Methods: NG isolates collected in Baltimore, Maryland between 2014 and 2016 were evaluated by Gyrase A (gyrA) PCR and E-test for susceptibility to ciprofloxacin. Clinical characteristics and demographics were evaluated by multivariate regression analysis to identify correlates of ciprofloxacin-resistant NG infections. Results: 510 NG isolates from predominately African American (96.5%), heterosexual (85.7%), and HIV-negative (92.5%) male subjects were included in the study. The overall percentage of isolates with mutant gyrA sequences, indicative of ciprofloxacin resistance, was 32.4%, and significantly increased from 24.7% in 2014 to 45.2% in 2016 (p < 0.001). Participants older than 35 years of age were 2.35 times more likely to have a gyrA mutant NG infection than younger participants (p < 0.001). Race, sexual orientation, symptomology, or co-infection the HIV or syphilis were not associated with a particular NG gyrA genotype. Conclusions: Resistance to ciprofloxacin in Baltimore is lower than other regions and indicates that in this environment, use of ciprofloxacin may be appropriate for targeted treatment provided utilization of enhanced surveillance tools. The targeted use of ciprofloxacin may be more beneficial for individuals under 35 years of age. Point-of-care tests for NG diagnosis and susceptibility testing are urgently needed to identify individuals who can be treated with this targeted approach.

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“…(7) Multiple PCR-based assays for resistance markers have been developed, e.g. (8,9), however the diversity of resistance mutations and variants within N. gonorrhoeae make developing comprehensive panels challenging. (7) In contrast, clinical metagenomics has the potential to detect N. gonorrhoeae and any antimicrobial resistance determinants present via direct sequencing of all DNA present in a clinical sample.…”

Section: Introductionmentioning

confidence: 99%

Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples

et al. 2020

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Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

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Molecular Characterization of Markers Associated With Antimicrobial Resistance in Neisseria gonorrhoeae Identified From Residual Clinical Samples

Cited by 11 publications

References 30 publications

Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates in Baltimore, Maryland, 2016: The Importance of Sentinel Surveillance in the Era of Multi-Drug-Resistant Gonorrhea

Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates in Baltimore, Maryland, 2016: The Importance of Sentinel Surveillance in the Era of Multi-Drug-Resistant Gonorrhea

Can Ciprofloxacin be Used for Precision Treatment of Gonorrhea in Public STD Clinics? Assessment of Ciprofloxacin Susceptibility and an Opportunity for Point-of-Care Testing

Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples

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