Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes
ABSTRACT With looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MGparC-AsyHRM, based on the com...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-08-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.01014-22 |
| _version_ | 1811188299724750848 |
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| author | Yamei Li Leshan Xiu Liqin Wang Lulu Zhang Feng Wang Junping Peng |
| author_facet | Yamei Li Leshan Xiu Liqin Wang Lulu Zhang Feng Wang Junping Peng |
| author_sort | Yamei Li |
| collection | DOAJ |
| description | ABSTRACT With looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetric high-resolution melting (HRM) technology and unlabeled probes, which simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. These enhancements expand the traditional HRM from the conventional detection of single-position mutations to a method capable of detecting short fragments with closely located AMR positions with a high diversity of mutations. Based on the results of clinical sample testing, this method produces an accordance of 98.7% with the Sanger sequencing method. Furthermore, the specificity for detecting S83I, S83N, S83R, and D87Y variants, the most frequently detected mutations in fluoroquinolone resistance, was 100%. This method maintained a stable and accurate performance for genomic copies at rates of ≥20 copies per reaction, demonstrating high sensitivity. Additionally, no specific cross-reactions were observed when testing eight common sexually transmitted infection (STI)-related agents. Notably, this work highlights the significant potential of our method in the field of AMR testing, with the results suggesting that our method can be applied in a range of scenarios and to additional pathogens. In summary, our method enables high throughput, provides excellent specificity and sensitivity, and is cost-effective, suggesting that this method can be used to rapidly monitor the molecular AMR status and complement current AMR surveillance. IMPORTANCE Mycoplasma genitalium was recently added to the antimicrobial-resistant (AMR) threats “watch list” of the U.S. Centers for Disease Control and Prevention because this pathogen has become extremely difficult to treat as a result of increased resistance. M. genitalium is also difficult to culture, and therefore, molecule detection is the only method available for AMR testing. In this work, we developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetrical HRM technology and unlabeled probes, and it simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. The MGparC-AsyHRM method is a high-throughput, low-cost, simple, and culture-free procedure that can enhance public health and management of M. genitalium infections and AMR control, providing a strong complement to phenotypic AMR surveillance to address the spread of fluoroquinolone resistance. |
| first_indexed | 2024-04-11T14:17:18Z |
| format | Article |
| id | doaj.art-a07195a6ea844aaea69d7b0679254ada |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-11T14:17:18Z |
| publishDate | 2022-08-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-a07195a6ea844aaea69d7b0679254ada2022-12-22T04:19:14ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-08-0110410.1128/spectrum.01014-22Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled ProbesYamei Li0Leshan Xiu1Liqin Wang2Lulu Zhang3Feng Wang4Junping Peng5NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaSchool of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaShenzhen Center for Chronic Disease Control, Shenzhen, ChinaNHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, ChinaABSTRACT With looming resistance to fluoroquinolones in Mycoplasma genitalium, public health control strategies require effective antimicrobial resistance (AMR) diagnostic methods for clinical and phenotypic AMR surveillance. We developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetric high-resolution melting (HRM) technology and unlabeled probes, which simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. These enhancements expand the traditional HRM from the conventional detection of single-position mutations to a method capable of detecting short fragments with closely located AMR positions with a high diversity of mutations. Based on the results of clinical sample testing, this method produces an accordance of 98.7% with the Sanger sequencing method. Furthermore, the specificity for detecting S83I, S83N, S83R, and D87Y variants, the most frequently detected mutations in fluoroquinolone resistance, was 100%. This method maintained a stable and accurate performance for genomic copies at rates of ≥20 copies per reaction, demonstrating high sensitivity. Additionally, no specific cross-reactions were observed when testing eight common sexually transmitted infection (STI)-related agents. Notably, this work highlights the significant potential of our method in the field of AMR testing, with the results suggesting that our method can be applied in a range of scenarios and to additional pathogens. In summary, our method enables high throughput, provides excellent specificity and sensitivity, and is cost-effective, suggesting that this method can be used to rapidly monitor the molecular AMR status and complement current AMR surveillance. IMPORTANCE Mycoplasma genitalium was recently added to the antimicrobial-resistant (AMR) threats “watch list” of the U.S. Centers for Disease Control and Prevention because this pathogen has become extremely difficult to treat as a result of increased resistance. M. genitalium is also difficult to culture, and therefore, molecule detection is the only method available for AMR testing. In this work, we developed a novel AMR detection method, MGparC-AsyHRM, based on the combination of asymmetrical HRM technology and unlabeled probes, and it simultaneously performs M. genitalium identification and genotypes eight mutations in the parC gene that are responsible for most cases of fluoroquinolone resistance. The MGparC-AsyHRM method is a high-throughput, low-cost, simple, and culture-free procedure that can enhance public health and management of M. genitalium infections and AMR control, providing a strong complement to phenotypic AMR surveillance to address the spread of fluoroquinolone resistance.https://journals.asm.org/doi/10.1128/spectrum.01014-22antimicrobial resistanceMycoplasma genitaliumunlabeled probehigh-resolution melting technology |
| spellingShingle | Yamei Li Leshan Xiu Liqin Wang Lulu Zhang Feng Wang Junping Peng Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes Microbiology Spectrum antimicrobial resistance Mycoplasma genitalium unlabeled probe high-resolution melting technology |
| title | Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes |
| title_full | Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes |
| title_fullStr | Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes |
| title_full_unstemmed | Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes |
| title_short | Rapid Detection of Antimicrobial Resistance in Mycoplasma genitalium by High-Resolution Melting Analysis with Unlabeled Probes |
| title_sort | rapid detection of antimicrobial resistance in mycoplasma genitalium by high resolution melting analysis with unlabeled probes |
| topic | antimicrobial resistance Mycoplasma genitalium unlabeled probe high-resolution melting technology |
| url | https://journals.asm.org/doi/10.1128/spectrum.01014-22 |
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