Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains
ABSTRACT Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections. Vancomycin intermediate S. aureus (VISA) variants can arise th...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-02-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02728-22 |
| _version_ | 1811164351631982592 |
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| author | Abraham G. Moller Robert A. Petit Michelle H. Davis Timothy D. Read |
| author_facet | Abraham G. Moller Robert A. Petit Michelle H. Davis Timothy D. Read |
| author_sort | Abraham G. Moller |
| collection | DOAJ |
| description | ABSTRACT Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections. Vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here, we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. Primers were designed to generate 10 amplicons covering 16 genes associated with the VISA phenotype. We sequenced amplicon pools as long reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. Each amplicon in the pool was sequenced to high (>1,000×) coverage, and no relationship was found between amplicon length and coverage. We also were able to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of the average minor allele frequency (6.5%) at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic resistance genotype inference using rapid nanopore sequencing from clinical samples. IMPORTANCE Bacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia that is also commonly antibiotic resistant. In this work, we develop a method to accelerate detection of a complex, polygenic antibiotic resistance phenotype in S. aureus, vancomycin-intermediate resistance (VISA), through long-read genomic sequencing of amplicons representing genes most commonly mutated in VISA selection. This method both rapidly identifies VISA genotypes and incorporates the most comprehensive database of VISA genetic determinants known to date. |
| first_indexed | 2024-04-10T15:20:14Z |
| format | Article |
| id | doaj.art-376d62c5359e46f696d2af205e123962 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-10T15:20:14Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-376d62c5359e46f696d2af205e1239622023-02-14T14:15:51ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-02-0111110.1128/spectrum.02728-22Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus StrainsAbraham G. Moller0Robert A. Petit1Michelle H. Davis2Timothy D. Read3Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia, USADivision of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia, USADivision of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia, USADivision of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia, USAABSTRACT Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections. Vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here, we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. Primers were designed to generate 10 amplicons covering 16 genes associated with the VISA phenotype. We sequenced amplicon pools as long reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. Each amplicon in the pool was sequenced to high (>1,000×) coverage, and no relationship was found between amplicon length and coverage. We also were able to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of the average minor allele frequency (6.5%) at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic resistance genotype inference using rapid nanopore sequencing from clinical samples. IMPORTANCE Bacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia that is also commonly antibiotic resistant. In this work, we develop a method to accelerate detection of a complex, polygenic antibiotic resistance phenotype in S. aureus, vancomycin-intermediate resistance (VISA), through long-read genomic sequencing of amplicons representing genes most commonly mutated in VISA selection. This method both rapidly identifies VISA genotypes and incorporates the most comprehensive database of VISA genetic determinants known to date.https://journals.asm.org/doi/10.1128/spectrum.02728-22Staphylococcus aureusamplicon sequencingantibiotic resistanceclinical microbiologydiagnosticsgenomics |
| spellingShingle | Abraham G. Moller Robert A. Petit Michelle H. Davis Timothy D. Read Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains Microbiology Spectrum Staphylococcus aureus amplicon sequencing antibiotic resistance clinical microbiology diagnostics genomics |
| title | Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains |
| title_full | Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains |
| title_fullStr | Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains |
| title_full_unstemmed | Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains |
| title_short | Development of an Amplicon Nanopore Sequencing Strategy for Detection of Mutations Conferring Intermediate Resistance to Vancomycin in Staphylococcus aureus Strains |
| title_sort | development of an amplicon nanopore sequencing strategy for detection of mutations conferring intermediate resistance to vancomycin in staphylococcus aureus strains |
| topic | Staphylococcus aureus amplicon sequencing antibiotic resistance clinical microbiology diagnostics genomics |
| url | https://journals.asm.org/doi/10.1128/spectrum.02728-22 |
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