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 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.

Keywords: Staphylococcus aureus; amplicon sequencing; antibiotic resistance; clinical microbiology; diagnostics; genomics; nanopore sequencing; vancomycin resistance; vancomycin-intermediate Staphylococcus aureus.

FIG 1

Gene coverage analyses for simulation (blue), DNA (amplicon; green), and cell (culture; red) parent/mutant mixtures. Average coverage was calculated for each gene and condition based on SAMtools mpileup counts of aligned bases in each gene. (A to C) Coverage distributions represent average coverages for all samples separated by mixture type (simulation, DNA, or cells) and amplicon (A) or just mixture type (B and C). (A) Coverage distribution for each of the 10 amplicons visualized as a violin plot; coverage for each amplicon is compared among mixture sets with a nonparametric Wilcoxon test. (B) Coverage distribution for each mixture set (simulation, DNA, or cell) visualized as a violin plot; coverage is compared between mixture sets with a nonparametric Wilcoxon test. (C) Coverage distribution against amplicon length for each mixture set presented with correlation and P value.

FIG 2

Evaluating the limit of detection over a range of mutant/parent mixes (cultures, amplicon DNA, and simulated sequence reads). (A) Introduced mutant proportion (x axis) versus detected mutation proportion (y axis) for culture (olive-green), amplicon DNA (blue-green), or simulated (purple) sequence read mixtures along with the diagonal expected if the introduced mutant proportion matched the detected mutation proportion (defined here as a “perfect” correlation; red). (B) Introduced mutant proportion (x axis) versus detected mutation Z-score (y axis) for cell (red), amplicon DNA (green), or simulated (blue) sequence read mixtures.

FIG 3

Overview of the VISA amplicon sequencing process, from sample isolation to SNP calling and comparison to known VISA mutations. Steps 1 to 5 represent sample isolation, DNA extraction, PCR, nanopore sequencing, and variant calling, respectively. In step 1 bacteria are grown in a blood bottle (left), from which multiple colonies are cultured (right), including both the two individual VISA mutants (red and green) and the VSSA parental strain (blue). Step 2 represents DNA extraction from the pooled colonies cultured at the end of step 1, while step 3 represents PCR amplification of VISA amplicons from the extracted bacterial DNA. Step 4 shows the loading of an amplicon nanopore library on an Oxford Nanopore MinION instrument. Finally, step 5 shows the calling of the two VISA mutations in the sequenced nanopore library against reference gene sequences. Two VISA mutations are illustrated in red and green (steps 2, 3, and 5), while their parental strain is in blue.