Abstract
ABSTRACTEscherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and β-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using high-resolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRM-capable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.
Highlights
Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and -lactam resistance
The derivative melt curves from multiplexed highresolution melting (HRM) reactions were compared between ST131 and non-ST131 E. coli
A direct comparison of the derivative melt curves between strains showed that all ST131 isolates shared similar profiles that were distinct from the profiles of non-ST131 isolates
Summary
Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and -lactam resistance. One strain that has emerged as an international multiresistant high-risk clone is sequence type 131 (ST131) Escherichia coli [2] This sequence type has been associated with fluoroquinolone resistance and the global dissemination of CTX-M antibiotic resistance genes and has recently been shown to harbor mcr-1bearing plasmids [3, 4]. Tracking the spread of this particular clonal complex has been made possible through ST identification by whole-genome sequencing (WGS) and multilocus sequence typing (MLST) [7,8,9] These methods, while effective, can be expensive, labor intensive, and time consuming. HRM is a technique used to determine whether two PCR amplicons of similar size have identical sequences This technique is similar to quantitative PCR (qPCR) in that it amplifies a gene target in the presence of a fluorescent reporter dye. The purpose of this study was to develop and validate the single-nucleotide polymorphism (SNP)-level discriminatory power of HRM to enable rapid and cost-effective differentiation of ST131 E. coli from non-ST131 E. coli
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