Integrated Bacterial Identification and Antimicrobial Susceptibility Testing Using PCR and High-Resolution Melt

Abstract

Accurate and timely diagnostics are critical for managing bacterial infections. The current gold standard, culture-based diagnostics, can provide clinicians with comprehensive diagnostic information including bacterial identity and antimicrobial susceptibility, but they often require several days of turnaround time, which leads to compromised clinical outcome and promotes the spread of antibiotic resistance. Nucleic acid amplification tests such as PCR have significantly accelerated the detection of specific bacteria but generally lack the capacities for broad-based bacterial identification or antimicrobial susceptibility testing. Here, we report an integrated assay based on PCR and high-resolution melt (HRM) for rapid diagnosis for bacterial infections. In our assay, we measure bacterial growth in the presence or absence of certain antibiotics with real-time quantitative PCR or digital PCR to determine antimicrobial susceptibility. In addition, we use HRM and a machine learning algorithm to identify bacterial species based on melt-curve profiles of the 16S rRNA gene in an automated fashion. As a demonstration, we correctly identified the bacterial species and their antimicrobial susceptibility profiles for multiple unknown samples in blinded tests within ∼6.5 h.

Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae

“…Another recent work by Athamanolap et al demonstrated the use of ΔCq measurement and high-resolution melt analysis as a way of identifying and testing antimicrobial susceptibility in urinary tract pathogens. 14 While the preceding work highlights the methodology of obtaining ΔCq for drug susceptibility testing, our work presented here drastically simplifies the assay workflow for N. gonorrhoeae detection by incorporating direct-PCR and N. gonorrhoeae -specific primers. The high level of correlation demonstrated against a gold standard comparator is a unique contribution highlighting the potential utility of the direct-qPCR assay for clinical applications.…”

“…Athamanolap and co-workers recently developed an assay for combined identification and AST by targeting the 16S rRNA gene in a panel of urinary tract pathogens including Escherichia coli , Enterococcus faecalis , Proteus mirabilis , and Staphylococcus aureus via PCR and high-resolution melt analysis. 14 Although this approach uniquely enables both species-level identification and phenotypic drug resistance in a single reaction, a high level of agreement with established methods for minimum inhibitory concentration (MIC) determination is necessary to demonstrate clinical utility. An essential component to facilitate this in gonorrhea diagnostics is a robust liquid growth medium for N. gonorrhoeae culture directly from clinical swab specimen, capable of growth under low inoculum and in the presence of nongonococcal flora.…”

Machine Learning-Assisted Digital PCR and Melt Enables Broad Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Test

“…Following relative Tm-based classification, to enable the identification of digital melt curves of interest based on their shapes, we normalize the area under the curve of all digital melt curves and then align them to a single point. Finally, using an adapted in-house developed digital melt curve classification tool based on one-versus-one support vector machine (ovoSVM) algorithm [26,48,53] , the shape of each digital melt curve of interest is compared to the digital melt curves in the Tm-classified group to identify the bacterial species represented by the digital melt curve of interest.…”

“…"Pheno-molecular AST" is an emerging approach that achieves reliable AST and concurrent bacteria detection by combining phenotypic characterization of antibiotic susceptibility with quantitative, nucleic-acids-based molecular detection of bacteria. [22,[40][41][42][43][44][45][46][47][48][49][50] In pheno-molecular AST, bacteria are first briefly incubated in the presence and absence of antibiotics. The amounts of bacterial nucleic acids -serving as surrogates of bacterial growths -between antibiotic-treated samples and no-antibiotic controls are then quantitatively detected and compared to determine the antibiotic susceptibilities.…”

“…Schoepp et al [45,46] developed microfluidic-based, digital pheno-molecular AST assays that shortened the antibiotic exposure time to 15 to 30 min, though they only focused on E. coli as the target organism. Meanwhile, although we and others [47,48] have combined phenomolecular AST with real-time, quantitative universal PCR and HRM analysis to achieve broad identification, these bulk-based assays have a limited ability of analyzing polymicrobial samples because composite melt curves from multiple bacteria species cannot be easily decoupled and resolved.…”