Thirty-minute screening of antibiotic resistance genes in bacterial isolates with minimal sample preparation in static self-dispensing 64 and 384 assay cards

• Published in: Applied microbiology and biotechnology Vol. 99; no. 18; pp. 7711 - 7722
• Main Authors: Kostić, Tanja, Ellis, Michael, Williams, Maggie R, Stedtfeld, Tiffany M, Kaneene, John B, Stedtfeld, Robert D, Hashsham, Syed A
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2015; Springer; Springer Nature B.V
• Subjects: Analysis; Antibiotic resistance; Antibiotics; Automation; Bacteria; Bacterial genetics; Bacteriological Techniques - methods; Biomedical and Life Sciences; Biotechnology; Design; Drug resistance; Drug resistance in microorganisms; Drug Resistance, Bacterial; Genes; Genes, Bacterial; Genetic aspects; Genetic testing; Genetic Testing - methods; Genetics; Health aspects; Infections; Life Sciences; loop-mediated isothermal amplification; Methods and Protocols; Microbial Genetics and Genomics; Microbial Sensitivity Tests - methods; Microbiology; Nucleic Acid Amplification Techniques - methods; quantitative polymerase chain reaction; Sample preparation; screening; Staphylococcus aureus; Staphylococcus aureus - genetics; Staphylococcus infections; Studies; Time Factors; Tuberculosis; United States; Austria; Gene-Z; Rapid genetic testing; LAMP; Point of care; Antibiotic resistance
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6774-z

In a clinical setting, molecular assays such as polymerase chain reaction offer a rapid means to infer or confirm identity and therapeutic decisions. Accordingly, a number of molecular assays targeting identity and antibiotic resistance (AR) genes have been developed; however, these methods can be technically complex and relatively expensive. Herein, we describe a diagnostic concept utilizing isothermal amplification technology with non-purified heat-lysed cells and self-dispensing cards for testing multiple primers in parallel. This proof-of-concept study, performed with Staphylococcus aureus isolates and associated AR genes, was compared with culture-based susceptibility and quantitative PCR (qPCR). Results demonstrate reduced sample processing steps resulting in a turnaround time (starting from bacterial culture to ending in the antibiotic resistance gene profile) in less than 30 min. For antibiotics tested in which an associated AR gene was targeted on the Gene-Z card, 69 % (18/26) of culture-based resistance events were positive for related AR genes. A comparison of loop-mediated isothermal amplification (LAMP) and qPCR assays targeting the same antibiotic resistance genes showed a 98.2 % agreement in terms of presence and absence calls. Identity-based discrepancies between conventional (phenotypic) and molecular (genotypic) results were further resolved, and we were able to demonstrate higher accuracy in identification with the molecular analysis.