A rapid and simple detection method for phenotypic antimicrobial resistance in Escherichia coli by loop-mediated isothermal amplification

Abstract

In infectious disease therapy, administration of adequate antimicrobial agents is essential for preventing the emergence and spread of resistant bacteria. However, conventional antimicrobial susceptibility testing (AST), based on bacterial growth, is time consuming; therefore, a rapid, simple assay is needed for the timely selection of appropriate antibiotics in clinical laboratories. Here, we established a simple, cost-effective, time-saving and highly sensitive AST assay based on loop-mediated isothermal amplification (LAMP). The targeted bacteria were cultivated for a short period with or without antibiotic before the LAMP reaction. The time to detect a positive reaction with LAMP was used to generate a threshold time (Tt) value, and subtraction of the Tt value for an antibiotic-free sample from the Tt value in an antibiotic-exposed sample generated the ΔTt value, which was used as a marker of antimicrobial susceptibility. The ΔTt value generated using the LAMP-based assay simply and quickly detected antimicrobial resistance in clinical Escherichia coli isolates. Detection of susceptibility to levofloxacin using the ΔTt value perfectly matched with the results of the conventional assay. In addition, the sensitivity and specificity for the detection of ampicillin, trimethoprim-sulfamethoxazole and fosfomycin resistance were 100 %, 93.8 %, 100 % and 80.0 %, 93.3 %, 97.6 %, respectively. These results showed that this LAMP-based AST has high sensitivity and specificity for detecting resistant strains and a significant time advantage compared with the conventional method.