Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick

Abstract

The worrying emergence of multiple resistance genes to last-resort antibiotics in food animals and human populations throughout the food chain and relevant environments has been increasingly reported worldwide. Enterobacteriaceae pathogens are considered the most common reservoirs of such antibiotic resistance genes (ARGs). Thus, a rapid, efficient and accurate detection method to simultaneously screen and monitor such ARGs in Enterobacteriaceae pathogens has become an urgent need. Our study developed a recombinase polymerase amplification (RPA) assay combined with a lateral flow dipstick (LFD) for simultaneously detecting predominant resistance genes to last-resort antibiotics of Enterobacteriaceae pathogens, including mcr-1, blaNDM-1 and tet(X4). It is allowed to complete the entire process, including crude DNA extraction, amplification as well as reading, within 40 min at 37°C, and the detection limit is 101 copies/μl for mcr-1, blaNDM-1 and tet(X4). Sensitivity analysis showed obvious association of color signals with the template concentrations of mcr-1, blaNDM-1 and tet(X4) genes in Enterobacteriaceae pathogens using a test strip reader (R2 = 0.9881, R2 = 0.9745, and R2 = 0.9807, respectively), allowing for quantitative detection using multiplex RPA-LFD assays. Therefore, the RPA-LFD assay can suitably help to detect multiple resistance genes to last-resort antibiotics in foodborne pathogens and has potential applications in the field.

Antimicrobial resistance and biotechnological potential of plastic‐associated bacteria isolated from an urban estuary

“…Additionally, the bla KPC ‐positive strains were further investigated for the phenotypical production of carbapenemases by modified Hodge test, revealing that seven out of the eight strains were able to produce these enzymes, namely A. hydrophila , A. caviae , and E. bugandensis . Carbapenems and colistin comprise one of the current last‐resort antibiotics and bacterial resistance to these compounds poses a significant risk to effective antimicrobial therapy (Lu et al, 2023). Overall, the detection of mcr variants and carbapenemases in plastic‐associated bacteria isolated from a recreational urban estuary reinforces the urgent need for a holistic approach to antimicrobial resistance and plastic pollution crises.…”

Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein

“…Compared to the SHERLOCKv2 system, the CRISPR-TMSD assay involves additional steps and more reagents. However, the system can be optimized through the implementation of multiplex RPA reactions and multiplex Cas12a cleavage reactions. , Furthermore, in order to avoid contamination associated with lid opening, optimization for a one-pot reaction can be implemented . These enhancements will render the current system to be much more applicable for molecular detection.…”

Editorial: Antimicrobial resistance and one health: from culture to genomics

“…Thus, AMR threatens human, animal, and environmental health globally ( Abia and Essack, 2023 ). Moreover, the widespread misuse and uncontrolled prescription of antibiotics aggravates the problem, diminishing antibiotic efficacy, including last-resort ones ( Lu et al., 2023 ). This problem is accentuated in rural settings in low- and middle-income countries where community members are small-scale farmers, often living with their livestock inside the yards and lacking adequate water and sanitation facilities ( Sulis et al., 2022 ).…”