Quantification of the mobility potential of antibiotic resistance genes through multiplexed ddPCR linkage analysis

There is a clear need for global monitoring initiatives to evaluate the risks of antibiotic resistance genes (ARGs) towards human health. Therefore, not only ARG abundances within a given environment, but also their potential mobility, hence their ability to spread to human pathogenic bacteria needs to be quantified. We developed a novel, sequencing-independent method for assessing the linkage of an ARG to a mobile genetic element by statistical analysis of multiplexed droplet digital PCR (ddPCR) carried out on environmental DNA sheared into defined, short fragments. This allows quantifying the physical linkage between specific ARGs and mobile genetic elements, here demonstrated for the sulfonamide ARG sul1 and the Class 1 integron integrase gene intI1. The method's efficiency is demonstrated using mixtures of model DNA fragments with either linked and unlinked target genes: Linkage of the two target genes can be accurately quantified based on high correlation coefficients between observed and expected values (R2) as well as low mean absolute errors (MAE) for both target genes, sul1 (R2 = 0.9997, MAE = 0.71%, n = 24) and intI1 (R2 = 0.9991, MAE = 1.14%, n = 24). Furthermore, we demonstrate that adjusting the fragmentation length of DNA during shearing allows controlling rates of false positives and false negative detection of linkage. The presented method allows rapidly obtaining reliable results in a labor- and cost-efficient manner.