Sequencing the fused PCR products provides accurate and sensitive associations between 16S sequence taxonomy and a given target gene. Alternatively, epicPCR ( Spencer et al., 2015) uses fusion PCR and two emulsion steps to associate a taxonomic marker with a functional gene. Several targeted methods using single-cell qPCR have been used to identify the hosts of specific genes however, each uses specialized microfluidic devices, is limited in bacterial taxa they can capture, and most do not allow direct sequencing of the PCR products ( Ottesen et al., 2020 Zeng et al., 2010 Tadmor et al., 2011). Culturing, applied broadly to capture the full diversity of the gut microbiome, is complicated by the need for wide-ranging media and growth conditions ( Zou et al., 2019 Poyet et al., 2019). Targeted methods, such as bacterial cell culture under antibiotic selection, require that the ARG is expressed, functional, and selective in all hosts. Alternatively, single-cell whole-genome sequencing offers an ideal solution to this problem, but may be lower throughput, more expensive and require specialized equipment ( Xu et al., 2016 Lan et al., 2017). Novel untargeted sequencing methods, such as bacterial Hi-C ( Kent et al., 2020) and methylation profiling ( Beaulaurier et al., 2018), provide broad reconstruction of plasmid–host relationships in metagenomes, as a trade-off for sensitivity. Standard molecular tools such as PCR and next-generation sequencing often fail to associate mobile ARGs with their bacterial hosts because they cannot capture the cellular context of extrachromosomal genes. ARGs are often carried on mobilizable plasmids that impose technical challenges to surveying the set of bacteria affiliated with these genes. The gut microbiome harbors a diverse repertoire of ARGs, and these genes have been proposed to serve as a reservoir for HGT with MDR pathogens ( Sommer et al., 2009). The emergence of multidrug-resistant (MDR) pathogens is a grave public health threat that occurs when pathogenic bacteria acquire antibiotic-resistant genes (ARGs) through horizontal gene transfer (HGT) with bacteria in their proximal environment. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization.
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