Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR.

Peter J Diebold,Michael Hovan,Felicia N New,Ilana L Brito,Michael J Satlin

doi:10.7554/elife.66834

Abstract

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. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. 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. 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. 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. 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. 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.

Highlights

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
The gut microbiome harbors a diverse repertoire of ARGs 31 and these genes have been proposed to serve as a reservoir for HGT with MDR pathogens[1]
One-step Isolation and Lysis PCR (OIL-PCR) applies established fusion PCR methods to fuse any gene of interest to the 16S rRNA gene using three primers: two primers hybridize to the target gene, and a universal 16S reverse primer hybridizes to the V4 region

Summary

Introduction

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. ARGs are often carried on mobilizable plasmids that impose technical challenges to surveying the set of bacteria affiliated with these genes 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. Single-cell whole genome sequencing offers an ideal solution to this problem, but may be lower throughput, more expensive and require specialized equipment[4,5] Targeted methods, such as bacterial cell culture under antibiotic selection, require that the ARG is expressed, functional, and selective in all hosts. Sequencing the fused PCR products provides accurate and sensitive associations between 16S sequence taxonomy and a given target gene This method can be challenging to execute, difficult to scale up for multiple samples, and utilizes toxic and difficult-to-acquire reagents. We 57 demonstrate the utility of this approach in examining the bacterial hosts of three extended spectrum beta[58] lactamase genes in the gut of neutropenic patients

Results

Methods

Computational Methods