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

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 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.

Wastewater surveillance of antibiotic-resistant bacterial pathogens: A systematic review

“…The detection of ARB and related genes in wastewater cannot indicate the possible source of contamination. However, monitoring ARB together with host-specific primers ( Hultman et al, 2018 ; Diebold et al, 2021 ), and using microbial source tracking methods may help in predicting the load from non-human sources ( Green et al, 2014 ; Harwood et al, 2014 ; Rytkönen et al, 2021 ). Detection of such sources is important for the remediation of contamination and can be helpful for the actual estimation of the prevalence of ARB at a population level.…”

Modulating Gene Expression within a Microbiome Based on Computational Models

“…Each sequence in the set will be engineered to have poor expression in the unwanted hosts organisms while having greater expression levels in the wanted hosts organisms (as seen in Figure A4 ), strengthening the ability of this tool to scale up and operate on more complex microbiomes. Validations: The OIL-PCR method by Peter J. Diebold et al [ 37 ] enables quantitative evaluation of bacteria carrying the plasmid, which can be used in order to assess the GOI abundance, we intend to extend this approach to integrate expression as well using the same concepts only relying on fusion-PCR of 16s rRNA genes and the mRNA products of GOI (detailed in Figure A5 ). This proposed method can be used to determine the overall effectiveness of the engineering technique in a real microbiome.…”

“…Validations: The OIL-PCR method by Peter J. Diebold et al [ 37 ] enables quantitative evaluation of bacteria carrying the plasmid, which can be used in order to assess the GOI abundance, we intend to extend this approach to integrate expression as well using the same concepts only relying on fusion-PCR of 16s rRNA genes and the mRNA products of GOI (detailed in Figure A5 ). This proposed method can be used to determine the overall effectiveness of the engineering technique in a real microbiome.…”

Single-cell resolution genetic association analysis of heterogeneous bacterial communities by utilizing droplet digital PCR

“…The advantage of the QX200 system is that, in addition to droplet fluorescence measurement, it also inspects droplet shape and size and excludes abnormal droplets from the analysis. In comparable methods, such as in epicPCR and OIL-PCR (Spencer et al 2016;Diebold et al . CC-BY-NC-ND 4.0 International license perpetuity.…”

“…The advantage of the QX200 system is that, in addition to droplet fluorescence measurement, it also inspects droplet shape and size and excludes abnormal droplets from the analysis. In comparable methods, such as in epicPCR and OIL-PCR (Spencer et al 2016;Diebold et al 2021), partitions of various sizes are formed, hence producing compartments with various ratios of PCR reagents to the target DNA. This, in turn, may result in notably differing amounts of amplified DNA that are produced from a single target cell.…”