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

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

doi:10.1101/2021.01.22.427834

Cited by 4 publications

(4 citation statements)

References 47 publications

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“…In particular, these methods require a combination of droplet reinjection, splitting, and merger steps which have high failure rates and are difficult to implement without extensive droplet microfluidics expertise 9,18,19 . As a result, compromises in efficiency are typically made to achieve cell lysis and molecular reactions simultaneously in a one-pot reaction 14 .…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand, emulsion PCR based methods, where multiple PCR amplicons from single cells are fused by PCR into a single amplicon for sequencing 12 , have been used to link genes of interest within cells in microbial populations 13,14 . However, a major limitation of these methods is that the linked amplicon is only generated when all target sequences of interest are present in the cell and correctly amplified.…”

Section: Introductionmentioning

confidence: 99%

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Massively parallel single-cell sequencing of genetic loci in diverse microbial populations

Lan

Saba

Ross

et al. 2022

Preprint

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Single cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology. However, we lack a broadly applicable and accessible method to study this heterogeneity at the single cell level. Here, we introduce a simple, robust, and generalizable platform for quantitative and massively parallel single cell sequencing of target genetic loci in microbes using ultrahigh-throughput droplet microfluidics (Droplet Targeted Amplicon Sequencing or DoTA-seq). Using DoTA-seq, we elucidate the highly diverse single cell ON/OFF states of the phase-variable capsule synthesis operons in the prevalent human gut species Bacteroides fragilis. In addition, we quantify the shifts in antibiotic resistance gene abundances in different species in a 25 member human gut microbial community in response to antibiotics. By sequencing tens of thousands of single-cells derived from a human fecal sample, we identify links between plasmid replicons and the taxonomic lineages of their associated hosts. In sum, DoTA-seq is an accessible and broadly applicable tool for profiling single-cell genetic variation in microbiomes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Massively parallel single-cell sequencing of genetic loci in diverse microbial populations

Lan

Saba

Ross

et al. 2022

Preprint

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“…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 nonhuman 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.…”

Section: Limitations Of Wastewater Surveillance and Future Directionmentioning

confidence: 99%

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

et al. 2022

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Infectious diseases caused by antibiotic-resistant bacterial (ARB) pathogens are a serious threat to human and animal health. The active surveillance of ARB using an integrated one-health approach can help to reduce the emergence and spread of ARB, reduce the associated economic impact, and guide antimicrobial stewardship programs. Wastewater surveillance (WWS) of ARB provides composite samples for a total population, with easy access to the mixed community microbiome. This concept is emerging rapidly, but the clinical utility, sensitivity, and uniformity of WWS of ARB remain poorly understood especially in relation to clinical evidence in sewershed communities. Here, we systematically searched the literature to identify studies that have compared findings from WWS of ARB and antibiotic resistance genes (ARG) with clinical evidence in parallel, thereby evaluating how likely WWS of ARB and ARG can relate to the clinical cases in communities. Initially, 2,235 articles were obtained using the primary search keywords, and 1,219 articles remained after de-duplication. Among these, 35 articles fulfilled the search criteria, and an additional 13 relevant articles were included by searching references in the primary literature. Among the 48 included papers, 34 studies used a culture-based method, followed by 11 metagenomics, and three PCR-based methods. A total of 28 out of 48 included studies were conducted at the single sewershed level, eight studies involved several countries, seven studies were conducted at national or regional scales, and five at hospital levels. Our review revealed that the performance of WWS of ARB pathogens has been evaluated more frequently for Escherichia coli, Enterococcus spp., and other members of the family Enterobacteriaceae, but has not been uniformly tested for all ARB pathogens. Many wastewater-based ARB studies comparing the findings with clinical evidence were conducted to evaluate the public health risk but not to relate with clinical evidence and to evaluate the performance of WWS of ARB. Indeed, relating WWS of ARB with clinical evidence in a sewershed is not straightforward, as the source of ARB in wastewater cannot be only from symptomatic human individuals but can also be from asymptomatic carriers as well as from animal sources. Further, the varying fates of each bacterial species and ARG within the sewerage make the aim of connecting WWS of ARB with clinical evidence more complicated. Therefore, future studies evaluating the performance of many AMR pathogens and their genes for WWS one by one can make the process simpler and the interpretation of results easier.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Dutra

Jalasvuori

Franz

et al. 2022

Preprint

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Microbial communities often respond to various challenges, such as the presence of antibiotics, as a whole. Dissecting these community-level effects into separate acting entities requires the identification of organisms that carry functional genes for the observed feature. However, unculturable microbes abound in various environments, hence making the identification challenging. Here we present a cultivation-free technique that can be utilized to link functional genes with carrying bacterial species at single-cell resolution. The developed protocol is relatively simple to use, utilizes commercially available droplet microfluidics devices, does not require toxic reagents (as compared to some previous methods), eliminates invalid signals emerging from extracellular DNA, and allows simultaneous analysis of community diversity via 16S rRNA gene sequencing. The method can be customized for any given genetic trait to accurately identify its hosting subpopulation from a heterogeneous and potentially uncultivable bacterial community.

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

Cited by 4 publications

References 47 publications

Massively parallel single-cell sequencing of genetic loci in diverse microbial populations

Massively parallel single-cell sequencing of genetic loci in diverse microbial populations

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

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

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