Download plasmid capture

MinION Sequencing of a Captured
Antibiotic Resistance Plasmid
Kevin Libuit*, Curtis Kapsak*, Erica F. Gehr*, Stephen Turner**, and James B. Herrick*
*James Madison University, Harrisonburg, VA
**University of Virginia, Charlottesville, VA
ABSTRACT
METHODS (cont.)
Plasmids in agriculturally-impacted bodies of water may play a significant role in
the dissemination of antibiotic resistance (AR). Previously, our lab captured
environmental plasmids without cultivation of host bacteria from stream sediment into
Escherichia coli. Individual plasmids were capable of conferring resistance to a
surprising array of antibiotics including aminoglycosides and extended-spectrum βlactams. In this investigation, our aim was to use high-throughput genome
sequencing to determine the genotypes responsible for the resistance phenotypes.
Plasmids were sequenced using both MinION and Ion Torrent PGM sequencers. An
assembly derived from only nanopore data was polished with Ion PGM reads,
correcting ~2000 sites. Known AR genes detected in assembled reads encoded
resistance to tetracycline, sulfonamides, aminoglycosides, fluoroquinolones, & betalactams. These results suggest that there may be a significant reservoir of AR genes
in streams capable of transmission to pathogenic Enterobacteriaceae.
METHODS
PLASMID CAPTURE
- Sediment samples taken from a stream heavily impacted by farm runoff
- Plasmids “captured” exogenously into E. coli (Fry and Day, 1990) (Fig. 1)
- Plasmids electroporated into 2nd E. coli strain and tested for decrease in
susceptibility to 12 antibiotics via Stokes Method (Collins, et al., 1989)
- Plasmid pEx 1-20 DNA extracted, visualized and quantified using a protocol
developed in our laboratory (Heringa et al., 2007) (Fig. 2)
SEQUENCING DATA ANALYSIS
Reads from both sequencers utilized for assembly, polishing and annotation (Fig. 5)
Annotation with
prokka
Manual Annotation
Figure 5. Sequencing and data analysis pipeline. Plasmid pEx1-20 was sequenced using
MinION and PGM DNA sequencers. MinION reads were assembled into a single contig (PBcR)
and polished with PGM reads (PiLON). Automated annotation was performed using prokka,
followed by further manual annotation of predicted CDS.
RESULTS
ANTIBIOTIC SUSCEPTIBILITY
- pEx1-20 conferred clinical levels of resistance to the following antibiotics:
tetracycline (tet), ticarcillin (tic), piperacillin (pip), kanamycin (kan),
& ciprofloxacin (cip)
ASSEMBLY AND POLISHING
Figure 1. Exogenous capturing of tetracycline resistance
plasmids from sediment. Plasmids captured from stream
sediment samples by releasing native cells from sediment and
conjugating with a rifampicin-resistant strain of E. coli.
Transconjugants selected on tetracycline-and rifampicinamended medium. Plasmids purified and electroporated into an
electrocompetent E. coli strain and tested for decreased antibiotic
susceptibility to 12 antibiotics relative to un-electroporated strain.
- Nanopore-only assembly to produce a single 91kb contig
- Polishing w/ PGM data corrected ~2,000 sites (mostly SNPs/small indels)
- Assembly circular with unresolved complex repeats at ends
GENE ANNOTATION
- Automated Annotation (prokka): 140 predicted CDS; 36 Annotated;
104 Hypothetical Proteins
- Manual Annotation of predicted CDS: 74 Annotations added
- Putative regions identified (Fig. 6)
- 23 AR genes related to antibiotic resistance identified
Figure 2. Plasmid Preparation, Visualization, and DNA Quantification
Plasmid preparation procedure developed in our laboratory for the isolation of large, native, single-copy plasmids
was used to isolate multi-drug resistant plasmids for DNA sequencing
Outline of Plasmid Extraction Protocol
1. Electroporated E. coli cells grown in TSA broth overnight
2. 1.5 – 2ml cells pelleted by centrifugation, supernatant
discarded
3. Pellet resuspended in 100uL resuspension buffer (50mM
glucose/10mM EDTA/10mM Tris-Cl, pH 8.0)
4. 200uL lysis solution (0.2M NaOH/1% sodium dodecyl
sulfate [SDS]) added, mixed by inversion, incubated for
five minutes at room temperature
5. 150uL 7.5M ammonium acetate and 150uL chloroform
added, mixed by inversion, chilled on ice 10 minutes
6. Centrifuged at 10,000g 10 minutes, supernatant
transferred to 200uL precipitation solution (30%
polyethylene glycol 8000/1.5M NaCl), mixed by
inversion, chilled on ice 15 minutes
7. Centrifuged at 10,000g 15 minutes, supernatant
removed, resuspended in 100uL TE
8. Plasmid DNA treated with PlasmidSafe exonuclease
(Epicentre) according to manufacture’s protocol.
L
2
3
Linear DNA
25kb
Relaxed plasmid
Supercoiled plasmid
Plasmid Backbone
Function & Stability
Stability & Inheritance
Type VI Secretion
RNA
Genetic Load
Integrons
Plasmid DNA visualized on 0.7% Agarose gel
with GelRed (Biotium) before and after
Plasmid Safe treatment (Lanes 2 and 3,
respectively; L: 1kb ladder).
Transposons
PLASMID SEQUENCING & DATA PROCESSING
- Plasmid sequenced on the Ion Torrent PGM according to manufacturer’s protocol
- MinION library preparation and sequencing performed according to manufacturer’s
protocol (SQK-005) with one alteration: DNA sheared using
a low-cost restriction enzyme fragmentation method (Fig 3)
- Nanopore MinION data: extracted passing 2D reads using poretools
- Ion PGM data: filtered using ea-utils
for read lengths 100-400 bp,
Figure 3. Alternative fragmentation of gDNA
using restriction enzyme Sau3Ai
removing reads with ≥4 Ns and avg.
Plasmid DNA was fragmented without the use of the
Q<25 (Fig. 4)
Figure 6. Putative regions of the IncP-1b multiresistance plasmid pEx1-20. Both Prokka and manual
annotations were used to determine the putative regions of the plasmid backbone and genetic load. The
functional modules of the backbone include mate-pair formation & conjugative transfer (green) regulation
of plasmid function & stability inheritance (yellow) and a type VI secretion system (orange). The genetic
load includes two integrons (blue) and two transposons (red). Antibiotic resistance genes are marked in
purple arrows. The inner gray circle of the map indicates GC% throughout the plasmid with the dotted line
representing 50% GC content.
CONCLUSIONS
recommended g-TUBE (Covaris)
L
20kb
5kb
300bp
Figure 4: NGS read stats. Top: nanopore
throughput & length; bottom: Ion PGM
quality distribution.
2
3
A. Partial Digestion Protocol
1. 1U of Sau3Ai added per 200uL
plasmid DNA
2. Mixture incubated at RT for 1m
3. Enzyme immediately
inactivated at 70C
4. Fragmented DNA purified and
concentrated using 1 volume
equivalent of Agencourt
AMPure XP Beads (Beckmen
Coulter)
5. DNA quantified using Qubit BR
dsDNA kit (Thermofisher
Scientific)
Fragmented DNA visualized on 0.7%
Agarose gel with GelRed (Biotium) before
and after partial digestion (Lanes 2 and 3,
respectively; L: 1kb ladder).
- DNA sequencing revealed genes encoding resistance to multiple clinical antibiotics
on transmissible plasmids captured directly from stream sediment bacteria
- Suggests the presence of a significant reservoir of clinically-important antibiotic
resistance genes in stream
- GC% shifts in pEx1-20 and the identification of mobile elements indicates high
level of recombination
- Reservoir may be accessible to pathogenic Gram-negative bacteria such as
members of the Enterobacteriaceae
- MinION represents a cheap and efficient method for detecting resistance genes in
clinical and environmental samples
References:
Collins, C.H., Lyne, P.M. and Grange, J.M. (1989) Microbiological Methods, Oxford, UK, Butterworth-Heinemann.
Fry, J.C., & Day, M.J. (1990) Plasmid transfer in the epilithion. In: Bacterial Genetics in Natural Environments. pp. 5580. (Fry, J.C. & Day, M.J., Eds.). London: Chapman and Hall.
Heringa, S. D., J. Monroe, and J. Herrick. 2007. A simple, rapid method for extracting large plasmid DNA from
bacteria. Nature Precedings http://dx.doi.org/10.1038/npre.2007.1249.1
Special thanks to 4-VA for providing
funding for this project (4-va.org).