1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
Laboratory experimental evolution has been used to rapidly evolve
resistance to spherical silver nanoparticles (AgNP) and ionic silver
(AgNO3) in E. coli K-12 MG1655. Here we attempt this experiment
utilizing different types of silver (triangular silver NPs AgNPl, spherical
silver NPs; and AgNO3). In our first attempt, by generation 442 we
observed evidence of silver resistance in AgNP-selected, AgNO3-selected,
but not in AgNPl-selected populations. In generation 25, minimum
inhibitory concentration (MIC) for AgNP-selected, AgNO3-selected,
AgNPl-selected, and controls were equivalent at 12.5 mg/L. In generation
442, MIC for AgNP-selected, AgNO3-selected, AgNPl-selected, and
controls were 27.5 mg/L, 103.75 mg/L, 6.25 mg/L, and 17.5 mg/L
respectively. These results indicate that selection was effective in
increasing AgNP-selected and AgNO3-selected versus the controls, but was
ineffective for AgNPl-selected. This presentation reports the results of a
2nd attempt to evolve resistance against AgNPls. In generation 160,
AgNPl-selected populations are being cultured at 40 g/L of AgNPl,
showing resistance compared to controls. By generation 400, AgNPl-
selected lines had greatly surpassed control MIC at 250 mg/L versus 33
mg/L.
We have shown that while silver is highly toxic to E. coli in both ionic and
spherical nanoparticle forms that bacteria can rapidly evolve resistance.
However, the rate at which bacteria evolve resistance to triangular silver
nanoparticles suggests that these may be more efficacious in controlling
silver-resistant bacteria.
Abstract
Introduction
Bacteria culture
Escherichia coli K-12 MG1655 was cultured using Davis Minimum Broth
(DMB, DifcoTMSparks, MD) with Dextrose 10% (Dextrose, Fisher
Scientific, Fair Lawn, NJ), enriched with thiamine hydrochloride 0.1%
(Thiamine Hydrochloride, Fisher scientific, Fair Lawn, NJ) in 10 mL of total
culture volume maintained in 50 mL Erlenmeyer flasks. AgNO3-selected,
AgNp-selected, and AgNPl-selected populations had increasing
concentrations of these materials added to the flasks. All nanoparticles were
Biopure and obtained from Nanocomposix, San Diego, CA. In experiment 1,
all three silver selection treatments were maintained at 100 mg/L. In
experiment 2, the AgNPl treatment began at 50 mg/L and was increased to 90
mg/L after generation 160. The flasks were placed within a shaking incubator
at a maintained temperature of 37 o C for 24 hours. After 24 hours, 0.1 mL of
the culture within the incubated flasks were transferred to new flasks
containing 9.9 mL of the DMB and placed in the incubator for another 24
hours. This procedure was repeated daily. After 24 hours, all flasks exhibiting
obvious turbidity with the unaided eye on average will contain approximately
3,000,000,000 bacteria. Broth dilutions are conducted to standardize the
cultures in each flask to achieve effective results for susceptibility testing.
Antimicrobial Preparation
MIC tests were conducted in standard 96 well culture plates. Beginning with
1mg/mL stock solutions of the silver nitrate (AgNO3), serial dilutions using
Mueller-Hinton (MH) Broth are carried out to create a concentration gradient
across each of the well’s rows (12 x 8, 500mg/L, 250, 175, 100, 75, 50, 25,
12.5, 10.0, and 6.25).
Broth Microdilution
Using Mueller-Hinton (MH) Broth as our medium of choice, bacteria cultures
were diluted according to a standard inoculum density.[3,4] The suspension is
adjusted to achieve a turbidity and optical density equivalent to a 0.5
McFarland turbidity. This results in a suspension containing approximately 1
to 2 x 108 CFU/mL (Bacteria per mL). Said suspensions are then diluted by a
tenth (~2 x 107 CFU/mL). Each well will contain 95μl of MH Broth and
various AgNO3 dilutions and will be inoculated with 5μl of the adjusted
bacteria suspension resulting in a final inoculum concentration of 5 x 105
CFU/well.[4]
Determining MIC End Points
After an 18-24 hour 37oC shaking incubation period, MIC end points are
determined by the first non-turbid well seen with the unaided eye in each row.
For example, if in the row containing C4, columns 8 to 12 are clearly turbid
and columns 2 to 7 are clearly non-turbid (column 1 is a designated control
column and therefore should always be turbid), column 7 being the first non-
turbid well going up the row and up the antimicrobial agent gradient, would
be considered the well containing the minimum inhibitory concentration for
the C4 bacteria suspension. This would correspond to a minimum inhibitory
concentration of 5mg/L if following the concentration gradient mentioned
above.
Materials & Methods Results
Conclusion/Discussion
Our results have demonstrated that bacteria can rapidly evolve resistance to
silver in its various forms. However, it seems that the adaptive response to
triangular silver nanoparticles (AgNPl) is delayed relative to ionic silver and
spherical silver nanoparticles (AgNP). We conducted a > 400 generation
experiment in which no evidence of adaptation to AgNPl was found
(experiment 1). Indeed we found that many of the AgNPl-selected replicates
in that experiment went extinct. In experiment 2 we found that adaptation to
AgNPl had occurred in 6/8 replicates at generation 476. This can be
compared to our previous experiments in which we found evidence of
adaptation by generation 100. We are now conducting genomic analysis to
determine if the changes in the AgNPl-selected replicates repeat the findings
of Graves et al. 2015 and Tajkarimi et al. 2015 (see Table 1). It is possible
that the delayed result of the triangular nanoparticle selection was due to a
requirement of fixing mutations additional to those we found in earlier studies.
References
1. Graves JL. Tajkarimi M. Cunningham Q. Campbell A. Nonga H. Harrison SH. Barrick JE. Rapid
Evolution of Silver Nanoparticle Resistance in Escherichia coli. Frontiers in Genetics 2015. doi:
10.3389/fgene.2015.00042; Tajkarimi M. Sedighi R. Baghaee-Ravari S. Campbell A. Nonga H.
Cunningham Q. Hung A. Harrison SH. Barrick JE. Graves JL. Experimental Evolution and
Genomics of Silver Resistance in Escherichia coli. (under review) mBio. 2015.
2. Randall CP. Gupta a. Jackson N. Busse D. O’Neil AJ. Silver resistance in Gram-negative bacteria: a
dissection of endogenous and exogenous mechanisms. J. Antimicrob. Chemother. 70: 1037—1046,
2015.
3. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests
for Bacteria That Grow Aerobically; Approved Standard—Seventh Edition. Clinical and
LaboratoryStandards Institute document M7-A7 [ISBN 1-56238-587-9]. Clinical and Laboratory
Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA,
2006.
4. Ericsson HM, Sherris JC. Antibiotic sensitivity testing. Report of an international collaborative
study. Acta Pathol Microbiol Scand.1971;217(suppl B):1-90.
Acknowledgements
The following individuals gave aid to completing these experiments: Soodeh Baghaee-Ravari, Rachel Spencer,
Adrian Goodwin, and Thomas Morten.
Metallic and Metallic Oxide nanoparticles have been proposed as new
antimicrobials. It is argued that bacteria will have great difficulty evolving
resistance to these nanoparticles. Graves et al. (2015) and Tajkarimi et al.
(under review) utilized laboratory experimental evolution and showed that
Escherichia coli K12 MG1655 could quickly evolve resistance to 10 nm
citrate-coated spherical silver nanoparticles (AgNP) and ionic silver
(AgNO3) This E. coli strain was chosen it did not contain many known
silver resistant genetic elements. They found that increased resistance was
achieved within 100 generations. [1] Both of these studies found that the
genomic basis of resistance was relatively simple, involving mutations in
the cusS gene, ompR, and in RNA polymerase subunits (rpoABC).
Randall 2015 corroborated our findings relative to cusS and ompR [2].
This study examines the impact of nanoparticle shape on the evolvability
of silver resistance. It reports the Minimum Inhibitory Concentration
(MIC) for AgNO3-selected, AgNP-selected, and triangular silver NP-
selected (AgNPl) lines and their controls. We tested MIC for replicate
populations of the selected populations of bacteria (Ag1-Ag5); (AgNP1—
AgNP5); (AgNPl1—AgNPl5) and their non-silver resistant controls as (C1-
C5). Genomic analysis of the AgNPl-selected lines was conducted to
compare against previous studies of AgNP and AgNO3 resistance.
1Department of Nanoengineering, Joint School of Nanoscience & Nanoengineering, North Carolina A&T State University and UNC Greensboro, Greensboro, NC 27401
2Department of Nanoscience, Joint School of Nanoscience & Nanoengineering, North Carolina A&T State University and UNC Greensboro, Greensboro, NC 27401
Director, Nanotechnology Program, Forsyth Technical Community College, Winston Salem NC
4BEACON Center for the Study of Evolution in Action, Michigan State University MI 48825
Inmani Sharpe1, Adero Campbell1, Jaminah Norman1, Marjan Assefi2, Dr Mehrdad Tajkarimi3, and Dr. Joseph Graves Jr.1
Does Nanoparticle Shape Influence the ability of bacteria to evolve silver resistance?
Ctrl50mg/L2517.5107.552.51.25
625μg/L312156
MICT/C
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Figure 2
A standard 12 x 8 culture well plate used for the minimum inhibitory
concentration test. The first row provides an example of a possible result
expected from a typical susceptibility test. The MIC for the tested bacteria
suspension in this case would be 5mg/L, the first column only contains
bacteria suspended in broth for quality control purposes.
Figure 3
In the first selection experiment the AgNO3-selected lines (blue) increased their
MIC in AgNO3 relative to the controls (black). In generation 25 mean MIC for
AgNO3-selected lines was 12.5 mg/L +/- 0.0 and it had increased to 103.50 +/-
26.70 mg/L in generation 442. On the other hand, AgNP-selected lines
increased from 12.5 +/- 0.0 in generation 25 to only 27.5 +/- 1.89 mg/L in
generation 442. The AgNPl (triangular)-selected lines actually showed a
decrease in MIC 12.5 +/- 0.0 to 6.25 +/- 0.0 mg/L in generation 442. These
results suggest that resistance to silver nanoparticles need not result in general
ionic silver resistance, or that some lines achieved nanoparticle and ionic silver
resistance while others did not. This is illustrated in figure 4.
Figure 4
In the first selection experiment the AgNP-selected lines showed no consistent
response by replicate. In generation 300, AgNP1 and AgNP2 are performing best
relative to controls, but in generation 442 it is AgNP2 and AgNP3. In generation
442, the mean of AgNP-selected replicates are not statistically significantly
higher than the controls (27.5 mg/L +/- 1.89 SE versus 17.5 +/- 1.36 SE).
However the mean of AgNP2 and AgNP3 were statistically higher than the
controls (F = 15.23; p = 0.011.) This pattern was also observed in the AgNO3-
selected lines as well, two replicates (Ag1, Ag2) showed evidence of silver
resistance while Ag3,Ag4, and Ag5 were not statistically different from controls.
Figure 5
The means and standard errors for MIC of control (33.33 +/- 2.15) v. AgNPl-
selected (250.37 +/- 27.35) populations in generation 476 are shown. The
mean MIC of the AgNPl-selected were statistically significantly different as
tested by nested ANOVA; F =5.76, p = 0.033.
Table 1
Figure 1
SEM image of E. coli K-12 MG1655. Treated with 20 mg/L PVP coated
spherical silver nanoparticles. Silver nanoparticle accumulation is
apparent on the surface of the treated bacterial cell.
Silver nanoparticles
The number of anti-silver mutations are shown for experimentally evolved
E. coli K-12 MG1655 from Graves et al. 2015 and Tajkarimi et al. (under
review) are shown. The largest number of mutations have occurred in the
cusS gene that encodes a sensory histidine kinase in a two-component
regulatory system (with CusR) that senses both copper and silver ions; rpoA,
rpoB, rpoC encode the alpha, beta, and beta prime subunits, respectively, of
RNA polymerase.
Controls
AgNPl-selected