Can silver nanoparticles be used to treat Methicillin-Resistant super bugs?

Travis D. Lambert
Ana L. Heully
Pathogenic Microbiology Laboratory (Biol 4415L)
Fall 2015
Department of Biology
Our Lady of the Lake College
November 3, 2015
In Partial Fulfillment of the Independent Project Component

Introduction:
Nosocomial infections are a big risk of extended hospital stays, and one of the microbes
causing these extended stays is Methicillin-Resistant Staphylococcus aureus. After 2000 hospital
stays due to MRSA tripled and by 2005 368,600 of the hospitalized patients were infected with
the bacteria (Elizhauser & Steiner, 2007). This microbe is resistant to methicillin, which is a
semisynthetic beta-lactamase-resistant penicillin (Freeman & Morris, 2014). MRSA was first
reported in Europe in 1961, and has since become a prominent nosocomial infection worldwide.
Penicillin was the first antibiotic used to treat the bacteria in the 1960s, and as it grew resistance
methicillin was used for treatment. There are only a few antibiotics known to treat this infection
with any success, but the bacteria are starting to become resistant to current treatment options
(Brown, et. al, 2005). The mecA gene that is found in bacteria, is responsible for the production
of beta-lactamase. Once produced, beta-lactamase degrades the beta-lactam ring found in
penicillin-like antibiotics which will prevent binding to the trans peptidases found on bacterial
cell walls allowing the bacteria to replicate normally, which will make the bacteria antibiotic
resistant (Hwang et. al, 2012). The purpose of this research is to test the synergistic effects that
antibiotics and silver nanoparticles can have against resistant bacteria. It has already been shown
in other studies that Ampicillin, an antibiotic that MRSA has been reported to be resistant
against, when combined with silver nanoparticles will exhibit a synergistic effect against
bacteria, this study will aid in the understanding of how this synergistic activity will occur
Methicillin-resistant Staphylococcus aureus will be the organism in question for this
project. MRSA is a known species that is resistant to many different antibiotics. Typically, one
could defeat a Staphylococcus aureus infection with a simple antibiotic like Ampicillin, but
because it is resistant to this antibiotic other routes must be taken (Brown et. al, 2005). Our

hypothesis is that silver nanoparticles will work synergistically with antibiotics to make them no
longer resistant.
It has been shown that nanoparticles can make MRSA susceptible to Ampicillin again,
but no research has been found on the mechanism. One possibility is that the nanoparticles will
down regulate the expression of the mecA gene in the bacteria, which is responsible for the
production of the beta-lactamase that breaks down the beta lactam ring and preventing the
antibiotic to break-down the cell wall. To test that hypothesis the bacteria were dosed in silver
nanoparticles and underwent a polymerase chain reaction test to look at the expression of mecA
versus the controls. The controls used were Vancomycin, an effective antibiotic against MRSA,
Ampicillin, a methicillin that MRSA is resistant to, and a sterile water control.
Materials and Methods:
Culturing
supplies
Table 1: Materials
Chromogenic agar
MSA plates
TSA plates

Nanoparticle synthesis
Silver nitrite (AgNO3)
Methyl cyclopentadienyl
manganese tricarbonyl
(MMT)
Sodium borohydride
(NaBH4)

PCR and Protein assay

Vancomycin
Ampicillin
Primers: The forward (59CATTTTGAGTTCTGCA
CTACC- 39) and reverse
(59GCAATACAATCGCACA
TACATTAATAG- 39)
primers (Ryffel et al.,
1992)
Mueller-Hinton agar
(MHA)
SYBR
Table 1: Materials needed to culture the bacteria for the research, to synthesize silver
nanoparticles and to perform PCR and protein assay
Samples were taken from everyday surroundings with a sterile swab that was kept in a
falcon tube with sterile water. Of the many samples taken, only three were selected for this study.
The location and time in which they were found is listed in Table 2.
Table 2: Sample Collection

Sample
Swab of a piece of titanium
kept in a garage

Date and Time

9:45 AM
09 September 2015

Swab of a door handle in a

public establishment

5:30 PM
12 September 2015

Hair Swab

4:00 PM
10 September 2015

Location
4110 Pasadena Dr.
Baton Rouge, La, 70814
Latitude: N 30 27' 2.6863"
Longitude: W 91 9' 16.3836"
5175 Government Street
Baton Rouge, La 70806
Latitude: N 30.450743
Longitude: W 91.154645

Table 2: Location, date and time of when each sample in the

study were collected.

Methods
Sampling and MRSA Identification
The table above shows that the samples were taken from different locations in a typical
students environments. A dichotomous flowchart (Figure 1) was constructed based on the
identification flowcharts in Bergeys Manual of Determinative Biology. In order to properly
identify the bacteria, a series of tests were done on the three samples: hair, a door, and a piece of
titanium. First, the three swabs that were taken from the samples were Gram stained and all three
gave a Gram-positive result. To insure that the stain was done correctly, the three swabs from the
samples were placed onto phenylethyl alcohol and mannitol salt agar plates. The first test
performed was the catalase test, which was done by placing 3% hydrogen peroxide onto a slide.
A colony was scraped off of the plate and placed in the hydrogen peroxide. Once placed on the
plate, bubbles formed indicating that the sample was Staphylococcus aureus. The next test used
for confirming the presence of S. aureus was mannitol fermentation. This test was done by
isolating a colony on a tryptic soy agar (TSA) plate and then streaking for isolation on a mannitol
salt agar plate. The mannitol fermentation test showed whether the species was able to tolerate
the mannitol salt and can also differentiate between staphylococcus and streptococcus (Leboffe

& Pierce, 2012). The final step of culturing out MRSA is streaking on chromogenic plates. The
MRSA colonies will appear to be blue, and they were selected to be tested against the
nanoparticles and antibiotics (Freeman & Morris, 2015). Each of the samples that were cultured
on the chromogenic media had the expected color change (Figure 2), which further indicated the
presence of Methicillin-Resistant Staphylococcus aureus

Figure 1: Dichotomous
flowchart

Figure 1: Dichotomous flowchart of tests conducted to properly identify the samples

Antibiotic Susceptibility Tests

The variables were the different combinations of antibiotic and nanoparticle. The controls were
Vancomycin, a known antimicrobial against MRSA and ampicillin, a known antibiotic that
MRSA is resistant to, before they are treated with silver nanoparticles. These were tested with the
disc diffusion method and then DNA/protein extracted from bacterial samples.
Absolute Quantification of MRSA Concentrations
Detection of the mecA gene. Broth cultures of S. aureus were grown over night. Once cultured
50 microliters was combined with 200 microliters of Purezol and allowed to incubate for 24
hours to allow for the breakdown of the gram positive bacteria. The RNA was then quantified
using the Nanodrop spectrophotometer. of cDNA, which was used in combination with a
mastermix to run qPCR, which measured the mecA gene expression.
Results

Methicillin-resistant Staphylococcus aureus was found in three samples that were taken. The
PEA agar plates yielded Gram-positive results and the MSA plates all had good, yellow growth,
which is indicative of Staphylococcus aureus. A catalase test was done on a colony of bacterial
growth from each plate. All of the samples were positive to catalase production, which proves all
of the samples Staphylococci.
Table 3: Test results
Test
Gram stain
PEA plate
MSA plate

Hair swab
Door swab
Titanium swab
Gram Positive
Gram Positive
Gram Positive
Gram Positive
Gram Positive
Gram Positive
Good, yellow
Good, yellow
Good, yellow
growth
growth
growth
Chromogenic agar Strong growth
Strong growth
Strong growth
Table 3: Results of test ran on samples to properly identify them as MRSA.
Figure 2

qPCR mecA expression

Control (no antibiotic)

Ampicillin

Vancomycin

Nanoparticles

Ampicillin + nanoparticles

Figure 2: This figure is a representation of the data obtained from the quantitative PCR samples.
This shows the relationship between cycles it took to reach threshold. The group which has a
control closest to the S. aureus positive control showed that nanoparticles and Vancomycin took
longer to reach threshold. This could be indicative of mecA down regulation.

Figure 3

Cq values normalized to positive control

Cq Control

Cq Ampicillin

Cq Ampicllin + nanoparticles

Cq Vancomycin

Cq Nanoparticles

Figure 4

Cq values normalized to E. coli mecA

Cq

0.80
0.60
0.40
0.20
0.00
Doorknob

Hair

Titanium

Axis Title
Cq control
Cq Amplicillin
Cq Ampicillin + nanoparticles
Cq Vancomycin
Cq Nanoparticles

Figures 3 and 4 show the Cq and Cq values respectively. Using these graphs expression can
be determined by looking at the samples vs. the control lines. In Doorknob and Hair
samples the sample values are higher than those of the control. However, the Titanium sample
values are opposite those of the first two sample groups. The control has a higher value than the
samples, and the Ampicllin + nanoparticles sample has the lowest value and the highest
delineation from the control.

These results were the number of cycles for the samples to reach the cycle threshold of the mecA
gene expression. In the Hip samples the control was very close if not exactly what the positive
control s. Aureus showed. The Vancomycin and Ampicillin samples showed a down regulation
of the mecA gene as compared to the control. The Ampicillin plus nanoparticle samples actually
showed a slight up-regulation. In figures 3 and 4 the Ct values were compared to that of the
positive control S. aureus and the non-template control E. coli. In the figures it is determined
mecA gene expression overall has a trending downward slope away from the control as the lines
move across the different groups. In the Vancomycin and Nanoparticle samples they stay with
the highest vales as compared to the S. aureus control. This is further confirmed with the Cq
values.
Discussion:
MRSA was cultured from a sample that was swabbed from hair. Tests were performed as stated
above, and discovered that Methicillin-resistant Staphylococcus aureus had been cultured and
confirmed by growing the sample on a chromogenic agar plate. With this positive result
susceptibility tests could be done using Vancomycin, Ampicillin, Ampicillin treated with silver
nanoparticles, and silver nanoparticles by themselves. These results have the potential to be
significant and important because one of the journal articles that was reviewed showed that
susceptibility had gone up when disc diffusion was done with nanoparticles and Ampicillin
combined against MRSA. While by itself, Ampicillin is not able to achieve this, and is not
effective due to the beta lactamase. The current study examined the inactivation of the mecA
gene, which codes for beta-lactamase and penicillin-binding protein 2 which, is responsible for
the microbes main resistance. The results did not show a lot of statistical significance, but in the
HIP samples it can be seen that the control was equal to that of the standard and the

nanoparticles by themselves had the same effect on the mecA gene as Vancomycin. The research
done was to measure expression of this gene. This means that the organism had to be alive and
actively transcribing this gene. The higher number of average cycles is because the gene was not
active. This could be because the organism was dead or because it was specifically effected.
More research will need to be done to vindicate what the actual mechanism might be to make
MRSA susceptible to ampicillin by adding silver nanoparticles. Once the mechanism can be
determined then there could be another line of antibiotics that could successfully treat the super
bugs.
Conclusion:
Through the completion of this study it has been found that the expression of the mecA
gene was not down regulated by the Ampicillin and silver nanoparticle mixture as was expected,
but instead up regulated. This is assumed to be a cause of the technique used for the extraction of
RNA from the bacterial samples. The technique used is commonly used to extract RNA from
Caenorhabditis elegans, but it is suggested that in future research a method that is more
compliant with Gram-positive bacteria be used.
The next speculation that could be tested if the mecA gene is not down-regulated in the sample
treated with Ampicillin and silver nanoparticles, that the nanoparticles might be attaching to or
degrading the beta-lactamase. To test this hypothesis, a protein assay of all of the samples and
their concentrations of beta lactamases must be done. If the expression remains unchanged but
the Ampicillin is still activated, then one could assume that the silver nanoparticles are
essentially forming a pore in the cell wall allowing the Ampicillin to enter the cell.

References:
Brown, D., Edwards, D., Hawkey, P., Morrison, D., Rigway, G., Towner, K., & Wren, M. (2005).
Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant
Staphylococcus aureus (MRSA). Journal of Antimicrobial Chemotherapy, 56(6).
doi:10.1093/jac/dki372
Elixhauser, A., & Steiner, C. (2007, July 1). Infections with Methicillin-Resistant Staphylococcus
Aureus (MRSA) in U.S. Hospitals, 19932005. Retrieved December 9, 2015, from
http://www.hcup-us.ahrq.gov/reports/statbriefs/sb35.jsp
Freeman, J., & Morris, A. (2014, March 13). Rapid detection of methicillin-resistant
Staphylococcus aureus. Retrieved December 3, 2015, from
http://www.uptodate.com/contents/rapid-detection-of-methicillin-resistantstaphylococcus-aureus
Hwang, I., Hwang, J., Choi, H., Kim, K., & Lee, D. (2012). Synergistic effects between silver
nanoparticles and antibiotics and the mechanisms involved. Journal of Medical
Microbiology, 61, 1719-1726. doi:10.1099/jmm.0.047100-0
Identification flow charts from Bergeys Maunual of Determinative Biolovy excerpt provided on
Moodle
Leboffe, M., & Pierce, B. (2012). Microbiology: Laboratory theory & application (2nd ed.).
Englewood, CO: Morton Pub.
Shameli, K., Ahmad, M. B., Zargar, M., Yunus, W. M. Z. W., Rustaiyan, A., & Ibrahim, N. A.
(2011). Synthesis of silver nanoparticles in montmorillonite and their antibacterial
behavior. International Journal of Nanomedicine, 6, 581590.
http://doi.org/10.2147/IJN.S17112
Solomon, S., Bahadory, M., Jeyarajasingam, A., Rutkowsky, S., Boritz, C., & Mulfinger, L.
(2007, February 2). Synthesis and study of silver nanoparticles. Retrieved November
10, 2015, from http://pubs.acs.org/doi/abs/10.1021/ed084p322
Stiernagle, T. (2006, February 11). Maintenance of C. elegans. Retrieved December 10, 2015,
from http://www.ncbi.nlm.nih.gov/books/NBK19649/