Speciation of Methicillin-Resistant Staphylococci Isolated
from Ecuadorian Hospitals and Communities
Student Researcher: Beatrice R. Soderholm; Faculty Advisor: Daniel P. Herman, PhD
Department of Biology, University of Wisconsin – Eau Claire
Original PCR
Conditions
Background
Staphylococcus aureus is a bacterium that commonly causes infections in
the human population. Many patients’ prognoses are worsened because some
strains have acquired antibiotic resistance. Surveillance of methicillin-resistant
Staphylococcus previously focused on the well characterized aureus species
(MRSA).
Community and hospital samples collected from various regions of Ecuador
from 2010-2012 revealed the majority of methicillin-resistant Staphylococcus
(MRS) are not of the aureus species. These species are speculated to be
important in the acquisition of methicillin resistance in S. aureus by serving as a
reservoir for the SCC-mec cassette, which is the genetic element responsible for
methicillin resistance [1].
We tested a PCR protocol to identify the species of Staphylococcus that are
resistant to methicillin. The protocol identifies species based on an intergenic
region between the 16S and 23S rDNA regions on the chromosome [2]. The
banding pattern from this intergenic region is specific to each species. It is our
hope that speciation of the methicillin-resistant Staphylococcus isolates will allow
us to develop a better understanding of how antibiotic resistance is transferred
between Staphylococcus species.
Materials & Methods
Sample Collection: Nasal swabs were collected using StarSwab II ™ Platinum
Series swabs (Starplex Scientific, Inc.) from community members and hospital
staff and patients age 12 or older. These samples were collected from various
regions in Ecuador from 2010-2012.
Final
Component
Concentration
Flexi Buffer
1X
(5X)
dNTP
2 mM
(100 mM)
MgCl2
1 mM
(25 mM)
G1 Primer
0.5 mM
(10 µM)
L1 Primer
0.5 mM
(10 µM)
DNA Template
0.63 ng/µl
(.025 µg/µl)
Taq
Polymerase
0.05 u/µl
(5u/µl)
Samples were run on a 3%
agarose gel with 0.5X TBE at 45V
for 4 hours.
Modifications to PCR
Conditions
Component
Working
Concentration
DNA Template
2 ng/µl
(.1 µg/µl)
Samples were run on a 2.5%
agarose gel with 1X TBE at 60V
for 2.5 hours.
Samples were loaded with 2 µl
1X TBE buffer and 2 µl of
additional loading buffer.
Tables 1 & 2. Original PCR recipe was adapted from
Mendoza et al. (1998). The recipe was
optimized through trial and error until
adequate separation of the expected
bands was obtained. The G1 primer
selects for highly conserved region of
16S RNA gene and the L1 primer
selects for a conserved sequence of
the 23S RNA gene. For all recipes H2O
was added for a final volume of 50 µl.
4
5
6
7
8
9
2
3
4
While PCR is identified as being an appropriate method for Staphylococcus
speciation, further optimization can be obtained. The electrophoresis conditions
can be further modified to get better resolution of the bands. The most obvious
adjustment that should be made in subsequent experiments is running the gel
longer, potentially up to 6 hours, to allow more separation of the bands. If
necessary, it may be appropriate to switch to a higher quality agarose gel medium
to allow distinction between the bands in the intergenic region.
Continuing to optimize this protocol is important because MRS species
identification will increase the understanding of the spread of methicillin
resistance among staphylococci. This insight may be used to further characterize
the SCC-mec cassette, specifically its transference among Staphylococcus species.
Speciation by PCR Analysis: PCR was performed on the characterized MRS
isolates in order to develop a reliable speciation protocal. Isolated DNA from
MRS samples was used as a template in the PCR recipe (Tables 1 & 2). These
samples were originally run on a 3% agarose gel containing 2 μl ethidium
bromide at 45V for 4 hours. The gel used was adjusted and tested until optimal
separation was obtained consistently (Figures 2 & 3).
3
1
Discussion
MRS Characterization: Positively identified MRS isolates were sent to
Marshfield Clinic for further characterization using matrix-assisted laser
desorption/ionization-time of flight mass spectrometry (MALDI-TOF).
2
Preliminary results reveal PCR is a promising method in the speciation of
Staphylococcus samples. The PCR protocol used can be found in Tables 1 and 2.
Most of the PCR recipe is retained; however the concentration of template DNA
used is increased by roughly 300%.
Additionally, adjustments have been made to the electrophoresis
conditions. It was found that a 2.5% agarose gel gives better resolution than a 3%
agarose gel. Since the gel is being run for a prolonged period in one direction, the
bands are prone to warp. These warping issues are resolved by increasing the
buffer from 0.5X TBE to 1X TBE. Samples are also loaded with 2 µl of 1X TBE
buffer to further counter warping. Diluting the samples with the buffer decreases
the density of the samples. To compensate for this decrease in density, samples
are loaded with an additional 2 µl of loading buffer.
Figure 3. Agarose gel electrophoresis for speciation of MRS isolates with a molecular ladder
(lane 1), negative control (lane 2), Staphylococcus warneri, and Staphylococcus
haemolyticus samples (lanes 3 & 4, respectively).
Primary PCR Analysis: Polymerase chain reaction (PCR) using three increasingly
selective primers was performed on isolates that passed catalase, Gram stain,
and latex agglutination tests. Samples were run on a 2% agarose gel containing
2 μl ethidium bromide. Samples were compared to a positive S. aureus control,
a positive MRSA control, and a PCR negative control in order to accurately
identify each isolate (Figure 1).
1
Results
References
10
[1] Berglund, C. & Sӧderquist, B. (2008). The origin of a methicillin-resistant Staphylococcus aureus isolated from a
neonatal ward in Sweden – possible horizontal transfer of a staphylococcal cassette chromosome mec between
methicillin-resistant Staphylococcus haemolyticus and Staphylococcus aureus. Clinical Microbiology and Infection,
14, 1048-1056.
[2] Mendoza, M., Meugnier, H., Bes, M., Etienne, J. & Freney, J. (1998). Identification of Staphylococcus species by
16S-23S rDNA intergenic spacer PCR analysis. International Journal of Systematic Bacteriology, 48, 21049-1055.
16S
FemB
MecA
Figure 1: Ethidium bromide gel with a molecular ladder (lane 1) and negative, MRSA, and S.
aureus controls (lanes 2-4, respectively) and isolates of interest. Lane 9 contains
an MRS isolate.
Acknowledgments
Figure 3. Schematic representation of PCR-amplified 16s-23S rDNA intergenic region from Staphylococcus
strains. Figure adapted from Mendoza et al. (1998).
Special thanks to the UW – Eau Claire Office of Research and Sponsored
Programs and to the Center for International Education for their financial support
of this project. Also, thank you to Marshfield Clinic for their assistance in the
characterization of MRS isolates.