Examinations of Methicillin – Resistant Strains of S. aureus
1
Introduction
Staphylococcus aureus is a bacteria present in humans as natural flora of the body. It is
often isolated from the nose, respiratory tract and skin of a healthy person. However, S. aureus
is also a common causative agent of skin diseases.
, Feifei, Beiler, 2002), originating from the hospital and later became as one of the increasing
community acquired infections due to its ability to spread through person to person direct contact
(Parvin, 2015). Now, one of the major causes in spreading nosocomial infections, MRSA has the
ability to survive in different types of β- lactam antibiotics such as ampicillin and oxacillin and
able to resist other types of antibiotics (Timothy, Aleigh, Blake, Joen et. al, 2013)
Although MRSA is commonly acquired in hospitals and health facilities, there are cases
and studies reporting that MRSA infection is also found in healthy individuals without any
recognized risk factor. Since MRSA infection can already be acquired in the community, this
strain is now called Community – acquired (CA) MRSA (Vandenesch, et al., 2003).
Transmission can be by physical contact, such as hand contaminations. Lacking access
of proper hand hygiene can increase the cases of transmission. The bacteria can also live for days
or months on inanimate objects, contaminated surfaces and shared objects (Neeley and Maley,
2000; Baggett et al., 2004).
Although there are many prevention and control programs for the transmission of MRSA,
poor implementations contributed to the global spread of MRSA causing it to become one of the
alarming problems of health. In the Philippines, there are rare published reports on the
efficiency of implemented mitigation and control programs for MRSA, both hospital and
community – acquired.
Examinations of Methicillin – Resistant Strains of S. aureus
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Furthermore, it is becoming more difficult to combat because of its emerging resistance
to all current antibiotics. There are no rational nomenclature and no clear evolutionary origins of
MRSA and so it is poorly understood. (Enright, 2002). Although literature says that the clinical
use of methicillin has led to the appearance of MRSA. This has become a major human
pathogen in which it has the ability to acquire to most antibiotics. Moreover, it has the ability to
produce constant emergence of clones. Hence, making S. aureus a “superbug”. (Lakhundi &
Zhang, 2018)
At present, MRSA is a serious problem in health management and medicine. The rise of
MRSA infection may be one of the significant setbacks in medicine. To date, there are no
published studies in Magalang, Pampanga. Monitoring of MRSA could be a breakthrough in
`addressing the problem in medicine. Thus, this study examined the presence of Methicillin –
Resistant strains of Staphylococcus aureus on contact surfaces at Rodolfo V. Feliciano Memorial
High School in San Pedro 2, Magalang, Pampanga It determined the presence and percentage of
putative MRSA on selected contact surfaces. Furthermore, it identified the preventive measures
for the school to reduce the risk of disease transmission and other infections in the school. The
results of the study would serve as a basis for improvement of the students and teachers’ personal
hygiene and to enhance the existing DepEd program of the school such as the Water Sanitation
& Hygiene in School (WinS) Program.
Examinations of Methicillin – Resistant Strains of S. aureus
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Materials and Methods
Study Site
Swab samples were collected from selected surfaces at Rodolfo V. Feliciano Memorial
High School, San Pedro 2, Magalang, Pampanga. These contact surfaces will be the biometrics
of teachers, hand railings of the Junior and Senior High School buildings and computer
peripherals in the computer laboratory.
All samples were transported immediately to the
Microbiology Laboratory of the College of Arts and Science in Pampanga State Agricultural
University.
Research Design
The study utilized the experimental method of research with an extension program for
mitigating the transmission of Methicillin – Resistant Staphylococcus aureus in the school.
An area of 40 sq. cm (4cm x 10cm) was swabbed from the following samples:
1- Biometrics
2- Hand Railing #1
3- Hand Railing #2
4- Hand Railing #3
5- Hand Railing #4
6- Hand Railing #5
7- Hand Railing #6
8- Computer Peripheral: Keyboard 1
9- Computer Peripheral: Keyboard 2
10- Computer Peripheral: Keyboard 3
Examinations of Methicillin – Resistant Strains of S. aureus
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11- Computer Peripheral: Keyboard 4
12- Computer Peripheral: Keyboard 5
13- Computer Peripheral: Keyboard 6
14- Computer Peripheral: Keyboard 7
15- Computer Peripheral: Keyboard 8
16- Hand Railing #7
17- Hand Railing #8
Research Instruments
The laboratory equipment and reagent used in the study were the following: Biosafety
cabinet Level 2, pippetors and tips, sterile petri dishes, cotton swabs, microcentrifuge tubes,
Muller – Hinton Broth (MHB), Mannitol Salt Agar (MSA), and Oxacillin Antibiotic.
Sample Collection
Using a dry sterile cotton swab, each sampling area was wiped in rolling motion. The
cotton swab was immediately placed in a sterile microcentrifuge tube containing Muller – Hinton
Broth (MHB) with 4% Sodium chloride. Each sample was labelled properly indicating the area,
and replicate number.
Determination of Staphylococcus aureus and Methicillin – Resistant Staphylococcus aureus
Each swab sample, inoculated into MHB containing 4% NaCl, was incubated aerobically
for 24 hours at 370C. An inoculum from each sample with observed turbidity was streaked onto
two sets of Mannitol Salt Agar (MSA), one containing oxacillin with 8 µg/mL concentration and
Examinations of Methicillin – Resistant Strains of S. aureus
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another free of the antibiotic. MSA is a medium selective for Staphylococcus species. The MSA
plates were incubated aerobically for 24 hours at 370C. Colonial growth was observed after
incubation. Bacteria that produced yellow, round, pinhead convex colonies were considered as
Staphylococcus aureus. Other species of Staphylococcus showed pink or red colonies (Warren et
al., 2004). Other species of bacteria on the other hand, could not grow on MSA due to high salt
concentration and presence of phenol.
Staphylococcus aureus that grew on MSA containing oxacillin were considered as
putative Methicillin – resistant Staphylococcus aureus.
Isolates that grew on both media were further characterized to determine the percentage
of putative MRSA among isolated S. aureus. Ten colonies, characteristic of S. aureus were
selected from MSA plates, subcultured individually into MHB-4%NaCl and incubated overnight.
Each of the broth cultures were spot plated onto MSA-Ox, observed for growth and computed
for the percentage.
Waste Disposal
All materials contaminated with S. aureus or putative MRSA, and the reference
microorganisms, were decontaminated using an autoclave at 15 psi, 1210C for 30 to 45 minutes
prior to disposal (CDC, 2009).
Examinations of Methicillin – Resistant Strains of S. aureus
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Results and Discussion
Detection of putative Methicillin – Resistant Staphylococcus aureus from contact surfaces
A total of 17 samples from the selected contact surfaces of Rodolfo V. Feliciano
Memorial High School—biometrics, building railings, and computer peripherals—were
collected. The samples were individually inoculated in microcentrifuge tubes containing MHB
with 4% NaCl and incubated for 24 hours at 370C. The turbidity was observed in all tubes after
incubation; Figure 1 shows turbidity growth of suspected Staphylococcus.
Figure
1.
Photograph showing growth of the samples in medium (MHB-4% NaCl)
compared to the blank medium (tube on the left of each photo).
The samples with positive growth in MHB-4% NaCl were subcultured in pure MSA and
MSA with 8 micrograms per millimeter oxacillin. After incubation, the growth of colonies on
MSA were observed. Colonies approximately 2–3 millimeters in size, round shaped, with
smooth texture, convex elevation and regular margins were considered as putative
Staphylococcus aureus. They can ferment mannitol in the medium producing yellow colonies.
Colonies with similar morphology but were pink were other species of Staphylococcus; they are
Examinations of Methicillin – Resistant Strains of S. aureus
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unable to ferment mannitol, maintaining the pH and color of the medium. Figure 2 shows the
growth of putative Staphylococcus aureus on MSA.
Figure 2. MSA plate containing isolated creamy white colonies with pinpoint size,
circular margin and convex elevation. Colonial characteristics suggest the
presence of S. aureus in swab samples.
Table 1 presents the result of the screening of Methicillin–Resistant Staphylococcus
aureus on the selected surfaces of the school. Isolates from all the selected surfaces were found
to have Staphylococcus aureus; colonies characteristic of S. aureus were observed on MSA
plates. While five out of 17 surfaces were found to have putative Methicillin – Resistant
Staphylococcus aureus. These surfaces were Sample 3 (Railing #2), Sample 5 (Railing #4),
Sample 6 (Railing #4), Sample 7 (Railing #6) and Sample 15 (Computer Peripheral: Keyboard
#8). Colonies of S. aureus isolated from swab samples grew on MSA containing oxacillin,
indicating that these were resistant to oxacillin or methicillin.
Examinations of Methicillin – Resistant Strains of S. aureus
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Table 1. Screening of Methicillin – Resistant Staphylococcus aureus on selected area surfaces
MSA
MSA - OX
Samples
LOCATION
1
Biometrics
√
X
2
Railing 1
√
X
3
Railing 2
√
√
4
Railing 3
√
X
5
Railing 4
√
√
6
Railing 5
√
√
7
Railing 6
√
√
8
Computer Peripheral: Keyboard 1
√
X
√
X
9
Computer Peripheral: Keyboard 2
√
X
10
Computer Peripheral: Keyboard 3
√
X
11
Computer Peripheral: Keyboard 4
√
X
12
Computer Peripheral: Keyboard 5
√
X
13
Computer Peripheral: Keyboard 6
√
X
14
Computer Peripheral: Keyboard 7
√
√
15
Computer Peripheral: Keyboard 8
√
X
16
Railing 7
√
X
17
Railing 8
√
X
Wild-type S. aureus
X
X
Blank Medium
√: presence of growth
X: absence of growth
Table 2 shows the percentage of surfaces containing putative MRSA. The Biometrics had
Staphylococcus aureus but the isolates were not resistant to oxacillin, thus, are not MRSA.
Among the eight investigated railings of the school, fifty percent (50% or 4/8) contained
suspected MRSA while only 12.5% 0r 1/8 of the investigated computer keyboards were positive
with the putative MRSA.
Table 2. Percentage of Surfaces containing putative MRSA
Location
Biometrics
Percentage
0.00 % (0/1)
Railings
50.00 % (4/8)
Computer Peripheral: Keyboards
12.50 % (1/8)
Examinations of Methicillin – Resistant Strains of S. aureus
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To determine the approximate percentage of the putative MRSA from the number of
isolated S. aureus in the swab samples, isolates that grew on both MSA and MSA-ox were
subcultured; ten colonies from MSA plates were tested against oxacillin. Table 3 presents the
approximate percentage of putative MRSA from isolated S. aureus. Results suggest that, among
the isolated S. aureus from building railings, approximately 28.57% are putative MRSA while
14.29% or less of the isolated S. aureus from computer keyboards are putative MRSA.
Table 3. Approximate Percentage of putative MRSA from isolated Staphylococcus aureus
Location
Percentage
Railings
≈ 28.57%
Computer Peripheral: Keyboards
≤14.29 %
Mitigation Program Implemented to reduce the acquisition of MRSA in the School
Awareness campaign was executed by the researchers to the students through posters and
room–to–room campaign.
The content of the posters discussed information on MRSA—
description, pathogenecity, sources, transmission and prevention. Three (3) posters were placed
at different areas of the school (Figure 3).
Examinations of Methicillin – Resistant Strains of S. aureus
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B
A
Figure 3. The researchers posting information dissemination materials (A) and discussing
information on MRSA—description, pathogenecity, sources, transmission and prevention.
Discussion
The results of the study suggest that all samples from the selected contact surfaces of
Rodolfo V. Feliciano Memorial High School contained Staphylococcus aureus. Swab samples
were first inoculated into MHB-4%NaCl to select salt tolerant bacteria such as species of
Staphylococcus, then inoculum from the cultures was subcultured onto the selective-differential
Mannitol Salt Agar (MSA) to isolate and differentiate S. aureus from other species of
Staphylococcus. Yellow colonies—pinpoint, circular, opaque, convex—are mannitol fermentors
and are S. aureus (Sharp and Searchy, 2006). The isolates from all samples, in this study,
exhibited similar colony characteristics on MSA, and may be identified as Staphylococcus
aureus.
Moreover, there were five samples out of the seventeen, which were positive for putative
MRSA. Isolates from these samples were able to grow on MSA with oxacillin antibiotics,
suggessting the presence of MRSA among Staphylococcus aureus isolates. Btabyal, Kundu and
Examinations of Methicillin – Resistant Strains of S. aureus
11
Biswas (2012) explained that MRSA is any strain of Staphylococcus aureus that has developed
recalcitrance to beta-lactam antibiotics, including penicillins (methicillin, dicloxacillin, nafcillin,
oxacillin, etc.) and the cephalosporins. Hence, isolates in this study that were able to resist
oxacillin and grew on MSA containing the antibiotic may be classified as MRSA and confirmed
by detecting the presence of a gene responsible for the antibiotic resistance. While the detection
of this gene was the delimitation of the study, the isolates are described as putative MRSA.
Furthermore, there were no putative MRSA on the biometrics sample. This suggest that
the transmission of MRSA is prevented probably due to good personal hygiene of the teachers.
The biometrics is used only by teachers and is located near the faculty office where students have
limited acces. However, 50% or four (4) out of eight (8) railings of the school buildings were
positive with putative MRSA. These surfaces are often touched by the students when not in
class, waiting or looking at the school premises. Likewise, 12.5% of the computer keyboards in
the Computer Laboratory were positive for putative MRSA. The computers are used for the
computer classes, hence, are often in contact with the students. With a significant number of
students in contact with this surfaces, MRSA may be spread from one area of the school to
another.
As Stefani et al. (2012) stated in their study, MRSA strains are spreading quickly
across the globe.
With the results of the study, detecting MRSA on different areas, an initial preventive
measure was implemented in the school; to reduce the risk of transmitting diseases such as
MRSA infection among students. The researchers conducted an awareness campaign on MRSA
to the students through posters and room–to– room discussion. Following these campaign, good
hygiene practices will be monitored and implemented in the school, including the installation of
Examinations of Methicillin – Resistant Strains of S. aureus
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pump bottles containing antiseptic solution, and detection of MRSA will be performed quarterly
to determine the effectiveness of the campaign to prevent the transmission of MRSA.
Limitations on the Research Design and Materials
The study is limited only in identifying the presence of putative MRSA on the selected
contact surfaces at Rodolfo V. Feliciano Memorial High School. There were only 17 samples in
the study from the most used contact surfaces of the students and teaches. Also, it determined
the percentage of putative MRSA in isolated S. aureus and the preventive measures of the school
in transmitting MRSA. However, characterization and molecular identification of the putative
MRSA were not done in the study.
Conclusions
Based on the results, Staphylococcus aureus was present on all selected contact surfaces
of Rodolfo V. Feliciano Memorial High School. Among the seventeen (17) contact surfaces,
five (5) may have putative MRSA. Also, there was no putative MRSA present on the biometrics,
there were 50% positive for putative MRSA in railing samples and 12.5% in computer keyboard
samples.
Hence, preventive measures were conducted in the school to reduce the risk of spreading
the bacteria and the infections it may cause. Posters and classroom campaign were done to raise
the awareness of students about MRSA, the diseases it may cause, and the importance of proper
washing of hands.
Examinations of Methicillin – Resistant Strains of S. aureus
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References
Batabyal, Biswajit, Gautam K R Kundu, and Shibendu Biswas. 2012. “Methicillin-Resistant
Staphylococcus Aureus: A Brief Review.” International Research Journal of Biological
Sciences I. Res. J. Biological Sci.
Cabrera, Esperanza C. 2012. “Methicillin Resistant Staphylococcus Aureus (MRSA).” In
Multidrug Resistance A Global Concern.
https://doi.org/10.2174/978160805292911201010130.
Chambers, Henry F., and Frank R. DeLeo. 2009. “Waves of Resistance: Staphylococcus Aureus
in the Antibiotic Era.” Nature Reviews Microbiology. https://doi.org/10.1038/nrmicro2200.
David, Michael Z., and Robert S. Daum. 2010. “Community-Associated Methicillin-Resistant
Staphylococcus Aureus: Epidemiology and Clinical Consequences of an Emerging
Epidemic.” Clinical Microbiology Reviews. https://doi.org/10.1128/CMR.00081-09.
Enright et. al. The Evolutionary history of Methicillin - resistant Staphylococcus aureus
(MRSA). Proceedings of the Ntaional Academy of Sciences 99 (11), 7687-7692,2002
Lakhundi & Zhang. Methicillin - resistant Staphylococcus aureus: molecular characterization,
evolution, and epidemeiology. Clinical Microbiology reviews 31 (4), e00020-18,2018
Miller, Loren Gregory. 2010. “Community-Associated Methicillin Resistant Staphylococcus
Aureus.” In Antimicrobial Resistance. https://doi.org/10.1159/000298753.
Sharp, Susan E., and Cindy Searcy. 2006. “Comparison of Mannitol Salt Agar and Blood Agar
Plates for Identification and Susceptibility Testing of Staphylococcus Aureus in Specimens
from Cystic Fibrosis Patients.” Journal of Clinical Microbiology.
https://doi.org/10.1128/JCM.01129-06.
Stefani, Stefania, Doo Ryeon Chung, Jodi A. Lindsay, Alex W. Friedrich, Angela M. Kearns,
Examinations of Methicillin – Resistant Strains of S. aureus
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Henrik Westh, and Fiona M. MacKenzie. 2012. “Meticillin-Resistant Staphylococcus
Aureus (MRSA): Global Epidemiology and Harmonisation of Typing Methods.”
International Journal of Antimicrobial Agents.
https://doi.org/10.1016/j.ijantimicag.2011.09.030.
Yoshikawa, Thomas T., and Larry J. Strausbaugh. 2006. “Methicillin-Resistant Staphylococcus
Aureus.” In Infection Management for Geriatrics in Long-Term Care Facilities, Second
Edition.
Examinations of Methicillin – Resistant Strains of S. aureus
PLATES
15
Examinations of Methicillin – Resistant Strains of S. aureus
Plate 1.Swabbing of the Biometrics
Plate 2. Swabbing of the railing
Plate 3. Swabbing of the railing #2
Plate 4. Swabbing of the computer keyboard
16
Examinations of Methicillin – Resistant Strains of S. aureus
Plate 5. Heating of the microcentrifuge tube
Plate 7. The microcentrifuge tube with the
control
Plate 6. Cutting of the swab
Plate 8. Weighing of the Mannitol Salt Agar
powder
17
Examinations of Methicillin – Resistant Strains of S. aureus
Plate 9. Boiling the MSA
Plate 11. Pouring of the MSA in petri dishes
Plate10. Autoclaving the MSA and other
apparatus to be used
Plate 12. Pouring of the MSA in petri dishes
18
Examinations of Methicillin – Resistant Strains of S. aureus
Plate 13. .Inoculating the cultured bacteria
from the microcentrifuge
Plate 15. The culture media
19
Plate 14. Streaking of the inoculated bacteria
Plate 16. .Incubation of the cultured media
Examinations of Methicillin – Resistant Strains of S. aureus
Plate 17. Observing the results
Plate 19. The sample with the blank control medium
20
Plate 18. The cultured bacteria in pure MSA and MSA - OX
Plate 20. Comparison of the results of the pure MSA and
MSA - OX
Examinations of Methicillin – Resistant Strains of S. aureus
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Plate 21. The results of the 2nd isolation of putative MRSA
Plate 22. The result of the sub culturing from the Sample
#15
Plate 23. The results of the 3rd subculturig of the putative
MRSA
Plate 24. A sample medium which shows the growth of the
putative MRSA
Examinations of Methicillin – Resistant Strains of S. aureus
Plate 25. Posting posters on the wall of the
school campus
Plate 27. The researchers with the poster
campaign in the washing area of the campus
22
Plate 26. The researchers with the poster
campaign in the SHS building
Plate 28. The researchers during the classroom
campaign
Examinations of Methicillin – Resistant Strains of S. aureus
Plate 29. The Lysol and Alcohol in the school
Plate 30. A reminder on the importance of hand washing
Plate 31. JHS girls at the washing area
Plate 32. JHS boy at the washing area
23
Examinations of Methicillin – Resistant Strains of S. aureus
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Plate 33. Streaking training – workshop by the Qualified
Scientist
Plate 34. Streaking training – workshop using agar plate by the
Qualified Scientist
Plate 35. Pouring agar in petri dishes training – workshop by
the Qualified Scientist
Plate 36. Inoculating and Streaking training – workshop by the
Qualified Scientist
Examinations of Methicillin – Resistant Strains of S. aureus
25
INTER-MEMO
FOR:
FROM:
SUBJECT:
DATE:
All Advisers, Subject – Teachers and Students
School Principal
PROPER HYGIENE AND REITERATION ON THE IMPORTANC OF WASHING OF
HANDS
September 26, 2019
It has been investigated that selected contact surfaces such as hand railings and
keyboards in the school has SUSPECTED antibiotic- resistant bacteria (i.e. Methicillin – resistant
Staphylococcus aureus/MRSA). In line with this, the need to wash hands and improve one’s
personal hygiene are reiterated. Washing of hands must be done before taking your recess and
lunch in the afternoon. Lysol Antibacterial soap will be in the washing area as an additional
safety precaution to lessen the transmission of MRSA.
Approved by:
ALBERT V. DATU
School Head