1. RESULTS
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
SPRING 2013 FALL 2013 SPRING 2014 FALL 2014 SPRING 2015 FALL 2015 SPRING 2016
Penicillin
Cefoxitin
Erythromycin
Clindamycin
Tetracycline
METHODS
Swabs of the anterior nares and pharynx of pre-nursing and nursing students at UNCW were collected over seven semesters from
Spring 2013 to Spring 2016. Participants were encouraged to contribute samples once per semester starting the semester before
entering the BA nursing program until their graduation from the two-year program (5-6 semesters total). Not all 624 samples,
however, were collected from different people. A total of 342 different participants were sampled, meaning that some students
participated in the study for two or more semesters. Participants filled out a consent form and brief questionnaire (Table 1). Of the
624 participants, 36 males and 584 females participated (3 students did not fill out surveys). Their average age was 22.8 years with a
minimum age of 18 years and maximum age of 62 years. Protocol #H1213-137 was approved by the UNCW Institutional Review Board
in March 2013.
Antibiotic resistance profiles of S. aureus isolates collected from
the noses and throats of nursing students
Zachary P. Johannesson, Ashton L. Honeycutt, Matthew C. Mason, Caroline L. Jones,
Aaron T. Kesinger, Nicholas C. Loekman, and Kevin B. Kiser
Department of Biology and Marine Biology, University of North Carolina Wilmington, 601 S. College Road, Wilmington, NC 28403
Staphylococcus aureus (S. aureus) is an opportunistic bacterial pathogen that can cause a range of skin, soft tissue and invasive infections in humans. It is also carried harmlessly in the nose of 30% of the population as well as throat in unknown amounts. S. aureus is of concern in healthcare settings, where compromised hosts and
invasive procedures allow the bacteria to bypass host defenses, causing infection. These infections are made worse by antibiotic resistant strains, particularly methicillin-resistant S. aureus which are difficult to treat with antibiotics. Studies have shown that MRSA is carried by 6.25% of healthcare workers, possibly contributing to the
spread in hospitals. To test whether the rates of MRSA carriage will increase from the 1-2% with an increase in clinical exposure, the noses and throats of UNCW nursing students were tested for the presence of MRSA. The nose and throat swabs were incubated in a selective culture then transferred to CHROMagar Staph aureus
plates. S. aureus colonies were identified, isolated, and then subjected to antibiotic susceptibility testing. Over seven semesters the S. aureus carriage rates have been 52% (36% nose/31% throat), 43% (23% nose/29% throat), 41% (24% nose/31% throat), 62% (46% nose/34% throat), 50% (22% nose/36% throat), 47.8% (29%
nose/39% throat), and 41% (21% nose/32% throat). Only nine students (2.3%) tested positive for MRSA, indicating the increased clinical exposure in nursing school does not lead to increased carriage rates. However, this study highlights the importance of testing the throat in addition to the nose, as several semesters saw more
throat than nasal carriers.
FIGURE 5. Rates of S. aureus carriage in nursing students across
seven semesters. Mean carriage rates: Total carriers, 47.8%;
nose carriers, 29.2% (exclusive nose carriers, 15.0%); throat
carriers, 32.7% (exclusive throat carriers, 18.5%).
ACKNOWLEDGEMENTS
UNCW students William Brock, Brandon Ludlum, Meghan Dalziel, Anna Fairfax, Ashley VanDerlip, Austin Martini, Camry Wagner, Rheanna McKnight, Lindsey DeSandre, Robert Schilke, Cortney Castine, Taylor
Morrisette, Samuel Gmuca, Garrett Hutchings, Haleigh Nelson, Erin DeYoung, and Megan Davitt-Loysen also contributed to this study. We would like to thank the students and faculty of the UNCW School of
Nursing for their overwhelming cooperation and support. Special thanks to Dr. Anne-Marie Goff, the Association of Nursing Students advisor, for helping us to coordinate sample collections. This research was
supported with intramural funds from UNCW including an Experiencing Transformative Education through Applied Learning (ETEAL)-supported pedagogy initiative, Center for the Support of Undergraduate
Research and Fellowships (CSURF) Research Supplies Awards and Travel Grants, and applied learning funds from the Department of Biology and Marine Biology.
INTRODUCTION
Staphylococcus aureus is an opportunistic bacterial pathogen that is harmlessly carried in the nose of 30% of the population. S. aureus
can cause a range of skin, soft tissue and invasive infections, which is of concern in healthcare settings, where compromised hosts and
invasive procedures allow the bacteria to bypass host defenses, causing infection. These infections are made worse by antibiotic-
resistant strains, particularly methicillin-resistant S. aureus (MRSA), which are difficult to treat with antibiotics. In 2013 alone, 80,461
severe infections and 11,285 deaths were caused by MRSA.[1] Healthcare-associated infection (HAIs) account for 86% of invasive
MRSA infections [2] and carry a mortality rate of 20-50%.[3] It is estimated that a patient that contracts an infection is usually exposed
to the pathogen within 48 to 72 hours when admitted into the hospital.[4] Studies have shown that MRSA is carried by 6.25% of
healthcare workers [5], possibly contributing to the spread in hospitals. An additional study testing the nares and axilla of both nursing
students with clinical exposure and non-nursing students in a microbiology class found a statistically significant difference in MRSA
carriage between nursing and non-nursing students.[6] While this study indicates an increased risk of MRSA carriage associated with
increased clinical exposure, other studies testing just the nares of nursing students showed no increase in MRSA carriage rates as
clinical exposure increased.[7,8] Emphasis is placed on the nose, as 80% of invasive nosocomial infections originate from nasal
carriers.[9] However, a 2007 study showed that 37.1% of tested individuals were nasal carriers, but 12.8% were solely throat carriers,
which emphasizes the importance of not only checking the nose but also checking the throat for carriage.[9] The goal of our study is to
observe the MRSA carriage rates in both the nose and throat of nursing students over the course of several semesters. By
continuously testing nursing students throughout their nursing school career, we will be able to discern carriage rates of MRSA and
observe whether S. aureus carriage changes over time.
REFERENCES
1. Center for Disease Control and Prevention. (2013). FY15 Detect and Protect against Antibiotic Resistance Budget Initiative. Drug resistance. Retrieved from http://www.cdc.gov/drugresistance/threat-report-2013/pdf/FY15-DPAR-budget-init.pdf
2. Jernigan J., Kallen A. (2010). MRSA tool kit [PowerPoint slides]. Retrieved from http://www.cdc.gov/HAI/pdfs/toolkits/MRSA_toolkit_white_020910_v2.pdf.
3. Hanberger, H. (2011). Increased mortality associated with methicillin-resistant Staphylococcus aureus (MRSA) infection in the Intensive Care Unit. National Center for Biotechnology Information. U.S. National Library of Medicine, 38(4), 331-5. doi: 10.1186/cc9654
4. Radhakrishna, M., Monalisa, D., Subbannayya, K., Vishwas, K., & Shashidar. M. (2013). Prevalence of methicillin resistant Staphylococcus aureus carriage amongst health care workers of critical care units in kasturba medical college hospital, mangalore, India. Journal of Clinical and Diagnostic Research, 7(12),
2697-2700. doi: 10.7860/JCDR/2013/5160.3735
5. Mattieu E., Yolaine M. (2004). Carriage of Methicillin-Resistant Staphylococcus aureus Among Hospital Employees: Prevalence, Duration, and Transmission to Households Infection Control. Infection Control and Hospital Epidemiology, 25(2), 114-20. doi: 10.1086/502360
6. Gillen, A., Daycock, W., Serafin, A. (2014). High MRSA Carriage Rate Among Nursing Microbiology Students. Advances in Microbiology, 4(13), 871-7. doi: 10.4236/aim.2014.413096
7. Rohde, Rodney E., Cheryl Rowder, Tom Patterson, Gerald Redwine, Bob Vasquez, and Emillio Carranco. (2016). Methicillin Resistant Staphylococcus Aureus (MRSA): An Interim Report of Carriage and Conversion Rates in Nursing Students. Clinical Laboratory Science, 25(2), 94-102. Retrieved from
http://www.ascls.org/continuing-education/publications
8. Rohde, R. Patterson, T., Covington, B., Vasquez, B., Redwine, G., Carranco, E. (2014). Staphylococcus, not MRSA? A final report of carriage and conversion rates in nursing students. Clinical Laboratory Science, 27(1), 21-31. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/24669443
9. Dominik M., Reno F., Barbara J. (2007). Throat Swabs Are Necessary to Reliably Detect Carriers of Staphylococcus aureus. Clinical Infection Diseases, 45(4), 475-7. doi: 10.1086/520016
DISCUSSION
Out of 624 individuals tested during the past seven semesters, 47.4% were carriers of S. aureus in their nares and/or pharynx. Of those, there
were more students who were strictly throat carriers than strictly nose carriers. This is higher than expected based on other studies [9], and
highlight the importance of testing the throat in addition to the nose. Furthermore, penicillin resistance was lower than expected with an
average of 67.2% of isolates exhibiting resistance to the antibiotic. Studies have shown that over 80% of staphylococcal infections displayed
penicillin resistance. [5] Additionally, of the clindamycin resistant isolates 52.7% had inducible resistance, indicating the importance of testing for
inducible resistance with clindamycin. S. aureus isolates that were resistant to cefoxitin, a methicillin surrogate, were defined as MRSA. Of the
total participants, only 2.29% were MRSA carriers, which coincides with the data found in other studies of nursing students. [7,8] The data
suggest that exposure to clinical environments does not increase MRSA carriage in nursing students. However, there is potential for healthcare
workers and nursing students to become passive carriers, which was difficult to quantify with this study. The overall low percentage of MRSA
carriers may be attributed to the increased awareness and education of safe healthcare practices in the UNCW School of Nursing.
FIGURE 6. Rates of S. aureus resistance to selected antibiotics
across seven semesters. Percentages include intermediate
resistance to erythromycin, clindamycin, and tetracycline and
inducible resistance (D-test) to clindamycin. Mean % of S. aureus
resistance to antibiotics: penicillin 64.37%, cefoxitin 2.29%,
erythromycin 29.26%, clindamycin 23.15%, tetracycline 21.71%.
*Tetracycline resistance not determined in Spring 2013.
FIGURE 2. CHROMagar
Identification of S. aureus. Each
swab sample was spotted on
CHROMagar™, a medium which
allows S. aureus to exhibit a pink-
to-red hue after a 24-hour
incubation period at 35˚C.
Samples that showed other colors
or no growth were not tested
further and were concluded to be
negative for S. aureus.
FIGURE 1. Sample
Collection. Nose and
throat swabs were
collected from each
participant and placed
in tubes of tryptic soy
broth supplemented
with 7.5% NaCl.
Samples were
incubated for 24 hours
at 35˚C.
FIGURE 3. Coagulase
Test. Isolates were
confirmed as S. aureus
by testing for
coagulase. After a 24-
hour incubation period
in rabbit plasma with
EDTA at 35˚C, the
samples were observed
for coagulation.
FIGURE 4. Disk Diffusion Test for
Antibiotic Susceptibility. Mueller-
Hinton agar was swabbed with a
suspension of bacteria. Five
antibiotic disks were placed on the
agar. After incubation for 24 hours
at 35˚C, zones of inhibition were
measured to distinguish between
sensitivity, intermediate resistance,
and resistance. A “D-zone” indicated
inducible-clindamycin resistance.
TABLE 1. Questionnaire data. 1) Percentage of
participants from each semester that have been
hospitalized in the past 12 months (*Spring 2013
participants were asked “have you ever been
hospitalized.” This may explain the higher percentage
of positive responses compared to the other
semesters). 2) Percentage of participants each
semester that have ever been diagnosed with a staph
or MRSA infection. 3) Frequency of antibiotic use
among the participants over given periods of time
(**Spring 2013 participants were only asked if they
were prescribed antibiotics in the past 6 months: 37%
yes, 61.5% no). 4) Percentage of participants that self-
reported nose picking. This is being investigated as a
contributing factor to nursing students becoming
carriers of S. aureus.
CENTER FOR THE SUPPORT
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UNDERGRADUATE RESEARCH AND FELLOWSHIPS
CSURF
*
QUESTION
SPRING
2013
FALL
2013
SPRING
2014
FALL
2014
SPRING
2015
FALL
2015
SPRING
2016
1) Previous hospitalization
Yes 32.30%* 11.22% 5.30% 3.80% 4.55% 6.82% 2.82%
No 66.10%* 88.77% 94.70% 96.20% 95.45% 93.18% 97.18%
2) Previous staph/ MRSA infection
Yes 10.70% 4.10% 1.80% 3.80% 4.55% 6.82% 2.82%
No 87.70% 95.90% 98.20% 96.20% 95.45% 93.18% 97.18%
3) Frequency of antibiotic use**
Never ---- 4.10% 1.80% 0.00% 3.03% 3.41% 7.04%
Not in 12 months ---- 38.80% 46.90% 44.80% 34.85% 56.82% 36.62%
Once in 12 months ---- 30.60% 30.00% 35.20% 30.30% 26.14% 32.39%
More than once in the past
12 months
---- 26.50% 21.20% 20.00% 31.82% 19.32% 23.94%
4) Self-reported nose picking
Yes 20.30% 43.60% 47.30% 53.33% 72.73% 59.10% 53.52%
No 79.70% 56.40% 52.70% 46.66% 27.27% 40.90% 46.48%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
SPRING
2013
FALL 2013 SPRING
2014
FALL 2014 SPRING
2015
FALL 2015 SPRING
2016
None
Throat Only
Nose and Throat
Nose Only