The document summarizes a study conducted by a biology student that found antibiotic-resistant bacteria present in higher numbers in February compared to October at a local daycare center in North Carolina. Swabs were taken from various surfaces in a toddler room in the fall and winter. Five bacterial species showing resistance to multiple antibiotics were identified in October, compared to fourteen species in February. The student used microscopy, biochemical testing, and DNA sequencing to identify the bacteria isolated. The results indicate antibiotic-resistant bacteria pose a legitimate health threat in daycare centers.

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Prevalence of antibiotic-resistant bacteria at a local daycare center in coastal
North Carolina
Rachael M Cannon, B.S. Biology student
Supervisor: Dr. Kevin Kiser, PhD
University of North Carolina Wilmington Department of Biology and Marine Biology
Abstract
Due to our society’s previous carelessness with antibiotic overprescribing and the relatively recent anti-vaccination trends
amongst uneducated parents, I have become increasingly concerned with the possible threat of antibiotic-resistant
bacteria in our daycare centers. I swabbed the Toddler 2 Room, frequented by approximately ten 18 to 24 month-old
children, of a daycare center located in the coastal North Carolina region during the months of October and February as a
seasonal comparison. Swabs of high-traffic areas were transferred directly onto nutrient agar containing penicillin. Five
bacterial species (n = 12) found in the October swabbing and fourteen bacterial species (n = 19) found in the February
swabbing exhibited multiple antibiotic resistances. These results illustrate that the threat of multiple antibiotic drug
resistance at daycare centers is a legitimate concern that warrants further study.
T
Figure 2a. Isolated Staphylococcus aureus Figure 2b. Mueller-Hinton II agar plate Figure 2c. Differential Gram stain of Figure 2d. Growth of S. aureus on
on nutrient agar plate illustrating multiple antibiotic-resistance S. aureus CHROMagar designed for S. auerus
of S. aureus
Figures 2a, b, c, and d all originate from samples taken in October 2013. Multiple antibiotic-resistant Staphylococcus aureus was collected and isolated (Figure 2a), and
determined to be a gram-positive cocci (Figure 2c). Growth on CHROMagar designed for S. aureus growth confirmed the species (Figure 2d), which exhibited multiple
antibiotic drug resistance (Figure 2b). Antibiotic codes are as follows: S (streptomycin 10µg), E (erythromycin 15µg), P (penicillin 10 IU), T (tetracycline 30µg), AM (ampicillin
10µg).
Results
Using the determinative bacteriology methods described, the following bacteria were
found from the October 2013 swabbing: Staphylococcus aureus was on the toilet floor,
Shigella sonnei was on some of the books, Staphylococcus sp. was on the changing
table, and Pseudomonas sp. was on the back door. Sphingomonas sp. was identified
using DNA sequencing of 16S rRNA gene. The bacterial species in Table 2 were
identified using DNA sequencing of 16S rRNA gene, to include Pseudomonas fluorescens,
Acinetobacter calcoaceticus, Acinetobacter radioresistens, Rhodococcus equi, and
Massilia timonae. All of the bacteria identified exhibited resistance to at least two of the
antibiotic drugs they were tested against (Tables 1 and 2). Time constraints prevented
identification of all bacteria in the winter sampling.
Figure 1. Surface swabs from the fall sampling onto
three mediums (from the left): plain nutrient agar,
nutrient agar plus penicillin (to remove all
penicillin-susceptible bacteria), and MacConkey
agar (to determine if there was a significant
presence of Enterobactericeae.)
Methods
Study Location
Surface sampling was conducted in the Toddler 2 Room at a daycare center in Jacksonville,
NC. It is frequented by approximately ten 18 to 24 month-old children on a daily basis and
is the first room in the daycare center where potty training is available.
Surface Sampling
All surface sampling was conducted on a random basis. Surface samples were collected
with sterile cotton swabs and then transferred onto agar plates. For the fall sampling,
three cotton swabs were used simultaneously on each surface to swab onto three
different agar plates: nutrient agar, nutrient agar plus penicillin (10 units/ml), and
MacConkey agar (Figure 1). After incubation, each unique colony on the NA + pcn plates
was transferred to a fresh nutrient agar (NA) plate for isolation and further analysis. For
the winter sampling, all swabs were transferred onto NA + pcn plates only (Figure 2a). The
same isolation and analysis procedures were used thereafter.
Resistance Profiling via Disc Diffusion Method
Several colonies of each isolate were suspended in sterile saline to match the turbidity of
a 0.5 MacFarland standard. A 3-way streak was performed on Mueller-Hinton II agar, and
then the antibiotic discs were placed. After incubation, the zones of inhibition were
measured and compared to cutoff values as set forth by the Clinical and Laboratory
Standards Institute (See Figure 2b) (6). See Tables 1 and 2 for the antibiotics tested.
Determinative Bacteriology
Each bacterial isolate was identified by Gram stain and brightfield microscopy at
1000x magnification (See Figure 2c). Based on preliminary identification, each
isolate in the fall sampling was subjected to further biochemical testing.
DNA Sequencing of 16S rRNA Gene
Several colonies of each isolate on nutrient agar were suspended in 50L of
nuclease-free water and DNA was obtained in the supernatant following boiling and
centrifugation. The supernatant was used for PCR amplification of a 16S rRNA gene
sequence. Each PCR reaction contained the following: 5L DNA (supernatant), 0.5
µM each primer (1509R and Eco8F), 1x GoTaq Green FlexiBuffer, 1.5 mM MgCl2, 0.5
units GoTaq (Promega), 0.2 mM dNTP (2). PCR products were purified by spin
column (EZNA Cycle Pure Kit, Omega Bio-Tek) and submitted for DNA sequencing
with the Eco8F primer (Operon). DNA sequences were submitted to BLAST (NCBI)
to identify bacterial species.
Discussion
For the purposes of this discussion, all bacteria showing intermediate or resistant zones
of inhibition for two or more antibiotics were defined as possessing multiple antibiotic
resistances. It appears that there are more antibiotic-resistant bacteria present in this
daycare center during the winter months, but there are several sources of potential
error that could be contributing to these results. Most importantly, I had never
performed the surface swabbing and experimental techniques that were required
before collecting the bacteria in October 2013. Also, due to the low number of trials, I
cannot claim that there is a statistically significant difference between the numbers of
antibiotic resistant bacteria found between the two collections.
What I found particularly worrisome about my results was the sheer number of
antibiotics that some of the bacterial isolates were either resistant to or exhibited
intermediate resistance to. Five of the isolates were resistant to five of the 12 antibiotics
they were tested against, two were resistant to six antibiotics, and two were resistant to
seven of the antibiotics. Even more unsettling is the utility of these laboratory resistance
profiles in clinical practice. For example, the Staphylococcus aureus bacterium that I
isolated showed resistance to penicillin, erythromycin, and ampicillin. According to the
Clinical and Laboratory Standards Institute (CLSI), if this bacterium were to be oxacillin-
or methicillin-resistant, which was not tested for, its laboratory susceptibility to
imipenem would also be clinically insignificant (6). In this case, one would merely hope
that the physician prescribes one of the few appropriate options left, four of which are
commonly stocked in pediatric suspensions.
Finally, I believe that the easiest thing that we can do in the fight against antibiotic
resistance is to educate our daycare providers. Bacteria can utilize the same mechanism
to resist both antibiotics and household cleaners. For example, the adaptations of
AcrAB-TolC efflux pump, located in the cell membrane of E. coli, are largely responsible
for the development of its resistance to many of today’s antibiotics (1). It functions by
essentially returning whatever substance has crossed the cell membrane, to include
both antibiotics and antibacterials such as pine oil, back into the extracellular space (5).
If we can convince daycare centers to stop using household cleaners such as Pine-Sol®,
perhaps we can slow the rate of microbial evolution at the lowest level so that many of
the issues that are occurring in the scientific world, such as lack of funding and
resources for the creation of new antibiotics, will be less detrimental in the future.
Table 1. Antibiotic Resistance Profile for Fall 2013 Surface Samples
Table 2. Antibiotic Resistance Profile for Winter 2014 Surface Samples
Tables 1 and 2. Abbreviations for antibiotics are as follows: PCN = penicillin 10 IU, DOX = doxycycline 30µg, CIP = ciprofloxacin 5µg, AMC = amoxicillin20µg/clavulanic acid
10µg, SXT = sulfamethoxazole 23.75µg/trimethoprim 1.25µg, CLN = clindamycin 2µg, RAM = rifampin 5µg, IPM= imipenem 10µg, AMP = ampicillin 10µg, ERY =
erythromycin 15µg, TET = tetracycline 30µg, STP = streptomycin 10µg. Note: For clarity, if the bacteria was susceptible to the antibiotic, no notation has been made.
Color coding provides visual representation for number of antibiotics the bacteria showed either resistance or intermediate resistance to. Resistance to 7 species was
coded with red, 6 was coded with orange, 5 was coded with yellow, 4 was coded with green, 3 was coded with blue, and 2 was coded with purple. Source: Clinical and
Laboratory Standards Institute (2007).
References
1. Brunel, J.M., Lieutaud, A., Lome, V., Pages, J., Bolla, J. 2013. Polyamino geranic derivatives as new
chemosensitizers to combat antibiotic resistant Gram-negative bacteria. Bioorganic and Medicinal Chemistry.
21:1174-1179.
2. Erwin, P.M., Olson, J.B., Thacker, R.W. 2011. Phylogenetic diversity, host-specificity and community profiling of
sponge-associated bacteria in the Northern Gulf of Mexico. PLoS ONE. 6(11):1-16.
3. Frieden, T. 2013. Antibiotic resistance threats in the United States, 2013. US Department of Health and Human
Services Centers for Disease Control and Prevention. 1-113.
4. Guidos, R.J., 2004. Bad bugs, no drugs. Infectious Diseases Society of America. 1-35.
5. Marshall, B.M., Robleto, E. Dumont, T., Levy, S.B. 2012. The frequency of antibiotic-resistant bacteria in homes
differing in their use of surface antibacterial agents. Current Microbiology. 65:407-415.
6. Wikler, M.A., Cockerill, III, F.R., Craig, W.A., Dudley, M.N., Eliopoulos, G.M., Hecht, D.W., Hindler, J.F., Low, D.E.,
Sheehan, D.J., Tenover, F.C., Turnidge, J.D., Weinstein, M.P., Zimmer, B.L., Ferraro, M.J., Swenson, J.M. 2007.
Performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. Clinical
and Laboratory Standards Institute. 27(1):1-177.
Acknowledgements
I would like to thank the Children’s Castle Daycare Center in Jacksonville, NC for allowing
me to utilize their facility. This research was supported by applied learning funds from
the Department of Biology and Marine Biology at UNCW.
Introduction
Although the discovery of antibiotics was a major success in the medical field, the overuse
and misuse of such valuable medications have led to the evolution of increasingly resistant
strains of bacteria. The threat of multiple antibiotic-resistant bacteria to public health was
first recognized in the 1980’s, but only recently has attention been given to the presence
of these bacteria on surfaces outside of hospitals or other healthcare-centered facilities
(1).
While many government agencies understand the gravity of the situation, the typical US
adult is not aware that microbes are increasingly present on everyday surfaces (4).
Unfortunately, the hygiene habits that we assume keep these dangerous microbes at bay
could in fact be contributing to the development of their resistance. Using antibacterials
such as triclosan or pine oils to clean surfaces will leave residues that can cause microbes
to evolve over time and develop mechanisms of resistance (5). However, the situation is
much more complex than the simple overuse of household and industrial cleaners.
Another major cause of multiple antibiotic resistance is the overprescribing and
inappropriate prescribing of antibiotics over the decades. The CDC recognized this issue
several years ago and organized the national Get Smart campaign, which focuses on
improving the appropriate use of antibiotics (3). In this manner, both the prescribers and
the patients can be working in tandem to contribute to the issue. These risk factors could
culminate in daycare center environments, where employees frequently use agents such
as Pine-Sol® to clean the floors, and children are frequently being treated with antibiotics
for common ailments such as ear infections and strep throat.
This study focuses on the identification of multiple antibiotic-resistant bacteria in a local
daycare center near the University of North Carolina Wilmington. I swabbed the Toddler 2
Room during the months of October 2013 and February 2014, where I found five and
fourteen multiple antibiotic-resistant species, respectively. After a species was identified
as having multiple antibiotic resistances, a mixture of microscopy, biochemical testing and
DNA sequencing were used to identify the bacteria.
Resistance Profile
Surface Bacteria PCN DOX CIP AMC SXT CLN RAM IPM AMP ERY TET STP
Books Shigella sonnei R R R I
Toilet Floor Staphylococcus aureus R R R
Back Door Pseudomonas sp. R R R
Changing Table Staphylococcus sp. R R
Toddler Sink Sphingomonas sp. R R
Resistance Profile
Surface Bacteria PCN DOX CIP AMC SXT CLN RAM IPM AMP ERY TET STP
Toilet Floor Pseudomonas fluorescens R R I R R R R
Toilet Floor Acinetobacter calcoaceticus R R I R R R I
Toilet Floor Acinetobacter radioresistens R R R R I
Toilet Floor Rhodococcus equi R R R R
Toilet Floor gram (+) coccus R R R
Toilet Floor gram (+) coccus R R I
Toilet Floor Massilia timonae R R
Toilet Floor gram (+) coccobacillus R I
Toilet Floor gram (+) bacillus R R
Toys Acinetobacter radioresistens R R R R R I
Toys Acinetobacter radioresistens R R R I R
Toys gram (-) bacillus R I R R R
Toys gram (+) coccus R R R R I
Back Door gram (+) bacillus R R R I