1

Bringing Work Home: The Occurrence of Methicillin-Resistant Staphylococcus aureus (MRSA)

and Micrococcus luteus on the Cell Phones of Healthcare Workers and on Those of Their
Cohabiting Partners
Autumn Rounds-Knox
BIOL 4415L: Pathogenic Microbiology Laboratory
Fall 2015

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I.

INTRODUCTION

When working in any occupation, there will be times when bringing work home is necessary. For health
care workers, bringing work home can be entirely involuntary. This because health care workers are exposed to a
myriad of pathogenic microorganisms (such as Methicillin-resistant Staphylococcus aureus (MRSA) and
Micrococcus luteus) throughout in the workplace, and these nosocomial (hospital-acquired) pathogens are known to
not only be transmitted via direct (person to person) contact, but also via indirect contact. In fact, some bacteria
associated with nosocomial infection can survive on fomites (inanimate objects) for up to months on dry surfaces.
Research shows that bacteria associated with nosocominal infection, such as Staphylococcus aureus(including
MRSA), Clostridium difficile, and Candida albicans can surive for months on fomites (Kramer et. al, 2006).
Though most would not consider a personal cell phone when considering possible fomite reservoirs for
nosocomial infections, cell phones are exposed to virtually the same amount of bacterial contamination as are the
hands of its owner. Studies investigating the microbial overlap of personal cell phones and their owners hands have
shown as much as 20% of bacterial taxa were identical and that cell phones represent a sample of their owners
microbiome (Meadow et. al, 2014).
Tambekar et. al (2008) found that 83% of cell phones owned by doctors participating in the study harbored
Methicillin-resistant Staphylococcus aureus (MRSA), and 18% of cell phones harbored Micrococcus luteus (M.
luteus). This is significant, since it is known that these bacterial are highly virulent and show a high survival rate on
outside of a host (Tambekar et. al 2008).
Furthermore, research shows that this fomite transmission occurs not only in hospital settings, but also in
homes. Indeed, it is known that cohabiting family members share microbiota with one another (Song et. al, 2013). In
this study samples were taken from the dogs and humans, fecal, oral, forehead, and right and left palm communities/
or all four paws if applicable (Song et. al, 2013). This study found that there are significant differences in
communities between the dogs paws and forehand compared to their owners palms and foreheads (Song et. al,
2013). However the bacterial communities present in the tongue and gut are very similar between the dogs and
owners (Song et. al, 2013). This study also noticed a common trend between age group and bacterial communities
(Song et. al, 2013).
The current study aimed to expand upon the previous research into cohabitation and prevalence of bacteria
on cell phones in order to test the hypothesis that these factors show correlation. That is, the cell phones of

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healthcare workers would be contaminated with (MRSA) and/or M. luteus), as would those of their cohabitating
partners. Samples were collected from five volunteer couples, cohabitating for the appropriate length of time. A
survey was giver to gather information about the couple to account for any uncontrollable variables. The couples
touch surfaces of their phones were swabbed. Preliminary testing was done. This was followed by extraction of
DNA and end-point PCR.
II.

MATERIALS AND METHODS

A. SAMPLE SELECTION
The participants were chosen based on relationship length and occupations. Surveys were given to all the
volunteers, which included questions such as, age, gender, length of time cohabitating, and how often cohabitants
share a personal cell phone. Since this experiment will be looking at the length of time cohabitating and the presence
of Methicillin-resistant Staphylococcus aureus and Micrococcus luteus on the couples individual cell phones, the
survey is used to address the potential contaminates of the variables. The volunteers were asked to provide the
following information : Select the correct age range (Less than 20, 20-30, Great than 30); Gender (Male, Female);
Length of time cohabitating with your significant other (None, Less than 6 months, 1<2 years); and How often does
your significant other use your personal cell phone (Always, Sometimes, Every Once in a While, Rarely, Never), Do
you work in health care field (Yes, No) (Refer to Table 1). Individuals where asked to circle what applied to them.
The amount of time spent on their significant others individual cell phones could affect the similarities between
specific pathogens present and give false results.
Samples were collected from five volunteer couples (10 total samples). These couples consisted of two
couples cohabitating for <6 months and two couples cohabitating for 1<2 years, and a male and female whom have
never cohabitated. The male and female whom have never cohabitated was used as a control in this experiment.

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Table 1: Sample Survey Results

Sample
Control M
Control F
1M
1F
2M
2F
3M
3F
4M
4F

Age
>30
20-30
20-30
20-30
20-30
20-30
20-30
20-30
20-30
20-30

Time
cohabitating
None
None
1< 2 Years
1< 2 Years
> 6 Months
> 6 Months
1< 2 Years
1<2 Years
> 6 Months
> 6 Months

How often does your

significant other use your
phone?
/
/
Every Once in a While
Every Once in a While
Every Once in a While
Sometimes
Always
Rarely
Always
Always

Do you work in health care

field?
No
Yes
No
Yes
Yes
No
No
Yes
No
Yes

Table 1. Sample survey. Note: F (Female) and M (Male). The numbers corresponds to cohabitating male and female
pairs

A. SAMPLE COLLECTION
Using a sterile swab prepared in distilled water, the touch surfaces of each mobile phone was swabbed for
approximately 1 minute. The swabs were placed into sterile tubing. The procedure for obtaining an environmental
sample with a cotton swab is described by Leboffe & Pierce (2012). All samples were collected on September 22 nd
2015 at the individual couples residence. The male and female controls were collected on September 24 th 2015 at
Our Lady of the Lake College campus. All samples were collected in Baton Rouge, LA, USA (30.471165,
-91.147385).
B. TARGET ORGANISMS
Target organisms included Micrococcus luteus, and Methicillin-resistant Staphylococcus aureus. According
to De Vos (2009), Staphylococcus aureus are nonmotile, nonspore-forming, gram positive, cocci, observed in pairs
or clusters, colonies are raised, smooth, shiny, and translucent, color is grey to yellow and is grown best under
aerobic conditions. Further S. aureus include, positive mannitol fermentation, and coagulase production. Methicillinresistant Staphylococcus aureus will show growth on chromogenic agar and is used to differentiate between
Staphylococcus aureus Micrococcus luteus conditions for identification (Fig. 1) include: gram-positive cocci,
negative mannitol formation, and positive glucose fermentation (Leboffe & Pierce, 2012).

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C. PRELIMIARY TESTING
Once samples were collected, a glycerol stock was prepared (50% glycerol and 50% of collected sample
and distilled sterile water), and a spread plate on Nutrient Agar was preformed to culture the bacteria. These
procedures are described by Leboffe & Pierce (2012). Once the samples were cultured on the Nutrient Agar, gram
staining was performed. Once gram stain and morphology were evaluated, a dichotomous flow chart was created.
The dichotomous flow chart outlines the tests needed to confirm target species were present. The samples where
streaked for isolation on Mannitol Salt Agar (MSA) to confirm gram-positive cocci, salt tolerance, and to test for
mannitol fermentation. A coagulase test was preformed to further confirm Staphylococcus aureus was presence. To
differentiate between Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus, chromogenic agar
was used. Growth on chromogenic agar indicates the presence of MRSA. Micrococcus luteus conditions for
identification The samples that had yellow pigmented colonies and grew on Mannitol Salt Agar were subjected to a
glucose test was to confirm that Micrococcus luteus was present (see Figure 1).

Figure 1: Dichotomous Flow Chart

Figure 1: Dichotomous Flow Chart. Outline of the steps taken to identify Staphylococcus aureus and Micrococcus
luteus in the preliminary stages of the experiment

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E. DNA Extractions and PCR
Sample was first brought to room temperature (15-25C). QIAamp DNA Mini Kits were used to isolate
DNA from the bacterial species using the Qiaquick mini kit. . Manufacture protocol was followed (Qiagen). Samples
inoculated overnight. Once DNA was extracted, NanoDrop operating protocol found in the user manual was
preformed (2005). Once the presence of DNA was confirmed, the samples were prepared for end point PCR. End
point PCR was used to detect the presence of MRSA (using primers mecA_F: 5-CATTTTGAGTTCTGCACTACC3and mecA_R: 5-GCAATACAATCGCACATACATTAATAG-3) (Klaschik et. al, 2002) and Micrococcus luteus
(using primers recA_F: 5-GCCCTGGACCCGGTCTACGCCCG-3 and recA_R:
5CGCCGATCTTCTCGCGCAGCTGG-3) (Liu, Dongyou, ed. 2009) in bacterial sample. The PCR mixture (20 l)
contained 0.5 l of MgCl, 1 l of Taq DNA Polymerase (5 units/ l), 5 l of template DNA (10ng-500ng), 10 l
sterile dH2O, 0.5 l dNTP mix, 1.5 l of forward primer, and 1.5 l of reverse primer.
PCR protocol was as follows: 2 cycles of denaturation at 95 C for 30 seconds, primers were annealed for 1
minute on a temperature gradient (61.4, 63.3, 64.5 and 65C), and DNA extension for 1 minute for 70C. These
steps were repeated for 35 cycles. The last extension of DNA was at 70C for 5 minutes. To check the size and
concentration of PCR product the 2 l of the samples were ran on a 1% agarose gel.
III.

RESULTS

Table 2 summarizes the results from the preliminary tests. The results from these tests indicated that CM,
2M, 2F, 3M, 3F, and 4F all had the presence of MRSA when cultured and isolated in the lab. 2M, 3F, 4M, and 4F all
had the presence of Micrococcus luteus when cultured and isolated in the lab. CF did not culture in the lab on
nutrient agar. This may indicate that the pathogens present may not culture well in the lab. Since, CF did not culture
in the lab a Nano Drop procedure was performed. Table 3 shows that all samples have genetic material present. The
260/280 ratios in the last column, shows that samples can be evaluated for purity. A ratio approximately 1.8 is
considered pure for DNA. Figure 3 show that PCR was able to identify that all four of the male samples had the
presence of MRSA. Figure 4 also shows that PCR was able to identify that all four samples has the presence of
MRSA except for F4. No bands were present in the first run so F4 was ran again. Figure 5 includes the F4 rerun for
mecA gene in lane 1, which concludes the same result. F4 proves to be the only sample without the mecA gene
present. The following lanes are for the recA gene. CF and 3F show bands for the recA gene. Figure 6 is the results

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for recA gene in males. When these samples were first san the males did not show any bands on the gel image. So
the gel was ran again at lower amperage and voltage for a longer time. Even at these settings the only band shown
was CM sample and very small in size at approximately 100-125 bp.
Table 2: Preliminary Results
CM
Gram
Stain
Morphol
ogy
MSA

Glucose
Fermenta
tion
Coagulas
e
Chromog
enic
Plates
Identifie
d

C
F

1M

1F

2M

2F

3M

3F

4M

4F

Cocci,
Rods,
Endospore
s
Growth

Cocci

Cocci

Cocci

Cocci

Cocci

Cocci

Cocci

Growth
and
Mannitol
Fermentati
on

Growth
and
Mannitol
Fermentati
on

Growth

Growth
and
Mannitol
Fermentati
on

Growth
and
Mannitol
Fermentati
on

Growth

Cocci
Rods,
Endospo
res
No
growth

Growth

No growth

No growth

Methicilli
n-resistant
Staphyloc
occus
aureus
and
Micrococc
us varians

Staphyloc
occus
aureus

Staphyloc
occus
aureus

Growth

Growth

Growth

Growth

Growth

No
Growth

Little
Growth

Methicilli
n-resistant
Staphyloc
occus
aureus
and
Micrococc
us luteus

Methicilli
n-resistant
Staphyloc
occus
aureus

Methicilli
n-resistant
Staphyloc
occus
aureus

Methicilli
n-resistant
Staphyloc
occus
aureus
and
Micrococc
us varians

Microco
ccus
luteus

Methicilli
n-resistant
Staphyloc
occus
aureus
Micrococc
us luteus

Table 2: Summary of the results from the preliminary tests.

Figure 2: Chromogenic Plates

Figure 2: Samples streaked on chromogenic agar. Growth indicates the presence of MRSA.

Table 3: Nanodrop (Quality of DNA)

Sample ID

Nucleic Acid

A260 (Abs)

A280 (Abs)

A260/A280

CON-M AR

37.5

0.750

0.564

1.33

CON-F AR

199.3

3.987

2.986

1.34

1M- AR

27.7

0.553

0.461

1.20

1F-AR

151.8

3.037

2.272

1.34

2M-AR

99.8

1.996

1.481

1.35

2F -AR

30.7

0.614

0.433

1.42

3M-AR

237.4

4.748

3.048

1.56

3F -AR

130.1

2.602

1.945

1.34

4M-AR

16.6

0.332

0.244

1.36

4F -AR

114.4

2.289

1.815

1.26

Table 3: NanoDrop Units: ng/l shows that the Nanodrop shows that all samples had the presence of genetic
material. The 260/280 ratios in the last column, shows that samples can be evaluated for purity. A ratio
approximately 1.8 is considered pure for DNA.
Figure 3: PCR Presence of mecA Gene in Males

Figure 3: PCR Males mecA gene. The PCR bands on 1% agarose show that all male samples showed the presence of
the mecA gene.
Figure 4: PCR Presence of mecA Gene in Females

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Figure 4: PCR Females mecA gene The PCR bands on 1% agarose show that all female samples except 4F showed
the presence of the mecA gene.
Figure 5: PCR F4 Rerun for mecA and Presence of recA in all Females

Figure 5: PCR, F4 in lane one is the rerun for mecA gene, and the following lanes are the females for recA gene. The
PCR bands on 1% agarose show that F4 does not have the presence on mecA gene, and CF and F3 show the
presence of the recA gene.
Figure 6: PCR Presence of recA Gene in Males

Figure 6: PCR recA gene. The PCR bands on 1% agarose show that all males except for CM do not have the recA
gene present. Note that the CM sample band is very small in size. 26

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IV.

DISCUSSION AND CONCLUSION

The significance of these results include that cell phones are carriers of MRSA. It is important to raise
awareness of the potentially harmful pathogens on cell phones since cell phones are a growing necessity of our
culture. It is important to be made aware of the potential pathogens we are exposed to everyday and the people in
our day to day life we could also be exposing. This experiment concludes that cell phones are carriers of MRSA and
no other conclusions from this experiment can be made due to the results be inconstant and potential errors. Due to
lack of time and experience in the lab the potential sources of error include; contamination, inconstancies in the
uniformity of procedures performed on each sample, multiple people running preliminary test on samples, and
finally lack of experience pipetting. Referring back to Table 3 you can see 260/280 ratios are below 1.8. This shows
that my samples were not consider pure which will affect the results. It is important to have pure DNA samples
when running PCR. Other molecules that have not been extracted out will affect your gel results.
This experiment hopes to raise awareness of the spread of nosocomial infections not only by health care
workers, who are exposed to theses pathogens on a day to day basis, but also the individuals that cohabitate with
health care workers. This experiment does lead to further research ideas for this field, specifically looking at
occupations and the types of pathogens present. In future research, I hope to show how much occupation plays a role
in the pathogens families are exposed to.

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V.

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