Identification and Characterization of Bacteria Present on Mobile Phones

By Skylar Capriola, Stacy Huang, Kim Nguyen

Abstract
Background:
Mobiles phones are invaluable devices that have become integrated into the daily life of
individuals worldwide. These devices are constantly in use in a variety of environments where
they are exposed to many bacteria, which makes mobile phones reservoirs for pathogenic bacteria.
Objective:
This study aimed to characterize and identify the bacteria present on cell phones and examine the
properties of these bacteria to determine whether they were pathogenic.
Materials and Methods:
Bacterial samples were collected by swabbing five mobile phones. The samples were cultured and
isolated on BHI, R2A and Schaedler agar. The morphological characteristics of the isolates were
evaluated with gram stain and microscopy. The identity of the isolates was determined by partial
16S rRNA gene sequencing.
Results:
Counts of average colony forming units (CFU/ml) showed that both the back and front surfaces of
mobile phones consist of significant contamination. The back of the mobile device had an average
of 546 ± 94.03 CFU/ml and the front had an average of 593.33 ± 159.44 CFU/ml, but this
difference was not statistically significant. Among the isolates cultured, 11 were part of the
Pseudomonadaceae family, 9 were Enterobacteriaceae bacteria (6 were Klebsiella species) and 1
was a Comamonadaceae bacteria (Variovorax species).
Conclusion:
Pathogenic species of bacteria are present on the surfaces of mobile phones, thus mobile phones
may act as vehicles of transmission of diseases and may pose as a health risk to humans.
INTRODUCTION

With increased dependence and improvements in technology in recent years, mobile

phones are among the many devices that have made a significant impact worldwide. The number

of mobile phones in use is almost equivalent to the number of humans there are on the planet [4].

In 2013, there were more than 1.6 billion smartphones used worldwide. This number is expected

to approximately double in the next 4 years [2]. Contributing to the increased popularity of mobile

phones is that they can be used for almost everything, including both personal and professional

activities. Many people own a mobile phone now and they have been integrated into the daily of

life of people all around the world.

Cell phones are used by their owners all day long in many different settings, and more often

than not, they are not properly cleaned. Phones come into contact with body areas such as the

hands, face, and ears, as well as a variety of surfaces and environments (e.g. bathroom, work,

school) where they can be placed. The human skin is always exposed to microbes and it is easily

colonized by certain microorganisms, possessing the ability to support approximately 1012

bacterial cells/person [5]. The average individual touches his/her phone screen about 153 times a

day [3]. Therefore, it can be presumed that use of mobile phones can cause colonization of bacteria

from phones onto the human skin and vice versa, or there could be migration of bacteria from

surfaces to humans or from environments to humans via phones. As a result, mobile phones can

carry many bacteria and may act as potential vehicles for spreading pathogenic microorganisms

and thus pose as health risks to users.

Despite mobile phones being so important in people’s daily lives, there is limited

knowledge about their impact on human health. Interestingly, it has been suggested that cell

phones might act as fomites. Not many studies on the microbes present on cell phones exist, and
most of these studies focus on cell phone contamination among workers in healthcare settings.

Brady et al. reported that 9-25% of mobile phones are contaminated with pathogenic species of

bacteria. Studies have also shown that in hospitals, this number can be as high as 94.5% [2].

Only a few studies have examined the microbial community present on mobile phones

outside of healthcare settings. There, the microbial content of phones may be different due to

different environments or different human activities that phones are exposed to. However, given

the constant daily use of phones, they are likely to still possess pathogenic species of bacteria.

Therefore, this study aimed to identify and characterize the bacteria present on cell phones, as well

as to evaluate the properties of the bacteria to determine if they are pathogenic species. Bacterial

samples were obtained from five cellular phones, from which a total of 21 isolates were cultured

on BHI, R2A and Schaedler plates. Gram staining and microscopy were used to determine the

morphological characteristics of the isolates. Also, the isolates were identified by partial 16S rRNA

gene sequencing. Then the properties of these identified bacteria were investigated to determine

whether they had pathogenic attributes.

The results demonstrate that the number of bacteria on the front and back surfaces of cell

phones is not very different. According to average colony forming units counts (CFU/ml), the back

had an average of 546 ± 94.03 CFU/ml and the front had an average of 593.33 ± 159.44 CFU/ml,

but this difference was not significant. Consistent with this observation, there was a relatively even

distribution of isolates on the front and back surfaces of the phones; there were 10 out of 19 isolates

from the front and 9 from the back. Among the isolates, 11 were determined to be part of the

Pseudomonadaceae family. There were 9 isolates that were Enterobacteriaceae bacteria (6 were

Klebsiella species) and 1 was a Comamonadaceae bacteria (Variovorax species). Variovorax from

the Comamonadaceae family does not cause disease in humans, however, the families
Enterobacteriaceae and Pseudomonadaceae consist of pathogenic species that can cause

opportunistic infections in humans. This indicates that mobile phones may serve as potential health

risks to humans.

MATERIALS AND METHODS

Sample Collection

Five iPhones from students and instructors in a graduate teaching lab at the University of

Michigan were voluntarily chosen for study. An Ultimaker 3D printer (Cambridge, MA) was used

to print a 3 cm x 3 cm rectangular area that was used to help maintain consistency in defining the

swabbing area for collection of bacterial samples. Bacterial samples on the front and back surfaces

of phones were collected using swabs moistened in sterile pH 7.4 PBS buffer, and using the 3D

printed apparatus. A 3 cm x 3 cm area on the bottom half of the front and back surfaces of the

phones were swabbed. The swabs were then placed in 1 ml of PBS buffer. Between the collection

of each sample, the 3D printed apparatus was sterilized using 70% ethanol to prevent cross-

contamination between the different phones and phone surfaces.

Bacterial Culturing

The bacterial samples suspended in PBS were serially diluted (up to 10-5 dilution) in PBS

buffer. Portions (0.1 ml) of the dilutions (10-3 to 10-5) were plated onto three types of agar: Becton,

Dickinson and Company (BD) BBL BHI agar (Franklin Lakes, NJ), BD-Difco R2A agar (Franklin

Lakes, NJ), and BD-BBL Schaedler agar (Franklin Lakes, NJ). These plates were incubated at

23°C for 2 days. Colonies were differentiated based on morphotype characteristics, including

colony color, size, and morphology. Then the differentiated colonies were isolated and streaked
onto plates containing the appropriate agar type from which the colonies were selected from. The

plates were incubated at 23°C for 2 days.

Characterization of Distinct Bacterial Colonies

Unique colony morphotypes were determined using a Motic SMZ-168 Stereo Zoom

Microscope (Hong Kong) and a Motic B1-252 Binocular Microscope (Hong Kong). Stereo

microscopy was used to examine colony color, size, form, elevation, and margin of differentiated

colonies. Light microscopy was used to visualize morphology as well as gram reaction results of

the isolated colonies.

Identification of Bacterial Isolate Using partial 16S rRNA PCR Amplification and

Sequencing

To prepare for 16S rRNA sequencing, one colony from each isolation plate was picked

off with a sterile pipette tip and was transferred to a PCR Eppendorf tube (1.5 ml) containing 50

ul of sterile nanopure water. The cell suspensions were then boiled for 10 minutes. Five

microliters of each of the boiled cell suspension was used as the template for PCR amplification

of the 16S rRNA gene. PCR was performed using a master stock PCR mix consisting of 25 ul of

GoTaq Green Master Mix (Promega, Madison, WI), 15 ul of nanopure water, 2.5 ul of forward

primer (8FPL), and 2.5 ul of reverse primer. DNA amplification was performed following

standard PCR protocol with a My Cycler TM thermal cycler (Hercules, CA). PCR products were

purified according to the QIAquick PCR Purification Kit Protocol (Qiagen, Valencia, CA).

Sequencing was completed by the DNA Sequencing Core at the University of Michigan (Ann

Arbor, MI). The partial 16S rRNA gene sequences were analyzed using CHROMAS

(Technelysium Pty. Ltd., Australia) and then were compared to known sequences available in the
National Center for Biotechnology (NCBI) database using the Basic Local Alignment Search

Tool (BLAST).

Phylogenetic Analysis

A neighbor-joining phylogenetic tree was created using CLUSTALX v. 2 to align the

partial 16S rRNA gene sequences with closely related strains in the NCBI database. Sequence

analysis was done using the TreeView software. The 16S rRNA gene sequence from Thermus

thermophilus (EMBL accession no. X07998) was chosen as an out-group.

RESULTS

Distribution of CFU on the front and back of mobiles phones is not significantly different

Microbial growth was only observed among cultures of bacterial samples swabbed from

two of the five cell phones (phones 1 and 2). Therefore, only these two phones were included in

analyses, while the remaining three phones were considered outliers and were excluded from the

analyses to avoid skewness in the data. The average colony forming units (CFU) (million

CFU/ml) of the front and back surfaces of the two phones were compared. The back surface had

an average of 546 ± 94.03 CFU/ml, while the front surface had an average of 593.33 ± 159.44

CFU/ml (Fig. 1). The distribution of average CFU among the front and back surfaces of the

phones differed slightly between the surfaces, however, this difference was not statistically

significant (0.10 < P < 0.25). These are both still large numbers and indicate a high amount of

bacterial contamination on mobile phones.

Uneven distribution of unique colony morphotypes of the front and back of mobile phones

A total of 21 isolates with distinct colony morphologies were obtained from the two

phones across all the agar plates. Characterization of these isolates by gram-staining revealed a
differential distribution of unique colony morphotypes on the back and front surfaces of the

phones (Fig. 2). The front surfaces appeared to have a relatively even distribution of the number

of unique colonies characterized as gram-positive and gram-negative bacteria. In contrast, the

back surfaces had approximately twice the number of distinct colony type characterized as gram-

negative bacteria than gram-positive.

Pathogenic and non-pathogenic bacterial species are present on mobile phones

Although a total of 21 isolates were obtained from the surfaces of the phones, only 19

were sequenced (because there may not have been enough DNA in two of the samples). On

phone 1, five isolates were cultured from the front and 6 isolates were cultured from the back.

On phone 2, 4 isolates were cultured from both the back and front surfaces. Identification based

on 16S rRNA sequencing identified the isolates as belonging to three families:

Enterobacteriaceae, Comamonadaceae and Pseudomonadaceae (Fig. 3). Only the isolate

belonging to the family Comamonadaceae was exclusive to phone 1 (front surface), while

isolates belonging to the families Enterobacteriaceae and Pseudomonadaceae were found on the

front and back surfaces of both phones. There were 11 Pseudomonadaceae isolates found across

the surfaces of both phones, with 5 obtained from the front (45.5%) and 6 from the back

(54.55%) (Table 1). Seven of the isolates on the surfaces of phones 1 and 2 were classified as

being in the family Enterobacteriaceae, with 3 from the front surface (42.86%) and 4 (57.14%)

from the back surface (Table 1). Six (85.71%) of the Enterobacteriaceae were members of the

genus Klebsiella and one (14.29%) was of an unidentified genus (Table 1). Two of the Klebsiella

(33.33%) were found on the front surface and four (66.67%) were obtained from the back

surface (Table 1). The remaining Enterobacteriaceae isolate was obtained from the front surface
of phone 1. Finally, the one isolate of the family Comamonadaceae, which is a member of the

genus Variovorax, was found only on the front surface of phone 1.

DISCUSSION

The work presented here shows that there was an approximately even distribution of

bacterial isolates obtained from the back and front surfaces phones 1 and 2. Nine isolates were

from the back and ten were from the front. This observation was consistent with results from the

average CFU counts, which suggested that the difference in the amount of bacteria found on the

front and back surfaces of phones is likely to be insignificant. As expected, for these isolates, the

difference was only one, however, comparing this to the actual large numbers of bacteria

contamination on phones, the difference between the front and back of phones would be even

more insignificant.

Most of the bacteria (11 out of 19 isolates) found on the phones were identified as

belonging to the Pseudomonadaceae family, and they were located on both the back and front

surfaces of phones 1 and 2. The next most common type of bacteria found were those of the

Enterobacteriaceae family (7 out of 19 isolates), which were on both surfaces of phones 1 and 2,

and they included species of Klebsiella and one of an unidentified genus. The remaining isolate

obtained from the front surface of phone 1 was part of the Comamonadaceae family, specifically

the Variovarax genus.

The Pseudomonadaceae family contains the Pseudomonas genus of gram-negative

bacteria that are usually found in soil or water, and may act as opportunistic pathogens in

humans, plants, and animals [9]. Pseudomonas aeruginosa is a species that causes infections in

humans, typically in those who are immunocompromised. It often causes infections with a high
mortality rate, which is partly due to its high resistance to antimicrobials. For example, P.

aeruginosa is involved in cystic fibrosis and is considered one of the most important pulmonary

pathogens and the main cause of mortality in cystic fibrosis [1].

Enterobacteriaceae bacteria are gram negative facultative anaerobes that are causes of

many diseases, including urinary tract infections, bloodstream infections, pneumonia,

nosocomial infections, and intra-abdominal infections [7]. There has been emerging resistance

and spread of resistance in Enterobacteriaceae that make them significant problems, further

complicating diseases. [7]. Klebsiella pneumoniae, which was found on both surfaces of phones

1 and 2, is one such species of Enterobacteriaceae that has received significant attention for its

role in causing disease. K. pneumoniae often causes infections in immunocompromised

individuals, but the emergence of hypervirulent strains have made the number of people

susceptible to disease increase, including healthy individuals [6]. Like other Enterobacteriaceae

species, K. pneumoniae has become more and more resistant to antibiotics which make the

bacteria difficult to treat [6]. The combination of hypervirulent strains and resistant strains could

potentially lead to multidrug resistance and persistence of the bacteria, which can complicate

infections and make treatment even more challenging.

There was also an Enterobacteriaceae strain found on the front surface of phone 1 that

was closely related to the genus Pantoea. Pantoea is a relatively new genus that was defined

approximately 25 years ago, and it currently is composed of 20 recognized species that have

been isolated from many different environments [10]. As the neighbor-joining phylogenetic tree

in this study demonstrates, the Pantoea genus is closely related to species of Klebsiella (this has

been revealed in prior studies). Similar to Klebsiella, some species of Pantoea are known to

cause disease since Pantoea have frequently been isolated from nosocomial environments.
Currently, there is debate on the pathogenic abilities and characteristics of Pantoea to cause

disease in humans. Some Pantoea such as P. septica, P. calida, and P. dispersa, have been

frequently isolated from human wounds, fractures, blood, stool, cysts, and abscesses [10]. It has

been suggested that contamination of medical instruments, intravenous nutrition, or wounds can

lead to exposure and colonization of the bacteria onto patients.

One of the isolate was identified as part of the family Comamonadaceae, which are gram-

negative aerobes, and most are motile with flagella. They are often found in water and soil

habitats [11]. The isolate found on phone 1 was a Variovorax strain. Three species of Variovorax

have been identified thus far, and they are chemoorganotrophs that are associated with important

catabolic processes, such as the degradation of toxic or complex chemical compounds. They are

also involved in mutually beneficial relationships with plants and other bacterial species [8]. It

has been suggested that Variovorax strains may potentially be used for bioremediation and other

biotechnical applications [8]. Presently, there is very minimal knowledge on Variovorax, and the

limited numbers of studies that exist are about its environmental role and catabolic properties.

Therefore, it is not yet known, and it seems that Variovorax species are not harmful to humans.

To conclude, the Enterobacteriaceae and Pseudomonadaceae that were discovered on

phones 1 and 2 possess pathogenic capabilities and are known to cause infections in humans,

while the Comamonadaceae isolate does not, or is not yet known to cause disease in humans.

Furthermore, Comamonadaceae bacteria are not usually found on humans, so the reason for its

presence on phone 1 and not phone 2 may just be due to different exposures the owners of the

phones subject their devices to. This study shows that bacteria found on humans, or in the

surrounding environment are also found on cell phones. This means it is likely that the bacteria

on phones may have been transferred from humans, objects surfaces, or atmospheres to phones,
and this exchange is likely to occur in the reverse direction as well. Given that there can be

pathogenic bacterial species present on phone surfaces and they can be transferred to humans,

mobile phones have the potential to pose as health risks to humans and society, especially to

individuals who are immunocompromised.

CONCLUSION

In summary, the sampled mobile phones considered in this study (phones 1 and 2) were

contaminated with species of bacteria, Enterobacteriaceae and Pseudomonadaceae, that have

pathogenic characteristics, as well as bacteria that is not harmful to humans, Comamonadaceae.

Additionally, the average CFU count of the phone surfaces produced large numbers that were

based on dilutions, so the actual numbers are presumed to be much larger. Though the findings

observed here are based on two sampled cell phones, they are consistent with prior studies on

this subject that revealed cell phones carry a great number of bacteria, including those that are

pathogenic and resistant to antimicrobials. In a review by Bloomfield et al. (2012), it was

suggested that regularly touched surfaces play a key role in the transmission of diseases in both

clinical and domestic settings [2]. This applies to mobile phones, which are constantly in use all

day and every day by their owners, making them possible vehicles for transmission of bacterial

diseases.
Figures and Data

Figure 1: Average colony forming units (CFU) by counting the number of colonies present on
agar plates that were diluted to 10-5. Units on the y-axis is CFU (million)/mL after accounting for
the serial dilution.
Figure 2: Number of unique colony morphotypes (n=21) that were identified using gram staining
techniques. The overall distribution of the front of both phones was compared to the overall
distribution present on the back surface of both phones.
Figure 3: Neighbor-joining phylogenetic tree was constructed using partial 16S rRNA gene
sequences of the cell phone bacterial isolates and closely related strains available through NCBI.
Thermus thermophilus was used as the outgroup. The bar at the bottom represents one nucleotide
difference for every ten nucleotides in the sequence. Red text indicates that the sample strain was
from the front surface and the blue text indicates that the strain was from the back surface. A key
table was provided to identify the origin of the strain, the media that was used, and characteristic
of the colony type.
 Family
Front Count (%) Back Count (%)
Genus (%) % of Genus on Front % of Genus on Back
5 (45.45) 6 (54.55)
Pseudomonadaceae

Pseudomonas spp.(100) 100% 100%

Enterobacteriaceae 3 (42.86) 4 (57.14)

Enterobacteriaceae spp. (14.29)* 1 (100%) 0 (0%)

Klebsiella (85.71) 2 (33.33%) 4 (66.67%)

Comamonadaceae 1 (100) 0 (0)

Variovarax (100) 1 (100%) 0 (0%)

Table 1: From the information of the neighbor-joining phylogenetic tree, the distribution of the
identified bacterial isolates was compared between the front and back surfaces. The family name
used bolded text while the genus name was not bolded. If the genus was specified, the text was
italicized.
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