Lip products possess potentiality to function as bacterial reservoirs for enteric

Gram-negative bacteria in hospital settings

Chance A. Bennett
Our Lady of the Lake College
5414 Brittany Drive
Baton Rouge, LA 70808
chancebennett@ololcollege.edu

Lip products possess potentiality to function as bacterial reservoirs for enteric

Gram-negative bacteria in hospital settings
Chance A. Bennett
Our Lady of the Lake College, Louisiana, United States of America

Abstract
This experiment investigates the role of frequently used personal items, in
particular lip color products, in substantiating the growth of enteric, Gram
negative bacteria

that are known

to

cause nosocomial

gastroenterological

infections. The research was conducted by performing a series of isolation

techniques, using selective and differential media, biochemical tests, serial
dilutions as a means of quantifying target bacteria. I predicted that the solid
lipstick would have a greater potential to sustain bacterial growth, due to its
stable consistency and solid composition. The results proved my hypothesis to be
incorrect; the solid and liquid lipstick were equally capable of substantiating Gram
negative bacterial growth. The results of the research implies the need to enforce
established guidelines for minimal use of cosmetic items, such as lipsticks and
similar cosmetics, by healthcare staff in a hospital setting and may assist in
determining the patterns of transmission, as well as measures which can be taken
to prevent the spread of harmful microbial communities within hospitals.

Introduction

Nosocomial infections are infections acquired in a hospital setting, due to extensive use of antibiotics, lengthened hospitalization, and the
utilization of implantable and invasive medical devices, equipment, and biomaterials during medical treatment and procedures. Five out of
ten of the top pathogens associated with nosocomial blood infections are Gram-negative bacteria (Murray, 2002, p. 753). Out of those five
pathogens, Escherichia coli exhibited the highest percentage of isolates.

In a typical hospital setting, inanimate surfaces, uniforms and PPE, devices, and other medical related items are cleaned and sterilized
regularly. In contrast, the seemingly insignificant objects and places are disregarded as potential bacterial reservoirs. Due to the ubiquitous
nature of bacteria, even the most unlikely things have the potential to constitute an abundance of bacterial growth but those unlikely
objects are often overlooked. For example, consider cosmetic products and how frequently they are used in all settings. There are many

antibacterial products that can be used to wash ones hands or face but there are not many antibacterial products that can be used to clean
the cosmetic products that are used and touched by those same hands and face. This makes the use of the antimicrobial products
somewhat counterproductive.

Cosmetic products, such as lip gloss and lipstick, are constantly exposed to bacteria, such as E.coli, Staphylococcus aureus, and
Staphylococcus epidermis, on the face, lips, hands, and even the air, depending on the packaging of the product and the location at which
the product is stored (The Unhealthy Handbag, 2015). In general, the constituents of many lip products are wax-like substances, oils,
pigments, and dyes, all of which vary depending on the brand, color, and texture of the lipstick (Senese, 2015). Lip products water
content; approximately 0.88%, makes the products suitable environments for bacterial cultivation (Fischer, n.d.). Lipstick is worn by
many healthcare providers as well as patients and visitors frequenting hospital settings. Despite utilizing effective handwashing
techniques, something as simple as touching the surface of lipstick during reapplication or even touching lipstick on a persons lips could
expose an individual to the bacteria residing in the lipstick.

The purpose of this experiment was to investigate the capacity of an unlikely source, specifically lipstick, to substantiate bacterial growth.
The goal of the procedures performed in this experiment was to isolate Gram-negative bacteria that are commonly associated with
nosocomial pathogenicity. The pathogens that I decided to target were E. coli and Pseudomonas aeruginosa. These species are significant
because they are commonly associated with nosocomial infections. These species are often present in and on the surface of the body,
which are areas where lipstick is often exposed or in contact. This work addresses methods of prevention and the significance of minimal
makeup wear in hospital settings.

I predicted that the solid lipsticks would produce the broadest variety of microbial growth. I predict that, because of its uniform
consistency and solid composition, the solid lipsticks will serve as a more suitable environment for microbial growth in comparison to the
mercurial consistency of the liquid lipstick. In order to test my hypothesis, a series of diagnostic tests were performed to determine
phenotypic characteristics of the cultures obtained from the samples such as colony morphology, cell morphology, cultural characteristics,
and biochemical properties. The independent variables established for this experiment were the lipsticks consistency, constituents, and
composition, while the dependent variables were amount and range of microbial growth produced from each sample.

Materials and Methods

Sample Cultivation. Samples were obtained from three different lip products, two solid lipsticks and one liquid lipstick using sterile
cottons swabs. The surface of each of the solid lipstick and the opening of the liquid lipstick tube were swabbed, and the specimen was
then placed into a vial containing sterile distilled water. Next, the specimen were taken to the lab and used to prepare glycerol stocks
(50:50 ratio of liquid specimen to 100% glycerol) and broth cultures (4ml Tryptic Soy Broth held in sterile glass culture tubes and

inoculated with ~100l of liquid specimen). The glycerol stocks were stored at -80C, and the broth cultures were incubated at 37C for 24
hrs prior to being stored at 4C. The samples were labeled as follows: solid lipstick 1 was titled BB in correspondence to its brand name,
Brazen Berry, solid lipstick 2 was labeled LF, in correspondence to its brand name, Lilac Flush, and the third lip product, L.A. girl Lip
paint liquid lipstick in Whimsical, was labeled Li in correspondence to its consistency.
Selective and Differential Media. Selective and differential media was used to isolate species from one another and target Gram
negative bacteria. The broth cultures used to streak selective agar, including mannitol salt agar (MSA), MacConkey agar (MAC), as well
as blood and nutrient agar (NA). For each plate, the samples were incubated at 37 C for 24 hours. The results for each sample were
collected Thursday, September 25, 2015, at 30.4023993,-91.1108173.
Biochemical Tests. Gram-negative species were isolated and used to perform further biochemical tests, including phenol red tests to
determine whether or not the isolated Gram-negative species fermented lactose. Oxidase test was performed to further narrow the possible
identities of the Gram-negative species. Finally, specimen were subjected to a battery of tests using the API 20e testing strips
(BioMerieux) to finally determine the identification of the enteric Gram-negative bacteria found in the sample.

Quantification of Target Bacteria. Serial dilutions were performed to enumerate the number of species in each sample as well as
determine the concentration of the species in the original sample. Dilutions were performed using micropipettes, nutrient agar, and
glycerol broths containing original samples.

Results
Selective and Differential Media. All of the samples produced growth on MSA plates, leading to the conclusion that all of the lipstick
products had one or more types of Gram-positive bacteria present. The BB sample exhibited substantial growth on the MSA plate after
being incubated, indicating the presence of gram positive bacteria. Although there was no Gram-negative bacteria found in the BB
sample, based on the results of the MSA plate (Fig. 1A), there is high probability that the Gram-positive species was that of S. aureus, one
of the leading causes of nosocomial infections. The LF sample produced a significant amount of slightly green pigmented growth on
MSA, indicating mannitol fermentation and halo-tolerance. The Li sample produced growth on both MAC and MSA (Fig. 1C).

The BB sample did not produce any growth on the MAC agar, indicating the absence of Gram-negative bacteria. Only two of the samples,
LF and Li displayed growth on the MAC agar plates. The LF sample also produced growth on the MAC agar, indicating the presence of
lactose fermenting bacteria as well as Gram-negative bacteria. The growth on MAC agar also displayed isolated colonies. The Li sample
growth on MAC agar also displayed isolated colonies, similar to those produced from the LF sample.

Biochemical Tests. The BB sample exhibited beta hemolysis on blood agar while the LF sample produced alpha hemolysis on the blood
agar plate. The LF sample on blood agar appeared wormlike, with filamentous structures around the edges, a dry consistency, and an
opaque, creamy color. The Li sample produced beta hemolysis on the blood agar plate, characterized by mucoid growths, irregular in
shape, and an opaque, creamy color.

When tested for lactose fermentation using the Phenol Red indicator, the LF sample tested positive for lactose fermentation in addition to
acid and gas production; the phenol turned red and gas and acid formed in the small tube inside of the larger test tube. The Li sample
tested positive for lactose fermentation and exhibited gas and acid production during the Phenol Red test.

Since the BB sample did not exhibit any Gram-negative growth, further biochemical tests specific for Gram-negative bacteria were not
performed using the BB sample. Using the results depicted in Fig. 1B and 1C., and the analytical profile index for the API 20E test strips,
I determined that the LF sample most likely contained bacteria from the Aeromonas spp. I also determined that the Li sample most likely
contained bacteria from the Pseudomonas spp.

Quantification of Target Bacteria. There were two colony morphologies on the Li and LF nutrient agar plates, indicating that there were
two species. The larger colonies were from one species, while the smaller colonies were from a different species. The LF sample
contained 0.0000003 cfu/ml concentration in the original sample while the Li sample contained 0.0000004 cfu/ml.

Discussion
Significance of Results. There are a number of ways that lip products become contaminated. Lip products stored at the bottom of a purse
placed on the ground is just one hypothetical situation in which a lip product can be exposed to lethal pathogens. The product packaging
can also play a major role in determining the level of contamination the product will be subjected to. If a product has minimal packaging
that allows easy exposure of the product to the environment, the product will most likely become contaminated before its shelf life has
expired. Another factor to consider would be when and how the product is applied. If the product is touched by unwashed hands or if it is
applied after eating, there is a major possibility that cross-contamination can occur.

LF Sample. According to the API 20E index table, the LF sample most likely contained bacteria from the Aeromonas spp. Aeromonas is
an anaerobic, Gram-negative rod whose genus is composed of 21 species (Murray, 2002,p. 322 ). Of the 21 species, one of the most
prominent species in the Aeromonas genus is Aeromonas hydrophila. The species in the Aeromonas genus are capable of causing diarrheal
disease, wound infections, and opportunistic disease. Some of the virulence factors exhibited by this genus of bacteria include endotoxins
and hemolysins. The most dangerous aspect of this species is the lack of knowledge known in regards to the species role in its disease
manifestations, making it exceptionally hard to target and treat in some instances.

Li Sample. Based on the results of the experiment, I concluded that the Li sample was most likely a species from the Pseudomonas
genus. Of the 200 species in the Pseudomonas genus, Pseudomonas aeruginosa, is the most commonly associated with nosocomial
infections (Murray, 2002, p. 334-338). It is often located in soil, water, and the surface of the skin (Nall, 2015). It is an extremely lethal
pathogen, particularly in hospital settings, because it has the capacity to cause sepsis. In addition to causing sepsis, this species is also
capable of causing severe rashes and inflammation. Pseudomonas aeruginosa is an aerobic, Gram-negative rod that often appears mucoid

as a results of one its virulence factors, a polysaccharide capsule, and is highly resistant to several types of antibiotics (Murray, 2002, p.
334-338). It produces a number of enzymes that are capable of inactivating antibiotics such as penicillin and cephalosporins. In addition
to the capsule, other virulence factors possessed by Pseudomonas aeruginosa include toxins, adhesins, enzymes, and antimicrobial
resistance. One of its most important virulence factors is exotoxin A. Exotoxin A is toxin instrumental in causing tissue damage and it is
responsible for inhibiting protein synthesis in eukaryotic cells. Pseudomonas aeruginosa is manifest as several diseases, including but
not, limited to, bacteremia, endocarditis, respiratory tract infections, urinary tract infections, skin and soft tissue infections, and ear and
eye infections. Those at risk for infection are primarily hospital patients who have been administered broad spectrum antibiotics. Due to
its high level of antibiotic resistance, Pseudomonas aeruginosa is capable of mutation that enables an even broader range of resistance
upon exposure to broad spectrum antibiotics. It is particularly abundant in water supply and on moist surfaces.

Preventative Measures. The FDA has not established stipulations regarding expiration dates for lip products, but, if the product is
continuously used past its estimated shelf life, the product is more likely to harbor an abundance of bacteria (Dahl, 2011). Although there
are no solid regulations designating the appropriate disposal date for lip products, there are other clues that may indicate that the product
is contaminated. If the smell or color of the product appear to change, the product should be thrown away immediately. Most importantly,
makeup use in general, including all types of cosmetic products, should be kept at a minimal in hospital settings to minimize crosscontamination. If lip products are worn, they should be cleaned with cosmetic antimicrobial products (Ellis, 2010). Any products worn
within a medical facility should be kept in the medical facility, applied after hands and face are washed, and kept in a sterile storage
environment. Lipstick can also be stored in the freezer overnight to kill any bacteria.

To expand on the findings in this work, research should be conducted to determine ways to optimally sterilize all personal items entering
and leaving hospital facilities. Methods of sterilization should be able to effectively sterilize without compromising the integrity of the
items. Further research should also should focus on adjusting the properties of lipstick and other similarly composed personal items, such
as pH, as a means of decreasing their potentiality to substantiate microbial growth. Development of antimicrobial cleaning products for
different types of surfaces and objects, such as those effective for cleaning cosmetic products, could also prove effective.

REFERENCES

Dahl, M. (2011, May 14). Germs lurking in old makeup: It isn't pretty. Retrieved December 1, 2015, from
http://www.today.com/style/germs-lurking-old-makeup-it-isnt-pretty-2D80556139

Ellis, A. (2010, May 13). How Dirty Is Your Makeup? Retrieved December 1, 2015, from http://abcnews.go.com/GMA/OnCall/dirtymakeup-gma-found/story?id=10631498

Fischer, K. (n.d.). Water Content of Toiletry (2) [Lipstick]. Retrieved December 1, 2015, from http://www.kyotokem.com/en/pdf/industry/CosmeticsSoap/EKVX-01542.pdf

Murray, P. (2002). Medical microbiology (4th ed., 322, 334-338). St. Louis: Mosby.

Nall, R. (2015, January 28). Types of Bacteria Found in Makeup. Retrieved December 1, 2015, from
http://www.livestrong.com/article/68575-types-bacteria-found-makeup/

Senese, F. (2015, August 15). What is the chemical composition of lipstick? Retrieved December 1, 2015, from
http://antoine.frostburg.edu/chem/senese/101/consumer/faq/lipstick-composition.shtml

The Unhealthy Handbag - The top ways your purse can make you sick. (2015, June 30). Retrieved December 10, 2015, from
http://www.baystatehealth.org/news/2015/06/the-unhealthy-handbag

Fig. 1. A series of diagnostic tests were performed in order to determine the phenotypic and biochemical properties found in each lipstick
sample. (A) Based the results of the selective media, the Brazen Berry (BB) lipstick sample had Gram-positive bacteria but did not have
any Gram-negative bacteria present. (B) The Lilac Flush (LF) sample contained both Gram-negative and Gram-positive bacteria. (C) The
Whimsical (Li) sample contained both Gram-negative and Gram-positive bacteria

A
Product Name: Maybelline, Brazen Berry (Solid)
Gram Result

MSA
Performed 9/18/2015,

MAC

Blood Hemolysis

Phenol Red

Performed 9/18/2015,

Performed 9/24/2015,

results collected 9/25/2015

results collected 10/1/2015

results collected
9/25/2015

Sample contained gam + only;

microbial growth from sample limited

(+)

Fermentation
(+) Halo-tolerance

to MAC agar ONLY

Mannitol

(-)Lactose

Fermentation
No growth at all on

Beta hemolysis

MAC agar
API Test Strips Results

Tests
Results

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

OX

B
Product Name: Maybelline, Lilac Flush (Solid)
Gram Result

MSA
Performed 9/18/2015, results

MAC
Performed 9/18/2015,

Blood Hemolysis
Performed 9/24/2015,

Phenol Red

collected 9/25/2015

results collected 9/25/2015

results collected 10/1/2015

Alpha hemolysis

Filamentous,

Performed 9/24/2015

Sample contained gram and

gram + bacteria; Result

(+)

Fermentation
(+) Halo-tolerance

determined based upon growth

present on MAC and MSA media

(+)

Fermentation
Isolated
colonies

Mannitol

Lactose

wormlike appearance
around edges
Dry
consistency;

present

(+) Lactose Fermentation

Phenol red turned yellow
Produced acid and gas
bubbles in tube

opaque,

creamy

yellow color
API Test Strips Results

Tests
Results

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

OX

C
Product Name: L.A. Girl Glazed lip paint, Whimsical (Liquid)
Gram Result

Sample contained gram and gram +

MSA
Performed 9/18/2015,

MAC
Performed 9/18/2015,

Blood Hemolysis
Performed 9/24/2015,

Phenol Red

results collected

results collected

results collected

Performed 9/24/2015

9/25/2015

9/25/2015

10/1/2015

bacteria; Result determined based upon

(+)

Mannitol

Fermentation

growth present on MAC and MSA media

(+)

(+)

Lactose

Fermentation

Halo-

tolerance

Isolated colonies

Beta hemolysis
Mucoid

growth,

irregular

shape;

opaque,

creamy

(+)Lactose Fermentation
Phenol red turned yellow
Produced acid and gas
bubbles in tube

present
yellow color
API Test Strips Results

Tests
Results

ONPG

ADH

LDC

ODC

CIT

H2S

URE

TDA

IND

VP

GEL

GLU

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

OX