BACTERIAL CHALLENGE TEST OF SELECTED COSMETICS USING CONSORTIUM OF

E COLI AND S AUREUS

 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the study

According to the EC Regulation 1223/2009 of the European Parliament and of the Council of
30th of November 2009, a cosmetic product is “any substance or mixture intended to be applied
on the outer surfaces of the human body (epidermis, hair, nails, lips) or on teeth, on the mucous
membranes of the mouth, with the purpose of cleaning, smelling, modifying, protecting,
maintaining them in good condition or correcting bodily odors” (Regulation EC No 1223/2009).
Substances or mixtures applied A cosmetic product is a substance or mixture intended to be
applied on the outer surfaces of the human body or on teeth, on the mucous membranes of the
mouth, with the purpose of cleaning, smelling, modifying, protecting, maintaining them in good
condition (Moshahid et al., 2019). In order to prevent microbial proliferation in cosmetics,
substances with antimicrobial activity are used, to inhibit the development of microorganisms.
Among the most commonly used cosmetic contaminants, there are spore-forming bacteria,
molds, yeasts and bacteria on the surface of the body with other purpose than those indicated by
the Regulation, including all drugs and top medical devices (e.g. disinfectants, insect repellents)
are not cosmetics. Cosmetics can be classified as creams, emulsions, lotions, gels and oils, soaps,
prepared for baths and showers, deodorants and antiperspirants, tooth and mouth products,
depending on the function, and the area where they are applied (Lee, et al., 2013; Ogle, et al.,
2013; Adebooye and Opabode, 2014; Ayodele, 2015). The commitment of the cosmetic industry
and competent authorities is aimed at ensuring the safety of cosmetics and at protecting
consumers’ health (Scientific Committee on Consumer Products, 2016). The current Regulation
(1223/2009) stipulates that all cosmetics must be manufactured, handled, packaged and sold
under such conditions as to avoid damaging human health. The same regulation stipulates that
the manufacturer must assess marketed product safety. In this regard, cosmetic company must
evaluate the safety of the cosmetics, using a qualified expert, internal or external to the company
(Jimenez, 2014). Evaluation procedures must consider both the intrinsic properties of each used
component and the amount to which the consumer is exposed in his actual use of the product, to
obtain an estimate of the risk associated with the use of the product (Szeto, et al., 2012; Jimoh, et
al., 2014).

The microbial challenge test is a critical test used to assess the safety and efficacy of various
products, including cosmetics, pharmaceuticals, and food products. This test evaluates the ability
of microorganisms to survive and grow in the product over a specified period. The test typically
involves inoculating a product with a known number of microorganisms and incubating the
product under specified conditions. After incubation, the product is examined to determine if the
microorganisms have survived and grown (Gibbons et al., 2013).. The number of
microorganisms recovered is then compared to the initial inoculum to assess the efficacy of the
product. Microbial challenge testing is particularly important for cosmetics as they are used on
the skin, which is a natural habitat for microorganisms. In addition, cosmetics can be
contaminated during manufacturing, packaging, and use, which can lead to microbial growth and
potential harm to the user (Prescott and Klein, 2022). The acceptance criteria for microbial
challenge testing vary depending on the type of product being tested. However, it is generally
accepted that products should not support the growth of pathogenic microorganisms, and the
number of microorganisms recovered should be within acceptable limits (Roberts, 2016).
Overall, the microbial challenge test is a valuable tool for evaluating the safety and efficacy of
various products. It is an essential component of product development and helps ensure that
products meet regulatory requirements and are safe for consumers (Abalaka, 2016).

escherichia coli is a bacterium with a special place in the microbiological world since it can
cause severe infections in humans and animals but also represents a significant part of the
autochthonous microbiota of the different hosts. Of major concern is a possible transmission of
virulent and/or resistant E. coli between animals and humans through numerous pathways, such
as direct contact, contact with animal excretions, or via the food chain. E. coli also represents a
major reservoir of resistance genes that may be responsible for treatment failures in both human
and veterinary medicine. An increasing number of resistance genes has been identified in E.
coli isolates during the last decades, and many of these resistance genes were acquired by
horizontal gene transfer. In the enterobacterial gene pool, E. coli acts as a donor and as a
recipient of resistance genes and thereby can acquire resistance genes from other bacteria but can
also pass on its resistance genes to other bacteria. In general, antimicrobial resistance in E. coli is
considered one of the major challenges in both humans and animals at a worldwide scale and
needs to be considered as a real public health concern.

Staphylococcus aureus is a mammalian commensal and opportunistic pathogen that colonizes

niches such as skin, nares and diverse mucosal membranes of about 20-30% of the human
population. S. aureus can cause a wide spectrum of diseases in humans and both methicillin-
sensitive and methicillin-resistant strains are common causes of nosocomial- and community-
acquired infections. Despite the prevalence of literature characterising staphylococcal
pathogenesis in humans, S. aureus is a major cause of infection and disease in a plethora of
animal hosts leading to a significant impact on public health and agriculture.

1.2 Statement of research problems

The cosmetic industry is constantly expanding, and consumers demand products that are both
safe and effective. Microbial contamination of cosmetic products is a major concern as it can
cause skin infections and other health issues. To ensure the safety of cosmetic products, it is
essential to conduct bacterial challenge tests to assess their efficacy against various
microorganisms. These bacteria can cause infections and health problems in consumers.
Therefore, it is essential to evaluate the antimicrobial efficacy of cosmetic products to ensure
their safety and efficacy. Bacterial challenge testing is one of the most commonly used methods
to evaluate the antimicrobial efficacy of cosmetic products. Microbial contamination of
cosmetics poses a great problem to the Cosmetics manufacturing process, especially from an
economic point of view. Cosmetic products need not to be sterile but may contain low levels of
microbial load during use. Commercial cosmetic products have been found to be responsible for
serious overt and covert skin infections, which were often ignored as the sources or vehicles of
transmission of pathogens. Microbial contamination of cosmetic products is very crucial because
of their daily use and direct contact with the skin. These products are at high risk for microbial
contamination from various sources such as environment, consumer's hands, and body sweat and
during the time of manufacturing. Therefore, good manufacturing practices (GMP) and hygiene
must be carried out by manufacturers and personnel, cosmetic products should be stored in an
aseptic environment to avoid contamination before vending in the markets. The pharmaceutical
manufacturer should assure that product-specific knowledge and expertise are available for the
development of an effective plan.therefore this research tend to bacterial challenge test of
selected cosmetics using consortium of e coli and s aureus

1.3 AIM AND OBJECTIVES OF THE RESEARCH

1.3.1 Aim

Bacterial challenge test of selected cosmetics using consortium of e coli and s aureus

1.3.2 Objectives

 To evaluate the antimicrobial efficacy of selected cosmetic products against a consortium

of E. coli and S. aureus.
 To determine whether there are any differences in the antimicrobial efficacy of the
selected cosmetic products against E. coli and S. aureus.
 To determine how the presence of preservatives in the selected cosmetic products affects
their antimicrobial efficacy against E. coli and S. aureus.

1.4 SIGNIFICANCE OF THE STUDY

the bacterial challenge test of selected cosmetics using a consortium of E. coli and S. aureus is a
significant study that contributes to the safety and efficacy of cosmetic products. This study will
help to ensure that cosmetic products are safe for use, meet regulatory requirements, and provide
better protection against harmful bacteria. The bacterial challenge test of selected cosmetics
using a consortium of E. coli and S. aureus is significant for several reasons:

Consumer Safety: The presence of harmful bacteria in cosmetic products can cause infections
and health problems in consumers. By evaluating the antimicrobial efficacy of cosmetic products
using a consortium of E. coli and S. aureus, this test can help ensure the safety of cosmetic
products and protect consumers from potential harm.

Regulatory Compliance: The regulatory agencies, such as the US FDA, require cosmetic
manufacturers to ensure the safety of their products. Bacterial challenge testing is a commonly
used method for evaluating the antimicrobial efficacy of cosmetic products. This test can help
manufacturers ensure that their products meet regulatory requirements.

Quality Assurance: The bacterial challenge test of selected cosmetics using a consortium of E.
coli and S. aureus can be used as a quality control measure for cosmetic products. This test can
help manufacturers identify products that do not meet the required antimicrobial efficacy and
take corrective actions to ensure the quality of their products.

Development of Effective Cosmetics: By evaluating the antimicrobial efficacy of cosmetic

products using a consortium of E. coli and S. aureus, this test can help manufacturers develop
more effective and safer cosmetic products for consumers. The findings of this test can help
inform the selection of ingredients and preservatives in cosmetic products to ensure their efficacy
against harmful bacteria.
 CHAPTER TWO

RELATED LITERATURE REVIEWS

Most cosmetic products are susceptible to microbiological spoilage due to contaminations that
could happen during fabrication or by consumer’s repetitive manipulation. The composition of
cosmetic products must guarantee efficient bacterial inactivation all along with the product shelf
life, which is usually assessed by challenge-tests. A challenge-test consists in inoculating specific
bacteria, i.e. Staphylococcus aureus, in the formula and then investigating the bacterial log
reduction over time. Each year around the world, official authorities in Europe (Rapid Alert
System for Non-Food Products) or USA (US Consumer Product Safety Commission) notify
many recalls for cosmetic products due to microbiological contamination (Li et al. 2018).
Cosmetic formulas are complex and are susceptible to microbiological spoilage due to their
composition, containing water and nutrients such as lipids, polysaccharides, proteins (Kimmi et
al., 2017). Contamination of cosmetic products could happen during their fabrication but also by
consumer’s repetitive manipulations (Kandiah et al. 2020). The main pathogens frequently found
in cosmetic formulas are Pseudomonas aeruginosa, Escherichia coli, Burkholderia
cepacia, Candida albicans, Klebsiella oxytoca, Enterobacter gergoviae, Serratia marcescens and
Staphylococcus aureus (Ramonaite et al. 2019). S. aureus has been found in various cosmetic
products such as shaving cream, moisturizing cream, face care cream and depilatory cream (Nair
et al. 2017). It is a Gram-positive bacterium present on human skin and mucous membranes in
30% of the population. Many S. aureus strains produce exfoliative toxins secreted on the skin
that cause a wide range of clinical infections, including abscesses, furuncles or impetigo
(Wood,2020).

2.1 Definition and Classification of Cosmetics

The term ‘cosmetics’ derives from the Greek “Kosm tikos” meaning ‘having the power to
arrange, skilled in decoration’, to give “kosmein”, to adorn, and “kosmos”, order, harmony
(Wood, L 2019). The Council of European Union regulation gave the following definition:
“cosmetic product means any substance or mixture intended to be placed in contact with the
external parts of the human body (epidermis, hair system, nails, lips, and external genital organs)
or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly
to cleaning them, perfuming them, changing their appearance, protecting them, keeping them in
good condition, or correcting body odours” (Neza, E et al., 2008). Generally, a cosmetic product
is used in the direct treatment of the external surface of the human body in order to perform the
following four functions:

(1) maintenance in good condition;

(2) change in appearance;

(3) protection; and

(4) correction of body odor

The term “cosmeceutics” (or active cosmetics) was popularized by the dermatologist Albert
Kligman in the 1980s. This term means a combination of cosmetics and pharmaceuticals, used to
define products that can have a beneficial effect on skin, but cannot be considered as having a
clear biological therapeutic effect (e.g., retinol, certain bleaching agents, etc.). However, the
cosmeceutic term remains controversial without legal status and has not been generally accepted
by all researchers (Centini, M et al., 2016). Cosmetics can be classified according to their use,
fields of application, functions, form of preparation, consumer’s age or gender, among others
(Martini 2006). The most appropriate classification is as follows:

(1) cosmetics for personal cleansing (soaps, deodorants, shampoos);

(2) cosmetics for the skin, hair, and integument care (toothpastes, products for external intimate
care);

(3) cosmetics for embellishment (perfumes, lip colors);

(4) protective cosmetics (solar products, anti-wrinkle products);

(5) corrective cosmetics (beauty masks, hair dyes);

(6) maintenance cosmetics (shaving cream, moisturizing creams); and

(7) active cosmetics (fluoridated toothpastes, antiseptics).

2.2 Cosmetic Products with Antimicrobial Effect

Cosmetic products with antimicrobial effect can be described as preparations with the ability to
provide consumer’s protection against the presence of antimicrobial compounds, having
bactericidal effect. Products like mouthwashes, skin disinfectants or antibacterial soaps present
this characteristic. Currently, the limit between drugs and cosmetic products with antimicrobial
effect is increasingly indistinct. Sometimes the difference between a cosmetic product and a drug
lies in the concentration of the active ingredient in the product (e.g., mouthwash). There is also
an unclear distinction between the definition of cosmetic and dermatological treatment (e.g.,
acne treatment). As a result, some modern cosmetics are in an increasingly grey zone and can
almost be defined as drugs or over-the-counter (OTC). This fact confers a heavy responsibility
on the various international regulation agencies (Toler, J.C 2018). In all cases, a decision on
product qualification must be made by the competent national authorities on a case-by-case
basis, and taking into account all relevant factors, such as their appearance, the type of active
ingredient, length of use, mode of action, and claims (Siquet et al., 2016).

2.3 Microbial contamination

Microbiological contamination in a product can originate from one of two sources:
Contamination during production and filling: or from entering the product via the consumer
when the product is being used. From the moment the packaging is broken open up to the
complete consumption of the product, various microbiological contamination is continually
introduced from the environment and from the consumer themselves (hands, body)( tomasi et
al.,). Possible impurities during the production and filling processes will be tested by regular
routine microbiological controls of the batches. Bacteria getting into the product while it is being
used are confronted by the use of preservatives (Connor 2019). Contamination of
microorganisms in cosmetics may cause spoilage of the product and when pathogenic, they
represent a serious health risk for consumers. Most of the cosmetics are not sterile and they are
made of non-sterile raw material. Although cosmetics do not have to be sterile, limit values have
been reported according to the type of the cosmetics. The ability of microorganisms to grow and
reproduce in cosmetic products has been known for many years. (Butler, H et al., 2013)
Following are the cosmetic products used by men and women, which need to be
microbiologically safe. Talcum powders are cosmetic product used all over the world to prevent
rashes and keep skin free of moisture. Creams are external preparations, usually for application
to the skin. Creams may be considered as pharmaceutical products, as even cosmetic creams are
based on techniques developed by pharmacy. Creams are liable to microbial contaminations
either in the course of their preparation, transportation and/or accidentally, during use by the
consumers which may lead to their spoilage (Siemer, E. 2013). Body lotions are a low viscosity
topical preparation intended for application to unbroken skin. Hair straightened otherwise known
as ‘‘relaxer’’ is a type of lotion or cream generally used by people with "afro textured hair", to
make hair less curly, easier to straighten or to create perms by chemically "relaxing" the natural
curls by breaking down the proteins bonds of hair, temporarily or permanently(Sasseville, 2004).
Cosmetic eye preparations are liable to microbial contamination either in the course of their
preparation, by the personnel, storage environment, during transportation and/or use by the
consumers which may lead to their spoilage. Lipsticks fall under the face care cosmetics
category and are composed of waxes, oils, emollients, emulsifiers, pigments/colorants, and
binders in varying concentrations, which determine the characteristics of final products(Alvarez-
Lerma et al., 2008).. Product contamination may arise from raw materials or water used in
formulation. This spoilage may lead to alteration in organoleptic properties of creams which may
manifest in terms of changes in color, odor and/or taste; as well as biodegradation of active
constituent of such creams. The growth of bacteria that produces alcohols or degrades
emulsifiers may lead to instability, splitting of the emulsion and eventual spoilage. Microbial
growth can produce enzymes that cause degradation of active ingredients and changes in the
pH(Behravan et al., 2005; Campana et al., 2006).

2.4 Microbiological Safety of Cosmetic Products

Generally speaking, all products, including cosmetics, containing water and organic/inorganic
compounds under appropriate physicochemical conditions, are exposed to microbial
contamination. This justifies why these products require effective and adequate protection
against microorganism proliferation(Behravan et al., 2005; Campana et al., 2006). An ideal
preservation system (intrinsic or extrinsic) should protect the product from microbial
degradation, both in its original closed packaging until use, and in an open container throughout
its use. In recent years, the safety record for personal care products has been excellent, resulting
in a scarce occurrence of infections due to contaminated products. Studies have shown that the
mostly frequent microorganisms found in cosmetics comprise Pseudomonas aeruginosa,
Klebsiella oxytoca, Burkholderia cepacia, Staphylococcus aureus, Escherichia coli, Candida
albicans, Enterobacter gergoviae, and Serratia marcescens, but also other bacteria, fungi and
yeasts(Brannan, 2006). The skin and mucous membranes are protected against microorganisms;
however, their presence in these products can increase the risk of microbial infection. Microbial
contamination may occur during manufacture (primary contamination) and/or during consumer
use (secondary contamination) (Brannan, 2006; Tenenbaum, 1967). Moreover, all potential
sources of contamination must be identified and monitored. In order to do so, four steps must be
considered:

(1) inspection and control of raw materials;

(2) manufacturing process;

(3) delivery of the final product and; finally;

(4) its use by the consumer.

2.5 bacterial challenge test

A bacterial challenge test is a type of test that is used to evaluate the efficacy of antimicrobial
agents or other substances against bacterial infections (Scott et al., 2005; Beaufort et al., 2014).
In the context of cosmetics, bacterial challenge tests are used to evaluate the effectiveness of
preservatives and other antimicrobial agents in preventing the growth of bacteria in cosmetic
products. Bacterial challenge tests are an essential part of the cosmetic manufacturing process
(Beaufort et al., 2014). They help to ensure that cosmetic products are safe and free from
harmful bacteria that can cause infections or other adverse reactions in consumers. By subjecting
cosmetic products to bacterial challenge tests, manufacturers can ensure that their products meet
the highest standards of safety and quality. Microbiological challenge testing is a useful tool for
determining the ability of a food to support the growth of spoilage organisms or pathogens.
Microbiological challenge tests also play an important role in the validation of processes that are
intended to deliver some degree of lethality against a target organism or group of target
organisms. Quite often, with this latter purpose, there is an associated performance standard that
the process must deliver (Health Canada, 2012). Microbial challenge studies are an important
tool in the development of a new product formulation or process, or in the validation of a control
measure as part of establishing a food safety plan. For example, microbial challenge studies may
be performed to determine if a product is capable of supporting the growth of pathogenic
organisms, to test the efficacy of a natural preservative, or to provide data supporting a scheduled
process for a cold-filled acidified food (Scott et al., 2005).

2.6 The principle of microbial challenge Test

The principle of microbial challenge test procedure is to inoculate a product with microorganism
and then evaluating the product at a defined time interval to see whether there is a reduction in
the microbial population, or not, to desired levels in a given time. Neutralizing studies for the
preservative must be carried out before or in parallel with the preservative efficacy test, to ensure
the neutralization of biologically active agents, and if the neutralization study fails, alternate
neutralization approaches like membrane filtration may have to be used. The preservative
properties of the product are considered adequate if there is either a fall or no increase in the
number of microorganisms inoculated into the preparation. The organisms specified for use in
the tests are intended to be representative of those that might be expected to be found in the
environment in which the preparation is manufactured, stored and used. Environmental isolate
from manufacturing side also considered for this test. The fundamental principle of the
microbial challenge is based on the concept of measuring the survival ability of selected
microorganisms that are purposely introduced into a preserved test product system.
Conventional preservative efficacy tests or preservative challenge test methods generally
require microbial assays at multiple test points over extended periods of time.

2,7 Types of microbial challenge Testing

The results of a microbial test challenge are crucial in determining the safety of cosmetic
products and ensuring they are suitable for consumer use. All new product formulations must be
tested before market launch. The FDA requires cosmetics should be free of pathogenic
microorganism contamination and have a low density of non-pathogenic organisms. However,
they do not specify acceptable levels of “low”. As a result, the cosmetic industry generally
follows the guidelines of the Personal Care Products Council (PCPC) (formerly the Cosmetic,
Toiletry, and Fragrance Association (CTFA)) regarding the level of microbial contamination and
the absence of pathogens. Due to the unique set up and requirements of the Preservative Efficacy
Test, cosmetic testing is often conducted in a dedicated laboratory. Several test methods are
available to address PET criteria including:

 U.S. Pharmacopeia (Antimicrobial Effectiveness Testing)

In the United States, the USP guidelines require testing of Escherichia coli in addition to
Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger, and Candida albicans.
Spoilage microbes are not needed for PET. In contrast to the pharmacopeia tests, which only
evaluate pathogenic microbes. USP preservative efficacy testing evaluates cosmetic products by
exposing them to a single microbial strain at a time. 

 European Pharmacopeia

In the European Union, Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus niger, and
Candida albicans must be tested for all cosmetic products. In addition to the microbes above,
testing with microbes known to lead to spoilage of cosmetic products is recommended but not
required

 International Organization for Standardization (ISO) 

 Personal Care Products Council (PCPC)  

Although they are similar, each one calls out different criteria to ensure product microbiological
stability, and in turn, safety. 

2.8 Test organisms

The specific strains recommended to be used in these tests can be obtained from official cell
culture collections, such as the American Type Culture Collection (ATCC). The most common
test strains are potentially pathogenic representatives of Gram-positive bacteria (Staphylococcus
aureus), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa (Varvaresou et
al., 2009).

2.8.1 Staphylococcus aureus

Staphylococcus aureus is a major pathogen of increasing importance due to the rise in antibiotic
resistance (Lowy, 2018). It is distinct from the CoNS (e.g. S. epidermidis), and more virulent
despite their phylogenic similarities (Waldvogel, 2016; Projan and Novick, 2017). The species
named aureus, refers to the fact that colonies (often) have a golden colour when grown on solid
media, whilst CoNS form pale, translucent, white colonies (Howard and Kloos, 2016). To date
the S. aureus genome databases have been completed for 7 strains. The average size of the S.
aureus genome is 2.8Mb (Kuroda et al., 2019). The cell wall of S. aureus is a tough protective
coat, which is relatively amorphous in appearance, about 20- 40 nm thick (Shockman and
Barrett, 2013). Underneath the cell wall is the cytoplasm that is enclosed by the
cytoStaphylococcus aureus represents Gram-positive cocci in many tests. It is a part of normal
nasal and cutaneous microflora. Although rare, its presence in cosmetic products may be
indicative of human contamination (Geis, 2006). plasmic membrane. Peptidoglycan is the basic
component of the cell wall, and makes up 50% of the cell wall mass (Waldvogel, 2018). It is
integral in the formation of the tight multi-layered cell wall network, capable of withstanding the
high internal osmotic pressure of staphylococci (Wilkinson, 1997). Another cell wall constituent
is a group of phosphate-containing polymers called teichoic acids, which contribute about 40%
of cell wall mass (Knox and Wicken, 2016). There are two types of teichoic acids, cell wall
teichoic acid and cell membrane associated lipoteichoic acid; bound covalently to the
peptidoglycan or inserted in the lipid membrane of the bacteria. Teichoic acids contribute a
negative charge to the staphylococcal cell surface and play a role in the acquisition and
localisation of metal ions, particularly divalent cations, and the activities of autolytic enzymes
(Wilkinson, 2017). Peptidoglycan and teichoic acid together only account for about 90% of the
weight of the cell wall, the rest is composed of surface proteins, exoproteins and peptidoglycan
hydrolases (autolysins). Some of these components are involved in attaching the bacteria to
surfaces and are virulence determinants. Finally, over 90% of S. aureus clinical strains have been
shown to possess capsular polysaccharides (Karakawa and Vann, 2009; Thakker et al., 2015).
The growth and survival of bacteria is dependent on the cells ability to adapt to environmental
changes. S. aureus has evolved many mechanisms to overcome such changes, particularly in an
infection. A growth curve of S. aureus grown under ideal conditions can be divided into three
phases: lag, exponential, and stationary. During exponential phase, bacterium metabolism is
rapid and efficiently to ensure constant growth. As the bacteria age and stop growing (post-
exponential), cellular metabolism is re-organised for long-term survival under unfavourable
conditions. S. aureus has three well characterised global regulators of virulence determinant
production, agr (Recsei et al., 2016; Morfeldt et al., 2018), sar (Cheung et al., 2012), and sae
(Giraudo et al., 2014) that regulate the expression of surface proteins, exoproteins, and other
proteins essential for growth. Studies have shown that the accessory gene regulator (agr) up-
regulates the production of many exoproteins, including TSST-1, enterotoxin B and C, and V8
protease (sspA); and down-regulates the synthesis of cell wall associated proteins, including
fibronectin-binding proteins, and fibrinogen-binding proteins during post-exponential and
stationary growth phase (Foster et al., 2020; Lindberg et al., 2019). Cheung et al. (2012)
identified a second regulatory locus called staphylococcal accessory regulator (sarA), and is
distinct from the agr locus. A sarA mutant decreases the expression of several exoproteins, such
as α-, β-, and δ-haemolysin, and increases others such as proteases (Cheung et al., 2014; Chan
and Foster, 2018). Studies have also shown that sarA is essential for agr-dependent regulation
(Heinrichs et al., 2016; Lindsay and Foster, 2019).
 Fig 2.1:

2.8.2 Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative bacilli. It is a well-known and highly pathogenic

ubiquitous bacteria. It also shows high resistance against many preservatives. Escherichia coli is
a Gram-negative bacilli of the family Enterobacteriaceae. It is considered as an indicator of fecal
contamination. Like most coliform bacteria, it can easily develop resistance to
preservatives(Oishi, 2002; Darbre et al., 2002). Pseudomonas aeruginosa is among the more
common causes of infections in the hospital setting. These infections are associated with
significant morbidity and health care expenditures, especially when receipt of appropriate
antibiotic therapy is delayed. Antibiotic selection for patients with P. aeruginosa infections is
challenging because of the pathogen’s intrinsic resistance to many commercially available
antibiotics. Multidrug-resistant strains are prevalent, and often require treatment with novel or
“last resort” agents (Berthelot 2001). Infectious diseases pharmacists can help provide optimal
care for patients with P. aeruginosa infections by being familiar with key aspects of its
microbiology, epidemiology, pathogenesis, innate and acquired mechanisms of resistance, and
clinical presentation. In addition, pharmacists providing care to patients with P. aeruginosa
infections should be able to proactively identify patient populations at greatest risk of having an
infection caused by multidrug-resistant (MDR) strains, detail the available treatment options for
its varying clinical presentations, and provide timely evidence-based treatment
recommendations, especially for patients with suspected or documented MDR P. aeruginosa
infections. The conservation of strains is an important factor. For example, most bacteria and the
yeast Candida remain viable for one month under refrigerated conditions, while Pseudomonas
aeruginosa cannot be useful after two weeks (depending on specific conditions) (Pang 2019). An
effective way to keep mold spores is to store them at room temperature on slanted agar. Weekly
or periodic transplanting may be done to ensure the viability of microorganisms, but this practice
increases the risk of resistance loss. Alternatively, the cultures can also be frozen or lyophilized,
in order to maintain the stability of the microorganism and avoid the need of frequent
subcultures. The main advantage of these storage media is the prevention of genetic resistance
factors loss (Oishi, 2002; Darbre et al., 2002).

Fig 2.2:
2.9 Importance of challenge tests in cosmetics

A bacterial challenge test is a type of test that is used to evaluate the efficacy of antimicrobial
agents or other substances against bacterial infections. In the context of cosmetics, bacterial
challenge tests are used to evaluate the effectiveness of preservatives and other antimicrobial
agents in preventing the growth of bacteria in cosmetic products.

 Bacterial challenge tests are an essential part of the cosmetic manufacturing process.
They help to ensure that cosmetic products are safe and free from harmful bacteria that
can cause infections or other adverse reactions in consumers. By subjecting cosmetic
products to bacterial challenge tests, manufacturers can ensure that their products meet
the highest standards of safety and quality.
 There are several reasons why bacterial challenge tests are so important in cosmetics.
First and foremost, bacterial challenge tests help to ensure the safety of consumers. By
testing cosmetic products for bacterial growth, manufacturers can identify potential
sources of contamination and take steps to prevent it from occurring. This is critical in
ensuring that consumers are not exposed to harmful bacteria that can cause infections or
other health problems.
 Another important reason why bacterial challenge tests are important in cosmetics is that
they help to ensure the efficacy of preservatives and other antimicrobial agents.
Preservatives are added to cosmetics to prevent the growth of bacteria and other
microorganisms. By subjecting cosmetics to bacterial challenge tests, manufacturers can
evaluate the effectiveness of these preservatives and ensure that they are working as
intended.
 In addition to ensuring the safety and efficacy of cosmetic products, bacterial challenge
tests can also help to reduce the risk of product recalls and other costly disruptions to the
manufacturing process. By identifying potential sources of contamination early on,
manufacturers can take steps to prevent contamination from occurring and avoid the need
for costly recalls or other disruptions.
 CHAPTER THREE

MATERIAL AND METHODS

3.0 Study area

Lapai is a Local Government Area in Niger State, Nigeria, adjoining the Federal Capital
Territory. Its headquarters are in the town of Lapai on the A124 highway in the west of the area
at 9°03′00″N 6°34′00″E / 9.05000°N 6.56667°E. It has an area of 3,051 km2 and a population of
110,127 at the 2006 census. The area is roughly coterminous with the Lapai Emirate. The postal
code of the area is 911.

3.1 Sample collection

The unused cosmetic products were obtained from a local supermarket in Lapai Market , and
transferred to Ibrahim Badamasi Babangida University faculty of natural sciences department of
microbiology laboratory intact and analyzed as soon as possible.

3.2 Materials and reagents

The materials and reagents used in this study are distilled water,, analytical balance, water bath,
porcelain cup, glass stirrer, measuring flask beaker glass. three different cosmetic products E.
coli and S. aureus culture Sterile nutrient agar plates Sterile swabs Incubator set at 37°C
Antibacterial agent (e.g. alcohol)( All equipment used in the bacterial challenge test will be
disinfected with an antibacterial agent such as alcohol.)

3.3 Tested cosmetics

three commercial cosmetic products were used. These included an ultra-hydrating facial cream, a
tonic biphasic and a watery sunscreen. Cosmetics were preserved with phenoxyethanol and
ethylhexyglycerin having a broad-spectrum activity against bacteria, fungi and yeast. Each
cosmetic was analyzed for the determination of total bacteria count, in order to establish possible
initial contamination. One gram of samples was aseptically placed into a 9 mL sterile diluent
(NaCl 0.9%). A volume of 100 µL of samples and its decimal dilution were spread on Triptone
Soya Agar plate and incubated at 37 °C for 24-48 h. After incubation, results were reported as
number of colony forming unit for mL (CFU/mL)

3.4 Test Microorganism

The following bacterial strains, recognized as pathogen species for human, were employed in the
challenge test: Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC 6538).
Inoculum were prepared from each strains according to manufacturer’s instructions. Broth
suspension were maintained in specific growth medium until reaching a concentration about 108
CFU/mL. Bacteria concentration was determined spectrophotometrically

3.5 Microbiological analysis

Total bacterial count The collected samples of cosmetic products (intact, in-use and ending
product) were analysed for the determination of total bacterial count. Each sample, serially
diluted (from 10)1 to 10)4 cell ml)1 ) in physiological buffer and opportunely homogenized, was
spread on Tryptone Soya Agar (TSA; Oxoid, Milan, Italy) and incubated at 37C. The plates were
observed after 24 h and after 5 days and the number of colony-forming units (CFU ml)1 ) was
determined. Each assay was performed in duplicate.

3.5.1 Media and isolation of pathogenic micro-organisms

To determine the presence of pathogenic micro-organisms, 1 ml of each 10)1 diluted samples

was spread on mannitol salt agar (Oxoid), McConkey (Oxoid) and cetrimide agar (Oxoid) to
allow the growth of Staphylococcus spp., enterobacteria and Pseudomonas spp. respectively. The
plates were then incubated at 37C for 24 h. Isolates were identified by conventional biochemical
tests (Barrow and Feltham 1993; Murray et al. 1995).

3.6 Laboratory efficacy test methods (challenge tests)

The following challenge tests, CTFA test (Cosmetics, Toiletries, and Fragrance Association,
Inc. 2001), FUI test (Official Italian Pharmacopeia: Anon 1994), and the Rapid Challenge Test
(RCT) (Cagliani et al. 1990), were performed on two cosmetic products, a body cream and a bath
foam, selected among the assayed cosmetics, which contained the same mix of preservatives
(phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben, isobutylparaben)
differing only for the presence of imidazolidinyl urea in the body cream. Uninoculated samples
of each cosmetic served as control.

3.6.1 CTFA test

Two reference strains, S. aureus (ATCC 4338) and P. aeruginosa (ATCC 9027), and two
isolated from the analysed products, Pseudomonas putida and Staphylococcus epidermidis, were
used to perform the CTFA test, with slight modification. The cultures were maintained in TSA
and transferred twice in Tryptone Soya Broth (TSB, Oxoid) (inoculum 10%) at 37C for 24 h.
The last transfer was the inoculum suspension (106 cell ml)1 ). At time zero, four samples (50 g)
of each test products were inoculated respectively with 0Æ1 ml of each inoculum suspensions.
Samples were shaken and maintained at room temperature (RT). After a contact time of 0, 3, 7,
14, 21 and 28 days, 1-ml aliquots were removed and placed onto 9 ml of neutralizing medium
Leethen broth (Difco, Milan, Italy) (Nostro et al. 2002). Cell viability was determined by the
plate count method on TSA and CFU were counted after 24 h incubation at 37C. All
determinations were performed in duplicate. A reduction in the number of each micro-organism
of 99Æ9% by 7 days was required in order for the formulation to pass the test.

3.6.2 FUI test

The same micro-organisms were utilized in the FUI test. The cultures, maintained in TSA
(Oxoid), were transferred in TSB (Oxoid) and incubated at 37C to reach an OD610 ¼ 0Æ13–
0Æ18, corresponding to 108 cell ml)1 (as measured by sprectrophotometer). Four samples (20 g)
of the two test products were inoculated respectively with 0Æ1 ml of each bacterial suspensions.
Inoculated samples, after shaking, were maintained at RT; after a contact time of 0, 2, 7, 14 and
28 days from challenge, sample aliquots (1 ml) were removed and counted according to the
method described above (Nostro et al. 2002). The acceptance criterion for the FUI test was a
logarithmic (log10) reduction of 3 of the viable micro-organisms by the 7th day from challenge.

3.6.3 Rapid Challenge Test (only for Gram-negative bacteria)

A 50-g portion of the two mentioned cosmetic products were challenged with 0.1 ml of broth
suspension containing more than 106 cell ml)1 of Gram-negative bacteria (P. putida isolated
from our cosmetic products and the reference strain P. aeruginosa ATCC 9027). Twenty grams
of the two test products were diluted in order to obtain three final concentrations: 100%, 90%
and 80% of the product. Aliquots (1 ml) were removed from each concentration of samples after
24 h and 7 days from challenge and counted according to the method described above (Nostro et
al. 2002). The acceptance criterion is established as follows:

i the product showing more than 102 CFU ml)1 after 24 h from challenge both in the undiluted
product and in the diluted product, is considered a high contamination risk product;

ii if a progressive decrease in the CFU values at the different dilutions (at least the 80% dilution)
after 24 h and absence of bacterial growth after 7 days from challenge was observed, the product
is considered a medium–low contamination risk product;

iii in complete absence of bacterial growth after 24 h and 7 days from challenge in all the
dilutions, the product is considered well preserved.