TABLE OF CONTENTS
TITLE PAGE
DECLARATION
ABSTRACT
ACKNOWLEDGEMENT
TABLE OF CONTENTS
LIST OF TABLES A
LIST OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER
1.0 INTRODUCTION
1.1 Background
1.2 Objective and specific objectives
2.0 LITERATURE REVIEW
3.0 MATERIALS AND METHODS
4.0 RESULTS
5.0 DISCUSSION
6.0 CONCLUSION
REFERENCES
APPENDICES
INTRODUCTION
Acne vulgaris, more commonly known as just acne, is a skin disease characterized by the occu
ence of blackheads, whitehead pimples and sca
ing. The disease is ranked the 8th most common disease worldwide, with an estimate of more than 600 million individuals affected. Acne most prevalent among adolescents, with a rate of occu
ence between 80% to 90% in teenagers that falls to less than half in age groups between 20 and 30. Acne is mostly found is areas with a higher density of oil glands, such as the face and the upper torso including the chest and back.
The bacterium Propionibacterium acnes has been widely linked in the formation of acne. However, its relationship with acne is still being studied. The bacterium is characterized as Gram-positive, non-motile and rod shaped. In vivo, the bacterium subsists on lipids in the form of sebum secreted from the pores of human skin. P. acnes converts the lipids found in sebum into fatty acids. Studies have shown that these by-products play a role in the inflammation of the pores and the formation of acne.
While P. acnes is classified as an anaerobic organism, its environment in vivo is one that is exposed to oxygen. There have been studies depicting satellitism when growing P. acnes on primary skin cultures. However, the relationship between P. acnes and other native skin flora have not been confirmed.
Antibiotics against P. acne are a common treatment for the diagnosis of acne in patients. The concentration and type of antibiotics prescribed is determined through minimum inhibitory concentration tests to determine the antibiotic type and concentration that will elicit the highest efficacy in treatment.
There have been reports of the appearance of antibiotic-resistant strains globally. The prevalence of strains resistant to each antibiotic is varied across different regions and countries. Data shown also has determined a co
elation between, the emergence of resistance toward a specific antibiotic, and the frequency in which it was prescribed for use in acne treatments. A number of gene mutations in the bacterium have been observed to co
elate to resistance against specific antibiotics. Point mutations have been analysed through whole genome sequencing to find that there is a strong link between antibiotic resistance and the existence of the specific base mutations at the same site.
Multiple challenges may arise through the conduct of this project. Firstly, many reports have shown difficulty in obtaining viable cultures of P. acne in vitro. The generation of P. acne cultures of sufficient yield may be difficult due to its slow growth, which may require long periods of time for incubation. Furthermore, P. acnes shares many characteristics with other common contaminants such a Staphylococcus sp. as well as other bacteria in its genus. Other challenges include the possibility of contamination as well as enumeration of concentration during the process of obtaining the MIC data. This can be circumvented through the use of various purity checks as well as positive and negative controls to ensure reliable results.
LITERATURE REVIEW
Acne Vulgaris
Acne vulgaris is an inflammatory disorder of the pilosebaceous unit leading to the formation of comedones, papules, pustules, nodules and/or cysts. It is a chronic skin disease. Acne is commonly classified as noninflammatory, characterised by open and closed comedones, or inflammatory with the presence of papules and pustules. Clinical, pathophysiological and histological features of acne vulgaris are well studied. In contrast, studies concerning the epidemiology of acne are relatively sparse. The pathophysiology of acnes begins with desquamation of hair follicle keratinocytes, forming microcomedones. The hyperproliferation of keratinocytes are caused by an increase in production of sebum from sebaceous glands. In turn, the increase in follicular epidermal cells increases sebum retention, creating a positive feedback loop. The follicles eventually distend and rupture, which releases pro-inflammatory chemicals stimulating inflammation. Multiple factors have been linked to the pathogenesis of the disease which play roles in four key development points. Points of interest are changes in sebum production, keratinocyte hyperproliferation, colonization of inflammation-inducing bacteria such as; Propionibacterium acnes, Staphylococcus epidermis and Malassezia furfur; and finally, inflammation. Acne severity can be classified into four grades. The first and least severe grade presents with comedones of both types. Open comedomes caused by sebum plugging of the pilosebaceous orifice on the skin surface, while closed comedomes are have plugging below the skin surface due to sebum and keratinization. Inflammation is not present at this grade of severity. The second grade is determined by inflammatory legions observed as small papules with erythema. The appearance of pustules characterises the third grade of severity. Finally, the most severe grade of acne is determined by the coalesce of numerous pustules, forming nodules and cysts.
Several risk factors have been linked to acne vulgaris. These include genetic predispositions, hormonal imbalances such as those occu
ing during menstrual cycles and puberty. Infections, diet, stress, environmental factors which cause mechanical obstruction of skin follicles, and certain medications have been implicated in playing a role in the disease.
Propionibacterium acnes
Propionibacterium acnes (P. acnes) is a bacterium that comprises one of the natural bacterial flora found on hair follicle organ. The bacterium is characterized as a rod-shaped, gram-positive anaerobe. P. acnes also exhibits aerotolerance. The bacterium typically has a commensal relationship with human skin. However, it has been widely linked to the skin condition acne vulgaris. The bacterium subsists on sebum, metabolic by-products as well as cellular detritus in its habitat within follicles, pores as well as the surface of skin.
P. acnes was originally coined Bacillus acnes and was later grouped under the genus of Propionibacterium due to its ability in generating propionic acid. The bacterium was recently reclassified under the novel genus Cutibacterium. Other species reclassified under this new genus include Propionibacterium avidum and Propionibacterium avidum. For the sake of clarity, the bacterium will be termed under its former name, Propionibacterium acnes throughout the course of this study.
P. acnes secretes extracellular proteins as part of its metabolic activity. Among them are digestive enzymes, which play a role in the desquamatisation of the epidermal layer of the follicles in which it inhabits. This along with other effects of its hyperproliferation, can trigger inflammation, leading to skin diseases such as acne vulgaris.
P. acnes has also been found in other regions such corneal ulcer, herniated discs and
onchioalveolar regions. P. acnes have been found to cause postoperative and post-neurosurgical infections where it has been found colonizing prostheses, shunts and prosthetic heart valves. The bacterium plays a role of an opportunistic pathogen and may cause complications due to its ability to elicit an inflammatory response.
Pathogenesis of P. acnes
Emergence and Prevalence of Antibiotic Resistance in P. acnes
Emergence of Antibiotic Resistance in P. acnes
Antibiotic resistance strains of P.acnes were first discovered in 1979, with a rate of resistance of 20%. This rate increased steadily to 34.5% in 1991 and peaked in 1997 with a rate of 64%. Cu
ent rate
Cu
ent Status of Antibiotic Resistance in P acnes globally
Antibiotic resistance strains of P.acnes were first discovered in 1979, with a rate of resistance of 20%. This rate increased steadily to 34.5% in 1991 and peaked in 1997 with a rate of 64%. Cu
ently reported rates of antibiotic resistance vary globally with a range between 50.8% and up to 93.6%.
Resistance towards erythromycin was the most commonly reported, followed by clindamycin. Cross resistance to clindamycin was also frequently observed in erythromycin resistant strains
Conventional Treatments for Acne Vulgaris
MATERIALS AND METHODS
STERILITY CONTROL
MEDIA AND SOLVENT PREPARATION
Culture Media
Mueller-Hinton agar was used in the culturing of P. acnes samples. The agar was prepared from powder form (INSERT BRAND HERE). The powder was diluted with distilled water at proportions in accordance to (BRAND) instructions in 1L Scott flasks. The medium was sterilized by autoclave at 121C for 15 minutes. The bottles containing the medium were stored in a water bath at 50C until the temperature was equalized. The Mueller-Hinton agar was then poured onto sterile plastic agar plates within a laminar flow cabinet and left to cool to room temperature. Agar plates were then stacked, packaged into bags and stored in a chiller at 4C until use.
Media and Solvent for Biochemical Testing
Indole testing was performed with tryptone soy agar due to incompatibilities of the test with Mueller-Hinton media. The tryptone soy agar was prepared from powder (INSERT BRAND HERE). Dilution with distilled water was performed in accordance with manufacturer instructions within 1L Scott flasks and sterilized by autoclaving at 121C for 15 minutes. The media was then cooled to 50C in a water bath. The molten agar was poured; in a sterile laminar flow cabinet; into universal bottles which were previously sterilized by autoclave at 121C for 15 minutes. The media was stored in the chiller at 4C until use.
Media for Broth Microdilution
Mueller-Hinton
oth was used as the solvent in the testing of Minimum Inhibitory Concentration. It was prepared from powder stock (BRAND HERE) and diluted with distilled water per manufacturer instruction within 1L Scott flasks. The medium was then sterilized through autoclaving at 121C for 15 minutes. The medium was stored at room temperature was used within one week of preparation.
SAMPLE COLLECTION
Samples for MIC testing were obtained previously from FYP student Negeeswary Marimuthu (FYP reference). The samples were procured from a population localized within Taylor’s University Lakeside (Address). The samples were stored in the form of pure cultures in Reinforced Clostridial Medium, within 1.5mL microcentrifuge tubes. The samples have been cryogenically preserved in -80C storage.
CULTURING OF SAMPLES
Samples were obtained from the -80 freezer and thawed in an ice box. Sterile swabs were dipped into the microcentrifuge tubes containing the samples and streaked onto agar plates containing Mueller-Hinton agar. The samples were then incubated at 37C for 72 hours. Anaerobic environments were generated for the cultivation of the samples by placing the agar plates in 10L anaerobic jars with 2 packs of 5L Anaerogen packs. After incubation, the morphology of the colonies was observed and recorded.
BIOCHEMICAL TESTING
Agar plate cultures displaying polymorphic colonies were excluded. Cultures displaying monomorphic cultures were subjected to further biochemical testing to confirm their identities. A negative control, in form of wild-type stain of P. acnes was included with each biochemical test
Gram staining
Single isolated colonies were selected from the sample plate cultures. The colonies were picked up with an inoculation loop sterilized by flame from a Bunsen burner. The culture was spread onto a glass slide containing a drop of