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...

“ANTIMICROBIAL SUSCEPTIBILITY OF DIFFERENT ANTIBIOTICS ON ISOLATED PROPIONIBACTERIUM ACNES”
by
Student Name:
Submitted in accordance with the requirement for the degree of doctor of philosophy
Taylor’s University
School of Science
November 2019
DECLARATION
I,_______________________ do hereby declare that the thesis entitled ‘Antimicrobial Susceptibility of Different Antibiotics on Isolated Propionibacterium acnes’ is developed solely by me under the guidance of ___________________________, associate professor of............................, .Taylor’s University.
I also declare that this work has not been earlier submitted in any college, for the award of any professional course or degree.
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Student Name
Date
ABSTRACT
Objective: The antibiotics have been used recurrently against P.acnes, however the resistance of this strain towards antibiotics is becoming a global concern. The aim of this study is to assess the antimicrobial susceptibility of P.acnes for five different antibiotics in Malaysia.
Design: The study was cross sectional. The samples from cutaneous lesions were collected from 60 volunteers. The samples were cultured in anaerobic medium to investigate the presence of P.acnes. The tests for susceptibility were performed for the antibiotics: Doxycycline, Azithromycin, Penicillin, Gentamycin and Tetracycline using Biochemical testing and Indole Test.
Results: Number of isolates against inhibitory concentration of Azithromycin= 64 ug/ml, doxycyline=0.5 ug/ml, Gentamycin =0.125 ug/ml, Tetracyline =1 ug/ml and penicillin= 64 ug/ml. It shows that the strain of P.acne were highly resistant to Azithromycin in 12 out of 20 participants (60%) and Penicillin showed 9 out of 20 paticipants (45%). The other three drugs showed less susceptibility.
Conclusion: P.acnes was highly sensitive to the macrolides like Azithromycin and Penicillin. It showed greater resistance to these antibiotics. One of the main reason behind these results is irrational use of antibiotics for the treatment of acne in Malaysia and other countries. The results reflect that more caution should be followed by the dermatologists while prescribing antibiotics for the problem of acne.
ACKNOWLEDGEMENT
I would like to express my deepest regards to Mr._________________, Professor, Taylors University, who provided me his valuable guidance to develop this project. Without his persistent guidance and support, this thesis would not have been possible.
I would like to express my sincere gratitude to Mr._________________________who encouraged me to select this topic and undergo valuable research on this topic. He made my time at the university more positive and worthy.
Lastly,I would like to thank my peers and everyone else who contributed help towards this project.
TABLE OF CONTENTS
TITLE PAGE........................................................................................1
DECLARATION.................................................................................2
ABSTRACT.........................................................................................3
ACKNOWLEDGEMENT ..................................................................4
TABLE OF CONTENTS.....................................................................5
LIST OF TABLES...............................................................................6
LIST OF FIGURES.............................................................................7
LIST OF ABBREVIATIONS.............................................................8
CHAPTER 1 INTRODUCTION........................................................9
1.1 Background
1.2 Objective
2.0 LITERATURE REVIEW.............................................................12
3.0 MATERIALS AND METHODS..................................................25
4.0 RESULTS......................................................................................33
5.0 DISCUSSION...............................................................................36
6.0 CONCLUSION.............................................................................40
REFERENCES.....................................................................................41
APPENDICES
LIST OF TABLES
Table 1. Susceptibility of P.acnes for different antibiotics
Table 2 Treatments for Acne vulgaris
Table 3 Demographic Information and Acne details of the Participants
Table 4 Minimum Inhibitory Concentration Testing conducted on 20 samples of P. acnes
LIST OF FIGURES
1. Immersion Oil Dermatoscope image
2. Pathophysiology of P.acne
3. Resistance of P.acnes towards the commonly used antibiotics in acne
4. 96 Well Plate Template
5. Pure culture of P.acnes from Nose Pore Strip
6. Test tube with Indole Positive test
7. No. of dispensed Prescriptions of Azithromycin and the No. Of combined cases of Chlamydia, Gonorrhooea and Pertussis
8. No. Of isolates against Inhibiting concentration of Azithromycin
9. No. Of isolates against Inhibiting concentration of Tetracycline
10. No. Of isolates against Inhibiting concentration of Gentamycin
11. No. Of isolates against Inhibiting concentration of Doxycycline
12. No. Of isolates against Inhibiting concentration of Penicillin
LIST OF ABBREVIATIONS
AV- Acne Vulgaris
RCM- Reflectance Confocal Microscopy
OCT- Optical Coherence Tomography
SNP- Single Nucleotide Polymorphism
OR- Odds Ratio
MIC- Minimum Inhibitory Concentration
FYP- Final Year Project
EUCAST- European Committee on Antimicrobial Susceptibility Testing
CLSI- Clinical and Laboratory Standards Institute
CHAPTER 1: INTRODUCTION
Acne vulgaris, more commonly known as just acne, is a chronic inflammatory skin disease characterized by the occurrence of blackheads, papules, pustules, scarring and comedones (Zhu,Wang, He & Sun,2019). The disease is ranked the 8th most common disease worldwide, with an estimate of more than 600 million individuals affected annually. Acne is most prevalent among adolescents, with a rate of occurrence in between 80% to 90% in teenagers 20 and 30 years old (Brook, 2019). Acne is mostly found in body parts 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 form of sebum secreted from the pores of human skin. P. acnes convert the lipids found in sebum into fatty acids. These by-products play an important role in the inflammation of the pores, follicular hyperkeratinisation and the formation of acne.
Acne therapy involves antimicrobial and anti-inflammatory medications (Dogan, 2017). Hence, the antibiotics are generally used to inhibit the colonisation of P. acnes and to mitigate the production of pro-inflammatory factors. Systemic and topical antibiotics are most commonly used in acne treatment. Since last 30 years, there has been identified a reduced susceptibility of P.acnes for these antibiotics globally.
Antibiotics against P. acne are a common treatment for the patients diagnosed with acne. 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. According to a study by Zhu, Wang, He & Sun (2019), the resistance rate as observed in China was: 98.7% patients were susceptible to Doxycycline, 96.9% to tetracycline, 44.5% were susceptible to Clindamycin, 44.93 were susceptible to Clarithromycin, 42.3% for Erythromycin and 41.4% to Azithromycin. Data shown also has determined a correlation 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 correlate 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.
1.2 Objective
To study the antimicrobial susceptibility of different antibiotics towards isolated Propionibacterium acnes.
The study involves Minimum Inhibitory Concentration Testing performed on 20 samples of P. acnes to examine its resistant to 5 different antibiotics.
CHAPTER 2. LITERATURE REVIEW
2.1 Acne Vulgaris
Acne vulgaris or Acne is an inflammatory disorder of the pilosebaceous glands of skin 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 inflammation with the presence of papules and pustules.
Image showing multiple open comedones (Grade 1, Acne)
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 such as 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 have plugging below the skin surface due to sebum and keratinisation ( George & Sridharan, 2018). 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 coalescence 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 occurring 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.
2.2 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 exhibit aero tolerance. 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 avidumand 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 secrete extracellular proteins as part of its metabolic activity. Among them are digestive enzymes, which play a role in the de-squamatisation 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 bronchioalveolar 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.
2.2.1 Other Species
The acne may be visibly similar to the infections from other species like Staphylococcus aureus, gram negative folliculitis, Trichophyton rubrum, Malassezia folliculitis or Microsporum canis (Dermnet, 2019).
2.3 Digital Dermatoscopy
It is a method of digital imaging of skin lesions using dermatoscopes like cameras, videos and adapters. These dermoscopic images are monitored for changes in appearance of suspicious lesions. The dermatoscopes involve a magnifying optic (with 10 X magnification) and a trans-illuminating light source. The device takes the images in three modes: Non polarized light, contact; Polarized light, Non contact; and Polarized light, Contact. The Non polarized light enables information retrieval for superficial skin lesions while the polarized light helps in imaging deeper skin layers.
Fig. 1 Immersion Oil Dermatoscope
Dermatoscopy helps in early identification of melanoma. A dermatoscope device involves a magnifier, polarized or non polarized source of light, a transparent plate and a liquid medium in between the skin and device (Thomas, 2017). Other techniques for cutaneous diagnosis of acne or skin lesions include radiological examinations like X Ray, MRI scans, CT scans, Wood Light Examination, Reflectance Confocal Microscopy and Optical Coherence Tomography.

2.3 Pathogenesis of P. Acnes
P.acnes mostly occurs due to opportunistic infection and is associated to multiple skin conditions like Progressive macular hypermelanosis, skin infections acne vulgaris, infections related to dental and medical devices, cervical disc disease, soft tissue infections, prostate cancer and sarcoidosis (Magdy & Ahmed, 2013). Other key requirements for P.acne to cause opportunistic infection are the extrinsic factors (trauma, medical devices, injury etc), the immune status of the host and his interaction with the other microbial communities.
Fig.2 Showing Pathophysiology of P.acne (Source: Contassot & French, 2014)
P.acnes initiate the secretion of Inter Leukin-1β in the monocytic cells by activating the Inflammasome NLRP3 (Contassot & French, 2014). The exposure of macrophages and P.acnes in dermal area may cause the release of IL-1β leading to substantial inflammation. Excessive production of sebum, altered keratinisation of follicles and growth of P.acnes in the sebaceous area disrupts the epithelium of follicles. It may lead to leakage of P.acnes from pilo sebaceous unit and facilitate its contact with macrophages while activating the NLRP3 inflammosome, and release of IL-1β.
Primarily the pathogenesis of Acne vulgaris is influenced by four interrelated factors: Hyperkeratinization and plugging of follicles, release of inflammatory mediators, excessive production of sebum and colonization of P.acnes (Dreno, 2017). However the main factor is based on Genetics. The excretion of sebum is regulated by multiple mediators and hormones.
Acne vulgaris involves a complex process of pathogenesis which includes abnormal hyperkeratinisation of follicles, elevated production of sebum, inflammation of pilosebaceous follicles and the presence of P.acnes. It leads to inflammation by metabolizing the sebaceous triglycerides into the fatty acids. These fatty acids attract the neutrophils chemotactically. The antibiotics mainly reduce the numbers of P.acnes over the pilosebaceous follicles and the skin.
P.acnes releases chemotactic elements which encourage the formation of acne by producing the proinflammatory cytokines IL-8, TNF-α and IL-1β. These elements cause the formation of papules, nodules and pustules. P.acne also initiates the innate immune system of the host by activating the receptors ‘Toll like’ while activating the nuclear factor NF-kB. The genetic expression encourages the cytokine, chemokine and development of adhesion along with NF-kB.
The P.acne rehabilitation also involves its suppression and CD4 neutrophils and lymphocyte enrolment through P.acnes. The proliferation of P.acnes occurs after perforation of the wall of follicles, which distorts the barrier and oozes out the lipid content in hyperkeratinized form. The accumulation of P.acnes in dermal area causes inflammation. Additionally te shortage eof linoleic acid may also develop hyperkeratinisation of the skin.
Factors Causing Acne vulgaris
The other factors which largely contribute in pathogenesis of acne are occupational exposures, occlusive cosmetics, dairy consumption, family history of severe acne, genetics, diet and environmental factors (Al-Natour, 2017). In adult women, the androgens play primary role during the hormonal treatments like Anti Androgen medications, Polycystic Ovary Syndrome and Oral contraceptives. This androgen triggers the production of sebum through androgen receptors over the sebaceous glands. These factors are explained in detail as follows:
1. Hormonal Factors: In adolescents the acne formation initiates with puberty and menstrual cycle. At this stage, the androgen hormone is found in higher concentration in body. It expands the sebaceous glands further elevating the production of sebum. In addition to Androgen, the other...