A COMPARATIVE STUDY OF THE IN VITRO ACTIVITIES OF Lactobacillus acidophilus AND TWO ANTIFUNGAL AGENTS ON Candida albicans

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A COMPARATIVE STUDY OF THE IN VITRO ACTIVITIES OF Lactobacillus acidophilus AND TWO ANTIFUNGAL AGENTS ON Candida albicans

TABLE OF CONTENT

TITLE PAGE ………………………………………………………………………………………………………………………………. i

CERTIFICATION …………………………………………………………………………………………………………..ii

DEDICATION ……………………………………………………………………………………………………………………………. ii

ACKNOWLEDGEMENTS …………………………………………………………………………………………………………. iv

TABLE OF CONTENTS …………………………………………………………………………………………………………….. xi

LIST OF TABLES ………………………………………………………………………………………………………………………. v

LIST OF FIGURES …………………………………………………………………………………………………………………… iix

LIST OF PLATES ………………………………………………………………………………………………………………………. x

ABSTRACT…………………………………………………………………………………………………………………..xi

CHAPTER ONE …………………………………………………………………………………………………………………………. 1

INTRODUCTION ………………………………………………………………………………………………………………………. 1

1.1: Background of Study …………………………………………………………………………………………….. 1

1.2: Statement of Problem…………………………………………………………………………………………….. 3

1.3: Aim and Objectives of the study……………………………………………………………………………… 4

1.4: Significance of Study …………………………………………………………………………………………….. 4

CHAPTER TWO ………………………………………………………………………………………………………………………… 5

LITERATURE REVIEW …………………………………………………………………………………………………………….. 5

2.1: Probiotics …………………………………………………………………………………………………………….. 8

2.2: Vaginal Lactobacilli …………………………………………………………………………………………….. 11

2.3: Mode of action of Probiotics …………………………………………………………………………………. 12

2.3.1: Growth inhibitory substances …………………………………………………………………………. 12

2.3.2: Adherence and competition ……………………………………………………………………………. 12

2.3.3: Growth, stability and resistance to antimicrobial agents and spermicidal ……………… 12

2.3.4: Other mechanisms…………………………………………………………………………………………. 13

2.4: Yeast Vaginitis ……………………………………………………………………………………………………. 13

2.5: Fungal Infections (Candida) …………………………………………………………………………………. 13

2.6: Understanding Fungal Infections …………………………………………………………………………… 14

2.7: Several areas of the body may be affected by fungal infection ………………………………….. 15

2.7.1. Urogenital tract …………………………………………………………………………………………….. 15

2.7.2. Skin …………………………………………………………………………………………………………….. 17

2.7.3. Mouth and throat …………………………………………………………………………………………… 18

2.7.4. Systemic infection …………………………………………………………………………………………. 19

2.7.5. Intestinal Candidiasis …………………………………………………………………………………….. 19

2.7.6. Fungal Sinusitis …………………………………………………………………………………………….. 20

2.7.7. Candida-Related Complex (CRC) …………………………………………………………………… 21

2.7.8. Conventional Treatment…………………………………………………………………………………. 22

2.7.9. Candidiasis of the skin …………………………………………………………………………………… 23

2.7.10. Fungal infections of the finger/toe nail plate (e.g., onychomycosis) …………………… 23

2.7.11: Vaginal Candida infections ………………………………………………………………………….. 23

2.7.12. Nutritional Therapy for Candida Infections ……………………………………………………. 24

2.7.13. Dietary modifications…………………………………………………………………………………… 24

2.7.14: Resveratrol …………………………………………………………………………………………………. 24

2.7.15: Goldenseal …………………………………………………………………………………………………. 25

2.7.16: Lactoferrin …………………………………………………………………………………………………. 26

17: Tea Tree Oil ……………………………………………………………………………………………….. 26

2.7.18. Other Essential Oils …………………………………………………………………………………….. 27

2.7.19. Garlic…………………………………………………………………………………………………………. 28

2.8: Additional Alternative Therapies…………………………………………………………………………… 29

2.8.1. AHCC …………………………………………………………………………………………………………. 29

2.8.2. Caprylic Acid ……………………………………………………………………………………………….. 29

2.8.3. Boric Acid ……………………………………………………………………………………………………. 29

Genetics of Candida albicans ………………………………………………………………………………… 30

2.9.1: Classical genetics ………………………………………………………………………………………….. 30

2.9.2: Mutant isolation ……………………………………………………………………………………………. 30

2.9.3: Parasexual genetics ……………………………………………………………………………………….. 31

Gene disruption strategies ……………………………………………………………………………….. 32

Gene reporter systems …………………………………………………………………………………….. 33

Isolation of albicans genes ………………………………………………………………………….. 34

Expression controlled systems …………………………………………………………………………. 34

A look into the future of albicans manipulation …………………………………………….. 35

Dimorphism …………………………………………………………………………………………………. 36

2.9.10: Experimental conditions for germ-tube formation …………………………………………… 37

2.10: Epidemiology: …………………………………………………………………………………………………… 38

2.11: Pathogenesis: ……………………………………………………………………………………………………. 39

2.11.1: Types of assays …………………………………………………………………………………………… 39

2.11.2: Systemic infection in mice ……………………………………………………………………………. 40

2.11.3: Mouse strains ……………………………………………………………………………………………… 41

2.11.4: Resistance to systemic Candidiasis………………………………………………………………… 42

2.11.5: Innate response …………………………………………………………………………………………… 42

2.11.6: Adaptative response …………………………………………………………………………………….. 43

CHAPTER THREE …………………………………………………………………………………………………………………… 45

MATERIALS AND METHODS …………………………………………………………………………………………………. 45

3.1 Sample Collection and Treatment …………………………………………………………………………… 45

3.2: Yeast Isolates Identification ………………………………………………………………………………….. 45

Gram Staining (Cheesbrough, 2006) ………………………………………………………………… 45

Culture Characteristics ……………………………………………………………………………………. 46

Germ tube test to identify albicans (Cheesbrough, 2006) ……………………………….. 46

3.3. Lactobacillus acidophilus PROBIOTICS ……………………………………………………………….. 48

Macroscopic appearance of cultured colonies on MRS Agar. ………………………………. 48

Microscopy …………………………………………………………………………………………………… 48

Biochemical tests …………………………………………………………………………………………… 49

Antifungal Assay (preparation of Lactobacillus acidophilus). …………………………………… 50

Preparation of fungal inocula ………………………………………………………………………………… 50

Susceptibility Test ……………………………………………………………………………………………….. 51

Minimum Inhibitory Concentration (MIC) Assay ……………………………………………………. 51

Statistical Analysis ………………………………………………………………………………………………. 52

CHAPTER FOUR ……………………………………………………………………………………………………………………… 53

RESULTS AND DISCUSSION ………………………………………………………………………………………………….. 53

4.1: Identification Tests …………………………………………………………………………………………….. 53

4.2 Results of confirmation of L. acidophilus probiotics…………………………………………………. 54

4.3: Susceptibility Test……………………………………………………………………………………………….. 57

4.4 MIC and MFC of L. acidophilus against C. albicans ………………………………………………… 65

4.5: DISCUSSION …………………………………………………………………………………………………… 101

CHAPTER FIVE …………………………………………………………………………………………………………………….. 103

CONCLUSION AND RECOMMENDATION ……………………………………………………………………………. 103

REFERENCES ……………………………………………………………………………………………………………………….. 104

APPENDIX 1 ………………………………………………………………………………………………………………………….. 128

APPENDIX 2 ………………………………………………………………………………………………………………………….. 133

ABSTRACT

The antifungal effect of Lactobacillus acidophilus probiotics against Candida albicans isolated from cases of recurrent vaginal candidiasis was investigated using standard microbiological procedures. All 250 isolates (100%) of C. albicans tested were susceptible (sensitive) to two broad spectrum antifungal agents, Nystatin and Fluconazole. The results of the testing of the C. albicans isolates showed that 240 of them (96%) were sensitive to the suspension of L. acidophilus probiotic with varying zones of inhibition at different concentrations. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the L. acidophilus suspensions showed higher inhibitory activity at the higher concentrations, with the maximum activity at 250 mg/ml. The commercial antifungal drugs were better than the suspension from the probiotic L. acidophilus. The results suggest that L. acidophilus could be a useful agent for development of antifungal against vaginal pathogens and may have great potential for use in external application and vaginal wash for preventing or reducing Candida growth.

Keywords:Lactobacillus acidophilus, probiotics, Candida albicans, antifungal, inhibit.

CHAPTER ONE

INTRODUCTION

1.1: BACKGROUND OF STUDY

Candida albicans is a virulent strain of yeast which is naturally present on human body (Oscar et al., 2000), often within areas of mucous membrane such as the inside of the mouth, on moist skin, vagina, intestines, lungs, on or under the fingers and toenails (Vazques and Sobel, 2000). Some common conditions that Candida overgrowth is responsible for include thrush, vaginal yeast infections and even diaper rash (Ping, 2002). Candidiasis or yeast hypersensitivity occurs when the yeast overgrowth becomes a systemic problem (Debbie et al, 2004). When the immune system is suppressed, the yeast can multiply rapidly, penetrate the intestinal lining and move into the bloodstream. Yeast populations are controlled by probiotics or “friendly” bacteria (Cheryl et al., 2001, Boonnaert and Rouxhet, 2000). The term, Probiotics, literally meaning “for life”, is used to describe organisms that are used medicinally including bacteria such as Lactobacillus which are naturally present in the small intestine and vagina, and have been shown to inhibit pathogenic ―unfriendly‖ organisms, and play an important role in maintaining human health (Rolfe, 2002). The primary role of Lactobacillus is to reinforce protective mucosal surfaces and prevent the entrance and attachment of harmful microorganisms and allergens (Wee et al., 2006). Strains of probiotics, like Lactobacillus acidophilus are of high value for protection against pathogenic yeast infections of the vagina, intestinal and oral cavity, and can be described as one of the body‘s primary defense mechanisms against Candida (Mercenier et al., 2003). Typically, probiotics are consumed as part of cultured foods such as acidophilus milk, yoghurt (Simin and Woel-Kyu, 2000). The topical application of such yoghurt products has been reported to control yeast and bacterial infections, and the ingestion of these preparations has been recommended to reduce the symptoms of sore mouth caused by Candida infections (Boonnaert and Rouxhet, 2000). Research has shown that L. acidophilus was the most popular species of probiotic bacteria that produces substances that slow or prevent the growth of Candida (Yang, 2000). The work was designed to study the inhibitory effect of L .acidophilus isolated from yoghurt as probiotics in reduction of C. albicans growth in vitro and in vivo.

This work was to study the effects of L. acidophilus probiotics and other fungicide agents on C. albicans comparatively. Several reports have shown that Lactobacilli produce metabolites that inhibit various bacteria (Mikolajcik and Hamaden, 1975, Shahani et al., 1976, Shahani et al., 1977), but few investigators have included fungi in their studies. Guillot (1958) reported that an inhibitor produced in casein broth by L. acidophilus inhibited C. albicans.

The term probiotics was first coined by Lilly and Stillwell in 1965 in reference to substances produced by protozoa, which stimulated the growth of other organisms. Probiotics are defined as the viable microorganisms that exhibit a beneficial effect on the health of the host by improving its intestinal microbial balance (Zerehpoosh et al., 2013, Kazemi et al., 2012). A probiotic organism should be nonpathogenic, non-toxic and also resistant to low pH to bile salts and also able to improve its chances of survival in the gastrointestinal tract (Suvarna et al., 2005). Probiotic microorganisms that have antagonistic activity against pathogens in vivo, have long been promoted as health enhancing in general and specifically are useful for vaginal problems (Mombelli and Gismondo, 2000). In addition to their usefulness are the prevention and/or treatment of many other diseases, such as antibiotics-associated diarrhoea (Saccharomyces boulardii), Helicobacter pylori infections, inflammatory bowel diseases, allergy, cancer, urinary tract infections and bacterial vaginosis (Falagas et al., 2006). Urogenital infections are a major reason for women to visit their family‘s physician and are generally referred to gastroenterology, gynaecology, urology, and infectious diseases specialists. Candida albicans is the main cause of the majority of fungal infection in oral cavity. Oropharyngeal candidiasis is a common infection and currently ranks as the most common human fungal disease. C. albicans is the major human pathogen of the genus Candida and is commensal yeast of the oral, gastrointestinal, and vaginal mucosa in healthy individuals. They are almost universally present. These microorganisms may change from commensal to pathogenic microorganism (Akpan and Morgan, 2002). There are several antifungal medications but the patients are mostly convenience using natural or herbal remedies. Several lactobacillus species are currently used as probiotics, i.e., live bacteria used as food supplements to provide beneficial effects to the host. Probiotic bacteria can produce bacteriocin-like compounds that inhibit infectious microorganisms (Reid et al., 2003). They can adhere to epithelium of the alimentary tract and block adherence of pathogens and they can stimulate host defense mechanisms (Miettinen et al., 1996, Wagner et al., 1997). Lactobacillus acidophilus is an important probiotic species, which has been reported to protect immunodeficient mice from orogastric candidiasis and protect humans from C. albicans vaginitis (Miettinen et al., 1996). In-vitro treatment with yoghurt or exogenous Lactobacillus species has been reported to alleviate the symptoms of vaginal candidosis, but few reports have investigated in-vitro effect of yoghurt on C. albicans (Wagner et al., 1997, Williams et al., 2001).

1.2: STATEMENT OF PROBLEM

acidophilus which is useful to human health in suppressing the growth of Candida albicans is killed off in cases where people overuse common antibiotics in treating infections such as candidiasis. It is therefore necessary to find other treatments that will allow the growth of naturally occurring L. acidophilus to further inhibit the growth of Candida albicans and reduce to the minimum the use of common antibiotics to avoid other complications such as reduced immunity due to overuse of antibiotics.

1.3: AIM AND OBJECTIVES OF THE STUDY

To determine and compare antifungal activity of Lactobacillus acidophilus probiotics and two antifungal agents on Candida albicans.

Objectives of the study

To isolate and characterize albicans from women suffering from recurrent vaginal candidiasis.

To use acidophilus probiotic as antifungal agent

To confirm the efficacy of acidophilus probiotic by comparing it with other commercial antifungal agents (fluconazole and nystatin).

To determine the minimum concentration of acidophilus probiotics that will inhibit the growth of C. albicans.

1.4: SIGNIFICANCE OF STUDY

The properties of Lactobacilli lead to use these bacteria as probiotic agent in addition to being member of normal flora and are generally regarded as safe for use in humans. So that in treatment of vaginitis, the normal flora disturbances must be restored by avoiding the use of wrong antibiotic those disturb the normal vaginal flora.

This study will be beneficial to humans in that probiotics can incorporated into powders, soaps, creams and diapers for the use of women and children. This will help maintain the normal flora balance of the body as it checks the overgrowth of C. albicans and enhances the presence of L.acidophilus because we believe the ability of lactobacilli to inhibit the growth of Candida cells.