GENOTYPING AND TREATMENT OF SECONDARY BACTERIAL INFECTIONS AMONG BURULI ULCER PATIENTS IN THE AMANSIE CENTRAL DISTRICT OF GHANA BY ELIZABETH GYAMFI (10442509) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A MASTER OF PHILOSOPHY DEGREE IN MEDICAL BIOCHEMISTRY JULY, 2015 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I ELIZABETH GYAMFI, do hereby declare that with the exception of references to other people’s work, which have been duly acknowledged, this thesis is the outcome of my own research conducted at the Department of Medical Biochemistry, University of Ghana Medical School, College of Health Sciences and the Department of Cell, Molecular Biology and Biochemistry, University of Ghana, College of Basic and Applied Science under the supervision of Dr. Lydia Mosi and Dr. Bartholomew Dzudzor. Neither all nor parts of this project have been presented for another degree elsewhere. ……………………………………………. Date: ………………………. ELIZABETH GYAMFI (Student) ……………………………………………. Date: ………………………… DR. LYDIA MOSI (Supervisor) ………………………………………….. Date: ……………………….. DR. BATHOLOMEW DZUDZOR (Supervisor) University of Ghana http://ugspace.ug.edu.gh ii ABSTRACT Background Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. BU is the third most common mycobacterial disease after tuberculosis and leprosy, but in Ghana and Cote d’ Ivoire, it is the second. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin which makes the infection painless. However, some patients have complained of painful lesions and delay healing. Painful ulcers and delay healing experienced by some patients may be due to secondary bacterial infections. Main Objective: To identify secondary microbial infections of BU patients, their genetic diversity as well as determine the levels of antibiotics resistance of these microorganisms. Methodology: The study was conducted at Biochemistry, Cell and Molecular Biology, University of Ghana. Subjects were recruited from Amansie Central District, Ashanti Region. Swabs of 51 BU patients were taken and immediately frozen for transport into the laboratory. Microscopy was performed using Ziehl-Neelsen and Gram staining techniques. The samples were also cultured on Luria Bertani, MacConkey, Mannitol, BPA and Sabouraud dextrose agar to identify bacteria and fungi. Antibiotic susceptibility tests were performed on selected bacteria species. DNA was extracted from the samples, after which Polymerase Chain Reaction (PCR) was performed using universal (16S rRNA), MSHA/PA (16S rRNA for mycobacterial) and IS2404 (insertion sequence specific to mycolactone producers) primers to find the different strains of organisms. Finally sequencing was performed on the DNA amplicons that were randomly selected to identify the kinds of microorganisms causing secondary infection. University of Ghana http://ugspace.ug.edu.gh iii Results: All the samples were positive for bacteria. However 49 and 40 positives were obtained from PCR products using the primers MSHA/PA, and IS2404 respectively, thus 40 BU patients were identified out of the total 51 patient samples. Majority of the bacteria identified after sequencing with universal primers for bacteria were Staphylococcus spp (aureus including MRSA, saprophyticus, and lentus), Alcaligene spp (aquatilis and faecalis), Pseudomonas spp (aeruginosa, stutzeri and koreensis) and bacilli cereus group of bacteria. Interestingly, 60% of the sequencing result for mycobacteria detected the presence of Corynebacterium spp (aurimucosum, diphtheria and striatum). Other bacteria identified were Brevibacterium iodinum and Rhodococcus erythropolis. Majority of these bacteria live in muddy areas and dirty water. The selected bacteria were less susceptible to rifampicin, clarithromycin and amikacin. Conclusion: Other bacteria beside M. ulcerans colonize and proliferate on BU lesions. The selected bacteria were less susceptible to clarithromycin and amikacin and rifampicin. The pains and healing delay experienced by some BU patients could be the result of these bacteria colonizing and proliferating on the ulcer or lesions. University of Ghana http://ugspace.ug.edu.gh iv DEDICATION This research work is foremost dedicated to the Almighty God for His undeserved kindness that has been showered upon me throughout my academic life. My profound appreciation goes to my mother for her love, care, support and encouragement over the years. University of Ghana http://ugspace.ug.edu.gh v ACKNOWLEDGEMENT I am most grateful to the Almighty God for his wisdom, guidance and protection throughout my academic years. I want to thank my supervisor; Dr. Lydia Mosi for her guidance and financial assistance throughout the project. I am very grateful for all the workshops she helped me to attend. I am also grateful to my co-supervisor, Dr. Bartholomew Dzudzor of the Department of Medical Biochemistry (U.G.M.S) for the trouble he took to frequently find out how the work was progressing. I appreciate the corrections and advice. I render an appreciation to Charles Narh and Charles Quaye for the wonderful skills they taught me during the field work. Your criticism and corrections kept me on my toes. My profound gratitude goes to the staffs and students of Molecular Biology Laboratory at the Department of Biochemistry, Cell and Molecular Biology (U.G) especially Mr. Jacob Kwaku Donkor, Adiza Abass, David Abana, Godwin Williams Ametsi, Senyo Gideon Amevinya and Georgia Naa K. Ghartey (Department of Animal Biology) for assisting me whenever help was needed. A big thank you also goes to the staff of the Department of Medical Biochemistry (U.G.M.S) especially, Dr. Nii Ayite Aryee for helping me in the completion of my thesis. Finally, I am grateful to my family and friends especially my mother, Grace Gyamfi, my sister, Esther Gyamfi and Ofori Adomako Ampaw and my colleagues Bismark Bimah, Hayford Narh and Elisha Kwaku Denkyirah for their advice and emotional support. University of Ghana http://ugspace.ug.edu.gh vi TABLE OF CONTENT DECLARATION ................................................................................................................. i ABSTRACT ........................................................................................................................ ii DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENT .................................................................................................. v TABLE OF CONTENT ..................................................................................................... vi LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ............................................................................................................. xi LIST OF ABBREVIATIONS ........................................................................................... xii CHAPTER ONE ................................................................................................................. 1 1.0 INTRODUCTION ..................................................................................................... 1 1.1 Background ............................................................................................................... 1 1.2. Justification/Relevance............................................................................................. 3 1.3. Hypothesis ................................................................................................................ 4 1.4. Aim ........................................................................................................................... 4 1.5. Objectives ................................................................................................................. 4 CHAPTER TWO ................................................................................................................ 5 2.0. LITERATURE REVIEW ......................................................................................... 5 2.1. Buruli Ulcer .............................................................................................................. 5 2.1.1. Mycolactone ...................................................................................................... 6 2.1.2. Epidemiology..................................................................................................... 7 2.2. Ecology and Transmission of the Disease.............................................................. 10 2.3. Clinical Presentation of Buruli ulcer ...................................................................... 11 2.4. Diagnosis ................................................................................................................ 14 2.5. Treatment of Buruli Ulcer ...................................................................................... 15 2.6. Suggested treatment other than antibiotics............................................................. 17 2.7. Immunology of the Disease.................................................................................... 18 2.8. Genetic Variation ................................................................................................... 19 2.8.1. Genetic Variation in Mycobacterium ulcerans ................................................ 19 2.8.2. 16S rRNA for Bacteria Detection .................................................................... 21 2.9. Microbial infections ............................................................................................... 22 2.9.1 Microbial infection of wounds ......................................................................... 22 2.9.2. Secondary infection of Buruli ulcer................................................................. 24 University of Ghana http://ugspace.ug.edu.gh vii CHAPTER THREE .......................................................................................................... 25 3.0 METHODOLOGY .................................................................................................. 25 3.1 Study Site ................................................................................................................ 25 3.2 Study Design ........................................................................................................... 25 3.4 Subjects ................................................................................................................... 26 3.4.1 Inclusion Criteria .............................................................................................. 27 3.4.2 Exclusion Criteria ............................................................................................. 27 3.5 Data on study samples ............................................................................................. 27 3.6 Sample collection from patients .............................................................................. 27 3.7 Sample processing for laboratory analyses ............................................................. 27 3.7.1 Sample Preparation ........................................................................................... 27 3.7.2 Gram Staining ................................................................................................... 28 3.7.3 Ziehl-Neelsen staining for acid-fast bacilli (AFB) ........................................... 28 3.7.4 Microscopic determination of fungal elements ................................................ 29 3.7.5 Culturing of bacteria ......................................................................................... 29 3.7.6 Culturing of fungi ............................................................................................. 30 3.7.7 Antimicrobial susceptibility test ....................................................................... 30 3.8 Molecular microbiology .......................................................................................... 31 3.8.1 DNA extraction................................................................................................. 31 3.8.2 Polymerase chain reaction (PCR) ..................................................................... 32 3.8.2.1 Nested PCR amplification using insertion sequence IS2404 as primers .. 33 3.8.2.2 PCR amplification using primers 16S rRNA (PA/MSHA) and 1_16SR/1_16SF .................................................................................................... 33 3.8.2.3 Gel electrophoresis and UV visualization................................................. 34 3.8.3 Sequence and data analyses .............................................................................. 34 3.9 Ethical clearance and community entry .................................................................. 34 CHAPTER FOUR ............................................................................................................. 36 4.0 RESULTS................................................................................................................ 36 4.1 General characteristics and demography of study population. ............................... 36 4.2. Isolation and characterization of bacteria isolates .................................................. 37 4.2.1. Gram staining of crude and unique colonies of bacteria identified on BU patients ....................................................................................................................... 37 4.2.2 Acid-Fast Positive bacteria on BU Patient ....................................................... 39 4.2.3 Non-acid fast rods identified on a crude bacterial of a BU patient .................. 40 University of Ghana http://ugspace.ug.edu.gh viii 4.2.4 Identification of Fungal Elements from crude microbial isolate of BU patients ................................................................................................................................... 42 4.2.5 Identification of Yeast Cells in 10% KOH ....................................................... 42 4.3 Culturing and Isolation of Bacteria ......................................................................... 43 4.3.1 Bacteria Culture on Luria-Bertani (LB) Agar .................................................. 43 4.3.2 Culturing and identification of Staphylococcus Mannitol Salt Agar ................ 44 4.3.3 Culturing on MacConkey Agar ........................................................................ 45 4.3.4 Culturing on Sabouraud Dextrose Agar for Detection of Fungal Element ...... 46 4.4. Antibiotic testing of selected bacteria strains after sequencing ............................. 48 4.5 Identification of bacterial strains using culture independent method ..................... 51 4.5.1. Detection of bacteria strains using 16S rRNA (universal primers) ................. 52 4.5.2 Identification of mycobacterial strains using 16S rRNA (Sequence specific for mycobacteria. ............................................................................................................. 53 4.5.3 Nested PCR Product of IS2404 on an Electrophoretogram .............................. 54 4.6 Sequencing of 16S universal bacterial, Mycobacteria and M. ulcerans.................. 55 CHAPTER FIVE .............................................................................................................. 58 5.0 DISCUSSION ......................................................................................................... 58 5.1 CONCLUSION ....................................................................................................... 70 5.2 RECOMMENDATION .......................................................................................... 71 5.3 LIMITATIONS ....................................................................................................... 71 REFERENCES ................................................................................................................. 72 APPENDICES .................................................................................................................. 98 University of Ghana http://ugspace.ug.edu.gh ix LIST OF FIGURES Fig. 2.1. A global map representing countries that have reported cases of Buruli ulcer disease as of 2009. .............................................................................................................. 9 Fig. 2.2. A. Early lesion of M. ulcerans disease on right arm of a 12-year-old girl; B. Ghanaian boy with a well-developed plaque of Buruli ulcer on the right flank. The ulceration is remarkably “stellate,” a feature of plaque lesions. ....................................... 14 Fig. 4.1. Gram stain from a crude bacterial isolate of a BU patient. ................................ 37 Fig. 4.2. Gram stain of unique colonies isolated from an LB plate after sub culturing of bacteria isolates from a BU patient. .................................................................................. 38 Fig. 4.3. Types of Gram positive bacteria, Gram negative bacteria and yeast cells after Gram staining of crude bacterial isolates. ......................................................................... 39 Fig. 4.4. Acid-fast bacteria identified from the crude bacteria isolate after Ziehl-Neelson stain ................................................................................................................................... 40 Fig. 4.5. Non-acid fast rods identified from the crude bacteria isolate of a BU patient. .. 40 Fig. 4.6. The number and types of acid and non-acid fast bacteria present after Zeihl- Neelsen staining. ............................................................................................................... 41 Fig. 4.7. Yeast cells identified from crude patients’ samples of BU patient after Gram and Ziehl-Neelsen stain. .......................................................................................................... 42 Fig. 4.8. Yeast cells viewed under an inverted microscope (x40) .................................... 43 Fig. 4.9. Growth of bacteria colony on LB agar plate from a BU patient ........................ 43 Fig. 4.10. Growth of both mannitol fermenters and non-mannitol fermenters of staphylococci spp from crude patients’ samples from the lesions of BU patients............ 44 University of Ghana http://ugspace.ug.edu.gh x Fig. 4.11. Distribution of Staphylococci spp from BU patients. ....................................... 45 Fig. 4.12. Growth of enteric or gram negative bacteria from a crude patients’ samples of a BU patient on a MacConkey plate .................................................................................... 45 Fig. 4.13. Distribution of Gram negative or Enteric bacteria from crude isolates among the BU patients .................................................................................................................. 46 Fig. 4.14. Growth of fungal elements from patients’ samples of BU patients on a Sabouraud dextrose agar plate .......................................................................................... 47 Fig. 4.15. Distribution of fungal elements present on the lesions of BU patients after culturing on Sabouraud Dextrose Agar............................................................................. 48 Fig. 4.16. Zones of inhibition of growth of B. anthracis isolated from the lesion of a BU patient using selected antibiotic ........................................................................................ 49 Fig. 4.17. 16S (universal bacteria), 16S (Mycobacteria) and IS2404 positivity among people with signs of BU. ................................................................................................... 51 Fig. 4.18. An electrophoretogram showing the PCR product of 16 samples using 1_16S primers (universal bacteria) .............................................................................................. 52 Fig. 4.19. An electrophoretogram of PCR products of 10 patients using MSHA/PA as primers for mycobacteria .................................................................................................. 53 Fig. 4.20. An electrophoretogram of PCR product of 27 patients using IS2404 nested (Pg 1 to Pg4) as primers .......................................................................................................... 54 Fig. 4.21. Sequence of one 16S universal bacteria compared to sequence nucleotide of Pseudomonas aeruginosa strain BN5. .............................................................................. 55 Fig. 4.22. Phylogenetic tree constructed for partial 16S rRNA gene of bacterial strains from BU Isolate................................................................................................................. 57 University of Ghana http://ugspace.ug.edu.gh xi LIST OF TABLES Table 3.1. List of primers used for PCR amplifications ................................................... 32 Table 4.1. The zones of inhibition (in mm) of 6 different antibiotics on susceptibility testing on selected bacteria ............................................................................................... 50 University of Ghana http://ugspace.ug.edu.gh xii LIST OF ABBREVIATIONS µL Micrometer BCG Bacille Calmette-Guerin BD Becton Dickinson Bp Base pair BU Buruli Ulcer CD62 Cluster of Differentiation 62 Cm Centimetre EDTA Ethylenediaminetetraacetic Acid ER Enoyl Reductase g Gram G-C Guanine-Cytosine KR Keto Reductase L Liter M. ulcerans Mycobacterium ulcerans Mb Megabase MIC Minimum Inhibitory Concentration ml milliliter NCBI National Center for Biotechnology Information nM nano Molar oC Degree Celcius University of Ghana http://ugspace.ug.edu.gh xiii PBS Phosphate Buffer Saline PCR Polymerase Chain Reaction PDT Photo Dynamic Therapy PLN Peripheral Lymph node rDNA Ribosomal Deoxyribonucleic acid rpm Revolutions per minute rRNA Ribosomal Ribonucleic acid S. aureus Staphylococcus aureus St. Saint Th Thymus- helper UV Ultra violet University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION 1.1 Background Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. It usually begins as a painless nodule or papule and may progress to massive skin ulceration (Duker et al., 2004b). It is the third most common mycobacterial disease after tuberculosis and leprosy (Johnson et al., 2005). Buruli ulcer infection has been reported in over 30 countries globally; Australia, South America, Asia and Africa with most cases in West and Central Africa including Ghana, Cote d’Ivoire and Benin with an estimated prevalence of 22% and 16% for Cote d’Ivoire and Ghana respectively (Merritt et al., 2010). The incidence of the disease keeps increasing over the last ten years but has recently started to decline. In Ghana, the number of new cases reported was 685 in 2003, 1021 in 2004 and 1097 in 2005 and to 1010 in 2006 (Williamson et al., 2008). In Ghana, active cases have been identified in all ten regions of the country and were present in 90 out of the then 110 districts of the country (Amofah et al., 2002). Buruli ulcer is more prevalent in the southern belt of Ghana: Ashanti, Central, Western and Eastern Regions. The infection has been observed to affect all sexes and age groups but has been found to occur more in children less than 15 years. (Van Der Werf et al., 2003). Buruli ulcer remains a priority health problem in Ghana especially in the Amansie West and Amansie Central District (Duker et al., 2004a). The pathology of M. ulcerans is different from other mycobacterial pathogens in that it is mainly extracellular and produces a plasmid- encoded toxin with a polyketide-derived macrolide structure, mycolactone (Stinear et al., University of Ghana http://ugspace.ug.edu.gh 2 2004). Mycolactone is responsible for the pathogenesis associated with the disease. It suppresses the host’s immune factors from eliciting inflammatory responses (Hong et al., 2008). The ulcers are therefore, painless unless there is secondary infection (Phillips et al., 2009). In cell culture experiments, mycolactone causes apoptosis and necrosis in many human cells (George et al., 2000). Destruction of subcutaneous fat of skin leads to lesion and ulcer could provide grounds for the colonization and proliferation of microorganism from normal skin flora and pathogenic species from the environment to establish secondary infection. Mycolactone, being an immunosuppressant toxin does not cause local inflammation (Van Der Werf et al., 2005). Thus, painful ulcers experienced by some patients may be due to secondary infections. According to Werf et al. (1999), there are usually severe complications in BU such as deformity. The presence of purulent secretion (pus) or at least two of the cardinal manifestation of inflammation could provide grounds for the growth of microorganism which may cause secondary infections in BU (Ohene, 1997). Secondary infections have also been reported after surgery (Johnson et al., 2005). Antibiotic treatment after confirmation of the disease is the recommended form of treatment. Wide excisional surgery may be performed if found necessary (Converse et al., 2011). There are also high reported cases of antibiotic resistance of M. ulcerans and this has contributed to BU treatment failure (Barogui et al., 2013). Unfortunately, extensive surgery may unnecessarily damage healthy tissues and it does not prevent recurrence of the disease (Merritt et al., 2010). Despite these setbacks, combination of rifampicin and dapsone showed marginal beneficial effect attributed to drug therapy (Espey et al., 2002). Also, it has been observed that antibiotics treatment combination University of Ghana http://ugspace.ug.edu.gh 3 containing aminoglycoside are more effective than those without (Van Der Werf et al., 2005). Research in Ghana reports that patients respond to streptomycin and rifampicin and in few cases, the lesions were completely healed (Barogui et al., 2013). A recent study identified the presence of secondary microbial infection associated with BU. These bacteria include S. aureus and P. aeruginosa (Yeboah-Manu et al., 2013). Drug resistance in some patients as well as functional limitations after treatment serves as a hindrance to the treatment of the disease (Chauty et al., 2007). 1.2. Justification/Relevance The issue of secondary infection has not received considerable attention. The pathogenesis of Buruli ulcer is like that of other infections that break barriers for bacterial infection (Silva et al., 2009). Both pathogenic and opportunistic microbes, stemming from normal skin flora or immediate environment, can colonize most skin ulcers and cause secondary infections in patients (Yeboah-Manu et al., 2013). Coincidentally, long term treatment recommended may cause antibiotic resistance for other opportunistic bacteria. Secondary microbial infection and its dynamics have however not received enough attention. Since BU was recognized as a health problem in Ghana, various researchers have investigated ways in which better treatment can be meted out to ailing persons. There have been challenges in identifying the best remedy to fight the infection due to antibiotic resistance and late case reporting. This study will aid in the identification of secondary microbial infections of BU, drug selection and the eradication of the disease. University of Ghana http://ugspace.ug.edu.gh 4 1.3. Hypothesis Buruli lesions may provide fertile grounds for colonization and proliferation of other microorganisms. There is high genetic variation within isolates that cause secondary infections which can be resolved using fine genetic fingerprinting techniques. The polymicrobial nature of secondary infected lesions confers synergy and adaptive features on these microorganisms which enable them resist some classes of antibiotics. 1.4. Aim To detect and characterize microorganisms causing secondary infections in Buruli ulcer lesions and to determine their level of antibiotic resistance. 1.5. Objectives  To culture and identify bacteria and other microorganisms from swabs from ulcerative lesions.  To determine the effect of certain antimicrobial drugs on selected secondary microorganisms from BU lesions and assess the efficacy of other class of antibiotics on these microorganisms.  To elucidate any genetic variations within isolated species causing secondary infections in BU. University of Ghana http://ugspace.ug.edu.gh 5 CHAPTER TWO 2.0. LITERATURE REVIEW 2.1. Buruli Ulcer Buruli ulcer is an emerging human disease caused by infection with a slow-growing pathogen, Mycobacterium ulcerans, which produces mycolactone, a cytotoxin with immunomodulatory properties (Demangel et al., 2009). The disease is characterized by a painless nodule, papule, plaque or edema, evolving into a painless ulcer with undermined edges, often leading to invalidating sequelae. In rare cases, osteomyelitis occurs (Walsh et al., 2011). These distinctive ulcers were first noticed in Uganda but described in Australia in 1948 and it was named Bairnsdale ulcer due to their focal distribution (Quek et al., 2007). The exact mode of transmission of BU is unclear. M. ulcerans is unique among mycobacterial pathogens in that it is mainly extracellular during human infection and produces a plasmid-encoded toxin with a polyketide-derived macrolide structure, named mycolactone (Stinear et al., 2004). Mycolactone is believed to play a central role in determining the extracellular localization of the bacteria and modulation of immunological responses to M. ulcerans (Adusumilli et al., 2005). Three features of M. ulcerans emerges as a determinant for the pathogenesis of BU. Low optimal temperature of growth that makes the skin its preferential target, its mycobacterial pedigree that at least at some phase of its cycle in the host should lead M. ulcerans to behave as an intracellular microorganism triggering inflammatory cell University of Ghana http://ugspace.ug.edu.gh 6 responses and the mycolactone–associated high cytotoxicity that contrasts with intracellular traits of its mycobacterial nature (Portaels et al., 2009a). M. ulcerans is an environmental microorganism. By using the insertion sequence IS2404 which is found in about 200 copies in a PCR, the bacterium has been found in environmental samples and wild animals and in pathology specimens from patients (Portaels et al., 2001). Interestingly, unlike the closely related M. marinum, it was noted that M. ulcerans is not capable of mounting a productive infection in fish and does not support the hypothesis based on epidemiological evidence shown by IS2404-PCR that fish may be a reservoir for M. ulcerans (Mosi et al., 2012). 2.1.1. Mycolactone Mycolactones are immunosuppressive and cytotoxic polyketides comprising five polyketide-derived macrolides. Besides its immunosuppressive effect, it usually diffuses beyond the site of M. ulcerans infection thus extending the lesion (Hong et al., 2008). After its secretion from the bacterium, it is diffused into the affected tissues and surrounding areas. The amount and precise distribution of the toxin in the lesion is always unknown (Portaels et al., 2009a). M. ulcerans mycolactone-negative mutants are nonvirulent and non-cytotoxic (Torrado et al., 2007). There are naturally occurring structural variants (A, B, C, D, E and F) produced by different species of very closely related mycobacteria including human pathogen. In M. ulcerans strain, Agy 99, mycolactone A/B is produced by 3 highly homologous type1 polyketide mega synthetase (PKS) whose gene (m/s A 1:51kb, m/s A 2:7.2kb and m/s B:42kb) are found on a 174kb plasmid, known as pMUM001 (Pidot et al., 2008). University of Ghana http://ugspace.ug.edu.gh 7 Evidence suggests that M. ulcerans isolates from Asia, Mexico and Australia may be less virulent than isolate from Africa. This could be explained from the heterogeneity in mycolactone variants. The toxin is eliminated by BU patients in a slow rate during treatment (Mve-Obiang et al., 2003). Mycolactone induces apoptosis in human keratinocytes, thus, contributing to Buruli ulcer lesions development (Bozzo et al., 2010). Mycolactone not only induces muscle damage but also prevents muscle regeneration to occur. These results may help to explain why patients with Buruli ulcer, experience muscle weakness and contracture (Houngbédji et al., 2009). This molecule serves an attractive candidate target for the diagnosis and monitoring of the BU. Assay based on detection of mycolactone in serum and ulcer exudate can form the basis of BU diagnostic tests and monitoring (Sarfo et al., 2011). 2.1.2. Epidemiology After tuberculosis and leprosy, BU is the third most common mycobacterial disease (Van Der Werf et al., 2005). In some West African countries, the number of BU cases may exceed those of tuberculosis and leprosy (Portaels et al., 2009a). In Africa, even though it was first observed in eastern Africa (Uganda), it is more endemic in the western wing and particularly more prevalent in Benin, Cote d’Ivoire, Ghana, Guinea, Liberia, Nigeria, Sierra Leone and Togo. Ivory Coast recorded 24,000 cases, Ghana recorded 11,000, and Benin has 7,000 confirmed cases by the year 2002 (Amofah et al., 2002). The overall crude national prevalence rate of active lesions was 20.7 per 100,000, but the rate was 150.8 per 100,000 in the most disease-endemic district (Amofah et al., 2002). Geographical distribution prevalence and incidence of BU may vary greatly by country and in localities within a country over time. These variations may be due to multiple University of Ghana http://ugspace.ug.edu.gh 8 factors such as geophysical activities that lead to soil turn over, climate and man-made environmental disturbances and other ecological changes such as alterations of the fauna and flora (Sopoh et al., 2007). Several studies have also suggested that there is no sex difference in the distribution of BU infection (Owusu & Adamba, 2012; Van Der Werf et al., 2005). According to Hamzat & Boakye-Afram (2011), the male to female ratio is 1:1, giving more evidence to show that there is no gender pattern in the prevalence of the disease. Nevertheless, men older than 59 years may have a higher chance of developing the disease than women. This may be due to work related activities and greater access to health care than the women (Debacker et al., 2004). In adults, legs are more frequently involved than arms. Children between 0 to 14 years tend to develop the disease more often on the truck, head and neck. Lesions at other sites such as genitalia are infrequent. Men have more lesions on the legs whiles arms are more frequently involved in women. Lesions on the palms and soles have not been described (Debacker et al., 2004). Unfortunately, information on the focal epidemiology of BU is sparse. Incidence, prevalence and other data are usually reported at the national or district level (Amofah et al., 2002). The disease is more prevalent in the tropics and the subtropics. It tends to affect people who live close to slow-moving water bodies such as swamps, rivers, ponds and lakes. However cases of the disease have been reported after flooding (Van Der Werf et al., 2005). Even though the disease is usually painless, most patients nonetheless, spend a significant amount of time in a hospital or rehabilitation setting. For instance a review of 102 University of Ghana http://ugspace.ug.edu.gh 9 patients in Ghana indicated that the average length of hospital stay was more than 100 days; and the Buruli infection led to 10 amputations, 12 joints contractures and 2 deaths arising from complications such as tetanus and sepsis of the ulcer (Amofah et al., 1993). A small but increasing proportion of African patients develop osteomyelitis, a complication that usually necessitates amputation (Sopoh et al., 2007). Such circumstances have economic implication which may be beyond the means of local health services in Ghana. Even in developed economies like Australia which has better access to health care, the cost and difficulty of treating M. ulcerans infections was observed to be considerably high (Drummond & Butler, 2004). Some under-developed countries usually have difficulties with the clinical diagnosis of the disease or the collection of specimens for laboratory testing. Due to this, the number of BU cases may be overestimated and worse off, patients with the disease other than BU can be mistreated (Portaels et al., 2009a). Fig. 2.1. A global map representing countries that have reported cases of Buruli ulcer disease as of 2009 (WHO) (Merritt et al., 2010). University of Ghana http://ugspace.ug.edu.gh 10 2.2. Ecology and Transmission of the Disease Molecular studies have shown the existence of M. ulcerans in water, mud, fish, aquatic insects, and snails from swamps in regions endemic for Buruli ulcer (Wallace et al., 2010). As most people who have Buruli ulcer live near these areas, material from swamps, ponds, or river regions may contaminate skin surfaces with M. ulcerans which can result in introduction of the causative agent into skin when it is broken by trauma or insect bites (Williamson et al., 2014). Trauma may be as slight as a hypodermic injection or as serious as a gunshot or a landmine wound (Meyers et al., 1974). Activities that are developed close to the water, such as farming constitute risk factors. A study in Uganda reported that families who use unprotected source of water like river and pond for domestic purposes had a higher prevalence rates of BU than those who used boreholes (Thangaraj, 1999). Interestingly, protective clothing appears to reduce the risk of contracting the disease (Van Der Werf et al., 2005). However unlike malaria and other tropical diseases, its mode of transmission is still unclear. Earlier evidence suggested that aquatic insects (Genus: Naucoris and Dyplonychus) may be involved (Wallace et al., 2010). But here still, the precise role of these water-insects, flies and mosquitoes is not known. M. ulcerans has been identified in the biofilms on insects. The method of transmission may likely be mechanical (Mosi et al., 2008). Merritt et al. (2010), reported that, M. ulcerans is found in biofilms of aquatic habitats and contacted by grazing or filter- feeding invertebrates that are then consumed by predators known to bite humans. Few if any patient recalls having being bitten by an insect prior to the development of the disease (Van Der Werf et al., 2005). Presently, it is unknown whether insect bites represent a route for transmission. In one report, severe M. ulcerans disease with University of Ghana http://ugspace.ug.edu.gh 11 osteomyelitis, followed a snake bite (Hofer & Hirschel, 1993). Again, the human- environment interaction suggested is not clear. Only two cases have been reported of human to human transmission (Debacker et al., 2002). Yet still, person-to-person transmission of the disease has not been properly understood, even though in some cases patients had relatives who were also infected (Sopoh et al., 2010). Clustering of cases among families has occasionally been observed. This may reflect an exposure to a common source of infection and also a common genetic susceptibility to infection with M. ulcerans (Stienstra et al., 2001). The disease has been occasionally diagnosed in tourists and other individuals who have only briefly visited countries where the disease is endemic (Semret et al., 1999). Although the mode of transmission is not quite understood, there are multiple reports which show that patients have had antecedent trauma at the site where the lesion or nodule occurred (Portaels et al., 2009a). Aerosols may also play a role in its transmission (Hayman, 1991). It is possible that M. ulcerans inhaled or ingested is then widely disseminated in the body and reactivates in low temperature areas at sites of trauma, but direct inoculation seems most likely. The incubation period is usually 13 weeks, but much longer, and occasionally shorter, incubation periods occur (Demangel et al., 2009). 2.3. Clinical Presentation of Buruli ulcer Even though the disease can affect any part of the body, it is more often found on the extremities (especially the limbs) as compared to the other parts. Research shows that the lower limbs are more likely to be affected than the upper limbs, and this may be because the lower limbs are frequent lying in contact with soil, water, plants, and insects (Toutous et al., 2013). University of Ghana http://ugspace.ug.edu.gh 12 The disease can be divided into four histopathological stages; non-ulcerative necrotic stage, ulcerated necrotic stage, initial healing stage and late healing stage (Boleira et al., 2010). Clinically, the disease can be divided into pre-ulcerative stage (papule, nodule, plaque, and diffuse oedema) as seen in Fig. 2.2A and ulcerative stage, which may be represented by ulcers smaller than 5 cm and ulcers larger than 5 cm as seen in Fig. 2.2B (Portaels et al., 2009b). It can also be classified into localized disease (papule, nodule and ulcer), and disseminated disease (plaque, diffuse edema and metastatis) (Adamba & Owusu, 2011). Patients may manifest clinical symptoms, such as plague and undermined oedema. Plaques are raised and hard, but are painless, with a certain degree of depigmentation or spotted erythema. They can be more than 2 cm in diameter, possibly reaching 15 cm. These can later develop into large ulcers with irregular borders (Boleira et al., 2010). The disease can also be present with a large area of marked indurations, diffuse oedema in the legs and arms or a well-demarcated plaque (Abalos et al., 2000). When there is only one oedema, the profile is more diffuse, and with poorly demarcated borders. Histological examination of excised skin lesions characteristically shows skin ulceration with extensive necrosis of subcutaneous fat. The ulcerating stage develops due to perforation of the necrosis above the epidermis. Initial ulcers may remain small, with 1 to 2 cm of diameter or become larger (larger than 5 cm) and destroy the skin around them. Necrosis frequently extends beyond areas where M. ulcerans is visible (Guarner et al., 2003). Acid-fast bacilli are present in enormous numbers within the necrotic area and are typically arranged in spherules. These clusters of bacteria extend along thickened septa in the subcutaneous tissue. The granulomatous inflammatory response normally associated University of Ghana http://ugspace.ug.edu.gh 13 with mycobacterial infection may be seen in long-standing lesions, but is strikingly absent in early lesions (Guarner et al., 2003). The disease may also affect the bones. Bone lesions may include osteitis or osteomyelitis beneath skin lesion or metastatic osteomyelitis from lympholematogenous spread of M. ulcerans (Walsh et al., 2008). More often, there are delays in reporting on the part of patients or late identification of cases. There is usually no pain and no systemic inflammatory response, although patients may complain of itch. Several factors may explain this, the main being that, the occurrence of a lesion is generally painless and with no systemic symptom (Kibadi et al., 2009). This is because mycolactone produced by M. ulcerans , appear to play a role in inhibiting the recruitment of inflammatory cells to the site of infection, which at least explains why inflammatory responses are poor in Buruli ulcer lesions (Adusumilli et al., 2005). Early stages of the disease are often ignored by the patients and may heal spontaneously. After variable periods of a week to several months, these forms ulcerate and often bring patient to the hospital (Portaels et al., 2009a). As a result, in many cases the disease progresses slowly, steadily and largely unnoticed. The type of disease that develops after the infection depends on many factors such as the immune status of the host, the size and depth of the inoculation, (superficial or deep) and the virulence of the M. ulcerans strain (Portaels et al., 2009a). University of Ghana http://ugspace.ug.edu.gh 14 Fig. 2.2. A. Early lesion of M. ulcerans disease on right arm of a 12-year-old girl (Wansbrough-Jones & Phillips, 2006); B. Ghanaian boy with a well-developed plaque of Buruli ulcer on the right flank. The ulceration is remarkably “stellate,” a feature of plaque lesions (Walsh et al., 2009). 2.4. Diagnosis Microbiological staining is usually done using Ziehl-Neelsen, fluorochrome or Harris Haematoxylin and eosin staining technique. The presence of large numbers of extracellular acid-fast bacilli in a swab from a necrotic ulcer makes the diagnosis likely positive, especially in countries with a low incidence of other mycobacterial infection. In culturing microorganisms associated with Buruli ulcer, Lowenstein-Jensen and Middlebrook 7H9, 7H10 and 7H11 agar medium can be used. M. ulcerans can be cultured from most cases provided the correct culture media and incubation temperature are used, and plates are kept for up to 12 weeks (Portaels et al., 2008). Microorganisms obtained can be confirmed not just by culture or staining but also through PCR (Mosi et al., 2008). Loop-Mediated Isothermal Amplification (LAMP) test has been identified as a rapid and sensitive way of detecting the DNA of M. ulcerans A B University of Ghana http://ugspace.ug.edu.gh 15 under field conditions based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome) (Njiru et al., 2010). A recent report describes a new dry-reagent based PCR for Buruli ulcer that could be used in small regional centres, and reports from Ghana suggest a sensitivity of 95% or more (Babonneau et al., 2015). 2.5. Treatment of Buruli Ulcer Treatment of the disease is relatively easy if identified early. Antibiotics have the potential of curing small lesions and limiting surgery for larger lesions. It has also been found to shorten the time for healing after surgery (Converse et al., 2011). Until this discovery, treatment was largely done through surgical means that often involved extensive excision, sometimes with skin transplantations. But if the identification of the disease is delayed and there is an extensive necrosis of the skin or an attack on the bone, the only option is extensive surgical treatment with skin grafting, and may involve amputation and protracted physiotherapy (Barogui et al., 2009). The use of antibiotics such as rifampicin and streptomycin is noted to have a positive impact on treatment outcomes (Etuaful et al., 2005). M. ulcerans is usually sensitive to clarithromycin, rifampicin and ethambutol. This microorganism is susceptible to several anti-mycobacterial drugs in vitro, but the most promising results in the mouse footpad model were obtained with a combination of rifampicin and amikacin (Dega et al., 2002). Furthermore, a human trial has recently shown that early nodular lesions may be rendered culture-negative after a minimum of four weeks therapy with rifampicin plus streptomycin (Evans et al., 2003). Healing takes months and can cause deep scarring, University of Ghana http://ugspace.ug.edu.gh 16 joint retractions, and deformity. Death is a rare complication (Sizaire, Nackers, Comte, & Portaels, 2006). Several anti-tuberculosis agents are active against M.ulcerans in vitro, but experienced clinicians favour surgery rather than drug therapy in initial management. Bacille Calmette-Guerin (BCG), the current vaccine for tuberculosis, has been shown to be partially protective for a few months in the treatment of the disease (Nackers et al., 2006) and in highly endemic areas BCG vaccination programs are likely to be cost-effective and relatively easy to implement (Portaels et al., 2004). Even though, it has been shown to offer protection against M. ulcerans osteomyelitis, it has controversial results regarding protection against subcutaneous BU (Tanghe et al., 2008). Several other vaccines are being put to test such as a preparation of mycolactone deficient-live M. ulcerans vaccine (Fraga et al., 2012), mycolactone polyketide based vaccine (Roupie et al., 2014), heat-shock protein based vaccine and Ag85a-based vaccine (Tanghe et al., 2008). Recently, protective effect of a dewaxed whole-cell vaccine is being research on as a vaccine in curing the infection (Watanabe et al., 2015). The lipidic nature of mycolactone complicates the development of immunization against the toxin. Nevertheless, an alternative would be to target enzymes involved in the biosynthesis of the toxin (Bali & Weissman, 2006). The term “macrolide” is used to describe drugs with a macro cyclic lactone ring of 12 or more elements (Cai et al., 2011). This class of compounds includes a variety of bioactive agents, including antibiotics, antifungal drugs, prokinetics, and immunosuppressant. The 14-, 15-, and 16-membered macrolides are a widely used family of antibiotics. They have excellent tissue penetration and antimicrobial activity, mainly against gram-positive cocci University of Ghana http://ugspace.ug.edu.gh 17 and atypical pathogens and as such, may be good drugs for curing the disease (Landersdorfer et al., 2009). There have been few cases of skin transplantation after treatment of the disease (Bechtle et al., 2010; Sizaire et al., 2006). To avoid social stigmatization, about 70% of Buruli ulcer patients in the Ga West District of Ghana consult traditional healers (Renzaho et al., 2007). Follow-up data on the rate of recurrence in hospital-treated BU patients is hardly reported. Amofah et al. (1998) found a local recurrence rate of 16% at the same site within a year of follow-up. Few patients had recurrence at more than one site. Others were not healed at all even after treatment. Thus treatment would require multiple interventions, including specific antibiotics, surgery, physiotherapy and follow-ups (Debacker et al., 2004). Although the disease can be managed at the early stages, beliefs and attitudes influence health-seeking behavior. Many patients and unaffected individuals in endemic areas believe that witchcraft and curses cause this indolent disease. Patients and their caregivers admit that the resulting stigma, a reluctance to seek treatment outside ones community, financial problems and fear of treatment effects, notably computation, caused delay in reporting (Stienstra et al., 2002). 2.6. Suggested treatment other than antibiotics Honey has been reported to have an inhibitory effect on aerobic and anerobic bacteria, yeast, fungi, and viruses (Al-Waili, 2004). Honey inhibited growth of Pseudomonas aeruginosa, Helicobacter pylori, Bacteriodes, and enteropathogens (Elbagoury & Rasmy, 1993). Honey has been used for treatment of respiratory diseases, urinary diseases, University of Ghana http://ugspace.ug.edu.gh 18 gastrointestinal diseases, and skin diseases including ulcers, wounds, eczema, psoriasis, and Dandruff (Zaghloul et al., 2001). Photo Dynamic Therapy (PDT) appears to represent an efficacious alternative modality for the treatment of localized microbial infections through the in situ application of the photosensitizer followed by irradiation of the photosensitizer- loaded infected area. Proposed clinical fields of interest of antimicrobial PDT include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral infections (Maisch, 2007). Some plants have been found to have good antimicrobial properties. Such plants include Spanish needle tree, lipstick tree, trumpet tree, onion and garlic plants. These plants have been shown to be effective against pathogenic microorganisms. It has been demonstrated that folk medicine can be as effective as modern medicine to combat pathogenic microorganisms (Srinivasan et al., 2001). Silver-based dressings appear to provide an effective alternative to antibiotics in the management of wound and ulcer infection (Chopra, 2007). Other topical treatments including application of heat (Radford, 2009), mineral clay (Haydel et al., 2008) nitrite ointment and hyperbaric oxygen have been suggested (Phillips et al., 2004) 2.7. Immunology of the Disease M. ulcerans produces mycolactone; an immune suppressive toxin. This toxin is known to exert its immunosuppressive effects at the systemic level. Assays that are based on mycolactone detection on circulating blood cells may be considered for diagnostic test of early disease manifestation (Hong et al., 2008). The immune mechanisms involved in protection against Buruli ulcer are also largely unknown at present. Mycolactone has University of Ghana http://ugspace.ug.edu.gh 19 been found to inhibit the ability to activate allogenic T-cell priming and to produce inflammatory molecules (Coutanceau et al., 2007). Guenin-Macé et al. (2011) identified that mycolactone injection led to a massive T-cell depletion in peripheral lymph nodes (PLNs) that was associated with defective expression of L-selectin (CD62-L). Pre- exposure to mycolactone impaired the capacity of T-cells to reach PLNs after adaptive transfer, response to chemotactic signals and expand upon antigenic stimulation in vivo in mouse. Interestingly, peripheral blood mononuclear cells obtained from people with a past or current M. ulcerans infection typically show a strong T helper (Th)–2 cytokine responses when exposed in vitro to M. ulcerans. In contrast, samples obtained from their household contacts (exposed healthy controls) exhibit a Th-1 immune response, suggesting that natural resistance may be determined by cell-mediated immune mechanisms directed against intracellular organisms (Gooding et al., 2002). In one fascinating case study, it has been shown that the development of ulcerative M. ulcerans disease is associated with a shift from the Th-1 to the Th-2 phenotype (Gooding et al., 2003). Interleukin-10 may be a key cytokine that mediates local Th phenotype switching within nodules and ulcers (Pidot et al., 2008). Further research can be carried out to help us clearly understand how host immunity is acquired. 2.8. Genetic Variation 2.8.1. Genetic Variation in Mycobacterium ulcerans Genomic analysis has revealed that M. ulcerans arose from M. marinum, a ubiquitous fast-growing aquatic species, through horizontal transfer of a virulence plasmid pMUM001 that carries a cluster of genes for mycolactone production, followed by University of Ghana http://ugspace.ug.edu.gh 20 reductive evolution (Demangel et al., 2009). M. ulcerans is distinguished from M. marinum by three major differences; loss of approximately 1 Mb of its genome; acquisition of a large virulence plasmid which encodes a toxic immunosuppressive macrolide, mycolactone and accumulation of 771 pseudo-genes, some inactivated by acquisition of 213 and 91 copies of the insertion elements IS2404 and IS2606 respectively. Thus the functional M. ulcerans genome [5.8 MB] is about 2 MB smaller than that of M. marinum (6.6 MB) (Stinear et al., 2007). M. ulcerans has two circular replicons. One is a 5,631,606-bp chromosome Agy99 and the other is a 174,155-bp plasmid, PMUM001 (Pidot et al., 2008). The plasmid contains 4 copies of IS2404 and 8 copies of IS2606 which are both used as molecular marker (Williamson et al., 2008). Interestingly, Stinear et al. (2007) noted that, a total of 209 copies of the insertion sequence IS2404 and 83 copies of IS606 have been found interspersed in the genome of the M. ulcerans reference strain Agy99 from Ghana. According to Janda & Abbott (2007), the mycobacterial 16S rRNA gene (encodes 16S rRNA) has been used to trace and differentiate strains of non-tuberculosis mycobacteria. SNP typing has also been successfully used to differentiate patients’ isolates of M. ulcerans from different regions of Ghana (Röltgen et al., 2010). A novel combined 16S rRNA RT/IS2404 qPCR assay has proved to be highly sensitive, specific, and efficient in detecting viable M. ulcerans in clinical samples. The assay is thus applicable for the classification of secondary lesions, monitoring of treatment success and provides a powerful tool for clinical research (Beissner et al., 2013). Two enzymes involved in biosynthesis of mycolactone; enoyl reductase (ER) and keto reductase (KR) are used as University of Ghana http://ugspace.ug.edu.gh 21 genetic markers having an average G+C content of 62.5% (Johnson et al., 2005). Further research can be done to enhance the knowledge of genetic variations in M. ulcerans. Although experimental infections allow the analysis of the evolution of M. ulcerans from the very beginning, human samples of the initial phases of the infections are not available, limiting our knowledge on the establishment of the infection (Portaels et al., 2009a). 2.8.2. 16Sr RNA for Bacteria Detection Genes used for bacteria detection include those that code for the 5S, the 16S (also called the small subunit), and the 23S rRNA and the spaces between these genes. The part of the DNA now most commonly used for taxonomic purposes for bacteria is the 16S rRNA gene (Tortoli, 2003). The 16S rRNA gene can be compared not only among all bacteria but also with the 16S rRNA gene of archeobacteria and the 18S rRNA gene of eucaryotes (Pace, 1997). The 16S rRNA gene is about 1,550bp long and is widely used as a target as it is a highly conserved gene, ubiquitous in all organisms and contains variable and hyper-variable regions of sequence. Molecular methods based on this gene are well established as a standard method for characterization and identification of bacteria (Arosio et al., 2008). Variable regions within the 16S rRNA gene have been used for discrimination between species and genera (Bertilsson et al., 2002). Targets containing conserved regions and areas of variability for specific identification of bacteria are the genes encoding 16S and 23S rRNA. In addition, a stretch of DNA between the 16S and 23S rDNA, the 16S-23S rDNA ISR, proved to be a comparably species specific segment. These three targets have already been used for molecular identification and differentiation of various streptococcal species (Hassan et al., 2003). Universal primers University of Ghana http://ugspace.ug.edu.gh 22 are usually chosen as complementary to the conserved regions at the beginning of the gene and at either the 540-bp region or at the end of the whole sequence (about the 1,550- bp region), and the sequence of the variable region in between is used for the comparative taxonomy (Rinttilä et al., 2004). Although 500 and 1,500 bp are common lengths to sequence and compare, sequences in databases can be of various lengths. 16S rRNA gene sequences allow bacterial identification that is more robust, reproducible, and accurate than that obtained by phenotypic testing. The gene’s analysis can identify uncultured bacteria, allowing independence from growth conditions (Clarridge, 2004). 2.9. Microbial infections 2.9.1 Microbial infection of wounds Common skin lesions that can be secondary infected with bacteria are scabies, psoriasis, poison ivy, atopic dermatitis and eczema (Brook et al., 1999; Brook et al., 2000). Microbial infections will have little impact on a minor wound that is healing rapidly but may establish large colonies on slowly healing chronic wounds (Sibbald et al., 2003). Many wound and skin infections that complicate skin lesions are caused by mixed bacterial flora. Bacteria rapidly colonize open skin wounds where they exist synergically. Microorganisms colonizing wounds usually originate from the patient’s endogenous skin and gastrointestinal and respiratory flora (Erol et al., 2004). Microorganisms may also be transferred to a patient’s skin surface via contact with contaminated external environmental surfaces, water, fomites, air, and the soiled hands of health care workers (Weber & Sheridan, 1997). Wound colonization by yeasts and fungi usually occurs later after treatment has usually begun (Katz et al., 2014). It is widely believed that aerobic or facultative pathogens such as S. aureus, P. aeruginosa and the beta-hemolytic University of Ghana http://ugspace.ug.edu.gh 23 Streptococci are primarily responsible for delayed healing (Bowler et al., 2001). Microorganisms transmitted from the hospital environment tend to be more resistant to antimicrobial agents than those originating from the patient’s normal flora (Cohen et al., 2004). Enteric gram-negative bacilli are usually isolated from leg and trunk lesions. Beta-haemolytic streptococci and S. aureus predominates in finger and hand lesions but S. aureus, the most prevalent aerobe, can be isolated from all body sites of lesions. Isolation of multiple organisms from patients illustrates the polymicrobial nature of secondary infected skin lesions and the potential for bacterial synergy between the different patients’ samples. Several studies have documented the synergic effect of mixtures of aerobic and anaerobic bacteria in experimental infections. Various hypotheses have been proposed to explain such microbial synergy. It may be the result of protection from phagocytosis and intracellular killing (Ingham et al., 1977), production of essential growth factors, or lowering of oxidation-reduction potentials in host tissue (Mills & O’Neill, 2013). The presence of β-lactamase-producing organisms in many secondary infected skin lesions not only survive penicillin therapy but also can protect susceptible bacteria from penicillin by releasing the enzyme into the adjacent tissues or abscess cavity (Brook et al., 1984). The exact pathogenic role of the organisms isolated from secondary infected skin lesions has not yet been determined. Treatment of serious skin infection should include systemic antimicrobial therapy. This could be important in the event of poor response to therapy or spread of the infection. Antimicrobial therapy for mixed aerobic and anaerobic bacterial infections is required when polymicrobial infection is suspected (Brook, 2002). University of Ghana http://ugspace.ug.edu.gh 24 2.9.2. Secondary infection of Buruli ulcer Limited research has been done concerning secondary infections found in Buruli ulcer. According to Yeboah-Manu et al. (2013), infections from other microorganisms such as S. aureus, P. aeruginosa, P. miriabilis among others can be found proliferating in BU infections. After treatment with Streptomycin with Rifampicin for 8 weeks (SR8), 75% of the lesions clinically suspected were microbiologically confirmed to be under infection with P. aeruginosa and P. miriabilis. This shows high resistance to antibiotics in Ghana. S. aureus serves as one of the main secondary microbial infection that may infest the ulcer thus patients usually received at least one course of antibiotics other than streptomycin and rifampicin during their hospital stay (Barogui et al., 2013). More research is thus needed in the field of secondary infections in BU and their susceptibility to drugs. University of Ghana http://ugspace.ug.edu.gh 25 CHAPTER THREE 3.0 METHODOLOGY 3.1 Study Site This study was an addition into an ongoing extensive study of zoonotic risk of Buruli ulcer between humans and mammals in Ghana and Cote d’Ivoire. For the purpose of this study, communities in the Amansie Central District of Ashanti region of Ghana which are known to be endemic for BU were used as study sites. The inhabitants are mostly farmers but some youth engage in surface mining in the Offin River basin which runs close to most of the communities. Even though there is at least one working borehole with a pump in each community, inhabitants still fetch water from surface water sources (ponds, streams, rivers) for domestic and agricultural activities. Saint Peters Hospital in Jacobu, the district capital where BU cases are usually treated was the main site for sample collection. This current study was approved by Ethical and Protocol Review Committee of the University of Ghana Medical School [MS-ET/M.11 – P 3.1/2013-2014] and permission was granted by the District Health Directorate and traditional leaders of the respective communities. 3.2 Study Design The study was a cross-sectional study which aimed at identifying microorganisms as well as genetic variation among isolates that cause secondary infection in Buruli ulcer. Samples were taken from February 2014 to June 2014. University of Ghana http://ugspace.ug.edu.gh 26 3.3 Sample Size Determination The minimum samples size was determined using the equation below: [Sample size, n; Prevalence, P; Standard score, Z (95% confidence level)] [Z=1.96; P= 0.0015 (Amofah et al., 2002) ; E=0.05] n= 2.13 The minimum sample size is approximately 2. However, a total of 51 samples were used for the study. This was to ensure statistically significant result and rule out chance variations. 3.4 Subjects All groups of patients of all ages and both sexes were enrolled in this study. Admitted BU patients with ulcer(s)-nosocomial secondary infections, BU patients from traditional healers with ulcer(s)-community acquired secondary infections and BU self-cared patients with ulcer(s)-community acquired secondary infections. All patients and parents on behalf of their children signed a consent form before enrolling into the study. Subjects were recruited from St. Peters Hospital in Jacobu. There was also a case search to find patients who were being treated by traditional healers as well as those under self – medication. University of Ghana http://ugspace.ug.edu.gh 27 3.4.1 Inclusion Criteria Suspected BU patients of all ages and sexes who may or may not have experienced pain in the ulcer or lesion and have given their consent. PCR positivity of IS2404 for BU criteria was also to be met. 3.4.2 Exclusion Criteria BU patients who refused to consent 3.5 Data on study samples Clinical information on the gender and age of randomly chosen subjects were recorded. Additional information on location, occupation, stage of BU and type of treatment were also taken. 3.6 Sample collection from patients There was a case search at the hospitals and individual communities for people with signs of BU, nodule, oedema and ulcer who experienced pain. Swabs from ulcers were taken by a well-trained Laboratory Technician from the St. Peters Hospital, Jacobu, Amansie Central District. Swabs were kept in properly labelled 15 ml conical tubes. The samples were kept on ice packs in a cooler and quickly transported back to St. Peters Hospital (Jacobu) for temporal storage in a refrigerator. 3.7 Sample processing for laboratory analyses All samples were processed prior to microscopy and DNA extraction 3.7.1 Sample Preparation Two milliliters of 1X Phosphate Buffer Saline (PBS) was pipetted into the 15 ml Eppendorf tube. The tube was then vortexed gently for 5 minutes to dislodge microbial University of Ghana http://ugspace.ug.edu.gh 28 cells and later transferred to a 2 ml screw-cap tube. Afterwards, 1 ml of the sample was pipetted into a new 2 ml screw-cap tube. The tube was then gently vortexed briefly and spun at 14,000 rpm for 5 minutes. Later, 800µl of supernatant was pipetted off and the remaining 200 µl (plus pellet) was used for DNA extraction. Of the remaining 1 ml, 200 µl was pipetted into another 2 ml screw-cap tube for decontamination. The remaining 800 µl was preserved at 4ºC in a refrigerator for later use. 3.7.2 Gram Staining Gram staining was performed according to the manufacturer’s (BD Gram Stain Kit, New Jersey, U.S.A) protocol. Briefly, about one drop of specimen was smeared on a glass slide (frosted slide glass, Thermo Scientific, Waltham, U.S.A), allowed to dry and gently passed over flame until smear was fixed. The slide was then flooded with Gram crystal violet staining reagent and left for 1 minute. The crystal violet was then washed off with distilled water and later flooded with Gram iodine for 1 minute followed by washing off with distilled water. The slide was again flooded with decolorizing agent for 15 seconds and counterstained with Gram Safranin for 1 minute. The stain was washed off completely and the slide was air-dried. Slide was viewed under a light microscope (Leica, Wetzlar, Germany), in oil immersion at a magnification of X1000. Pictures were taken using a camera (Sony, Tokyo, Japan). 3.7.3 Ziehl-Neelsen staining for acid-fast bacilli (AFB) Acid-fast Staining was performed according to manufacturer’s protocol (Oxoid, Hamphire, United Kingdom). Briefly, about two drops of specimen was smeared on a glass slide (Frosted slide glass, Thermo Scientific, Waltham, U.S.A) and gently passed over flame until smear was fixed. The slide was then flooded with carbol fuchsin and left University of Ghana http://ugspace.ug.edu.gh 29 for 5 minutes. The carbol fuchsin was later washed off with distilled water and stain was decolorized with acid alcohol (3% hydrochloric acid). The slide was then flooded with methylene blue and left for 2 minutes. Stain was washed off completely and the slide was air-dried. Slide was viewed under a light microscope (Leica, Wetzlar, Germany), in oil immersion at a magnification of X1000. Picture was taken using a camera (Sony, Tokyo, Japan). 3.7.4 Microscopic determination of fungal elements Fungal elements were detected as described (Garg et al., 2009) with slight modification. A solution of 10% KOH containing 10% glycerol was prepared. A volume of 100 µl of the solution was pipetted into a 24 well cell culture plates (NuncTm, New York, USA). A volume of 10 µl of each sample was added to the KOH solution. It was left to stand for 40 minutes and examined under an inverted microscope (ZEISS Promovert, Jena, Germany) at a magnification of x40 for the presence of fungal elements. 3.7.5 Culturing of bacteria Masses of 28.0 g, 55.0 g, 111.0 g and 63 g of Luria Bertani (LB) (Oxoid, Hamphire, United Kingdom), MacConkey (AccuminTm, New York, USA), Mannitol salt (BD, New Jersey, USA) and Baird-Parker (BP) agar were weighed respectively and dispersed in 1 L deionized water. They were allowed to soak for 10 minutes, swirled to mix and then heated to boil with frequent agitation until the media dissolved completely. This was followed by autoclaving at 121oC for 15 minutes. The mixture was allowed to cool to 47oC, mixed well and a volume of 25 ml was poured into each plate. For Baird Parker agar, it was supplemented with 1 vial of Rabbit Plasma Fibrinogen reconstituted with 10 ml of sterile purified water per 90 ml before dispersing in a petri dish. Serial dilutions of University of Ghana http://ugspace.ug.edu.gh 30 1 in 10 of each of the samples were streaked on the plate and sealed with parafilm M. This was followed by labeling and storing in an incubator (Fisher scientific, Pittsburgh, USA) at 30oC to allow growth of the bacteria. The plates were examined 16 to 48 hours for growth. 3.7.6 Culturing of fungi A mass of 65 g of Sabouraud dextrose agar with chloramphenicol (Oxoid, Hamphire, United Kingdom) was weighed and dispersed in 1 L deionized water. It was allowed to soak for 10 minutes, swirled to mix and then heated to boil with frequent agitation until the media dissolved completely. This was followed by autoclaving at 121oC for 15 minutes. The mixture was allowed to cool to 45oC and mixed well. A volume of 25 µl was poured into each plate. Serial dilutions of 1 in 10 of each of the samples were streaked on the plate and sealed with parafilm M. This was followed by labeling and storing in an incubator (Fisher scientific, Pittsburgh, USA) at 32oC to allow growth of the bacteria. The plates were examined 2 to 7 days for growth. 3.7.7 Antimicrobial susceptibility test This was performed according to the Kirby Bauer protocol with slight modification (Bauer et al., 1966). Amikacin (30µg), Kanamycin (30µg), Clarithromycin (30µg), Hygromycin B (30µg), Streptomycin (10µg) and Rifampicin (5µg), were obtained from Mast Diagnostics (Mast Group Limited, Bootle, United Kingdom). A mass of 38g of Mueller-Hinton agar was weighed and dispersed in 1 L deionized water. It was allowed to soak for 10 minutes, swirled to mix and then heated to boil with frequent agitation until the media dissolved completely. This was followed by autoclaving at 121oC for 15 minutes. The mixture was allowed to cool to 45oC and mixed well. A volume of 25 µl University of Ghana http://ugspace.ug.edu.gh 31 was poured into each plate. A total of 20 unique colonies of bacteria identified on the LB agar plates were subcultured on LB agar plates. Two colonies of each were picked. DNA was extracted from one set and sequenced after PCR using 16S primers. The second colony of each isolate was emulsified in 3ml sterile normal saline in bijou bottles and the density and turbidity was compared with a barium chloride standard (0.5 McFarland). Total cell count was performed before inoculation to prevent obtaining false positive and false negative results. A sterile swap was dipped into the standardized suspension of bacterial culture and used to evenly inoculate the entire surface of the Mueller-Hinton Agar (MHA) plate to obtain uniform inoculums. MH agar plates were allowed to dry. Each plate was divided into 6 sections and antimicrobial impregnated disks were placed on the middle of each section using a sterilized forceps. Each disk placed was gently pressed with the sterilized forceps to ensure complete contact with the agar surface. This was done until each sample had all antimicrobial impregnated disk placed over its plate. The plates were then covered and sealed with parafilm M, and stored at 32oC in an incubator (Fisher scientific, Pittsburgh, USA) for 18 hours, the zones of inhibition of each disk was then checked and recorded. 3.8 Molecular microbiology 3.8.1 DNA extraction DNA extraction was performed with the Qiagen Dneasy blood and tissue kit (QIAGEN, Venlo, Netherlands) following the manufacturer’s protocol. Briefly, 850 µl of 1X PBS was added to 150 µl of sample. From this, 500 µl was aliquoted into a 2 ml screw cap tube, centrifuged at 14,000 rpm for 15 minutes to pellet bacterial cells and 300 µl of supernatant was pipetted off. To the remaining solution, 180 µl of buffer ATL and 20 µl University of Ghana http://ugspace.ug.edu.gh 32 of proteinase K were added. The mixture was vortexed briefly and incubated at 56°C for 3 hours. Later, 200 µl of buffer AL was added. The mixture was vortexed briefly and incubated at 70°C for 30 minutes. To this, 250 µl of 100% ethanol was added. The mixture was vortexed briefly and transferred to a spin column. The column was then centrifuged at 8,000 rpm for 1 minute and flow-through was discarded. It was then washed by adding 500 µl of buffer AW1, centrifuged at 8,000 rpm for 1 minute and flow- through discarded. Washing was repeated but with buffer AW2. The column was dried by spinning at 13,000 rpm for 3 minutes. It was then put into freshly labelled 1.5 ml tubes, 150 µl of buffer AE added and centrifuged at 8,000 rpm to elute DNA. The DNA was stored at -20°C until further use. 3.8.2 Polymerase chain reaction (PCR) For each PCR run, negative and positive controls were included. A 2720 Thermal Cycler (Applied Biosystems, California, U.S.A) was used for all PCR reactions. All primers sequences used are listed in Table 1 below: Table 3.1. List of primers used for PCR amplifications Primers Forward and reverse sequences Expected sizes References 1_16SF F-AGGAGGTGATCCAACCGCA R-AACTGGAGGAAGGTGGGAT 350bp (Reysenbach et al., 2000) 16S rRNA MSHA-AAAAAGCGACAAACCTACGAG PA-AGAGTTTGATCCTGGCTCAG 600bp (Hughes et al., 1993) IS2404 (nest 1) pGp1: 5’AGGGCAGCGCGGTGATACGG-3’ pGp2: 5’CAGTGGATTGGTGCCGATCGAG-3 400bp (Ablordey et al., 2012) IS2404 (nest 2) pGp3: 5’GGCGCAGATCAACTCGCGGT-3’ pGp4: 5’CTGCGTGGTGCTTTACGCGC-3’ 200bp (Ablordey et al., 2012) University of Ghana http://ugspace.ug.edu.gh 33 3.8.2.1 Nested PCR amplification using insertion sequence IS2404 as primers Amplification for IS2404 loci was performed in a nested PCR, adapted from (Ablordey et al., 2012) with slight modifications. The first reaction was done in a 25 μl reaction containing 5X PCR buffer already containing 1 nM MgCl2 (Inqaba Biotech, Pretoria, South Africa), 300 μM each of deoxyribonucleotide and primers Pg1 and Pg2 (Inqaba Biotech, Pretoria, South Africa), and 2.5 µl of genomic DNA. In the second nested reaction, 3 μl of PCR product from previous reaction was used as template in a 25 μl reaction containing similar concentrations of reagents as in the previous nested reaction except for the primers; 500 nM each of Pg3 and Pg4 (described above). Preheating was done at 95°C for 2 minutes, followed by 40 cycles each of, denaturation at 94°C for 30 seconds, annealing at 64°C for 1 minute and extension at 72°C for 1.5 minutes. Final extension was at 72°C for 10 minutes and reaction held at 4°C. 3.8.2.2 PCR amplification using primers 16S rRNA (PA/MSHA) and 1_16SR/1_16SF Both PA/MSHA and 1_16SR/1_16SF genes were amplified separately as described (Williamson et al., 2008; Reysenbach et al., 2000) with slight modifications, using similar master mix concentration. This was performed in a 25 μl reaction containing 5X PCR buffer containing 2 mM MgCl2, 400 μM each of deoxyribonucleotide, 160 nM each of forward and reverse primers, 1U GoTaq polymerase and 2.5 μl of genomic DNA. Cycling was done at 95°C for 5 minutes, followed by 34 cycles each of, denaturation at 94°C for 45 seconds, annealing at 56°C for 45 seconds and extension at 72°C for 45 seconds. Final extension was at 72°C for 10 minutes and reaction held at 4°C. University of Ghana http://ugspace.ug.edu.gh 34 3.8.2.3 Gel electrophoresis and UV visualization For electrophoresis, 7 μl of PCR products were run on a 2% agarose (Sigma-Aldrich, Darmstadt, Germany) gel, stained with ethidium bromide (Sigma-Aldrich, Darmstadt, Germany), using 1X Tris Acetate EDTA (Thermo Scientific, Waltham, U.S.A) as running buffer, at 100V (Biometra, Gottingen, Germany) for 50 minutes. The gel was visualized under a UV transilluminator (UVP benchtop, Cambridge, U.K). For each run, 1 kb ladder was loaded alongside. The gel picture was taken (Kodak, New York, U.S.A) and band sizes were independently scored by two Laboratory Assistants. 3.8.3 Sequence and data analyses PCR products (30 µl), of all the DNA amplicons were sent for sequencing (Macrogen Inc, Amsterdam, Netherlands). Forward primers for all loci were used for sequencing. Sequences were aligned using NCBI basic local alignment of sequences tool (BLAST). Sequence similarity to database sequences were based on expect value (E), maximum identity and score, query coverage and total score. The nucleotide sequence was then used to construct a phylogenetic tree with the help of Chromas Lite, BioEdit and Mega 6 software. 3.9 Ethical clearance and community entry Ethical approval with number MS-ET/M.11 – P 3.1/2013-2014 was obtained from the Ethical and Protocol Review Committee of the University of Ghana Medical School. The study was explained in the local language by the assembly man or woman to all inhabitants within each community. Permission was sought from District Health Directorate, respective chiefs and opinion leaders. All patients and parents on behalf of their children signed a consent form before enrolling into the study. Patients who were University of Ghana http://ugspace.ug.edu.gh 35 confirmed positive for BU but were not on hospital treatment were put on 52 days of antibiotic treatment by the Amansie Central Health Directorate. University of Ghana http://ugspace.ug.edu.gh 36 CHAPTER FOUR 4.0 RESULTS 4.1 General characteristics and demography of study population A total of 51 participants were recruited from the Amansie Central District for the study. All participants had one or more signs indicating the presence of Buruli ulcer. All the 51 samples isolated from the participants were positive for bacterial infection. However, only 40 out of the 51 participants turned out to be positive for Buruli ulcer while 48 participants were positive for mycobacterial infection. All participants complained of pains on the lesion. A total of 26 (50.98%) of the participants were females and 25 (49.02%) were males. The participants were aged between 1-80 years. A total of 10 (19.6%) were below 18 years, 20 (39.2%) were between 18-50 years and 20 (39.2%) were above 50 years. The mean age was 38.2 with the modal age of 65 years. The youngest male and female were 1 and 10 years respectively. The oldest male and female were 80 and 75 years respectively. Available data on the residence of the patients indicate that majority of the patients reside in Jacobu, the capital town of the Amansie Central District where the district hospital St. Peters is located. Others live in near towns and villages such as Krofom, Donkuase, Homase, Mile 14, Afoako, Biribiwomang, Sukuumu, Numereso, Fahiakobo among others. Majority of the participants 18 (35.5%) were farmers. A total of 7 (13.7%), 4 (7.8%) and 2 (3.9%) were students, traders and miners respectively. One watchman, one seamstress University of Ghana http://ugspace.ug.edu.gh 37 and one mason also participated with each representing 1.96%. Participants who were not working were 17 (33.3%) in number. 4.2. Isolation and characterization of bacteria isolates 4.2.1. Gram staining of colonies of bacteria identified on BU patients For the isolation and identification of the different type of bacteria present on the lesions of BU patients, gram staining was performed on both the patients’ samples as well as isolated colonies after culturing the bacteria from the swab in LB agar. The result (Fig. 4.1) below shows different types of bacteria identified after gram staining. Both gram positive and gram negative cocci and rods were identified. Majority of the bacteria identified were gram positive single cocci in the crude sample and rods shaped bacteria from the isolated colonies. Fig. 4.1. Gram stain from a crude bacterial isolate of a BU patient (A) Gram positive packet of cocci, (B) Gram positive single cocci, (C) Gram positive chain of rod shaped bacteria, (D) Gram negative packet of cocci and (E) Gram negative single cocci. University of Ghana http://ugspace.ug.edu.gh 38 Fig. 4.2. Gram stain of unique colonies isolated from an LB plate after sub culturing of bacteria isolates from a BU patient A percentage bar graph was plotted to determine the proportions of each type of bacteria to the total types of bacteria identified after gram staining the crude bacteria isolates of the BU patients. Majority of the bacteria identified were Gram positive 90.2% (46/51) and Gram negative cocci 52.9% (27/51). Few 5.9% (3/51) Gram positive rods-shaped bacteria and Gram negative rod-shaped bacteria 7.8% (4/51) were identified. Yeast cells were identified in 23.5% (12/51) of the crude patients’ samples as shown Fig 4.3. University of Ghana http://ugspace.ug.edu.gh 39 Fig. 4.3. Types of Gram positive bacteria, Gram negative bacteria and yeast cells after Gram staining of crude bacterial isolates 4.2.2 Acid-Fast Positive bacteria on BU Patient In order to identify acid-fast bacteria such as M. ulcerans, Zeihl-Neelsen stain was performed on the crude bacterial isolates. Acid-fast positive cocci and rod shaped bacteria were both identified from the crude bacterial isolates of some BU patient. Fig. 4.4A shows different types of acid-fast bacteria identified after Ziehl-Neelsen stain. Fig. 4.4 B. shows acid-fast coccobacilli. This may likely be Rhodococcus spp confirmed after sequencing. University of Ghana http://ugspace.ug.edu.gh 40 Fig. 4.4. Acid-fast bacteria identified from the crude bacteria isolate after Ziehl- Neelsen stain A shows acid-fast single cocci whiles B shows acid-fast coccobacilli. 4.2.3 Non-acid fast rods identified on a crude bacterial of a BU patient Non-acid fast rods identified from the crude patients’ samples after Zeihl-Neelsen stain were very few. Unique club shaped rods were identified in two crude bacteria isolates from BU patients as shown in Fig. 4.5. Fig. 4.5. Non-acid fast rods identified from the crude bacteria isolate of a BU patient University of Ghana http://ugspace.ug.edu.gh 41 There are Gram positive rod-shaped bacteria that are also non-acid fast bacteria. These bacteria are usually from the suborder Corynebacterineae. Rhodococcus and Corynebacterium spp can be found in this group of bacteria. This club shaped bacteria shown in Fig. 4.5 above may likely be Corynebacteria which were confirmed after sequencing. Computational results of Zeihl-Neelsen stain shows that majority of acid-fast bacteria identified in all patients’ samples were single cocci (72.5%) representing 37 out of 51. Acid-fast rods were very few (5.9%) representing 3 out of 51 acid-fast bacteria. Coccobacillus (3.9%) representing 2 out of 51 and yeast cells (5.9%) representing 3 out of 51 were also identified as shown in Fig. 4.6. Fig. 4.6. The number and types of acid and non-acid fast bacteria present after Zeihl-Neelsen staining . University of Ghana http://ugspace.ug.edu.gh 42 4.2.4 Identification of Fungal Elements from crude microbial isolate of BU patients Yeast cells were also identified in the patients’ samples after Gram and Zeihl-Neelsen stain. Yeast cells were more prominent in Gram stains of the crude patients’ samples compared to that of Zeihl-Neelsen stain Fig. 4.7. Yeast cells identified from crude patients’ samples of BU patient after Gram and Ziehl-Neelsen stain (A) Yeast cells identified after gram stain whiles (B) Yeast cells identified after acid-fast stain. 4.2.5 Identification of Yeast Cells in 10% KOH Yeast cells were identified from the crude patients’ samples from BU patients when viewed in 10% KOH under the microscope with magnification of x40. Ten percent (10%) KOH was added to the crude microbial sample before and after culturing on Sabouraud dextrose agar. Fig. 4.8 shows yeast cells in the patients’ samples before and after culturing on Sabouraud dextrose agar. University of Ghana http://ugspace.ug.edu.gh 43 Fig. 4.8. Yeast cells viewed under an inverted microscope (x40) (A) Hyphal outgrowth of yeast cells from crude patients’ samples before culturing whiles (B) Yeast cells viewed after culturing on Sabouraud dextrose agar. 4.3 Culturing and Isolation of Bacteria 4.3.1 Bacteria Culture on Luria-Bertani (LB) Agar There were growth of bacteria on the LB agar plate. Fig. 4.9 shows the growth of bacteria on an LB plate after culturing for 24 hours. White and yellow unique colonies were sub cultured and characterized for identification through sequencing. Fig. 4.9. Growth of bacteria colony on LB agar plate from a BU patient University of Ghana http://ugspace.ug.edu.gh 44 4.3.2 Culturing and identification of Staphylococcus on Mannitol Salt Agar The patients’samples were cultured on mannitol salt agar to selectively and differentially identify staphylococcus spp as fermenters and non-fermenters of mannitol. Those that grow as yellow colonies were mannitol fermenters whiles those that form pink colonies were non-fermenters of mannitol. Fig. 4.10 shows the growth of both Staphylococci mannitol fermenters and non-fermenter from the crude patients’ samples of BU patients. Fig. 4.10. Growth of both mannitol fermenters and non-mannitol fermenters of staphylococci spp from crude patients’ samples from the lesions of BU patients (A) Growth of non-fermenters of mannitol (non-pathogenic staphylococci species) whiles (B) Growth of mannitol fermenters (pathogenic staphylococci species). (C) Absence of Staphylococcus. Fig.4.11. shows the distribution of Staphylococci spp from crude patients’ samples of BU patients. Out of the 51 patients, mannitol fermenters (36/51) were the majority representing 76.4% of the patient. 12 out of the 51 (23.5%) patients had no growth of Staphylococci spp. Non-mannitol fermenters were identified in only 2 out of 51 microbial isolate representing (3.9%) of the Staphylococci identified. University of Ghana http://ugspace.ug.edu.gh 45 0 10 20 30 40 50 60 70 80 90 Coagulase positive Coagulase negative No growth P ro p o rt io n o f St ap h yl o co cc i Mannitol Salt Agar Fig. 4.11. Distribution of Staphylococci spp from BU patients 4.3.3 Culturing on MacConkey Agar The patients’ samples were cultured on MacConkey agar to selectively identify gram negative and enteric bacteria and differentiate them into lactose fermenter and non- lactose fermenters. Fig. 4.12. Growth of enteric or gram negative bacteria from a crude patients’ samples of a BU patient on a MacConkey plate University of Ghana http://ugspace.ug.edu.gh 46 Fig 4.13 shows the distribution of Gram negative or enteric bacteria among the BU patients. Gram negative bacteria were detected in 48 crude patients’ samples. However, Lactose fermenters were identified in 47 out of the 51 (92.2%) culture plates, non- lactose fermenters were identified in 20 (39.2%) culture plates whiles 4 (7.8%) plates had no growth. This shows that some BU patients had both Gram negative lactose fermenters and non-lactose on their BU lesions. 0 20 40 60 80 100 120 Lactose fermenters Non-lactose fermenters No growth % o f gr am n eg at iv e b ac te ri a MacConkey agar Fig. 4.13. Distribution of Gram negative or Enteric bacteria from crude isolates among the BU patients Lactose fermenters and non- lactose fermenters were identified in 92.2% and 39.2% of the culture plates respectively whiles 4 (7.8%) plates had no growth. 4.3.4 Culturing on Sabouraud Dextrose Agar for Detection of Fungal Element The patients’ samples were cultured on Sabouraud dextrose agar to identify fungal elements (yeast and molds) present on the lesions of BU patients. Fig 4.14 shows the growth of fungal elements on a Sabouraud dextrose agar plate. Specimen taken from BU University of Ghana http://ugspace.ug.edu.gh 47 patient with ID 46, had no growth of fungal element, patient 47 had growth of yeast and patient 48 had growth of both yeast and few molds. Majority (74.3%) of the fungal elements were yeast cells (39/51). Molds were also identified in 3.9% (2 out of 51) of the crude patients’ samples. There was no growth in 10 patients’ samples (19.6%). Fig. 4.15 shows the distribution of fungal elements identified in the crude patients’ samples after culturing on Sabouraud dextrose agar. Fig. 4.14. Growth of fungal elements from patients’ samples of BU patients on a Sabouraud dextrose agar plate University of Ghana http://ugspace.ug.edu.gh 48 Fig. 4.15. Distribution of fungal elements present on the lesions of BU patients after culturing on Sabouraud Dextrose Agar 4.4. Antibiotic testing of selected bacteria strains after sequencing Bacterial susceptibility of isolates to antimicrobial agents was performed by the disk diffusion method using guidelines established by the Clinical laboratory services. The concentrations used as well as the zones of inhibitions were measured according to the Clinical Laboratory Services. Antibiotic testing was performed on selected bacteria (B. anthracis, B. infantis, B. cereus, B. aquimaris, B. thuringiesis, Staphylococcus. Sp M7, Staphylococcus sp MRSA and Staphylococcus aureus). All the selected bacteria were susceptible to clarithromycin and amikacin. The Bacillus cereus groups bacteria were found to be resistant to rifampicin, and streptomycin whiles kanamycin had the least susceptibility. University of Ghana http://ugspace.ug.edu.gh 49 Fig. 4.16. Zones of inhibition of growth of B. anthracis isolated from the lesion of a BU patient using selected antibiotic University of Ghana http://ugspace.ug.edu.gh 50 Table 4.1. The zones of inhibition (in mm) of 6 different antibiotics on susceptibility testing on selected bacteria Bacteria Clarithromycin (30µg) Kanamycin (30µg) Hygromycin B (30µg) Streptomycin (10µg) Amikacin (30µg) Rifampicin (5µg) B. anthracis 25mm (S) 23mm (I) 20mm (S) 25mm (I) 26mm (S) 27mm (S) Staphylococcus sp M7 23mm (S) 24mm (I) 18mm (S) 24mm (I) 24mm (S) 30mm (S) MRSA 28mm (S) 24mm (I) 18mm (S) 23mm (I) 28mm (S) 30mm (S) B. infantis 18mm (S) 24mm (I) 19mm (S) 22mm (I) 21mm (S) 18mm (R) B. cereus 20mm (S) 35mm (S) 22mm (S) 18mm (R) 30mm (S) 21mm (R) B. aquamaris 28mm (S) 25mm (I) 20mm (S) 18mm (R) 32mm (S) 22mm (R) S. aureus 30mm (S) 26mm (S) 20mm (S) 18mm (R) 28mm (S) 31mm (S) The isolated organisms were B. anthracis, B. infantis, B. cereus, B. aquimaris, B. thuringiesis, Staphylococcus. Sp M7, Staphylococcus sp MRSA and Staphylococcus aureus. The concentrations used as well as the zones of inhibitions measured were according to the Clinical Laboratory Services standards. (R-Resistance, S-Susceptible, I- Intermediary) University of Ghana http://ugspace.ug.edu.gh 51 4.5 Identification of bacterial strains using culture independent method PCR was run in order to detect bacterial strains that were not identified using the culture dependent method. For the purpose of identifying universal bacteria, mycobacteria and M. ulcerans, primers 1_16S, MSHA/PA and IS2404 nested (Pg1 to Pg4) with amplicon sizes 350bp, 600bp and 200bp respectively were used. All PCR reactions were carried out with at least a negative control. All samples (51) were first screened for bacteria spp using the 16S rRNA universal primers. Fig. 4.17 shows 1_16S positivity in all the patients’ samples indicating the presence of bacteria in all the patients’ samples. As expected, 16S positivity for mycobacteria was higher (48/51) compared to that of IS2404 (40/51). The presence of IS2404 discriminated Mycolactone Producing Mycobacteria (MPM) from Non-Tuberculous Mycobacteria (NTM) and hence positivity was reduced. Fig. 4.17. 1_16S (universal bacteria), 16S (Mycobacteria) and IS2404 positivity among people with signs of BU University of Ghana http://ugspace.ug.edu.gh 52 4.5.1. Detection of bacteria strains using 1_16S rRNA (universal primers) 16S rRNA was amplified directly from all DNA of crude patients’ samples as well as the 20 randomly selected bacteria colonies. As was expected, there was 100% detection of amplified DNA in all the samples showing the presence of bacteria DNA in all isolates from the BU patients. As shown in Fig 4.18 Lane 1 and 17 represents 1kb DNA ladder, lane 2-15, 18 and 19 represent PCR product from the BU patients. The size of the amplicon is 350bp. All turned out positive for bacterial infection. PC represents a positive control and NC for negative control. Fig. 4.18. An electrophoretogram showing the PCR product of 16 samples using 1_16S primers (universal bacteria) University of Ghana http://ugspace.ug.edu.gh 53 4.5.2 Identification of mycobacterial strains using 16S rRNA (Sequence specific for mycobacteria All patients’ samples were amplified with primers 16S rRNA specific for mycobacteria detection. A total of 48 were positive. However, amplification of the region and DNA sequencing led to the identification of Corynebacterium spp and Rhodococcus spp. Even though these bacteria are not mycobacteria, they have high G-C content with close phylogenetic relationship to mycobacteria. This may explain why they were amplified as mycobacteria. Fig 4.19 is a representative gel showing the results. Fig. 4.19. An electrophoretogram of PCR products of 10 patients using MSHA/PA as primers for mycobacteria Lane 1: 1kb DNA ladder (molecular weight marker); NC: negative control. Lane 2 to 11: PCR amplicons of 10 patients. The size of the expected amplicon is 600bp. All 10 patients were positive for mycobacteria. University of Ghana http://ugspace.ug.edu.gh 54 4.5.3 Nested PCR Product of IS2404 The patients’ samples were amplified with primers IS2404 specific for M. ulcerans detection. This primer is able to distinguish between Mycolactone Producing Mycobacteria (MPM) and Non Tuberculosis Mycobacteria (NTM). Amplicons were sequenced to identify the type of MPM amplified. Fig. 4.20 shows a representative gel showing positivity for IS2404. Fig. 4.20. An electrophoretogram of nested PCR product of 27 patients using IS2404 (Pg 1 to Pg4) as primers Lane 1 and 17: 1kb DNA ladder; lane 16 and 32: PCR marker (NEB). NC: negative control; lanes 2 to 15 and 18 to 30: patients with signs of BU. University of Ghana http://