COMPARISON OF DECONTAMINATION METHODS FOR ISOLATION OF 
MYCOBACTERIUM SPECIES FROM THE ENVIRONMENT 
 
BY 
 
NAKOBU ZULIEHATU 
(10358661) 
 
 
 
THIS THESIS IS SUMMITED TO THE UNIVERSITY OF GHANA, LEGON IN 
PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF 
MASTER OF PHILOSOPHY (MPHIL) DEGREE IN ZOOLOGY  
 
 
 
MARCH, 2014
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DECLARATION 
I, Zuliehatu Nakobu do hereby declare that except for references to other people‘s work which I 
have acknowledged, this thesis is the product of my own research, and it has not been presented 
in its entirety or part elsewhere for another degree.  
 
 
----------------------------------------------------------------             DATE --------------------------- 
Zuliehatu Nakobu (10358661)  
(Student)  
 
----------------------------------------------------------------                 DATE --------------------------- 
Prof. Dorothy Yeboah-Manu  
Bacteriology Department, NMIMR 
(Supervisor)  
 
-----------------------------------------------------------------                DATE --------------------------- 
Dr. Langbong Bimi  
DABCS, UG 
(Supervisor) 
 
 
 
 
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DEDICATION 
This thesis is dedicated to my late mother Mariama Iddirisu, my beloved husband Labram 
Massawudu Musah, my son Faisal Massawudu and my siblings: Kobshie Nakobu and Hawa 
Nakobu. Thank you for your support and love. 
 
 
 
 
 
 
 
 
 
 
 
 
 
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ACKNOWLEDGEMENT 
First and foremost I thank almighty Allah for showering his mercies upon me. 
I wish to acknowledge the important people who made this research possible. I am indebted to 
my supervisor, Prof. Dorothy Yeboah-Manu. I will like to show my heartfelt appreciation for her 
guidance and patience throughout this project. I thank her, for giving me the opportunity to 
undertake this thesis.  Her continued communications when I had even lost hope; the advice she 
gave me, her unstinting support, encouragement, understanding, tolerance and patience. I could 
not have done this without you. 
I am most grateful to my supervisor Dr. Langbong Bimi for giving me this opportunity and the 
encouragements.  
I will like to thank Head of Department of Animal Biology and Conservation Science and the 
entire department for giving me the opportunity. My appreciation goes to all the members of the 
STOP BU group most especially, Emelia Konadu Danso and Samuel Yaw Aboagye for their 
assistance throughout this work. I will like to thank Mrs. Adwoa Asante-poku Wiredu, Mr. Isaac 
Otchere Darko and Kobina Asan Ampah for their assistance. 
I will like to thank the Head of Department of Bacteriology for motherly assistance and her 
invaluable expertise in microbiology and the entire members of Department of Bacteriology of 
Noguchi Memorial Institute for medical research (NMIMR) for the opportunity and the various 
ways of support each member gave me during this work. 
I will like to thank Dr. Ekow Kaitoo the East Akim municipal health director and Mr. Cornelius 
Tenku of East Akim municipal health directorate, Mr. Lansah Asumah (MHIQ) of Suhum Sub-
municipal Health Directorate and Mr. Ebenezer Benyah of Akuapim South Municipal Health 
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Directorate for their support. I am most grateful to Optimus Foundation (UBS, Switzerland) for 
providing me with financial support. 
I will like to thank my husband, son, dad and siblings for their prayers and support. 
I acknowledge the assistance of Elias Asuming Brepong, Bright Kojo Azumah, Hector Samani, 
Prince Asare, Emmanuel Blay, Dora Obrepongmaa Odei, Christopher Adekah, Dorothea Osei, 
Edem Aku Kwamoa, Stephen Osei-Wusu, Grace S. Kpeli, Sarah Opoku, Victor Dery and all my 
friends who in their own small way helped in making this thesis a success. May God bless you 
all and good things come your way. Amen! 
 
 
 
 
 
 
 
 
 
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ABSTRACT 
The environment harbours many bacterial species, some of which include non-tuberculous 
mycobacteria which have recently become important in public health. Isolation of mycobacteria 
from the environment has not been easy because of the presence of other fast growing bacteria 
and fungi. For isolation of mycobacteria from the environment, decontamination methods that 
minimize contamination but maximize recovery of mycobacteria are needed. This study sought 
to compare three decontamination methods for isolation of mycobacteria from the environment. 
Sixty-five samples were collected from both Buruli ulcer disease endemic and non-endemic 
villages. Polymerase chain reaction (PCR) was done to detect the biomarker IS2404 as a first 
screening procedure. Direct microscopy was performed on the IS2404 positive samples and three 
decontamination methods were evaluated, namely; 4% NaOH/ 5% simplified OA  method, 0.3% 
malachite green/ 0.75 g/50 ml cycloheximide/ 4% NaOH and 3% SDS/ 4% NaOH 
decontamination methods. Three different media with antibiotic supplementation and one 
without antibiotic supplementation were used for isolation of mycobacteria from the 
environment. Thirty-seven out of the 65 samples were positive for IS2404 marker and 
5/37(13.5%) were acid-fast positive. Decontamination by NaOH/OA method gave the highest 
number of total tubes that confirmed mycobacterial growth (42/91, 46.1 %) and the least 
contamination. The medium containing PANTA-mycobactin-J (PM) was best among the four 
media used. Isolates obtained from this study were identified by Hain GenoType CM® line 
probe assay. Forty-four Mycobacterium species were identified and Mycobacterium chelonae 
was the most frequently isolated species. Decontamination with 4% sodium hydroxide/5% oxalic 
acid and L-J medium containing PANTA and mycobactin J (PM) were the most efficient in 
supporting the growth of mycobacteria and may be used as standard for isolating mycobacteria 
from the environment. 
 
 
 
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TABLE   OF CONTENTS 
DECLARATION .............................................................................................................................. i 
DEDICATION ................................................................................................................................ii 
ACKNOWLEDGEMENT ............................................................................................................. iii 
ABSTRACT .................................................................................................................................... v 
TABLE   OF CONTENTS ............................................................................................................. vi 
LIST OF TABLES .......................................................................................................................... x 
LIST OF FIGURES ........................................................................................................................ xi 
LIST OF ABBREVIATIONS .......................................................................................................xii 
CHAPTER ONE .............................................................................................................................. 1 
INTRODUCTION ........................................................................................................................... 1 
1.1 Background to Study ............................................................................................................ 1 
1.2 Problem Statement ................................................................................................................ 5 
1.3 Justification ........................................................................................................................... 6 
1.4 Objective of study ................................................................................................................. 7 
CHAPTER TWO ............................................................................................................................. 9 
LITERATURE REVIEW ................................................................................................................ 9 
2.1 Mycobacterium ..................................................................................................................... 9 
2.1.1  Mycobacterium Cell Wall ............................................................................................... 10 
2.1.2 Classification of Mycobacterium .................................................................................... 12 
2.2 Non-Tuberculous Mycobacteria (NTM) and Disease ........................................................ 14 
2.2.1 Mycobacterium chelonae ................................................................................................ 14 
2.2.2 Mycobacterium avium- intracellulare ............................................................................ 16 
2.2.3 Mycobacterium marinum ...................................................................................................... 18 
2.2.4. Mycobacterium ulcerans ..................................................................................................... 20 
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2.3.1 Epidemiology of Some Non-Tuberculous Mycobacteria ............................................... 22 
2.3.1.1 Mycobacterium avium – intracellulare complex ........................................................ 23 
2.3.1.2 Mycobacterium marinum ............................................................................................ 25 
2.3.1.3 Mycobacterium chelonae ............................................................................................ 26 
2.3.1.4 Mycobacterium ulcerans ................................................................................................... 27 
2.3.2 Laboratory Diagnosis ........................................................................................................... 29 
2.3.3 Treatment .............................................................................................................................. 31 
2.3.4 Isolation of Non-Tuberculous Mycobacteria ........................................................................ 34 
2.3.5 Decontamination Methods .................................................................................................... 36 
CHAPTER THREE ....................................................................................................................... 38 
MATERIALS AND METHODS .................................................................................................. 38 
3.1 Equipments and Reagents ........................................................................................................ 38 
3.1.1 Equipments ........................................................................................................................... 38 
3.1.2 Reagents ............................................................................................................................... 38 
3.1.3 Materials ............................................................................................................................... 39 
3.2 Study Design ........................................................................................................................... 39 
3.3 Study Site ................................................................................................................................. 41 
3.3 Sample Collection ................................................................................................................... 42 
3.4 Direct Detection of IS2404 Containing Mycobacterium from Environmental Samples ......... 44 
3.4.1 DNA extractions by FastDNA spin kit for soil protocol ...................................................... 44 
3.5 Polymerase Chain Reaction (PCR) ......................................................................................... 45 
3.5.1 Precaution to prevent contamination .................................................................................... 45 
3.5.2 Polymerase chain reaction procedure ................................................................................... 45 
3.5.3 Electrophoresis of PCR Product on 2% Agarose Gel .......................................................... 46 
3.5.3.1 Gel Preparation and Electrophoresis ................................................................................. 46 
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3.5.3.2 Visualization of Amplified Product ................................................................................... 46 
3.7 Microscopy .............................................................................................................................. 46 
3.7.1 Sample Processing ................................................................................................................ 46 
3.7.2 Direct smear microscopy ...................................................................................................... 47 
3.8 Isolation of Mycobacteria by Culture ...................................................................................... 48 
3.8.1 Decontamination Procedures ................................................................................................ 48 
3.8.1.1 Sodium hydroxide/Oxalic acid method ............................................................................. 48 
3.8.1.2 Malachite green/Cycloheximide/Sodium hydroxide method ............................................ 49 
3.8.2.3 Sodium dodecyl sulphate (SDS) / Sodium hydroxide (NaOH) ......................................... 49 
3.8.3 Cultivation of Mycobacteria ................................................................................................. 50 
3.8.3.1 Growth medium and Incubation ........................................................................................ 50 
3.8.3.2 Purification and Amplification of Microbial Growth by Sub-Culture .............................. 50 
3.8.4 Species Identification ........................................................................................................... 51 
3.8.4.1 PCR Amplification ............................................................................................................ 51 
3.8.4.2 Hybridization ..................................................................................................................... 52 
3.8.4.2.2 Evaluation and Interpretation ......................................................................................... 53 
CHAPTER FOUR ......................................................................................................................... 55 
RESULTS ...................................................................................................................................... 55 
4.1 Samples .................................................................................................................................... 55 
4.2 Direct Microbiological Analysis ............................................................................................. 55 
4.2.1 Detection of IS2404-positve Mycobacterium species .......................................................... 55 
4.2.2 Direct Smear Microscopy ..................................................................................................... 57 
4.2.3 Isolation of Mycobacterium species ..................................................................................... 57 
4.2.3.1 General Results .................................................................................................................. 57 
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4.2.3.5 Combined Performance of the Decontamination Methods and In-House Formulated 
Media. ............................................................................................................................................ 59 
4.2.3.6 Species Identification ........................................................................................................ 61 
4.2.3.5 The Sample Type and Mycobacterium species isolated .................................................... 63 
4.2.3.6 Organism Isolated and Decontamination Method ............................................................. 65 
4.2.3.7 Mycobacterium Species Identified and Type of Media ..................................................... 67 
CHAPTER FIVE ........................................................................................................................... 70 
DISCUSSION ................................................................................................................................ 70 
CHAPTER SIX ............................................................................................................................. 79 
CONCLUSION ............................................................................................................................. 79 
6.1 Limitation of the Study ....................................................................................................... 79 
6.2 Recommendation ................................................................................................................ 80 
REFERENCES .............................................................................................................................. 81 
APPENDIX A ............................................................................................................................. 102 
APPENDIX B .............................................................................................................................. 105 
APPENDIX C .............................................................................................................................. 106 
APPENDIX D ............................................................................................................................. 107 
APPENDIX E .............................................................................................................................. 108 
APPENDIX F .............................................................................................................................. 109 
APPENDIX G ............................................................................................................................. 110 
APPENDIX H ............................................................................................................................. 111 
APPENDIX I ............................................................................................................................... 112 
APPENDIX J ............................................................................................................................... 113 
APPENDIX K ............................................................................................................................. 114 
 
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LIST OF TABLES 
Table 2. 1: This is illustrated on the Runyon classification of non tuberculous mycobacteria ..... 13 
Table 3. 1: Samples analyzed in the study .................................................................................... 43 
Table 3. 2: The quantitative scale (IUALTD) for grading AFB in Smears ................................... 48 
Table 4. 1: Polymerase chain reaction positivity of respective samples analysed ........................ 56 
Table 4. 2: Acid-fast bacilli positivity of IS2404 positive samples by direct smear analysis ....... 57 
Table 4. 3: Performance of In-house formulated media and the decontamination methods ......... 61 
Table 4. 4: List of identified isolates obtained .............................................................................. 63 
Table 4. 5: Type of sample and Mycobacterium species identified .............................................. 64 
Table 4. 6: Fast growing mycobacteria isolates and sample type ................................................. 64 
Table 4. 7: Slow growing mycobacteria identified and sample type ............................................ 65 
Table 4. 8: Mycobacterium species identified from the three decontamination methods ............. 66 
Table 4. 9: Slow-growing mycobacteria obtained from the decontamination methods ................ 66 
Table 4. 10: Fast-growing mycobacteria obtained from the decontamination methods ............... 67 
Table 4. 11: Mycobacterium species identified and the in- house media used ............................. 68 
Table 4. 12: Effects of in-house selective media on fast-growing mycobacteria .......................... 68 
Table 4. 13: Effects of in-house selective media on slow-growing mycobacteria ........................ 69 
 
 
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LIST OF FIGURES 
Figure 2. 1: The Mycobacterium cell wall ---------------------------------------------------------------- 11 
Figure 2. 2: Mycobacterium chelonae lesion on the right lower limb -------------------------------- 16 
Figure 2. 3: Mycobacterium avuim leision on a patient‘s foot ----------------------------------------- 18 
Figure 2. 4: M. marinum lesion on patient‘s left hand -------------------------------------------------- 20 
Figure 2. 5: An illustration of changes in the skin caused by Buruli ulcer infection --------------- 21 
Figure 2. 6: Different clinical presentations of Buruli ulcer -------------------------------------------- 22 
Figure 2. 7: A world map showing the global distribution of Buruli ulcer worldwide in 2011 --- 29 
Figure 2. 8: Ziehl- Neelsen (ZN) stained smears of M. ulcerans observed under oil immersion 
x100 ------------------------------------------------------------------------------------------------------------ 30 
Figure 3. 1: Flowchart of the Study Design .................................................................................. 40 
Figure 3. 2: Map showing the district of the villages selected for the study ................................. 42 
Figure 3. 3: Evaluation chart for identifying Common Mycobacterial species ............................ 54 
Figure 4. 1: Gel electrophoresis analyses of some environmental samples .................................. 56 
Figure 4. 2: Culture tubes with AFB positive isolates: note the different colonial morphologies 58 
Figure 4. 3: Line probe hybridization analysis of isolates used for identification ........................ 62 
 
 
 
 
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LIST OF ABBREVIATIONS 
AFB - Acid- fast bacilli 
AIDS - Acquired immune deficiency syndrome or acquired immunodeficiency syndrome 
ATM -  Atypical mycobacteria  
ATS - American Thoracic Society  
BU - Buruli ulcer 
CDC  - Centers for Disease Control  
CPC - Cetylpyridinium chloride  
DNA - Deoxyribonucleic acid 
DNTPs -          Deoxynucleoside triphosphates 
EB - Ethambutol 
HIV - Human Immunodeficiency Virus 
H2SO4 - Sulfuric acid   
INH - Isoniazid 
IUALTD-       International Union against Tuberculosis and Lung Diseases 
LAM - Lipoarabinomannan 
L-J - Löwenstein-Jensen 
MAC - Mycobacterium avium complex  
MAI - Mycobacterium avium -intracellulare  
MTC - Mycobacterium tuberculosis complex 
MOTT - Mycobacteria other than tuberculolosis  
MU - Mycobacterium ulcerans 
NALC - N-acetyl cysteine  
NaOH - Sodium hydroxide   
NMIMR-        Noguchi Memorial Institute for Medical Research 
NTM - Non-tuberculous mycobacteria 
OA - Oxalic acid  
PANTA - Polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin 
PBS     -          phosphate buffered saline 
PCR - Polymerase chain reaction 
PIMs -  Phosphatidylinositol mannosides 
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PM - PANTA-Mycobactin J 
SDS - Sodium dodecyl sulphate 
SLS - Sodium lauryl sulphate 
TB -  Tuberculolosis 
WHO - World health organization 
ZN - Ziehl- Neelsen 
 
 
 
 
 
 
 
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CHAPTER ONE 
INTRODUCTION 
1.1 Background to Study 
The genus Mycobacterium belongs to the phylum of Actinobacteria and the family 
Mycobacteriaceae. The genus consists of more than 140 species (American Thoracic Society, 
2007; Slany et al., 2010). This genus is noted for the rigid and thick cell wall that allows them to 
resist many chemical solutions including acids and alcohol. Mycobacteria adapt easily on simple 
substrates and are ubiquitous (Falkinham, 2002).  They can be found in many natural and 
artificial environments; these include soil, rivers, treated water in distribution systems, biofilms, 
aerosols, equipment; bronchoscopes, catheters and food (Rahbar et al., 2010; Falkinham, 2002). 
Their distribution is also influenced by biotic factors such as soil type and local vegetation 
(Chilima et al., 2006; Rahbar et al., 2010). 
The genus includes pathogens known to cause serious diseases such as tuberculosis 
(Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The other members of the 
genus other than Mycobacterium tuberculosis (Mtb) and Mycobacterium leprae are referred to as 
non tuberculous mycobacteria (NTM), Mycobacterium species other than Mycobacterium 
tuberculosis (MOTT) or atypical Mycobacterium (ATM). Infections due to NTMs are 
increasingly becoming more of a public health problem. These NTMs are not so pathogenic, and 
because they are found ubiquitously in the environment than causing disease, they are also 
referred to as environmental pathogens. Non-tuberculous mycobacteria that were previously 
known to be non pathogenic have now been shown to cause infections. Some however can cause 
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serious infections in both immunocompetent (Mycobacterium ulcerans) and 
immunocompromised (Mycobacterium avium) persons. 
Disease such as Buruli ulcer which is an ulcerative skin disease mainly affects the skin and the 
subcutaneous tissue. Lymphadenitis which mostly affects lymph nodes in children is caused by 
M. avium – intracellulare complex (known as the MAI). From the stand point of human health, 
the most significant of the environmental Mycobacteria include MAI and M. ulcerans. 
Non-tuberculous mycobacteria have become a significant cause of infection with the emergence 
of HIV/AIDS. Mycobacterium avium – intracellulare is the predominant cause of disseminated 
mycobacteremia in about 25% to 50% of patients with HIV/AIDS in the United States and in 
Europe (Falkinham, 1996). In addition, Mycobacterium species that are members of the MAI 
also cause majority of NTM infections in developing countries (von Reyn et al., 1993).  
The environment may be the likely reservoir for these infections as there is no evidence of 
human to human transmission and the environment where NTMs occupy are shared by humans 
(Wolinsky et al., 1979; Griffith et al., 2007). Studies have implicated that Mycobacterium 
species such as Mycobacterium ulcerans could be transmitted from aquatic environments to 
humans.  Insects, aquatic plants, amoebae, and aquatic vertebrates and invertebrates have been 
suggested by various studies to be reservoirs of NTMs in natural environments (Heckert et al., 
2001; Marion et al., 2010). However, most of the studies on M. ulcerans ecology have been 
conducted through PCR based methods for detection. Different modes of transmission have been 
hypothesized for diseases caused by NTMs by different studies; none of them have been proven 
up to now (Griffith et al., 2007).  For instance, several different mechanisms have been proposed 
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for the transmission of M. ulcerans. These include contact with contaminated environment, 
aerosol resulting from vapourisation of contaminated water and insect bite (Marion et al., 2010). 
Therefore, there is the need to link data from isolates obtained from culture, genetic 
characterization and epidemiological studies to allow more inference on the actual sources and 
mode of transmission of these mycobacteria. 
Bacteria culture is considered the gold standard for the detection of Mycobacterium species as it 
proves viability of the bacteria in the sample. However, obtaining pure culture of mycobacterial 
species is a very difficult procedure due to several reasons. The environment contains other 
microorganisms other than mycobacteria such as fast growing bacteria and fungi. While other 
bacteria grow very fast such that within 18 hours macroscopic growth can be achieved, the fast 
growing mycobacteria grows within seven days, it can take the slow growers even more than six 
months to grow. Thus the cultures set for isolating Mycobacterium species from the environment 
is usually contaminated by these fast growing bacterium and fungi. Moreover, Mycobacterium 
which is grouped into fast and slow growers and the slow growing Mycobacterium species are 
the most pathogenic. 
The main factor that determines the growth rate is the number of rRNA operons; the slow 
growing mycobacteria possess one operon and rapid growers possess two while the other fast 
growing bacteria such as Escherichia coli have seven operons (Falkinham, 2008; Condon et al., 
1995). To arrest contamination of cultures, basic and or acidic reagents are introduced to remove 
all non-mycobacterial species from the samples and to improve the recovery of mycobacteria 
from both clinical and environmental samples, in a decontamination step during samples 
processing for culture. 
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Several decontamination solutions such as, cetylpyridinium chloride (CPC), sodium hydroxide 
(NaOH) (also known as Petroff's method), N-acetyl cysteine (NALC), sulfuric acid (H2SO4) 
oxalic acid (OA), centrimide, sodium dodecyl sulphate (SDS) and sodium lauryl sulphate (SLS) 
have been used for decontamination, for removing these fast growing bacteria from samples for 
mycobacteria culture before inoculation. Parashar et al., 2004 reported Mycobacterium species 
are not equally resistant to the different decontamination methods. Other studies have also shown 
that decontamination methods are known to be detrimental to mycobacteria, depending on 
concentration and length of treatment (Brooks et al., 1984; Jaramillo and McCarthy 1986). In a 
study, two decontamination methods and five media were compared for the isolation of 
mycobacteria from brook waters of different physical, chemical and bacteriological 
characteristics. Sodium hydroxide in combination with oxalic acid and sulfuric acid-
cycloheximide methods were used. The sodium hydroxide in combination with oxalic acid 
method was found to be better than the sulfuric acid - cycloheximide method (Livanainen et al. 
1997). In another study where pure cultures were tested, mycobacteria tolerated sulphuric acid 
better than sodium hydroxide (Jaramillo and McCarthy 1986). Palomino and Portaels in 1998 
used the BACTEC system to evaluate the effects of several decontamination methods (Petroff, 
reversed Petroff, oxalic acid, and mild hydrochloric acid) treatments and antibiotics on the 
viability of Mycobacterium ulcerans. From their results they concluded that, the decontamination 
methods used for isolation of M. ulcerans affected the viability of the bacteria and mild 
hydrochloric acid gave the best results.  
To further reduce contamination in cultures, antibiotics (example, PANTA is a cocktail of 
antibiotics containing polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and 
azlocillin), anti-fungal example cycloheximide and other chemicals (such as malachite green in 
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L-J) are added to culture media for isolating mycobacteria. While mycobacteria are resistant to 
the antimicrobials and decontaminating solutions, there seems to be variability among them in 
the level of resistance. 
It is therefore important to evaluate and standardize decontamination methods and selective 
media that will maximize the recovery of mycobacteria species of interest and at the same time 
reduce contamination usually found in mycobacteria cultures. This will enable further studies on 
the isolates from the environmental and the comparison of environmental and clinical isolates 
which will lead to knowing their mode of transmission and ecology for disease control 
 
1.2 Problem Statement 
Although Mycobacterium tuberculosis complex (MTC) are responsible for most mycobacterial 
infections worldwide, other infections due to NTMs are also increasingly becoming more of 
public health importance (Kankya et al., 2011). This is due to the increase in the number of 
immunocompromised individuals such as people living with HIV/AIDS, recipients of organ 
transplants (Zumla and Grange, 2002; Mok et al., 2007). Some of the NTMs such as M. xenopi, 
has been increasingly been identified as a cause of pulmonary infections among those with 
impaired immunity (Mangione et al., 2001; van Ingen et al., 2008; American Thoracic Society 
ATS, 1997; Zumla and Grange,2002; Ostroff et al., 1993; O‘Brien et al., 1987). In addition 
human environmental degradation activities have been shown to be a risk for emergence of some 
disease caused by NTMs, a typical example is Buruli ulcer (BU). Moreover NTMs infections are 
emerge unrecognized settings with new clinical manifestations (Griffith et al., 2007).  
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While NTMs have been recognized in recent times as important pathogen, large gaps still exist in 
our knowledge of the ecology and mode of transmission or how humans acquire infections. Thus 
more studies are needed in different geographic settings to understand the ecology and ultimately 
how humans acquire infection. Human disease is suspected to be acquired from the 
environmental exposures, although the specific source of infection cannot be identified (von 
Reyn et al., 2002).  Thus cultured mycobacteria are needed for molecular epidemiology analysis, 
since environmental specimens do not always contain sufficient bacilli to perform direct finger 
printing analysis. More importantly cultures prove the viability of the bacteria in the source.  
However, isolating mycobacteria from the environment is particularly very difficult for several 
reasons (1) environmental samples are contaminated by other microorganisms causing over 
growth in the culture (2) the most pathogenic NTMs have very slow growth rate. Thus removing 
the unwanted microbes from environmental samples before isolation culture is paramount. 
 
1.3 Justification 
Mycobacteria species that were previously regarded largely as saprophyte, non- pathogen and 
environmental are increasingly becoming important human pathogens, not only in immune-
compromised individuals but can affect immune-competent individuals. Globally, the picture of 
NTMs has changed drastically, affects also human and animal population in Africa. A study 
conducted in Uganda identified NTM from humans suffering from cervical lymphadenitis and 
cattle with lesion consistent with bovine TB (Kankya et al., 2011). Also a study by Asante-Poku 
et al in Ghana (personal communication) identified NTMs from lesions of carcass from cattle 
with macroscopic appearance of bovine TB. Thus in addition to the main mycobacterial diseases 
(TB and leprosy) efforts must be put in controlling disease caused by the NTMs. This 
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identification of risk factors for disease spread is crucial which requires a good understanding of 
the ecology of the causative agent.  
Moreover an important aspect in the control of infectious disease is identification of the risk 
factors that increase the chance that host will come into contact with the pathogen. Thus the 
ability to isolate viable bacteria, indicating reservoir makes it possible to identify preventable 
risk and implement public health measures. Currently there is very limited data on the 
environmental sources of NTMs in Ghana with few PCR-based studies looking at M. ulcerans. 
Thus the isolates that will be obtained from the study will be linked in future molecular 
epidemiological study involving both clinical and environmental study to allow inference about 
the real sources of NTM infection in the district studied. 
 
1.4 Objective of study 
The purpose of the study is to compare different decontamination methods for the isolation of 
Mycobacterium species from the environment.  
Specific objectives 
The specific objectives of this study are; 
I. To directly detect acid-fast bacilli from environmental samples. 
II. To detect IS2404 positive mycobacteria in environmental samples. 
III. To evaluate different in house selective media for isolating mycobacteria. 
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IV. To evaluate different methods for decontaminating environmental samples for isolating 
mycobacteria. 
V. To identify to the species level mycobacteria isolates from the environment. 
 
 
 
 
 
 
 
 
 
 
 
 
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CHAPTER TWO 
LITERATURE REVIEW 
2.1 Mycobacterium 
The genus Mycobacterium is a member of the phylum Actinobacteria, in the order 
Actinomycetales, with its own family Mycobacteriaceae (Ventura et al., 2007).  The genus 
comprise of pathogens known to cause important human diseases, notably; tuberculosis (M. 
tuberculosis), leprosy (M. leprae) and Buruli ulcer (M. ulcerans). The DNA of species of the 
genus Mycobacterium usually has a high guanine and cytosine (G+C) content in the range of 61 
to 71 % (except Mycobacterium leprae with G+C content of 54 to 57 %). 
Mycobacteria are generally aerobic, non-motile rods about 1-10 µm long, non-spore forming and 
they are able to enter into dormant states. Mycobacteria are usually considered gram positive, not 
necessarily as a result of gram reaction but the absence of an outer lipid membrane. The 
mycobacterial cell wall has unique characteristics which make them hydrophobic and retain dyes 
after acid or alcohol decolourization, this is known as acid fastness. The nature of the cell wall 
also makes them resistant to many hydrophilic compounds, disinfectants and common 
antibiotics. Optimum growth temperatures vary widely according to the species and range from 
25 °C to over 50 °C; thus some species can survive in hot water heaters and hot water pipes at 
temperatures of 50 to 55°C (Schulze-Röbbecke and Bucholtz 1992; Santos et al., 2007; 
Falkinham, 2009). 
 
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2.1.1  Mycobacterium Cell Wall 
Mycobacterium cell wall is characteristically thicker than the cell wall of many other bacteria. It 
is made up of two segments denoted upper (outermost) and lower (cell wall core). Directly 
attached to the membrane is the peptidoglycan, covalently attached to an arabinogalactan layer 
which is then linked to a thick layer of mycolic acids. These three layers make the cell wall core, 
known as Mycolyl arabinogalactan–peptidoglycan. The upper segment composed of free lipids 
interspersed such that the cell wall process lipoarabinomannan and other lipids. The components 
give the cell wall a hydrophobic nature wich contributes substantially to the hardiness of the 
genus, their resistance to common disinfectants and high intrinsic resistance of mycobacteria to 
many drugs (Hoffmann et al., 2008). 
The plasma membrane is the first layer from the inner side which also may contain substances 
such as carotenoids; which produce yellow to orange pigmentation in some non-tuberculous 
mycobacteria example, M. gordonae and M. kansasii (Brennan and Nikaido  1995 and Rastogi, 
1991).When the cell wall is disrupted, for instance extracted with various solvents, the free 
lipids, proteins, LAM, and PIMs are solubilized, and the mycolic acid–arabinogalactan 
peptidoglycan complex remains as the insoluble residue.  
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Figure 2. 1: The Mycobacterium cell wall 
Source:http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab12/diseases/mtuberculosis/images 
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2.1.2 Classification of Mycobacterium 
Mycobacteria can be classified into several groups by different criteria such as growth rates, 
pigmentation and pathogenicity. Medically mycobacteria are classified as tuberculous and non 
tuberculous mycobacteria (NTM). The NTMs are also referred to as mycobacteria other than 
tuberculolosis (MOTT) and atypical mycobacteria (ATM). The tuberculous mycobacteria 
include members of the M. tuberculosis complex and M. leprae (van Ingen, 2013). The NTMs 
were grouped into 4 broad groups by the Runyon classification in 1959. This classification was 
based on growth rates, colony morphology and pigment production. Some mycobacteria produce 
pigments without light while others require photo activation for pigment production. 
Mycobacteria that produce yellow – orange pigment after being exposed to light are 
photochromogens. Those that produce a pigment without exposure to light are also known as 
scotochromogens. Those that do not produce pigment are termed as non chromogens. 
The rate of growth of mycobacteria that is time to produce visible growth on standard solid 
media is also used to classify them; those that produce visible colonies within seven days are 
known as rapid growers and those that produce macroscopic growth after 7 days are termed slow 
growers. 
According to the Runyon classification, Groups I, II and III are slow-growing NTM and group 
IV are rapid growers.  Group I organisms are the photochromogens, group II organisms are the 
scotochromogens, the group III are the non-chromogenic (Butler and Guthertz, 2001; 
Jarzembowski and Young, 2008) (This is illustrated on Table 2.1 below). This classification 
system however could not define species within the Mycobacterium genus.  Although the 
Runyon classification system is out-dated, it provided laboratories with guidelines to identify 
individual species of NTM, resulting in better characterization of distinct diseases.  
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With advancement in molecular biology and availability of the genomes of most Mycobacterial 
species, specific probes or biomarkers have been identified and are now being used in 
mycobacteria laboratories for species identification. Yet these classification and specific 
biochemical assays are still in use for some mycobacteria species that research has still not 
advanced in them. 
 
Table 2. 1: This is illustrated on the Runyon classification of non tuberculous mycobacteria 
Runyon 
Class 
Description Growth Pigment production Examples 
I Photochromogens Slow growing Yellow-orange pigment 
production when 
exposed to light 
M. Kansasii, M. 
marinum 
II Scorochromogens Slow growing Yellow-orange pigment 
production with or 
without light 
M. scrofulaceum, M. 
gordonae, M. szulgai 
III Non-chromogens Slow growing None M. avium-intracellulare, 
M. xenopi, M. terrae, M. 
ulcerans 
IV Rapid growth Rapid growth 
(produces 
mature colonies 
in agar ≤ 7 days) 
Some do not produce 
pigment and others 
produce late 
pigmentation  
M. fortuitm, M. 
pereginum, M. 
abscessus, M. chelonae 
 (Source: Jarzembowski and Young, 2008). 
 
 
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2.2 Non-Tuberculous Mycobacteria (NTM) and Disease 
The NTMs cause various disease resembling tuberculosis, lymphadenitis, skin disease, or 
disseminated disease as described by O‘Brien et al., (1987). Pulmonary disease is most frequent 
followed by lymphadenitis in children, skin disease and disseminated infections in severely 
immunocompromised patients (Wolinsky, 1979). They also cause different diseases including 
nosocomial infections associated with outbreaks related to insufficient sterilization and 
disinfection of medical device. About 50 species of NTMs have been reported to be human 
pathogens (Wagner and Young, 2004) and some examples are described below; 
2.2.1 Mycobacterium chelonae 
Mycobacterium chelonae is a rapidly growing and ubiquitous NTM, classified as Runyon group 
IV organism. Mycobacterium chelonae has been found in natural and artificial sources including 
soil, medical instruments, foot baths in clinics and beauty salons, dust, sewage and water 
especially in tap water and water tanks (Levine et al., 1991; Larson et al., 2008; Sniezak et al., 
2003; Khan et al, 2005; Santos et al, 2005; Hay, 2009). 
Infections caused by M. chelonae clinically manifested as skin, bone and soft tissue disease 
which cause several different types of clinical syndromes. These include: lung disease, local 
cutaneous inflammations, osteomyelitis, joint infections, and ocular disease such as; keratitis or 
corneal ulcers. Mycobacterium chelonae is also involved in several different types of 
community-acquired infections (Brown-Elliott et al., 2002). It has also been implicated in eye 
infection cases associated with ophthalmologic procedures (Liu et al., 2007) and cosmetic 
surgeries and tattoos (Saha et al., 2006, Rajini et al., 2007; Munayco et al., 2008; Kennedy et al., 
2012).  
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Mycobacterium chelonae  rarely cause chronic lung disease, this was demonstrated in  a study by 
Griffith et al., 1993, which involved 154 patients with chronic lung disease due to rapidly 
growing mycobacteria (RGM), only 1 out of 146 isolates was identified to species level as M. 
chelonae. Diseases caused by M. chelonae are grouped into three basic types. The most common 
type is disseminated cutaneous disease which usually occurs in patients who are chronically 
immunocompromised such as AIDS patients (Azadian et al., 1981; Hassan et al., 2007). In 1992, 
a research carried out by Wallace et al reported that 53% of 100 clinical isolates of M. chelonae 
were from patients with disseminated cutaneous infections. Furthermore, these infections were 
seen in patients receiving long-term corticosteroids and/or chemotherapy, primarily because of 
underlying organ transplantation, rheumatoid arthritis, or other autoimmune disorders (Wallace 
et al., 1993).  
The second type of infection is acquired localized infections. These infections range from 
localized cellulitis, or abscess, to osteomyelitis. They are Health care-associated diseases, 
sporadic localized wound infections following medical or surgical procedures. They have been 
observed only in injection with contaminated syringes or needles, the implantation of 
contaminated porcine heart valves and the use of liposuction (Metcalf et al., 1981; Wallace et al., 
1999; Brown-Elliott et al., 2002).  
The third, and least common, type of infection caused by M. chelonae is the most common type 
of health care-associated disease, and is that of catheter-related infections. In 1992, Wallace and 
others reported that 8 out of 100 clinical isolates of M. chelonae were associated with 
intravenous catheters, an additional 3 involved chronic peritoneal dialysis catheters, and 1 
involved a haemodialysis shunt. They observed that both the use of corticosteroids and renal 
failure were risk factors for these catheter-related infections (Wallace et al., 1992). 
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Mycobacterium chelonae has been identified as the cause of approximately 10% of nosocomial 
outbreaks attributed to rapidly growing mycobacteria (Wallace et al., 1992). Figure 2.6 is an 
infection cause by M. chelonae. 
 
 
Figure 2. 2: Mycobacterium chelonae lesion on the right lower limb  
(Source: Ivan et al., 2008)  
2.2.2 Mycobacterium avium- intracellulare 
Mycobacterium avium and Mycobacterium intracellulare (MAI) are genetically closely related 
mycobacterial species referred together as M. avium complex (MAC) or M. avium- intracellulare 
complex (MAI) (Tortoli et al., 2004; Murcia et al., 2006; Guirado et al., 2012). Mycobacterium 
avium - intracellulare are slow growing bacilli that produce a yellow pigment in the absence of 
light (Inderlied et al., 1993 and Han et al., 2005). Mycobacterium avium- intracellulare are 
ubiquitous in nature and they are found in different environmental sources such as natural 
sources of water-salt and fresh water, pools, plants and bedding material, and dust and vegetation 
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(Ichiyama et al., 1988). Mycobacterium avium- intracellulare have also been known as 
opportunistic pathogens of humans and are the most frequently isolated NTM worldwide capable 
of causing disease in both humans and animals (Turenne et al., 2007; Thoen et al., 1981; Iseman 
et al., 1985). They cause pulmonary disease, mostly in patients with pre-existent pulmonary 
diseases, followed by lymphadenitis in immunocompetent children and disseminated disease in 
systemically immunocompromised patients (Griffith et al., 2007). 
The first case of human disease due to M. avium was reported in 1943 in a middle-aged 
underground miner from the Mesabi Iron Range of Minnesota in what became a classic 
description of pulmonary disease due to this organism (Feldman et al., 1943). Pulmonary disease 
due to M. avium predominantly involves white males 45 to 65 years of age with pre-existing 
pulmonary disease (Engbaek et al., 1981; Etzkorn et al., 1986; Rosenzweig and Schlueter, 1981) 
but there has been tremendous variation in the sex, age, and race of these patients. Predisposing 
conditions such as chronic obstructive pulmonary disease, bronchiectasis, chronic aspiration or 
recurrent pneumonia, inactive or active tuberculosis, pneumoconiosis, and bronchogenic 
carcinoma are present in 54% to 77% of patients with pulmonary MAI disease (Engbaek et al., 
1981). Differentiation of infection from the coexistent pulmonary disease may be difficult, and 
the clinical and radiographic presentation may be indistinguishable from tuberculosis (Ortbals 
and Marr, 1978). A positive tuberculin skin test may be helpful in differentiating the two 
processes; however, co-infection of M. tuberculosis and M. avium has been demonstrated 
(Tsukamura et al., 1981). Mycobacterium avium causes 95% of AIDS related MAI infections 
while M. intracellulare causes 40% of MAI infections in the immunocompetent patients 
(Koirala, 2010). Other reported MAI infections among patients with AIDS include mastitis, 
pyomyositis, cutaneous abscess and brain abscess. The symptoms vary and are nonspecific, 
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commonly including chronic productive cough, dyspnea, sweats, malaise, fatigue, and, less 
commonly, hemoptysis. Fever and weight loss are not common but may occur. Figure 2.3 shows 
M. avuim leision on a patient‘s foot. 
 
Figure 2. 3: Mycobacterium avuim leision on a patient’s foot 
Source: http://www.theaidsreader.com/binary_content_servlet The AIDS Reader. Vol. 18 No. 10  
2.2.3 Mycobacterium marinum 
Mycobacterium marinum is a slow-growing environmental Mycobacterium classified as group 
one by the Runyon classification. It is a photochromogenic and saprophytic mycobacteria that 
cause soft tissue infection in humans, usually acquired by inoculation with the bacterium through 
broken skin or by scratches or puncture wounds from fish, shrimp, and fins in an aquatic 
environment (Falkinham, 1996; Lewis et al., 2003), usually during swimming and in individuals 
employed in the fisheries industry (Zeligman, 1972).  It most often affects elbows, knees, feet, 
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knuckles or fingers (Figure 2.4) (Adams et al., 1970; Barrow and Hewitt 1971; Ries et al., 1990) 
Mycobacterium marinum infect not only humans can infect fish and amphibians worldwide 
(Clark et al., 1963; Laussucq et al., 1988).  Infections in humans result occasionally, in most 
cases as a granulomatous infection localized in the skin. Infection in immunosuppressed 
individuals can lead to chronic disease as the pathogen invades and colonise internal organs.  
In fish, the usual course of the disease is the appearance of weak and emaciated fish that 
eventually die over a long period (Connolly et al., 1985). Although sudden mass deaths can 
sometimes occur after new fish have been purchased, especially in aquaria (Bercovier and 
Vincent, 2001). In amphibians, example, frogs and toads, M. marinum infection can be localised 
as an ulcerative dermatitis and cellulitis (Done et al., 1993; Bercovier and Vincent, 2001) or can 
be invasive and fatal with the presence of disseminated granulomata (Alavi and Affronti, 1994). 
 
 
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Figure 2. 4: M. marinum lesion on patient’s left hand 
Source: http://dermnetnz.org/common/image.php?path=/bacterial/img/atyp-mb2.jpg 
 http://www.dermnetnz.org/bacterial/atypical-mycobacteria.html 
2.2.4. Mycobacterium ulcerans 
Mycobacterium ulcerans (MU) is an extremely slow growing Mycobacterium which causes 
mainly infections of soft tissues of the skin. It is an extracellular pathogen and the only 
mycobacteria that harbours plasmid called pMUM001 with a molecular size of 174-kb. Over half 
of the plasmid consists of genes that encode the enzymes required for synthesis of mycolactone, 
the cytotoxic lipid produced by M. ulcerans (Stinear et al., 2004). It is the only mycobacteria 
species that uses a toxin as the main virulent factor, which play an important role in the 
pathogenesis of the disease. In vivo studies, using a guinea pig model of infection suggested that 
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mycolactone is responsible for both the extensive tissue damage and immunosuppression which 
accompanies Buruli ulcer. A characteristic of an active lesion is the absence of inflammatory 
cells which is caused by cytotoxic activity of mycolactone (George et al., 1998). The cytoxicity 
of mycolactone has been linked to the activation of apoptosis; this was observed when several 
cell types were exposed in vitro to purified mycolactone. 
 
 
Figure 2. 5: An illustration of changes in the skin caused by Buruli ulcer infection 
(Van der Werf et al., 1999). 
 
There are theories on the mode of entry of M. ulcerans into the human body; the actual mode of 
transmission into the skin is not known (Duker et al., 2006). After a successful entry of the 
organism it confines under the skin. The incubation period may be 2-3 months, during which 
proliferation takes place within the dermis and produce mycolactone which then destroys the 
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adipocytes and cause necrosis that extends beyond the localized region to the lower dermis and 
subcutaneous fat (Guarner, 2003). 
In Australia, the presentation of Buruli ulcer disease differs from that in Africa. The first stage of 
M. ulcerans infection is a papule or pimple in the skin, while in African patients it usually starts 
as a firm nodule. Other early forms include plaque and the more serious form an oedema (figure 
2.3). When these early forms are not treated, extensive necrosis leads to a well demarcated ulcers 
with extensive undermined edges that often extend 15 cm or more which sometimes affects the 
underlying bones (figure 2.7). 
The extensive necrosis of the dermis result into sloughing off of the skin covering the pre-
ulcerative region (George et al., 1999). Ulcers may remain small and heal without treatment, or 
may spread rapidly, undermining the skin over large areas, even an entire leg, thigh, or arm. 
Important structures such as the eye, breast, or genitalia are sometimes severely damaged. Most 
lesions heal spontaneously but without appropriate therapy frequently leave extensive scarring, 
and deformity.  
 
 Nodule  Plaque   Oedema   Ulcer 
Figure 2. 6: Different clinical presentations of Buruli ulcer 
(WHO, 2012) 
2.3.1 Epidemiology of some Non-Tuberculous Mycobacteria 
Non-tuberculous mycobacteria are important human pathogens, yet little is known about their 
disease epidemiology and the true ecology of the pathogens. Non-tuberculous mycobacteria 
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cause infections of varying severity in both sporadic and epidemic form (Cassidy et al., 2009). 
There are numerous previously identified species of NTM the recent advancement in molecular 
methods more are being identified (Tortoli, 2003). Non-tuberculous mycobacteria diseases have 
been seen in most industrialized countries and the incidence rates vary from 1.0 to 1.8 cases per 
100,000 persons (Horsburgh et al., 1996; Griffith et al., 2007). The incidence of NTMs in the 
United States was 14.1 per 100,000 in 2003 (Marras et al., 2007; von Reyn et al,. 1993; 
Horsburgh et al., 1996; Falkinham, 2002). There have been several reports suggesting that the 
incidence of NTM diseases has increased over the past years. However, there has not been 
conclusively established due to the lack of comprehensive surveillance data. Moreover they are 
non-communicable, that is they are not suspected to be transmitted by person to person contact 
and therefore has not received much attention. Therefore there are no substantially more or better 
information about NTM disease epidemiology than that which was published in the 1997 by 
American thoracic society (ATS) statement on NTM. Therefore the epidemiology of some of the 
species is not known. Although there are variation in species isolation (Griffith et al., 2007), the 
NTM most frequently isolated and associated with disease are the Mycobacterium avium and 
Mycobacterium intracellulare (MAI). Other important human pathogens include Mycobacterium 
abscessus, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium marinum, 
Mycobacterium kansasii, Mycobacterium gordonae, Mycobacterium malmoense, Mycobacterium 
xenopi, Mycobacterium scrofulaceum and Mycobacterium ulcerans (Griffith et al., 2007). Below 
is epidemiology of some species.  
2.3.1.1 Mycobacterium avium – intracellulare complex 
Mycobacterium avium and M. intracellulare (MAI) are the most common and significant NTM 
species that are associated with human diseases, causing disseminated infection in patients with 
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AIDS, nodular bronchiectasis and other pulmonary infections, lymphadenitis, and skin infection 
(Inderlied et al., 1993; Han et al., 2005). 
The actual source of infection is unknown; however water and soil have been implicated as 
possible environmental source of MAI.  Thus, they have been found in different environmental 
sources (Brooks et al., 1984; Falkinham et al., 1980). Mycobacterium avium and M. 
intracellulare may be normal inhabitants of natural waters and drinking water, water droplets 
and soil (Falkinham et al., 1980; 2001; Brooks et al., 1984; Wendt et al., 1980; von Reyn et al., 
1994). In a study where DNA fingerprinting method was used, it was shown that isolates of M. 
avium obtained from AIDS patients were the same for those obtained from water consumed by 
the patients (von Reyn et al., 1994). This suggests a possible link between disease occurrence 
and drinking water source. Mycobacterium avium - intracellulare are responsible for pulmonary 
disease similar to tuberculosis in elderly patients (Wolinsky, 1979; Falkinham, 1996)  and in 
immunocompetent individuals with predisposing lung diseases such as silicosis and black lung 
(Wolinsky, 1979), cervical lymphadenitis in children (Wolinsky, 1995) and disseminated 
infection in AIDS and immunosuppressed patients (Zakowski et al., 1982; Kiehn et al., 1985). 
Thus, they have been found in different environmental sources (Brooks et al., 1984; Falkinham 
et al., 1980). Mycobacterium avium - intracellulare are normal inhabitants of natural waters and 
drinking water, water droplets, soil (Falkinham et al., 1980; 2001; Brooks et al., 1984; Wendt et 
al., 1980; von Reyn et al., 1994). 
Mycobacterium avium - intracellulare reportedly occurs worldwide but have been predominant 
in certain Northern temperate geographic areas, including the United States (Good and Snider, 
1982), Canada (Gill et al., 1987), Great Britain (Hunter et al., 1981), Europe (Debrunner et al., 
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1992), The Netherlands (Engbaek et al., 1981) and Japan (Miyachi et al., 1988). The disease also 
occurs in Australia (De Lalla et al., 1992) and South Africa (Nel, 1981). This may not be the true 
reflectionof endemicity but depicts countries with good laboratories to diagnose them. The 
incidence of laboratory isolation of MAI in the United States, based on a 1979 survey of 44 state 
public health laboratories, is estimated to be 3.2 cases per 100,000 population and was greatest 
for Hawaii (10.8 cases), Connecticut (8.9 cases), Florida (8.4 cases), Kansas (6.8 cases), North 
Carolina, Maryland, Rhode Island, and Arizona (Good and Snider, 1982) .  
In the United States, 40 to 50% of the clinical MAI infections in non-AIDS patients are caused 
by M. intracellulare, whereas in western Germany, 81% of the human infections are due to M. 
avium and only 19% are due to M. intracellulare (Meissner and Anz, 1977). In addition, serovar 
analyses suggest a shift in the proportion of human disease caused by M. avium relative to that 
caused by M. intracellulare in certain geographic areas (Miyachi et al., 1988). 
2.3.1.2 Mycobacterium marinum 
Mycobacterium marinum was first isolated from dead fish in a Philadelphia aquarium in 1926 by 
Arsonson and was first recognized to cause human disease in 1951 after isolation from 
granulomatous skin lesions in patients from Sweden (Huminer et al., 1986). It was also first 
described as a pathogen of fish under the names M. marinum, M. platypoecilus and M. balnei, 
before these species were recognised as synonyms and named M. marinum (Wolinsky, 1985). 
Mycobacterium marinum is distributed widely in aquatic environments especially stagnant water, 
such as in fish tanks and swimming pools, and in natural (fresh or salt) water bodies (Huminer et 
al., 1986; Hautmann et al., 1994; Falkinham et al., 2001 Gluckman, 1995).   
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Mycobacterium marinum infection was therefore called swimming pool granulomaas it was 
found to be an occupational hazard for aquarium cleaners and fishermen. Most often occur in 
people with recreational or occupational exposure to contaminated fresh or salt water. 
Temperature and water quality have demonstrated to be important factors for the development of 
M. marinum infection (Clark and Shepard, 1965). Infections in humans have been reported in 
coastal areas of the Middle East and the Far East (Evan-Paz et al., 1976; Iredell et al., 1992), in 
several countries in Europe (Collins et al., 1984) and the United States (Zeligman, 1972). 
2.3.1.3 Mycobacterium chelonae 
Mycobacterium chelonae was also described as M. chelonei or M. abscessus until 1972 
(Bercovier and Vincent, 2001). It is widely distributed in the environment in fresh water sources 
such as rivers, ponds, lakes, drinking water and aquaria. It is  principally a pathogen of fish, the 
bacterium was isolated in epidemics of fish tuberculosis (chronic inflammatory granulomatous 
disease) in freshwater fish such as the yellow perch (Perca flavescens), marine species such as 
the Atlantic salmon (Salmo salar) and ornamental fish (Daoust et al., 1989; Lansdell et al., 1993; 
McCormick et al., 1995; Bruno et al., 1998). Due to ubiquitous distribution of M. chelonae, 
infections in pigs, cats and dogs have been reported (Gross and Connelly, 1983; Thorel and 
Boisvert 1974; Thoen and Hime, 1977). Data shown by United State Centers for Disease Control 
and Prevention (CDC) in between 1993-1996 showed that 0.93-2.64 cases per million 
populations for M. chelonae related infection. 
 
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2.3.1.4 Mycobacterium ulcerans 
Mycobacterium ulcerans is the Mycobacterium species that causes Buruli ulcer (BU), the third 
most common mycobacterial disease after tuberculosis and leprosy (Johnson et al., 2005).  The 
disease was first described by Sir Albert Cook, a British physician, in patients from the Buruli 
County in Uganda; however the causative organism was first isolated by MacCallum and others 
in the Bairnsdale region of Victoria, Australia in 1948, hence the name the Bainsdale ulcer.  
The mode of transmission of M. ulcerans is not known and unlike M. leperae and M. 
tuberculosis which are transmitted by person-to-person (Merritt et al., 2010), it is thought that 
infection with M. ulcerans occurs through contact with the environment. Buruli ulcer cases are 
usually found in communities near wetlands such as swamps, marshes and slow moving rivers in 
areas that are prone to flooding. Also, there has been increasing number of cases reported in 
areas where the environment has been disturbed example, deforestation, eutrophication, dam 
construction, mining, population expansion, rice farming and construction of irrigation systems 
(Merritt et al., 2005; Aseidu et al., 2000; Duker et al., 2006; Hayman, 1991)   
The disease has been reported from more than 33 countries worldwide, mainly in tropical and 
subtropical regions (WHO, 2010); figure 2.5 shows the global distribution of Buruli ulcer. The 
worst affected areas are countries lying along the Gulf of Guinea in West-Africa, where BU 
prevalence exceeds that of leprosy, making it the second most important mycobacterioses. In 
West and Central Africa, the disease typically affects impoverished communities primarily 
children of remote areas where medical services are unavailable or too expensive. It is estimated 
that more than 7000 people develop BU annually, with the West African countries like Benin, 
Côte d‘Ivoire and Ghana having the highest incidence rates (WHO, 2008). Globally, 4,907 new 
cases of Buruli ulcer were reported in 2010 and of these Africa alone reported 4,846 cases. Re-
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emergence of cases over the last two decade and the increasing incidence of BU in certain parts 
of the world, as well as limited knowledge of the disease led the global Buruli ulcer initiative and 
the Fifty-Seventh World Health Assembly resolved to accelerate research to develop better tools 
for diagnosis, pathogenesis, and effective treatment (Walsh et al., 2008; Wansbrough-Jones et 
al., 2006). 
The first case of Buruli ulcer in Ghana was first reported in the Greater Accra in 1971 by 
Bayleyfrom patients living along the tributaries of the Densu River. In 1989, 96 cases were 
reported in Asante Akim North District of the Ashanti Region (Van der Werf., 1989). This was 
followed by the discovery of a major endemic focus in Amansie West in the same region 
(Amofah et al., 1993). Cases of the disease have been reported in all the ten regions of the 
country with the Ashanti Region reporting the highest number of cases of about 60 % of all 
cases. The overall national prevalence is 20.7 cases per every 100,000 population making BU the 
second most important mycobacterial disease after tuberculosis (Amofah et al., 2002). Globally, 
Ghana is the second most endemic country for Buruli ulcer after Cote d‘ivoire (WHO, 2012). 
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Figure 2. 7: A world map showing the global distribution of Buruli ulcer worldwide in 2011 
(WHO, 2011) 
2.3.2 Laboratory Diagnosis 
The importance of NTM in human pathology has increased due to the epidemic of AIDS 
(Bercovier and Vincent, 2001). Currently, with the inception of antibiotic treatment more cases 
are encouraged to be laboratory confirmed before initiation of therapy so that individuals are not 
unduly put on antibiotics. Despite advances in clinical and laboratory diagnosis of the different 
NTM diseases, diagnosing NTM diseases remains complicated (van Ingen, 2013). Smear 
microscopy, culture, polymerase chain reaction (PCR) and histopathology are usually the 
methods of diagnosis (Zakham et al., 2012; Wright et al., 1998). Smear microscopy mostly done 
in a two-step procedure by fluorochrome (auramine) and Ziehl-Neelsen staining. Microscopy is 
simple and less expensive method. Currently, it is the most extensive laboratory test used in 
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many developing countries. However, the sensitivity in confirming NTMs by microscopy is low; 
it is approximately between 20-60 per cent. More importantly acid-fast bacilli detection by 
micorsocopy is not specific as it cannot distinguish the different species within the NTMs. 
Culture, though is the final proof procedure; is a slow procedure for some of the species and like 
histopathology requires elaborate infrastructure and expertise (http://www.oie.int). The 
polymerase chain reaction has recently been used to detect and differentiate between members of 
the Mycobacterium tuberculosis complex and NTMs. The PCR assays are easily applicable and 
more rapid than culture (Kox et al., 1995; Kox et al., 1997; Sanguinetti et al., 1998). In addition 
it is very sensitive and primers can be designed to make it very specific, however PCR is 
expensive, required elaborate infrastructure and expertise to prevent contamination. 
 
 
 
 
Figure 2. 8: Ziehl- Neelsen (ZN) stained smears of M. ulcerans observed under oil 
immersion x100 
(http://www.pathologyoutlines.com/topic/stainsacidfast.html) 
Therefore PCR is widely used for the microbiological confirmation of diseases caused by NTMs; 
for example the gold standard method for laboratory confirmation of clinical diagnosis of Buruli 
ulcer is done by the detection of M. ulcerans specific insertion sequence IS2404. 
Acid-fast bacilli 
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2.3.3 Treatment 
Treatment of NTMs infections has been a major problem because of the resistance of NTMs to a 
wide range of antibiotic. There have been several studies on new and more effective 
antimycobacterials and new targets for antimycobacterial therapy (Falkinham, 1996). 
Furthermore, because most mycobacteria are intracellular pathogens and the mammalian host 
cells serve as a barrier to the delivery of drug to the intracellular environment. There are a 
number of published guidelines for prophylaxis and treatment of NTMs infections, more 
especially on M. avium (Griffith et al., 2007). 
Newer antibiotics example; macrolide antibiotics such as, clarithromycin are the most effective 
agents against NTMs and the ATS guidelines recommend that macrolides be part of all regimens 
for NTMs infections. Although effective, these agents should not be used as monotherapy to help 
prevent the risk of resistance. Although no specific macrolide has been shown to be better than 
the other, it is generally considered that clarithromycin may be more effective whereas 
azithromycin is usually better tolerated thus have been shown to be effective against 
Mycobacterium avium complex infection), and are more effective against intracellular 
mycobacteria than standard anti-tuberculosis drugs http://crohn.ie/archive/primer/mycodrug.htm. 
Patients with cavitary/fibronodular disease or severe symptomatic infection often require more 
aggressive treatment and recommended regimen for such patients  includes clarithromycin 1000 
mg daily (or 500 mg twice a day) or azithromycin 250 mg daily, plus rifabutin 150 to 300 mg 
daily or rifampin 10 mg/kg/day (maximum 600 mg/day) plus ethambutol 15 mg/kg/day. 
Intravenous amikacin or streptomycin for 2 to 3 months should be considered in severe or 
refractory cases (Griffith et al., 2007).    
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Surgery has been shown to improve outcomes in some patients with NTMs infection and is 
considered as an adjuvant or alternative treatment (Nelson et al., 1998; Shiraishi et al., 2002). 
Surgery, in combination with multidrug regimens, has been shown to be better treatment option 
than drugs alone in patients infected with macrolide-resistant organisms (Shiraishi et al., 2002). 
Mycobacterium chelonae is one of the most antibiotic- resistant species of the pathogenic rapidly 
growing mycobacteria. Treating M. chelonae infection is challenging because the organism is 
resistant or only partially susceptible to many antibiotics. Based on sensitivities, and to avoid the 
emergence of resistance, dual treatment with clarithromycin and another antibiotic that is 
effective against M. chelonae (e.g., amikacin, imipenem, or tobramycin) is recommended. 
Resistance to mono-therapy with clarithromycin has been reported (Nathan et al., 2000). 
Therefore treatment is continued for >6 months, which is difficult in terms of patient compliance 
(Kullavanijaya, 1999; Grandinetti et al., 2007). In serious disseminated infections involving M. 
chelonae, the injectable agents such as tobramycin plus imipenem are used for the first two to six 
weeks in combination with clarithromycin to prevent the development of drug resistance to the 
macrolide (Hassan et al., 2007).   
Generally, MAI infections are treated with 2 or 3 antimicrobials for at least 12 months. 
Commonly used first-line drugs include macrolides (clarithromycin or azithromycin), 
ethambutol, and rifamycins (rifampin, rifabutin). Aminoglycosides, such as streptomycin and 
amikacin, are also used as additional agents. In children MAI lymphadenitis is treated with 
surgical excision of the affected lymph nodes. 
Surgical debridement followed by skin grafting was the standard treatment for Buruli ulcer even 
though all bacilli were not completely removed (Rondini et al., 2006). Recurrence was common 
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with varying report rates between 6% and 47% (Amofah et al., 1998; Debacker et al., 2005). 
This method was not accessible to poor patients in rural areas of Africa. 
In order to limit surgical excision, other treatment options were explored which included the use 
of topical treatments such as Nitrites, Phenytoin powder, Clay and Heat, hyperbaric oxygen 
therapy, and antibiotics treatment (Sizaire et al., 2006). Many antimicrobial agents were tested as 
mono-therapy or combination therapy in search of effective antibiotic for the treatment of Buruli 
ulcer disease (Sizaire et al., 2006). Several of the antibiotics that demonstrated activity against 
M. ulcerans in vitro but their clinical efficacy for treatment of M. ulcerans disease was not 
proven (Chauty et al., 2007; Thangaraj et al., 2000; Portaels et al., 1998). A controlled trial was 
done  with Clofazimine, an anti-leprosy drug and  the result showed no improvement in healing 
process, no reduction in the number of surgical excisions and as well as no decrease in disease 
recurrences (Revill et al., 1973).   In Côte d‘Ivoire where different antibiotics were used in 
monotherapy for 1 month in a trial the results showed that the use of streptomycin had showed 
no significant effect on BU lesions (Darie et al., 1994). 
Studies with animal models showed that aminoglycosides (example, streptomycin and amikacin) 
and rifampicin had a strong bactericidal activity against M. ulcerans when used alone (Dega et 
al., 2000; Bentoucha et al., 2001). Following a successful pilot study from Ghana that confirmed 
that human lesions can be sterilized with antibiotics (Etuaful et al., 2005; WHO, 2004). 
This made WHO to issue a provisional guidance in 2004 recommending the combination of 
rifampicin and streptomycin for 8 weeks. The current recommendations for treatment are, a 
combination of rifampicin and streptomycin/Amikacin for eight weeks as a first-line treatment 
for all forms of the active disease; Nodules or uncomplicated cases can be treated without 
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hospitalization. Surgery to remove necrotic tissue, cover skin defects and correct deformities 
(WHO, 2003). 
The treatment protocol was supported by extremely encouraging reports of success, with this 
protocol in a case series from Benin where 47% of patients treated were completely healed after 
a year (Chauty et al., 2007) 
2.3.4 Isolation of Non-Tuberculous Mycobacteria 
Isolation of non-tuberculous mycobacteria involves cultivation on selective media after 
decontamination to remove other bacteria present in the sample being analysed. 
Cultivation of mycobacteria is considered to be the ―gold standard‖ for detection of 
mycobacteria in a sample. Culture of mycobacteria is done on solid or liquid media or both 
however solid media allow the observation of colony morphology. The recommended solid 
media include either egg based media such as Löwenstein-Jensen medium (L-J) and agar-based 
media such as Middlebrook 7H10 and 7H11 agar. Löwenstein-Jensen (L-J) media, containing 
malachite green dye to inhibit growth of contaminating organisms, is the traditional solid media 
for culture of mycobacteria. Newer media requires incubation between 2-8 weeks and even more 
than 6 months for the isolation of slow growing NTMs and by culture (Hosek et al., 2006; Sharp 
et al., 2000). However, with liquid media and modern culture systems such as the BACTEC 
AFB or Mycobacteria Growth Indicator Tubes, growth can typically be seen in approximately 2 
weeks. Many Mycobacterium species are also able to grow on very simple substrate, using 
ammonia as nitrogen source and glycerol as carbon source in the presence of mineral salts. 
Isolation of these organisms will enhance the progress of the molecular techniques for detection 
of mycobacteria from the environment. Since the development of techniques also depends on 
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isolates present, culture is necessary for achieving the highest possible isolation efficiency from 
the environment. 
The recovery of non-tuberculous mycobacteria from the environment is influenced by several 
factors including decontamination methods, climate conditions and their slow growth (Chilima et 
al., 2006; Ghaemi et al., 2006; Falkinham, 2002). The major problem encountered when 
isolating mycobacteria from the environment is the presence of high numbers of other fast 
growing microorganisms and fungi in the samples that overgrow on the media before the growth 
of mycobacteria. This leads to contamination of the medium by other fast growing bacteria 
which hinder the isolation of mycobacteria. 
Enrichment of culture media is needed to allow growth of mycobacteria. Mycobacterium 
haemophilum needs an iron source (ferric ammonium citrate or hemin) in the medium and is best 
incubated at 30°C (Saubolle et al., 1996) while for M. genavense it has been reported that media 
composed of blood, charcoal, caseine, yeast extracts and acidified to pH 6.0, is successful in its 
isolation (Realini et al., 1999).  Antibiotic supplements are added to the media for the isolation 
of some of species, for example PANTA (polymyxin B, amphotericin B, nalidixic acid, 
trimethoprim, and azlocillin) and cycloheximide can be added to the media for isolation of 
species such as M. ulcerans to reduce contamination. Most mycobacteria grow optimally 
between 28°C and 37°C. Mycobacterium marinum, M. ulcerans, M. chelonae and M 
haemophilum thrive better between 25 °C and 33°C.  
Although there have been many comparisons of different culture isolation methods for 
environmental samples, general guide for isolation cannot be deduced as a result of differences 
in sample   type , culture media for primary isolation, differences in geographic distribution of 
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mycobacteria species as well as differences in susceptibility to decontamination (Palomino et al., 
1998). There is the need for studies into selective media and decontamination methods that will a 
minimize contamination at the same time increases recovery of NTMs from different geographic 
regions. 
2.3.5 Decontamination Methods 
Most samples for environmental mycobacteria culture contain various amounts of other bacteria 
which usually grow faster. Therefore, recovery of mycobacteria is aided by a chemical 
decontamination process that effectively kills the contaminant to allow only the growth of 
mycobacteria of intrest. Decontamination process is a balance between maximizing recovery of 
mycobacteria, and minimizing contamination by other bacteria and fungi. A wide range of 
decontamination methods and culture conditions have been used by a variety of researchers to 
selectively isolate mycobacteria from the environment. However research has shown that, 
mycobacteria counts are found to be hundred times more in samples before treating with 
decontamination (Livanainen et al., 1997). Their isolation from environmental sample requires 
both selective decontamination of samples and cultivation on selective media due to the threat of 
overgrowth by more rapidly growing microbes (Falkinham, 1996). More also decontamination 
treatment should be at lower concentration. The stronger the alkali, the higher its temperature 
during the time it acts on the specimen, and the longer it is allowed to act, the greater will be the 
killing action on both contaminants and Mycobacterium. It is also important to note that a 
laboratory with no experience of contamination is probably using a method that kills too many of 
the bacilli. Strict adherence to the timed killing period is necessary to maximize recovery 
(Pfyffer et al., 2003).  
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Sodium hydroxide, oxalic acid, malachite green, benzalkolnium chloride and cetylpyridinium 
chloride (CPC) are the most commonly used decontaminants. In a study by Kamala et al., 1994, 
decontamination procedures for isolation of non-tuberculous mycobacteria from soil and water 
were evaluated, six decontamination methods were evaluated and it was found that treatment 
with 3% Soduim dodecyl sulfate (SDS) in combination with 1% sodium hydroxide (NaOH) was 
the most effective decontamination method for soil as well as water samples. When this 
procedure was used by Parashar et al., in a study reported in 2004, the decontamination method 
could not remove any contaminants. In another study, Engbaek et al., 1967 evaluated five 
methods for decontamination, and the 3% sodium lauryl sulfate with 1% Sodium hydroxide 
method was reported as most suitable. Thus decontamination methods that have been used in the 
isolation of mycobacteria from the environment and clinical samples, none has been universally 
accepted for the cultivation mycobacteria.  
 
 
 
 
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CHAPTER THREE 
MATERIALS AND METHODS 
3.1 Equipments and Reagents 
3.1.1 Equipments 
The following equipments were used: fast prep homogenizer (MP Biomedicals, U.S.A), mortar 
and pestle (CA Scientific Co., Inc, U.S. A.), eppendorf centrifuge 5415D (Marshall Scientific, 
U.S.A), 2720 thermal cycler (Applied Biosystems, Singapore), vortex (GENIE, U.S.A), 
twincubator (Hain Lifescience GmbH, Germany), water bath (Thermostat Io Shaking Water 
Bath, Thomas Kagaku Co. Ltd, Japan), incubator (Yamato, Japan), digital coagulator (Te-Her, 
Japan), class II microbiological safety cabinet (BioMAT2, U.S.A) 
3.1.2 Reagents  
The following reagents were used: sodium dodecyl sulphate (Sigma Aldrich, U.S.A), sodium 
hydroxide Anhydrous Pellets (Sigma Aldrich, U.S.A), oxalic acid dehydrate (Reagent Plus® 
>99% (Sigma, U.S.A)), malachite green (Sigma Aldrich, U.S.A), cycloheximide (Sigma, China), 
Genotype CM version 1.0 (Hain Lifesciences Nehren, Germany), fast DNA spin kit (MP 
Biomedical, U.S.A), l-Asparagine anhydrous (Fluka Biochemika, Italy), magnesium sulphate 
(Sigma, U.S.A.), magnesium citrate 14 hydrate (BDH (GPR), U.K.), potassium phosphate 
monobasic (KANTO chemical co. INC, Japan), agarose (Sigma, U.S.A.), immersion oil (Fluka 
analytical, Switzerland), glycerol (Riedel-de Haen, Sigma-Aldrich, U.S.A), sulphuric acid 
(Sigma-Aldrich, U.S.A), phosphate buffer saline (dulbecco A, OXOID, U.K), methylene blue 
(Sigma, U.S.A.), phenol crystals (Sigma Aldrich, U.S.A.), PANTA (a mixture of antibiotics, 
polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) (Becton Dickinson, 
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U.S.A), mycobactin J (Allied Monitor, U.S.A), isoniazid (Sigma, India), cycloheximide (Sigma, 
China),  ethambutol ihydrochloride (Sigma, India). 
3.1.3 Materials  
Scalpel blade (Swann-Morton, U.K), 50 ml centrifuge tubes (BD Falcon, U.S.A.), pipetting aid 
(Gilson, U.K), sterile pipette tips (Gilson, U.K), micropipettes (20µl, 200µl 1000µl) (Gilson, 
U.K),  conical flask (Pyrex, corning life sciences, U.S.A), measuring cylinders(Pyrex, corning 
life sciences, U.S.A), petri dishes (Pyrex, corning life sciences, U.S.A), pipetteman (Gilson, 
U.K), glass beads 3mm (MERCK, Germany), ground edge 90° frosted end microscope slides 
(Thermo scientific, U.S.A), 9 inch sterile pipette (Alpha laboratories, U.K.), serological pipettes 
(SARSTEDT, Germany), timer (Fisher scientific, U.S.A), DNA strip marker (Hain Lifesciences 
Nehren, Germany ), waste biohazard bag (SARSTEDT, Germany), gloves (Nitrile Bodyguard, 
U.K.), permanent tube marker (Stabilo, Germany). 
 
3.2 Study Design 
The study was an experimental study; samples were collected from three villages. Two of the 
communities were Buruli ulcer (BU) non endemic while one was endemic. Samples collected 
include, snail, soil, insects, water and vegetation. The water samples were placed in 50 ml falcon 
tubes while dried samples were placed in zip-lock bags and transported to the laboratory.  
Deoxyribonucleic acid extraction and PCR were performed on all collected samples for detection 
of IS2404 containing mycobacteria. Smears were then prepared directly and stained by Ziehl-
Neelsen procedure. Each of the IS2404 positive samples were decontaminated with three 
different decontamination methods and cultured on different in-house egg based selective media. 
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Acid-fast bacilli positive growths were further identified using line probe assay (HAIN 
hybridization). Figure 3.1 is the flowchart of the study design. 
 
Figure 3. 1: Flowchart of the Study Design 
 
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3.3 Study Site 
Three villages, Ntabea I, Ntabea II and Ashongkrom in the Eastern Region of Ghana were the 
sites for the study. These villages are located along the Densu River where most of the 
communities are known to be Buruli ulcer endemic. Ashongkrom is an endemic community 
located in the Akuapim South municipality (latitude: 5.83364, longitude: -0.30879) with a 
population of about 200 projected from the 2010 population census (Yeboah-Manu et al., 2012; 
National Buruli ulcer Control Programme (NBUCP)). The common disease conditions in this 
community are skin disease, malaria, Buruli ulcer and diarrhoea (Akuapim South municipality 
health directorate) 
Ntabea I is located in Suhum- Kraboa-Coaltar municipality under Obretema Suhum sub-
municipality (latitude: 6.12285, longitude: -0.42583). Ntabea I is a small community with a 
population of 215 projected from 2010 population census. The common disease conditions in 
this community are skin disease, malaria and diarrhoea (Suhum sub-municipal health 
directorate). 
Ntabea II is located in East-Akim municipal under Asafo sub-district on the coordinates, 
(latitude: 6.13825, longitude: -0.41788) with a population of 627 projected from 2010 population 
census. Their main occupation is farming and the common disease condition in this community 
is malaria (East-Akim municipal health directorate).  
However, inhabitants of these two non-endemic communities in the previous study showed 
exposure to M. ulcerans (Yeboah-Manu et al., 2012). This was evident by sero-positive reaction 
to the 18 kDa M. ulcerans specific protein as the endemic community. Map of the study site is 
shown below in Figure 3.2. 
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Figure 3. 2: Map showing the district of the villages selected for the study 
 
3.3 Sample Collection 
Sixty-five samples were collected randomly from locations such as along footpaths; small water 
bodies, compounds of houses, farms and along the river bank in all three communities.  Twenty 
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(20) and seventeen (17) samples were collected from Ntabea I and Ntabea II respectively, and 28 
from Ashongkrom. The samples collected included water, animal droppings, snails, vegetation 
(Both living and dead) and soil samples. Table3.1 below shows all the samples analyzed. Solid 
samples were put in individual zip lock bags and liquid samples were kept in tightly closed 50 
mL falcon tubes. Each sample was labelled with sample number, name of community, type of 
sample and date of collection. The name of the community, the type and identification number of 
sample was also recorded in a sample collection book. All the collected samples were kept in a 
transport bag and transported to the laboratory of the Noguchi Memorial Institute for Medical 
Research the same day.  The samples were stored at 4 °C until they were analyzed within a week 
of collection. 
Table 3. 1: Samples analyzed in the study 
Sample type Number (%)  
Millipede 2(3.08 %) 
Caterpillar 1(1.54%) 
Termite mount 1(1.54%) 
Snail 5(7.69%)   
Moss 3(4.62%) 
Web 1(1.54%) 
Algae 2(3.08 %) 
Insect  2(3.08 %) 
Vegetation 17(26.15%) 
Snail shell  1(1.54%) 
Animal dropping 9(13.85%) 
Soil  13(20.00%) 
Water  6(9.23%) 
Fungi  2(3.08 %) 
Total  65(100%) 
 
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3.4 Direct Detection of IS2404 Containing Mycobacterium from Environmental Samples 
3.4.1 DNA extractions by FastDNA spin kit for soil protocol 
Deoxyribonucleic acid was extracted from about 500mg portions of solid samples and about 500 
µL of liquid samples using the FastDNA spin kit according to the manufacturer‘s instruction. 
Samples were transferred into lysing matrix E tubes provided in the kit. Lysis buffer (800 µl) and 
200 µl of protein precipitation solution (PPS) were added to the sample and vortexed full speed 
for two minutes. The suspension was further homogenized to break the bacterial cell wall in the 
fast prep machine for 40 seconds at a speed of 6 m/s and centrifuged at 1400 x g for 10 minutes 
to sediment the soil, cell wall and other debris. Two hundred microlitres of the supernatant was 
transferred into 2ml microcentrifuge tube; 500 µl of binding matrix was added and vortexed 
briefly to bind the released DNA. The mixture was then transferred into a spin column provided 
in the kit using transfer pipette. It was then centrifuged for 1400 x g for 1 minute to wash away 
excess diluent. Five hundred microlitres of SEWS-M (contains ethanol) was added gently to the 
bound DNA in the column for further DNA purification and centrifuged at 1400 x g for 1 
minute.  The bound DNA in the column was centrifuged at 1400 x g for 2 minutes again without 
any solvent to dry the matrix after which it was air – dried for 5 minutes. Bound DNA was gently 
re-suspended in 100 µl of Dnase/Pyrogen-free water (DES) by flicking the sides and incubated 
for 5minutes.  Centrifugation was done for 1 minute at 1400 x g to elute the bound DNA 
suspension into a sterile catch tube. The DNA was stored at -20 °C until ready for PCR. 
 
 
 
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3.5 Polymerase Chain Reaction (PCR) 
3.5.1 Precaution to prevent contamination 
Four rooms were used for all the PCR assays to prevent contamination. The master mix and the 
addition of template were all done in sterilized safety cabinets. The function of each room is as 
follows. 
First room:   Preparation of Master mix 
Second room:   Addition of DNA template 
Third room:       Amplification of DNA 
Fourth room:  Electrophoresis and Visualization of amplified Products 
3.5.2 Polymerase chain reaction procedure 
Polymerase chain reaction (PCR) was done on the extracted DNA to detect IS2404 containing 
Mycobacterium species in the samples. Two primers used initiating amplification were forward 
MU1New (5′-GAT CAA GCG TTC ACG AGT GA-3′) and reverse MU2 (5′-GGC AGT TAC 
TTC ACT GCA CA-3′) (Fyfe et al., 2007). The reaction mixture consisted of 1 µl MU1and 1 µl 
MU2, 2 µl of 10x buffer, 4 µl of 5X Q-solution, 1 µl of 25mM MgCl2, 0.12 µl of 5units/ µl hot 
star tag polymerase, 0.4 µl of 10mM deoxynucleoside triphosphates (dNTPs), 6.48 µl nuclease 
free water and 4 µl of DNA, in a total volume of 20 µl (WHO, 2001). 
The amplification was done at initial denaturing temperature of 95 °C for 15 minutes; 30 cyles of 
denaturing at 95 °C for 30 seconds, annealing 60°C for 30 seconds, extension at 72 °C for 1 
minute; and followed by final extension at 72°C for 10 minutes. Each PCR run contained 
extraction negative control and IS2404 positive and negative controls. 
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3.5.3 Electrophoresis of PCR Product on 2% Agarose Gel 
3.5.3.1 Gel Preparation and Electrophoresis 
Clean gel trays well were placed on a flat table and combs used for creating wells placed at a 
centimeter from one end of the tray. Agarose gel was prepared by weighing 2g of agarose 
powder (Sigma, U.S.A) into a 500 ml sterile flask containing 100 ml 1X Tris-Boric acid-EDTA 
(TBE) buffer (see appendix). The mixture was dissolved by heating in a microwave, 2 µl of 
ethidium bromide (100mg/ml) was added and mixed well by swirling. The agarose gel was 
poured gently into the gel tray and was allowed to set. The comb was carefully removed and the 
solidified gel was placed in an electrophoresis tank. Electrophoresis buffer (1X TBE) was poured 
into the tank until the gel was completely covered. Amplified products (7µl) were loaded into the 
wells and run at 100 volts for 30 minutes. 
3.5.3.2 Visualization of Amplified Product 
In order to view the amplified product (band), the gel was placed in a gel logic system apparatus 
(UV transillumination) connected to a computer and viewed under UV light. Images were 
captured on the computer and saved for further analysis.  
 
3.7 Microscopy 
3.7.1 Sample Processing 
Hammer was used to break the shell of snails and samples such as leaves were diced with sterile 
disposable scalpels. They were further homogenized with a sterile porcelain and pestle and 
suspended in phosphate buffered saline (PBS).  Soil samples were shaken vigorously in sterile 
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distilled water and centrifuged at 600rpm for 5 minutes to sediment soil particles. Samples such 
as animal droppings were homogenized with a sterile porcelain and pestle and suspended in 
phosphate buffered saline (PBS). Water samples were vortexed to mix homogenously and 
centrifuged at 4,000 rpm for 30 minutes to sediment all suspended bacteria. The supernatant was 
decanted and the resulting pellet was suspended in PBS. 
3.7.2 Direct Smear Microscopy 
Frosted glass slides were labelled with sample identification number and date. Smear was 
prepared by transferring 100 µl each of processed onto respective labelled slide. The smear was 
air-dried in a level 2 biosafety cabinet to prevent risk of infection. The air-dried slides were heat 
fixed by passing the slide over flame 3 times. Heat fixed slides were arranged on a staining rack, 
leaving enough space between each slide to prevent cross contamination during the staining 
process. Slides were stained by Ziehl-Neelsen procedure as below: 
Each slide was flooded with filtered carbol fuchsin which contains phenol and heated underside 
to steam but not to boil; this was to break open the cell wall for the stain to penetrate the lipid 
rich thick cell wall of mycobacteria.  The slides were left at room temperature for 5 minutes. 
After rinsing off the excess carbol fuchsin stain with tap water, the smears were decolorized by 
flooding with 20% H2SO4 solution to remove the carbol fuchsin from the non acid-fast bacilli 
cell wall for 5 minutes and then gently rinsed in tap water. The slides were then counter stained 
with 0.1% methylene blue solution for 1 minute. The slides were air dried before observing 
under the microscope. Mycobacteria appeared bright reddish-pink in colour while other cells 
stained blue. The slides were observed using a light microscope under oil immersion and were 
graded using the International Union against Tuberculosis and Lung Diseases (IUALTD) grading 
scale (Table 3.2). A smear was declared negative only after reading at least 100 microscopic 
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visual fields and confirmed by a second reader. 
 Table 3. 2: The quantitative scale (IUALTD) for grading AFB in Smears 
 
3.8 Isolation of Mycobacteria by Culture 
Two millilitres each of the processed samples was transferred into separate 50 ml centrifuge 
tubes for decontamination. Figure 3.1 is a flow chart of isolation method employed within this 
project. 
 
3.8.1 Decontamination Procedures 
3.8.1.1 Sodium hydroxide/Oxalic acid method 
Decontamination was performed by treatment of the suspensions for 20 minutes with an equal 
volume of aqueous 4% sodium hydroxide (NaOH). It was incubated at room temperature for 20 
minutes; the suspension was vortexed intermittently.  The reaction was neutralized by adding 
Phosphate buffered saline (PBS) to the 45 ml mark and centrifuged at 3800 rpm for 30 minutes. 
The supernatant was carefully decanted, and the sediment was re-suspended in 2 ml of PBS and 
equal volume of 5% oxalic acid was added. It was incubated at room temperature for 30 minutes 
Report Number of fields to screen 
(Negative) No AFB found in at least 100 fields 
SCANTY (EXACT NO): 1-9 AFB found in 100 fields 
1+ 10-99 AFB found in 100 fields 
2+ 1-10 AFB found per field in at least 50 fields 
3+ More than 10 AFB per field in at least 20 fields 
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and neutralized with PBS to the 45 ml mark. This was then centrifuged at 3800rpm for 30 
minutes, the supernatant was decanted carefully. One milliliter PBS was added to the sediment 
and vortexed to mix thoroughly. Hundred microlitres of the concentrated sediment was 
inoculated onto respective growth medium (Yeboah-Manu et al.,   2004). 
3.8.1.2 Malachite green/Cycloheximide/Sodium hydroxide method 
Two and a half milliliter (2.5 ml) each of (0.3% of Malachite green, 0.075 g /50 ml of 
cycloheximide and 4% Sodium Hydroxide (NaOH)) solutions was added to 3 ml of the 
processed sample. It was incubated at room temperature for 30 minutes with intermittent 
vortexing. One normal hydrochloric acid (1N HCL) was used to neutralize the mixture. The 
mixture was then centrifuge at 3800 rpm for 30 minutes.  Supernatant was carefully decanted 
leaving the sediment. One milliliter PBS was added to the sediment and vortexed to mix 
thoroughly. Hundred microliters of the concentrated sediment was inoculated onto respective 
growth medium (Portaels et al., 1988). 
3.8.2.3 Sodium dodecyl sulphate (SDS) / Sodium hydroxide (NaOH) 
Decontamination was performed by treatment of the suspensions for 30 minutes with an equal 
volume of 3% Sodium dodecyl sulphate and processed sample. It was incubated at room 
temperature for 30 minutes; the suspension was vortexed intermittently.  The reaction was 
neutralised by adding PBS to the 45 ml mark and centrifuged at 3800 rpm for 30 minutes. The 
supernatant was carefully decanted, and the sediment was re-suspended in 2 ml of PBS and equal 
volume of 4% NaOH was added and incubated at room temperature for 30 minutes with 
intermittent vortexing. The mixture was then centrifuge at 3800rpm for 30 minutes and carefully 
decanted. One millitre PBS was added to the sediment and vortexed to mix thoroughly. Hundred 
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microliters of the concentrated sediment was inoculated onto respective growth medium 
(Parashar et al., 2004). 
3.8.3 Cultivation of Mycobacteria 
3.8.3.1 Growth medium and Incubation 
Four different growth media were evaluated. The media used for isolation were egg based; drug 
free Lowenstein-Jensen (L-J) and 3 L-J media containing drugs. The drug containing media were 
1) PANTA plus (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) and 
mycobactin J (PM), 2) ethambutol plus cycloheximide (EB+ cyclo) and 3) Isoniazid plus 
cycloheximide (INH+ cyclo).  
Inoculated media tubes were incubated at 31°C, observed daily for the first week and weekly 
thereafter until six months.  The colonial morphology and length of time before visible colonies 
appeared were recorded during culture reading. A culture was said to be positive if at least one 
tube from the same treatment had confirmed AFB growth; culture was said to be negative if none 
has microbial growth and contaminated when all tubes had more than half of the tube with other 
bacterial growth or liquefied.   
3.8.3.2 Purification and Amplification of Microbial Growth by Sub-Culture 
An inoculating loop was used to transfer a loopful of colonies into a sterile tissue culture tube 
containing six of three millimeter (3mm) glass beads and 2 drops of PBS. The mixture was 
vortexed for about two minutes until the colonies are completely emulsified. The tube was left 
for 5 minutes   to settle all created aerosol and 500 µl PBS added. A transfer pipette was used to 
transfer 100 µl of the inoculum onto 2 Lowenstein-Jensen (L-J) slants, incubated at 31 ºC and 
observed weekly until confluent growth was achieved. A drop of the prepared suspension was 
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also used to prepare a smear for ZN staining to confirm the acid-fastness of the microbial 
growth.  
3.8.4 Species Identification 
Acid-fast bacilli isolates were identified using the GenoType CM version 1.0. (Hian 
Lifesciences) identification kit. The GenoType CM version 1.0 permits the identification of M. 
tuberculosis and 24 most common NTMs such as the following species: M. avium, M chelonae, 
M. abscessus, M. fortuitum M. gordonae, M. peregrinum, M. marinum/M. ulcerans and M. 
xenopi. This technology is a line probe assay and follows three steps; DNA extraction, PCR 
amplification of specific genomic regions of the DNA and reverse hybridization of the 
amplicons.  DNA extraction was done by the boiling method.  
3.8.4.1 PCR Amplification 
The GenoType Mycobacterium CM line probe assay was performed as recommended by the 
manufacturer. The reaction mixture consisted of 2 μl of  MgCl2, 5 μl of 10 X buffer, 35 μl of 
primer (biotinylated) - nucleotide mix (PNM) (provided in the kit), 0.2 μl HotStarTaq 
polymerase (QIAGEN, Hilden, Germany), 2.8 µl of nuclease free water and 5µL of purified 
DNA, in a total volume of 50 μl. 
The amplification protocol was initial denaturation and enzyme activation of 95 ºC for 15 
minutes, 10 cycles performed as follows: 95ºC for 30 seconds, 58°C for 2 minutes, followed by 
additional 20 cycles of 95°C for 25 seconds, 53°C for 40 seconds and 70°C for 40 seconds and 
final extension at 70ºC for 8 minutes.  
 
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3.8.4.2 Hybridization 
The hybridization step was done by following the manufacturer‘s instruction. Hybridization and 
detection were performed in an automated washing and shaking machine known as the 
Twincubator.  
Amplicon denaturation was achieved by adding 20 µl of the amplicon and 20 µl of provided 
denaturing solution (DEN) (contains< 2% NaOH and dye) and mixed. It was incubated at room 
temperature for 5 minutes. 
One millitre of pre-warmed hybridization buffer (HYB) (contains 8-10% anionic tenside and 
dye) was then added to the trough containing the denatured amplicon. The solution was mixed by 
gentle tilting mixture up and down until a homogenous colour was developed.  The strip with 
immobilized oligonucleotide probe was labeled and placed in the trough. To make sure the strip 
was carefully covered by the solusion. The hybridization procedure was performed at 45 °C for 
30 minutes at 300 rpm using the twincubator. The strips were then washed twice to remove 
unbound DNA with RIN solution (< 1% anionic tenside and < 1%NaCl) (provided with the kit). 
One millitre pre-warmed stringent solution (STR) (contains >25% of quaternary ammonium 
compound, < 1% anionic tenside and dye) was added to each trough containing the strip 
incubated for 15 minutes to remove the non-specific bounded DNA. Excess stringent solution 
was removed completely by two washing steps using 1 ml rinsing solution (RIN) to and 
incubation at room temperature for 1 minute in each of the washing step. One millitre diluted 
conjugate solution (contains streptavidin-conjugated alkaline phosphate, 1% blocking reagent, 
1%NaCl) was then added to each strip and incubated for 30 minutes on the twincubator. 
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After rinsing twice for 1 minute each, one millitre of diluted Substrate solution (contains 
substrate concentrate (SUB-C) which contains dimethyl sulfoxide and (SUB-D) <1% MgCl2, < 
1%NaCl) was added to each and incubated for 5 minutes. The substrate was washed away twice 
with distilled water without shaking, this was done to stop the reaction and the DNA strips were 
removed and dried between two layers of absorbent paper.  
3.8.4.2.2 Evaluation and Interpretation   
The developed strips were pasted in the designated fields by aligning the conjugate control (CC) 
and universal control (UC) and genus control (GC) bands with the respective lines on the 
evaluation sheet.  
The Conjugate control (CC); this band must develop to show the efficiency of the conjugate 
binding and the substrate reaction.  
The universal control (UC) band detect all known Mycobacterium species and members of the 
group of Gram positive bacterium with high G+C content. If the universal control band, the 
conjugate control band and other bands are positive but pattern that cannot be assigned to a 
specific Mycobacterium species. Only those bands whose intensities are about as strong as or 
stronger than that of the universal control are to be considered.  
The genus control (G-C) band confirms the organism is a member of the genus Mycobacterium, 
the intensity of the band varies depending on the Mycobacterium species. 
Interpretation of the results was done based on the presence and absence of bands compared with 
reference provided in the kit. The organisms were identified based on the number and position of 
bands formed on the strip (Figure 3.4). 
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Figure 3. 3: Evaluation chart for identifying Common Mycobacterial species 
 
 
 
 
 
 
 
 
 
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CHAPTER FOUR 
RESULTS 
4.1 Samples 
Sixty-five samples were collected from three communities namely: Ntabea I (20 samples), 
Ntabea II (17 samples) and Ashongkrom (28 samples) in the Eastern region of Ghana. The 
samples collected included, soil 13 (20.00%), water 6 (9.23%), millipede 2 (3.08%), caterpillar 
1(1.54%), termitarium 1 (1.54%), snail 5 (7.69%), moss  3 (4.62%), spider web, 1 (1.54%), fungi 
2 (3.08%), algae 2 (3.08%), insect 2 (3.08%), vegetation other than moss 17 (26.15%), snail shell 
1 (1.54%) and  animal droppings 9 (13.85%). 
 
4.2 Direct Microbiological Analysis 
4.2.1 Detection of IS2404-positve Mycobacterium species  
The presence of IS2404 containing Mycobacterium species was confirmed in 37 (56.9%) of the 
samples by direct PCR analysis. This was evident by the detection of a 450bp amplification band 
after gel electrophoresis (Figure 4.1). The sample type and proportion that were IS2404 PCR 
positive is indicated in Table 4.1. 
 
 
 
 
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      Table 4. 1: Polymerase chain reaction positivity of respective samples analysed 
Sample type Sample positivity 
Moss 1/3(33.3%) 
Spider web 1/1(100%) 
Insect 2/2(100%) 
Snail shell 1/1(100%) 
Fungi 2/2(100%) 
Snail 5/5 (100%) 
Vegetation 12/17 (70.6%) 
Water 4/6 (66.7%) 
Millipede 1/2(50.0%) 
Animal dropping 4/9 (44.4%) 
Soil 4/13(30.8%) 
 
Direction 
of 
movement
200bp
100bp
300bp
500bp
 
Figure 4. 1: Gel electrophoresis analyses of some environmental samples 
 
MW is molecular weight ladder (100bp), Lanes 1-8 are analyzed environmental samples,  
EXT is extraction negative control, PCR is PCR negative control and POS is positive control. 
 
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4.2.2 Direct Smear Microscopy 
Smears were prepared from the thirty – seven samples that were IS2404 PCR positive. Acid- fast 
bacilli (AFB) were detected in 5 (13.51%) samples while thirty-two (82.49%) of the smears were 
negative as shown in table 4.2 
Table 4. 2: Acid-fast bacilli positivity of IS2404 positive samples by direct smear analysis 
Sample type Positivity for acid-fast bacilli (Number of 
positive / Total number of samples) 
Moss 1/1 (100%) 
Snail 1/5 (20.0%) 
Animal dropping 1/4 (25.0%) 
Soil 1/4 (20.0%) 
Vegetation 1/12 (8.3%) 
 
4.2.3 Isolation of Mycobacterium species 
4.2.3.1 General Results 
Thirty-seven (37) IS2404 PCR positive samples were cultured on 3 different drug-containing 
media and 1 drug–free media. The test was duplicated for the four media types (Drug free (DF), 
Isoniazid (INH), PANTA-Mycobactin-J (PM), Ethambutol (EB)) and the three different 
decontamination methods (sodium hydroxide/oxalic acid (NaOH/OA), malachite 
green/cycloheximide/sodium hydroxide (Malachite) and Sodium dodecyl sulphate/Sodium 
hydroxide (SDS/NaOH)). Thus 24 media tubes were inoculated per sample giving a total of 888 
inoculated tubes. Out of the 888 tubes inoculated, 91 (10.25%) had mycobacterial growth (acid-
fast bacilli), 700 (78.82%) had no bacterial growth and 97 (10.92%) were observed to be 
contaminated. Nine (24.32%) samples had macroscopic AFB growth after six months of 
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incubation while the remaining 28 (75.68%) samples had no mycobacterial growth. None of the 
37 inoculated samples had all 24 tubes contaminated. 
Some of the mycobacterial colonial morphologies observed were, smooth yellow dysgonic, 
smooth white dysgonic, rough buff dysgonic and smooth orange dysgenic (Figure 4.2). Acid- 
fast bacilli positive cultures were obtained from soil, 3/4 (75.0%), vegetation, 3/12 (25.0%), 
water, 2/6 (33.3 %) and snail, 1/5 (20.0%).  
 
Figure 4. 2: Culture tubes with AFB positive isolates: note the different colonial 
morphologies 
 
 
Smooth white dysgonic 
Smooth buff dysgonic 
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4.2.3.5 Combined Performance of the Decontamination Methods and In-House Formulated 
Media. 
 
Three different decontamination procedures and four different media either with or without 
antibiotic supplementation were evaluated. The decontamination procedures evaluated were 
sodium hydroxide/oxalic acid (NaOH/OA), malachite green/cycloheximide/sodium hydroxide 
(Malachite) and sodium dodecyl sulphate/sodium hydroxide (SDS/NaOH). For each of the three 
decontamination procedures 296 tubes were inoculated. The four different media evaluated were 
L-J-media with no antibiotic (drug free (DF), isoniazid +cycloheximide (INH+ Cyclo), PANTA-
mycobactin-J (PM) and ethambutol + cycloheximide (EB + Cyclo)). A total of 222 tubes of each 
media type were inoculated after sample processing. 
 
Table 4.3 shows the combined performance of the decontamination methods and the four in- 
house media types used for this study. The results of the analysis using ANOVA revealed that 
the three decontamination methods (NaOH/OA, SDS/NaOH and malachite) had a statistically 
significant effect on the performance of in-house selective media studied (P<0.05) (Appendix E) 
at 5% level of significance. In the case of NaOH/OA decontamination methods there was a 
significant difference in the mycobacteria growth (P=0.0001, F=355.667) and those that got 
contaminated (P=0.02, F=39.444) in relation to the four in-house selective media. The media 
with the highest number of mycobacterial growth was PM media whilst least was recorded in EB 
+ Cyclo. 
 
Using the SDS/NaOH decontamination methods, the results showed that there was a statistically 
significant difference in the mycobacterial growth (P=0.030, F=11.33) and the contamination 
(P=0.001, F=78) (Appendix E) with respect to the media. Drug free media was the highest in 
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terms of contamination whilst PM was least contaminated. Similarly, in the case of malachite 
green decontamination methods, the results showed that there was significant difference in the 
mycobacaterial growth (P=0.010, F=16.33) and those contaminated (P=0.010, F=16.3) at 5% 
level of significance.  
 
With the performance of in-house selective media used in the isolation of Mycobacterium 
species, PM recorded the highest mycobacterial growth value of 27 and the least value of 15 
recorded for EB+Cyclo. Isoniazid +cycloheximide and DF also recorded a mycobacterial growth 
value of 26 and 23 respectively. Statistical analysis using single factor Analysis of variance 
(ANOVA) at 95% confidence level (5% level of significant) showed that there were no 
significant differences in the performance of the four in-house selective media examined 
(F=8.056, P=0.008) in the growth of mycobacteria (Appendix G). However, with respect to 
contamination of the media, PM was the least contaminated and DF, the highest contaminated. 
Analysis of variance (ANOVA) at 95% confidence level (5% level of significant) showed that 
the contamination of the selected media differed significantly (P=0.0001) (Appendix G).When a 
post hoc analysis was conducted using the Least Significant Difference (LSD), it revealed that 
there were significant differences in contamination among the four media studied (Appendix F). 
The performance of the media in terms of contamination in a decreasing order of ranking are as 
follows; DF > INH + Cyclo > EB + Cyclo > PM. 
 
 
 
 
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Table 4. 3: Performance of in-house formulated media and the decontamination methods 
Media Decontamination Method 
NaOH/OA 
 
SDS/NaOH 
 
Malachite 
 
P N C P N C P N C 
DF 10 56 8 6 55 13 7 57 10 
INH+cyclo 13 57 4 8 55 11 5 58 11 
EB+cyclo 4 65 5 8 56 10 3 62 9 
PM 15 57 2 8 60 6 4 62 8 
Total 42 235 19 30 226 40 19 239 38 
P= Positive mycobacterial growth; N= No bacteria growth; C= contamination: DF =Drug 
free; INH + Cyclo = Isoniazid + cycloheximide; PM = PANTA-Mycobactin-J; EB + Cyclo = 
Ethambutol + cycloheximide. 
 
4.2.3.6 Species Identification 
Based on colony morphology (Figure 4.2), media and decontamination method as well as sample 
type, a total of 80 isolates were obtained from the nine positive cultured samples; 52 (65.0%) of 
the isolates were from the NaOH/OA decontamination method, 15 (18.75%) were from the 
SDS/NaOH decontamination method and 13 (16.25%) were obtained from the malachite green 
decontamination method. Figure 4.3 shows some of the line probe hybridization results of the 
isolates identified. Out of the 80 isolates 76 were identified as Mycobacterium species, 1 isolate 
identified as bacterium with high G-C content and 3 could not be amplified. As indicated in table 
4.4.  
 
 
 
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Figure 4. 3: Line probe hybridization analysis of isolates used for identification 
Bands 1-3 are Conjugate control, Universal control and Genus control respectively.  
The remaining 4-17 are species specific bands. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 
4 - 17 
3 
2 
Interpretation  
chart 
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     Table 4. 4: List of Identified isolates obtained 
Organism  Number of organisms isolated 
Mycobacterium species 32 
Mycobacterium chelonae 16 
Mycobacterium interjectum 7 
Mycobacterium fortuitum 6 
Mycobacterium avium 6 
Mycobacterium abscessus 4 
Mycobacterium malmoense 3 
Mycobacterium gordonae 1 
Mycobacterium peregrinum 1 
high G-C content bacterium 1 
Total  77 
 
 
4.2.3.5 The Sample Type and Mycobacterium species isolated 
As indicated in table 4.5 Mycobacterium chelonae was isolated from 3 soil, 1 vegetation and 1 
water samples. Mycobacterium avium was obtained from 3 soil samples, Mycobacterium 
fortuitum from 2 soil, Mycobacterium malmoense from 2 soil samples and Mycobacterium 
gordonae from 1 soil sample. Whereas Mycobacterium avium, Mycobacterium fortuitum, 
Mycobacterium malmoense and Mycobacterium gordonae were isolated from only soil, 
Mycobacterium abscessus was isolated from both soil and vegetation. On the other hand, 
Mycobacterium interjectum was isolated from only water. Other Mycobacterium species isolated 
from various samples are all indicated in Table 4.5. Some of the isolated mycobacteria were fast-
growing Mycobacterium species Table 4.6 and were isolated from soil, vegetation and water 
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samples where as others were slow growers (Table 4.7.) and were from isolated from only soil 
samples.  
Table 4. 5: Type of sample and Mycobacterium species identified 
Organism Soil Vegetation Snail Water Total 
Mycobacterium species 1 13 3 15 32 
Mycobacterium avium 6 - - - 6 
Mycobacterium abscessus 1 3 - - 4 
Mycobacterium chelonae 10 5 - 1 16 
Mycobactrium peregrinum - 1 - - 1 
Mycobacterium gordonae 1 - - - 1 
Mycobacterium malmoense 3 - - - 3 
Mycobacterium interjectum - - - 7 7 
Mycobacterium fortuitum 6 - - - 6 
Total 28 22 3 23 76 
 
Table 4. 6: Fast-growing mycobacteria isolate and sample type 
Organism Soil Vegetation Snail Water Total 
Mycobacterium abscessus 1 3 - - 4 
Mycobacterium chelonae 10 5 - 1 16 
Mycobactrium peregrinum - 1 - - 1 
Mycobacterium interjectum - - - 7 7 
Mycobacterium fortuitum 6 - - - 6 
Total 17 9 0 8 34 
 
 
 
 
 
 
 
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Table 4. 7: Slow-growing mycobacteria identified and sample type 
Organism Soil Vegetation Snail Water Total 
Mycobacterium avium 6 - - - 6 
Mycobacterium gordonae 1 - - - 1 
Mycobacterium malmoense 3 - - - 3 
Total 10 0 0 0 10 
 
4.2.3.6 Organism Isolated and Decontamination Method 
Out of the 44 identified isolates, 25 were obtained from the NaOH/OA decontamination method, 
followed by 10 from malachite green decontamination method and 9 from the SDS/NaOH 
decontamination method. Mycobacterium chelonae was the most isolated species from 
NaOH/OA. Out of the 8 species identified, 7 were isolated using NaOH/OA. Table 4.8 shows 
Mycobacterium species isolated and the decontamination methods employed. Table 4.9 and 
Table 4.9 shows the slow-growing and the fast-growing Mycobacterium species identified from 
the three decontamination methods respectively. Analysis of variance (ANOVA) at 95% 
confidence level (5% level of significant) showed that NaOH/OA, SDS/NaOH and Malachite 
green decontamination methods did not have any significant effect on the slow growing 
mycobacteria (P>0.05) (Appendix H) However, on the fast-growing mycobacteria, NaOH/OA 
(P=0.0001, F=148.5) and malachite green (P=0.002, F=26.167) were the decontamination 
methods that were found to have a significant effect (P<0.05) (Appendix I). NaOH/OA 
decontamination method was found to have the strongest effect on the fast-growing 
mycobacteria. SDS/NaOH decontamination method however did not have any significant effect 
on fast-growing mycobacteria (P=0.148, F=2.750). 
 
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Table 4. 8: Mycobacterium species identified from the three decontamination methods 
Organism NaOH/OA SDS/NaOH Malachite 
green 
Total 
Mycobacterium avium 4 1 1 6 
Mycobacterium abscessus 1 2 1 4 
Mycobacterium chelonae 14 2 - 16 
Mycobacterium peregrinum - 1 - 1 
Mycobacterium gordonae 1 - - 1 
Mycobacterium malmoense 3 - - 3 
Mycobacterium interjectum 2 2 3 7 
Mycobacterium fortuitum 1 - 5 6 
Total 35 9 11 44 
 
 
 
Table 4. 9: Slow-growing mycobacteria obtained from the decontamination methods 
Decontamination 
methods 
Organism isolated 
 
Mycobacterium 
avium 
Mycobacterium 
gordonae 
Mycobacterium 
malmoense 
Total P-value 
NaOH/OA 4 1 3 8 0.142 
SDS/NaOH 1 - - 1 0.650 
Malachite green 1 - - 1 0.164 
Total 6 1 3 10  
 
 
 
 
 
 
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Table 4. 10: Fast-growing mycobacteria obtained from the decontamination methods 
Decontamination 
methods 
Organism isolated 
 
M. 
abscessus 
M. 
chelonae 
M. 
peregrinum 
M. 
interjectum 
M. 
fortuitum 
Total P-value 
NaOH/OA 1 14 - 2 1 18 0.000 
SDS/NaOH 2 2 1 2 - 7 0.142 
Malachite green 1 - - 3 5 8 0.002 
Total 4 16 1 7 6 34  
 
4.2.3.7 Mycobacterium Species Identified and Type of Media  
In the case of in-house formulated media used, PM (P=0.001) and INH+ Cyclo (P=0.023) were 
found to be the only media that significantly influenced the growth of fast-growing mycobacteria 
(Appendix J). None of the four in-house selective media studied however had a significant effect 
on the slow-growing mycobacteria (P>0.05) at 5% level of significance (Appendix K). 
 
Seven different species were isolated from PM media. Mycobacterium chelonae was the only 
species isolated by all four media. Table 4.11 shows the Mycobacterium species identified from 
the in-house media used. Table 4.12 and Table 4.13 show the fast-growing and the slow-growing 
Mycobacterium species identified from the in- house media respectively. 
 
 
 
 
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Table 4. 11: Mycobacterium species identified and in- house selective media used 
Organism DF INH EB PM Total 
Mycobacterium avium 3 1 - 2 6 
Mycobacterium abscessus 1 - 1 2 4 
Mycobacterium chelonae 1 4 2 9 16 
Mycobactrium peregrinum - - 1 - 1 
Mycobacterium gordonae - - - 1 1 
Mycobacterium malmoense 1 - 1 1 3 
Mycobacterium interjectum 2 2 - 3 7 
Mycobacterium fortuitum - 1 - 5 6 
Total 8 8 5 23 44 
 
 
 
Table 4. 12: Effects of in-house formulated media on fast-growing mycobacteria 
Decontamination 
methods 
Organism isolated 
 
M. 
abscessus 
M. 
chelonae 
M. 
peregrinum 
M. 
interjectum 
M. 
fortuitum 
Total P-value 
DF 1 1 - 2 - 4 0.101 
INH+ Cyclo - 4 - 2 1 7 0.023 
EB+ Cyclo 1 2 1 - - 4 0.398 
PM 2 9 - 3 5 19 0.001 
Total 4 16 1 7 6 34  
 
 
 
 
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Table 4. 13: Effects of in-house formulated media on slow-growing mycobacteria 
Decontamination 
methods 
Organism isolated 
 
Mycobacterium 
avium 
Mycobacterium 
gordonae 
Mycobacterium 
malmoense 
Total P-value 
DF 3 - 1 4 0.142 
INH+ Cyclo 1 - - 1 0.385 
EB+ Cyclo - - 1 1 0.385 
PM 2 1 1 4 0.650 
Total 6 1 3 10  
 
 
 
 
 
 
 
 
 
 
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CHAPTER FIVE 
DISCUSSION 
The aim of the study was to compare decontamination methods for the isolation of 
Mycobacterium species from the environment. Many mycobacterial species that may be the 
cause of important diseases may escape detection and characterization as a result of 
inappropriate decontamination and growth conditions. Contamination of cultures by undesirable 
fast growing bacteria and fungi may hinder the isolation of desirable slow growing mycobacteria. 
More over infections due to NTMs are increasingly becoming more of public health importance 
(Kankya et al., 2011) in both immune-compromised individuals and immune-competent 
individuals. This makes it important to optimize decontamination methods for the isolation of 
non-tuberculous mycobacterial species from clinical and the environment. 
While most studies use genus specific biomarkers such as the heat shock protein for the detection 
of the species of the genus Mycobacterium, this study employed PCR to detect the biomarker 
IS2404 as a first screening procedure. This was done to give an assurance of the possibility of the 
sample containing M. ulcerans.  
The study was bias for M. ulcerans which causes BU, the most important non-tuberculous 
mycobacterial in Ghana and in West Africa. For the past decade, an average of 1,000 BU cases 
are diagnosed in various health facilities mainly in 6 of the 10 regions in Ghana, making BU the 
second most important mycobacterial disease and Ghana the second most BU endemic country 
after Ivory- Coast. At the same time the mode of transmission and the ecology of M. ulcerans is 
not known, a knowledge of which is greatly needed for the design of preventive strategies. 
 
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Thirty-seven of 65 (representing 56.9%) of the total samples collected were confirmed as IS2404 
positive. This was suggestive of the presence of IS2404-containing mycobacteria such as 
Mycobacterium ulcerans (Stinear et al., 1999; Stragier et al., 2007). While IS2404 is used in 
clinical samples for confirmation of M. ulcerans, this is not the case for environmental samples 
as other mycobacterial species such as Mycobacterium liflandii found in the environment contain 
the biomarker IS2404. 
 
Of the IS2404 positive samples, 17/28 (60.7%) were from the endemic community while 20/37 
(54.1%) were from the non-endemic communities. This suggests that IS2404 containing 
mycobacteria are ubiquitous and may be found in a BU non- endemic community in Ghana. In 
similar studies where environmental samples from both the endemic and non-endemic 
communities were analyzed using IS2404, samples from both BU endemic and non-endemic 
communities tested positive, however some studies recorded low  positivity when compared with 
this study (de Vandelannoote et al., (2010); Williamson et al., 2012; Yeboah-Manu et al., 2012). 
The differences in IS2404 positivity rates may be due to differences in sample sources and 
communities sampled. In a sero-epidemiology study by Yeboah-Manu et al. (2012) conducted in 
the same villages sampled in this study; it was found that the individuals in both endemic and 
non-endemic communities along the Densu River were exposed to M. ulcerans. Their findings 
and the findings from this study suggest that other factors such as host genetics, behavioral and 
nutrition may be important in converting sub-clinical infections to overt diseases. 
  
In this study all the five snail samples were IS2404 positive as shown in Table 4.1. Similarly, 
Marsollier et al. (2002) in a study collected ten snails from M. ulcerans endemic community and 
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found that 2 out of the 10 were IS2404 PCR positive. The findings of this study support the 
finding that certain aquatic snails harbor Mycobacterium species such as M. ulcerans after 
consuming aquatic macrophytes. Aquatic snails are the hosts of many organisms responsible for 
human infections such as schistosomiasis. In schistosomiasis snails are considered to be 
intermediate hosts because humans harbour the sexual stages of the parasites and the snails 
harbour the asexual stages. However humans serve as vectors by contaminating the environment 
and in transferring of the infection requires no direct contact between snails and human (Madsen, 
1992). However, more work needs to be done to confirm the role of snail in M. ulcerans 
transmission and ecology. Many of the vegetation samples 12/17 (70.6%) were positive for 
IS2404 PCR in this study (Table 4.1) and this compared with a study by Kazda et al., that 
isolated mycobacteria from plants (Kazda et al., 2009).  
 
Direct smears analysis of the 37 IS2404 PCR positive samples found only 5 (13.5%) samples as 
acid-fast bacilli positive by direct microscopy. The low percentage confirms the low sensitivity 
of Ziehl-Neelsen method compared to PCR (Table 4.1). Polymerase chain reaction has the added 
advantage of multiplying initial copy numbers (DNA) thereby increasing ability to detect 
mycobacteria. Thus, even though mycobacterial load may be low, mycobacteria can be detected 
by PCR. Although PCR has greater sensitivity, it is limiting in viability testing since it detect the 
presence of DNA of an organism which can either be alive or dead. For a sample to be 
microscopy positive, the mycobacterial load must be approximately 104/ ml, thus the low 
recorded positivity rate implies the mycobacterial load in the analyzed samples was low. Real 
time-PCR analysis confirmed that the samples had cycle threshold (CT) values higher than 30 
which implied that the mycobacterial load was low (communication with Mr. Samuel Yaw 
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Aboagye) (unpublished data). This finding probably suggests that while mycobacterial species 
may be ubiquitous in the environment, the load is quite low Three out of five smear positive 
samples were from the non-endemic communities and the other two from the endemic 
community. Mycobacteria were isolated from three out of the five the smear positive samples 
and there were no mycobacterial growth in the other two samples. Similar results were found in 
the previous studies conducted in Ghana, Benin and Ivory-Coast (Marsollier et al., 2002; 
Eddyani et al., 2004; Ngazoa-Kakou et al., 2011). 
Many decontamination methods have been used in the isolation of mycobacteria from both 
environmental and clinical samples but none of them have been universally accepted as the 
standardized method. The most important factors in isolation of mycobacteria from the 
environment are: (1) the decontamination technique and (2) recovery rate of viable mycobacteria 
bacilli. In this study, malachite green/cycloheximide/NaOH, sodium hydroxide/ oxalic acid and 
sodium dodecyl sulphate/sodium hydroxide decontamination methods were evaluated.  A good 
decontamination agent is that which effectively remove unwanted microorganisms 
(contaminants) and maximize recovery of wanted mycobacteria.  Decontamination by 4% NaOH 
followed by a simplified 5% oxalic acid (Yeboah- Manu et al., 2004) gave the highest number of 
total tubes that confirmed mycobacterial growth (42/91, 46.1 %) (Table 4.3). This method also 
gave the least number of contaminated culture tubes (19/97) (Table 4.3).  
In another study, 4% NaOH / 5% OA and H2SO4 were used to decontaminate natural water and 
the results obtained showed that NaOH/OA was the best (Livanainen et al., 1997). In contrast, a 
study by Livanainnen (1995) showed that decontamination with sodium hydroxide - malachite 
green-cycloheximide yielded the highest counts of mycobacteria and a low rate of contamination 
than decontamination with NaOH followed by oxalic acid. A similar result was obtained by 
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Portaels et al. (1988) when soil samples were pre-incubation in tryptic soy broth (TSB), followed 
by decontamination with malachite green, cycloheximide, and NaOH. The present study showed 
that sodium hydroxide/malachite green/cyclohiximide/ decontamination however had the least 
confirmed mycobacterial growth culture positivity rate 19/91(%).  Sodium dodecyl sulphate / 
sodium hydroxide decontamination had the highest number of contaminated cultures 40/97 
(13.5%) (Table 4.3) and least number of Mycobacterium species (Table 4.8). The differences in 
the results may be due to the differences in sample sources.  
Almost all slow growers were obtained from the NaOH/OA. If more samples are decontaminated 
using NaOH/OA, other important slow-growing mycobacteria may be isolated. Furthermore, 
using the data obtained from culture, characterization of the isolates with the available molecular 
tools and the use of ecological data may aid in elucidating the actual source and mode of 
transmission of some NTMs.   
Among the in-house selective media used, L-J containing PANTA and Mycobatin J (PM) was 
the most efficient in supporting the growth of both rapid (Table 4.12) and slow growing 
mycobacteria (Table 4.13).  The present study showed that addition of antifungal (mycobactin J) 
and the antibiotic PANTA to the medium was effective in preventing contamination by fungi and 
fast growing bacteria.  This was the first time both antifungal and antibiotic were used in a 
medium to isolate mycobacteria from different environmental samples. However, in another 
study on soil samples, it was shown that addition of mycobactin did not significantly enhance 
positivity (Portaels et al., 1988). 
Our observation was however supported by another study where the addition of mycobactin for 
the isolation of mycobacteria from specimens of clinical or animals significantly enhanced the 
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positivity and allowed the isolation of some strains which were missed when other media were 
used (Thoen et al., 1979; Portaels et al., 1982, 1985; Portaels et al., 1988). In addition, the 
addition of cycloheximide and INH did also have effect in reducing contamination as well as 
maximizing isolation rate (Table 4.3).  
Isoniazid (INH) and ethambutol (EB) were incorporated in selective media because some studies 
have shown that some NTMs are resistant to isoniazid (INH) and ethambutol (EB) (Portaels., 
1996; Portaels., 1998; Makarova and Freĭman 2009). Therefore addition of these drugs was to 
enhance isolation of such mycobacteria from the environment. From the results obtained, 
medium containing isoniazid supported the growth of four different species of Mycobacterium, 
namely: Mycobacterium chelonae, Mycobacterium interjectum, Mycobacterium fortuitum and 
Mycobacterium avium. Four different species of Mycobacterium were isolated from ethambutol. 
They include: Mycobacterium chelonae, Mycobacterium peregrinum, Mycobacterium abscessus 
and Mycobacterium malmoense (Table 4.11). This confirms our findings (not published) and that 
of other workers that some mycobacteria are resistant to both Isoniazid and ethambutol. 
Hian GenoType Mycobacterium CM assay was used to identify the isolates obtained from the 
study. Forty-four (55.0%) mycobacteria isolates could be identified to the species level, but 
32/80 (40%) confirmed as belonging to the genus Mycobacterium, the species could not be by 
Hian Genotype Mycobacterium CM assay. The assay has been reported to be 100% sensitive and 
94.4% -100% specific for identification of common mycobacteria (Padilla et al., 2004; Tortoli et 
al., 2003 Sarkola et al., 2004; makinen et al., 2002). However, in this study the specificity was 
76/80 (95.0%).  The assay was not discriminatory enough in the identification of some of the 
obtained isolates. Nevertheless Mycobacterium species of clinical importance such as 
Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium abscesscus, 
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Mycobacterium avium, Mycobacterium malmoense and Mycobacterium gordonae were 
identified. However, the assay is rapid, reliable, and easy to perform, it will be best for clinical 
samples. Future work on identification of isolates from the environment needs a more 
comprehensive assay such as sequencing of biomarkers including heat shock protein and 
16SrRNA. 
The data obtained from this study revealed that both fast growing and the slowly growing 
Mycobacterium species can be isolated from the environment. The most frequently occurring 
Mycobacterium species was Mycobacterium chelonae and was isolated from soil and vegetation 
samples (Table 4.5). 
The study also identified some NTM species that are common cause of skin and soft tissue 
infections in humans. These include M. fortuitum, M. chelonae, M. abscessus and M. avium 
(Figure 4.4). The NTMs are increasingly becoming important especially because infections can 
be either community acquired or nosocomial infections.   Mostly, infection occurs following an 
exposure of cut or abraded skin to organisms present in aquariums, pools, natural water supplies, 
vegetation and soil (Feldman, 1974; Wolinsky, 1979; ATS, 1997). A similar study was 
conducted by Thorel et al., (2004) where soil, peat, humus, tufa, sphagnum, and wood were 
collected in alpine and subalpine habitats and M. fortuitum, M. chelonae and Mycobacterium 
malmoense were the most isolated species from soil.  The presence of NTM species may be 
influenced by levels of organic matter in soil and surface water contributing to the mycobacterial 
flora (Parashar et al., 2009). Precaution on protection while working on or with the soil or 
ground by people should be encouraged. Children who play on the ground should be worn long-
sleeved shirts and trousers when outdoors. Cuts and abrasions on people who are always in 
contact with the soil or ground should clean and covered all the time till it heals. 
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Mycobacterium malmoense is one of the most clinically relevant NTM globally and a difficult 
species to isolate because of its exceptionally slow rate of growth and therefore need for special 
culture conditions. However, in this study three isolates were obtained from 2 different soil 
samples with NaOH/OA decontamination method. This confirms that our simplified NaOH/OA 
could be used to isolate Mycobacterium species that are difficult to isolate even though they are 
clinically relevant. Mycobacterium malmoense is the most common NTM isolated from 
suspected TB cases at the bacteriology department of Noguchi memorial institute for medical 
research (communication with Miss. Adwoa Asante-Poku). The increasing isolation from clinical 
cases and also the environment implicates M. malmoense as an important NTM in Ghana.  
 
Mycobacterium avium which was isolated from soil samples in this study also have been 
reported to be isolated from HIV/AIDS patients. It is one of the most significant NTMs 
associated with human diseases, causing disseminated infection in patients with AIDS and other 
pulmonary infections and skin infection (Han et al., 2005). Because NTM diseases in immune-
compromised individuals are mostly disseminated in many organs, concerns about the portal of 
entry of these mycobacteria have been raised as there is no evidence of person-to-person 
transmission of NTM. The environment has been considered a likely source of NTMs (O‘Brien, 
1989; Covert et al., 1999) and findings from this study support this hypothesis. Although there is 
evidence that the environment could be the source of NTMs that infect patients, further studies 
are needed to correlate the relatedness of patients‘ isolates to that of the environment from the 
same community.  
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The major finding from this study is that decontamination with NaOH/OA may increase the odds 
of isolating slow growing Mycobacterium species such as M. ulcerans from the environment. 
This will further be enhanced by inoculation on medium containing INH or PANTA and 
mycobactin J. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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CHAPTER SIX 
CONCLUSION 
This study confirmed the presence of acid-fast bacilli and IS2404 containing mycobacteria in 
both aquatic and non-aquatic sources. The study identified NTMs including Mycobacterium 
chelonae, Mycobacterium fortuitum, Mycobacterium abscesscus, Mycobacterium avium, 
Mycobacterium malmoense, Mycobacterium gordonae, Mycobacterium interjectum and 
Mycobacterium peregrinum in the environment. Decontamination with 4% sodium hydroxide 
and 5% oxalic acid was shown to be the best decontamination method for the isolation of 
environmental mycobacteria. The modified Lowenstein-Jensen media containing isoniazid and 
cycloheximide or PANTA and mycobactin J media when used for isolating mycobacteria from 
the environment can further inhibit the growth of fungi. Four percent sodium hydroxide and five 
percent oxalic acid decontamination method with Lowenstein-Jensen media containing PANTA 
and mycobactin J media may enhance the recovery of slow growing Mycobacterium species.  
 
6.1 Limitation of the Study 
The initial screening process for this study was done using only IS2404-specific biomarker for 
mycobacteria. This however is biased for only IS2404-containing mycobacteria. The use of heat 
shock protein (Hsp) specific for the genus mycobacteria is recommended to increase the isolation 
of mycobacteria from environmental samples. 
 
The line probe hybridization assay was not discriminatory enough to identify all the isolates. 
Therefore, a more comprehensive assay such as sequencing of biomarkers including heat shock 
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protein (hsp 65) and 16S rRNA should be used to obtain isolates identified to their species- 
specific level. 
 
6.2 Recommendation 
Environmental samples of diverse heterogeneity should be considered to test the robustness and 
efficiency of the modified NaOH/OA method.  
Two or more identification methods should be used in order to identify new mycobacteria 
species.  
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX A 
Samples collected from Ntabea I 
Sample Number Type of Sample 
1 Soil from community centre 
2 Sheep dropping 
3 Coconut shell 
4 Lizard dropping 
5 Algae from a house compound 
6 Mud at the bank of river Densu 
7 Decayed leaves at the bank of river Densu 
8 Water skates from river Densu 
9 Soil close to Densu 
10 Moss close to Densu 
11 Leaves in the river 
12 Vegetation along river path 
13 Soil under plantain tree 
14 Web on a plant 
15 Chicken dropping 
16 Fungi 
17 Snail shell 
18 Cocoyam leaves 
19 Millipede on a leaf 
20 Snail from a farm 
 
 
 
 
 
 
 
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Samples collected from Ntabea II 
Sample Number Type of Sample 
21 Water from Densu river 
22 Dead leaves from Densu river 
23 Dark soil around Densu river  
24 Cocoa pod husk 
25 Insects on decayed cocoa husk 
26 Caterpillar  
27 Mud from the Densu river bed 
28 Submerged leaves from river Densu 
28 Soil from farm 
30 Algae from the ground in a house  
31 Lizard dropping 
32 Snail 
33 Millipede 
34 Goat dropping 
35 Water in a bowl from a house 
36 Sheep dropping mixed with palm kernel shell  
37 Sand from school compound 
       
 
Table 3: Samples collected from Ashongkrom 
Sample Number Type of Sample 
38 Sheep dropping 
39 Vegetation along footpath 
40 Dried leaves along footpath 
41 Soil along footpath 
42 Cocoyam leaves 
43 Rotten plantain sucker 
44 Sand from house 
45 Lizard dropping 
46 Snails 
47 Water on a path  
48 Palm husk 
49 Mushrooms  
50 Moss from palm fronts  
51 Bigger snails 
52 Dead leaves in a stream 
53 Stagnant water on the road 
54 Dead leaves along the road 
55 Community swimming water  
56 Green vegetation along road 
57 Community swimming water and sand from the river 
58 Moss from community swimming water and underground water 
59 Snails from community swimming water 
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60 Dead leaves from community swimming water 
61 Termites mounts along the road 
62 Sand along the road 
63 Sand from community square 
64 Lizard dropping from community 
65 Soil from foot path in palm plantation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX B 
Reagents for Ziehl-Neelsen Staining Method 
 
Stock Alcoholic Fuchsin Solution; the stock solution was prepared by dissolving 3g of basic 
fuchsin in a 100 ml of a 95% ethanol. 
Carbol fuchsin solution; this was prepared by dissolving 10 ml of alcoholic fuchsin solution in a 
90 ml of 5% phenol solution. The resultant solution was filtered to remove fuchsin crystals 
before use. 
20% Sulphuric acid solution; 20 ml of concentrated sulphuric acid was slowly added to 80 ml of 
distilled water in a 100 ml volumetric flask.  
0.3% Methylene blue solution; 0.3g of Methylene blue was initially dissolved in a  50 ml 
distilled water contained in a 100 ml volumetric flask. The resultant solution was then topped to 
the mark with distilled water. Again the solution was filtered before use. 
 
 
 
 
 
 
 
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APPENDIX C 
Reagents for Decontamination 
4% Sodium hydroxide (NaOH); Weigh 4grams of sodium hydroxide and dissolve in 100ml of 
distilled water 
2% Sodium hydroxide; Weigh 2 grams and dissolve in 100 ml of distilled water 
0.3% Malachite green; Weigh 0.3 grams and dissolve in 100 ml of distilled water 
5% Oxalic acid (OA); Weigh 5 grams of oxalic acid and dissolve in 100 ml of distilled water. 
3% Sodium dodecyl sulfate (SDS) + 4% NaOH, 100ml 
Weigh 3 grams of SDS and weigh 4 grams of NaOH, dissolved in 100 ml of distilled    water. 
1Normal HCL; Add 8.6 ml of concentrate hydrochloric acid to 91.4 ml of distilled water 
contained in a 100 ml volumetric flask. 
Note: sterilize all the above solutions at 121 degree Celsius for 15min before use. 
Cycloheximide for decontamination; Weigh 0.075grams and dissolve in 50 ml of absolute 
ethanol and filter, dispense into 15ml Falcon tubes. 
 
 
 
 
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APPENDIX D 
Antibiotics for Media Preparation 
Cycloheximide for media; Weigh 150 mg or 0.15 grams +1ml ethanol and filter (For 150 ml 
media, add 0.5ml of cycloheximide) 
PANTA (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin); Add 3ml of 
sterile distilled water aseptically to PANTA (1 bottle). PANTA containing media; (For 1000ml 
of prepared media, add 20ml of PANTA) 
Mycobactin J; Dissolve mycobactin J by adding 0.5 ml of absolute ethanol (For 1000 ml of 
prepared media add all the 0.5ml dissolved mycobactin J) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX E 
ANOVA for performance of in-house formulation media and the decontamination 
methods. 
 
 
Sum of 
Squares 
df Mean Square F-ratio P-value 
NaOH Positive 
culture 
Between Groups 133.375 3 44.458 355.667 0.000 
Within Groups .500 4 .125   
Total 133.875 7    
NaOH No 
bacterial growth 
Between Groups 94.375 3 31.458 83.889 0.000 
Within Groups 1.500 4 .375   
Total 95.875 7    
NaOH 
Contamination 
Between Groups 44.375 3 14.792 39.444 0.002 
Within Groups 1.500 4 .375   
Total 45.875 7    
SDS/NaOH 
Positive culture 
Between Groups 8.500 3 2.833 11.333 0.020 
Within Groups 1.000 4 .250   
Total 9.500 7    
SDS/NaOH No 
bacterial growth 
Between Groups 34.000 3 11.333 22.667 0.006 
Within Groups 2.000 4 .500   
Total 36.000 7    
SDS/NaOH 
Contamination 
Between Groups 58.500 3 19.500 78.000 0.001 
Within Groups 1.000 4 .250   
Total 59.500 7    
Malachite 
Positive culture 
Between Groups 18.375 3 6.125 16.333 0.010 
Within Groups 1.500 4 .375   
Total 19.875 7    
Malachite    No 
bacterial growth 
Between Groups 36.375 3 12.125 32.333 0.003 
Within Groups 1.500 4 .375   
Total 37.875 7    
Malachite 
Contamination 
Between Groups 18.375 3 6.125 16.333 0.010 
Within Groups 1.500 4 .375   
Total 19.875 7    
 
 
 
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APPENDIX F 
Multiple Comparisons of the performance of in- house media used in the isolation of 
mycobacterium species. 
Dependent Variable (I) Medium (J) Medium Mean 
Difference 
(I-J) 
Std. Error P-value. 95% Confidence Interval 
Lower 
Bound 
Upper 
Bound 
Culture positive 
DF 
INH + Cyclo 1.00000 2.50555 .700 -4.7778 6.7778 
EB + Cyclo 8.33333* 2.50555 .010 2.5555 14.1111 
PM -3.66667 2.50555 .182 -9.4445 2.1111 
INH + Cyclo 
DF -1.00000 2.50555 .700 -6.7778 4.7778 
EB + Cyclo 7.33333* 2.50555 .019 1.5555 13.1111 
PM -4.66667 2.50555 .100 -10.4445 1.1111 
EB + Cyclo 
DF -8.33333* 2.50555 .010 -14.1111 -2.5555 
INH + Cyclo -7.33333* 2.50555 .019 -13.1111 -1.5555 
PM -12.00000* 2.50555 .001 -17.7778 -6.2222 
PM 
DF 3.66667 2.50555 .182 -2.1111 9.4445 
INH + Cyclo 4.66667 2.50555 .100 -1.1111 10.4445 
EB + Cyclo 12.00000* 2.50555 .001 6.2222 17.7778 
No bacterial growth 
DF 
INH + Cyclo -2.00000* .47140 .003 -3.0871 -.9129 
EB + Cyclo -15.00000* .47140 .000 -16.0871 -13.9129 
PM -11.00000* .47140 .000 -12.0871 -9.9129 
INH + Cyclo 
DF 2.00000* .47140 .003 .9129 3.0871 
EB + Cyclo -13.00000* .47140 .000 -14.0871 -11.9129 
PM -9.00000* .47140 .000 -10.0871 -7.9129 
EB + Cyclo 
DF 15.00000* .47140 .000 13.9129 16.0871 
INH + Cyclo 13.00000* .47140 .000 11.9129 14.0871 
PM 4.00000* .47140 .000 2.9129 5.0871 
PM 
DF 11.00000* .47140 .000 9.9129 12.0871 
INH + Cyclo 9.00000* .47140 .000 7.9129 10.0871 
EB + Cyclo -4.00000* .47140 .000 -5.0871 -2.9129 
Contamination 
DF 
INH + Cyclo 5.00000* .47140 .000 3.9129 6.0871 
EB + Cyclo 7.00000* .47140 .000 5.9129 8.0871 
PM 15.00000* .47140 .000 13.9129 16.0871 
INH + Cyclo 
DF -5.00000* .47140 .000 -6.0871 -3.9129 
EB + Cyclo 2.00000* .47140 .003 .9129 3.0871 
PM 10.00000* .47140 .000 8.9129 11.0871 
EB + Cyclo 
DF -7.00000* .47140 .000 -8.0871 -5.9129 
INH + Cyclo -2.00000* .47140 .003 -3.0871 -.9129 
PM 8.00000* .47140 .000 6.9129 9.0871 
PM 
DF -15.00000* .47140 .000 -16.0871 -13.9129 
INH + Cyclo -10.00000* .47140 .000 -11.0871 -8.9129 
EB + Cyclo -8.00000* .47140 .000 -9.0871 -6.9129 
*. The mean difference is significant at the 0.05 level. 
 
 
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APPENDIX G 
Performance of in-house media for isolation of mycobacteria 
Source of Variation Sum of 
Squares 
df Mean Square F-ratio P-Values 
Culture 
positive 
Between Groups 227.583 3 75.861 8.056 0.008 
Within Groups 75.333 8 9.417   
Total 302.917 11    
No bacterial 
growth 
Between Groups 462.000 3 154.000 462.000 0.00001 
Within Groups 2.667 8 .333   
Total 464.667 11    
Contamination Between Groups 350.250 3 116.750 350.250 0.00001 
Within Groups 2.667 8 .333   
Total 352.917 11    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX H 
ANOVA showing the effects of decontamination methods on slow growing mycobacteria. 
 
Source of variation 
(Decontamination methods) 
Sum of Squares df Mean Square F-ratio P-value 
NaOH/OA 
Between Groups 4.000 2 2.000 4.000 0.142 
Within Groups 1.500 3 .500   
Total 5.500 5    
SDS/NaOH 
Between Groups .333 2 .167 1.500 0.650 
Within Groups 1.000 3 .333   
Total 1.333 5    
Malachite green 
Between Groups 2.333 2 1.167 3.500 0.164 
Within Groups 1.000 3 .333   
Total 3.333 5    
 
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX I 
ANOVA showing the effects of decontamination methods on fast growing mycobacteria 
Source of variation 
(Decontamination methods) 
Sum of Squares Df Mean Square F-ratio P-value 
NaOH/OA 
Between Groups 237.600 4 59.400 148.500 0.000 
Within Groups 2.000 5 .400   
Total 239.600 9    
SDS/NaOH 
Between Groups 4.400 4 1.100 2.750 0.148 
Within Groups 2.000 5 .400   
Total 6.400 9    
Malachite green 
Between Groups 31.400 4 7.850 26.167 0.002 
Within Groups 1.500 5 .300   
Total 32.900 9    
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX J 
Effects Of In -House Selective Media on Fast Growing Mycobacteria 
Source of variation 
Selective Medium 
Sum of Squares Df Mean Square F-ratio P-value 
DF 
Between Groups 5.600 4 1.400 3.500 0.101 
Within Groups 2.000 5 .400   
Total 7.600 9    
INH+Cyclo 
Between Groups 12.400 4 3.100 7.750 0.023 
Within Groups 2.000 5 .400   
Total 14.400 9    
EB+Cyclo 
Between Groups 2.000 4 .500 1.250 0.398 
Within Groups 2.000 5 .400   
Total 4.000 9    
PM 
Between Groups 80.000 4 20.000 40.000 0.001 
Within Groups 2.500 5 .500   
Total 82.500 9    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX K 
Effects of In-House Selective Media on Slow Growing Mycobacteria 
 
Source of variation 
Selective medium 
Sum of Squares df Mean Square F-ratio P-value 
DF 
Between Groups 4.000 2 2.000 4.000 0.142 
Within Groups 1.500 3 .500   
Total 5.500 5    
INH+Cyclo 
Between Groups 1.333 2 .667 1.333 0.385 
Within Groups 1.500 3 .500   
Total 2.833 5    
EB+Cyclo 
Between Groups 1.333 2 .667 1.333 0.385 
Within Groups 1.500 3 .500   
Total 2.833 5    
PM 
Between Groups .333 2 .167 .500 0.650 
Within Groups 1.000 3 .333   
Total 1.333 5    
 
 
 
 
 
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