CHAPTER carried out to evaluate the anti-tuberculosis potential of

CHAPTER 1: ABSTRACT

In
view of urgent need for new tuberculosis (TB) drugs, which were effective,
cheaper and readily available from environment, thus research was carried out
to evaluate the anti-tuberculosis potential of metabolites from mangrove
actinobacteria against surrogate TB organisms (M.smegmatis. M. fortuitum and
M. kansasii) from Pulau Betong, Penang.
31 strains of actinomycetes were isolated and tested against surrogate TB
organisms in primary screening by using cross streak method. In secondary
screening, crude extract was produced through fermentation method, extracted by
organic solvent extraction method, and tested against surrogate TB organisms by
agar well diffusion method. The minimum inhibitory concentration (MICs) of the
crude extract against surrogate TB organisms were within 200 – 3200 ?g/mL. The highest activity was shown by crude extract against M.
kansasii with MIC of 200 ?g/mL. The findings of this study revealed
that mangrove actinomycetes appeared to have immerse potential source of antitubercular
compounds worthy of further investigation.

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CHAPTER
2: INTRODUCTION

Tuberculosis
(TB) which caused by Mycobacterium
tuberculosis was a highly prevalent infectious disease that resulted in
morbidity and mortality in human with almost one-third of global population
believed to be infected (Bratati & Ganguly, 2013; Manikkam, Venugopal,
Subramaniam, Ramasamy, & Kumar, 2014; Sanei Taheri et al., 2015). TB still remains as a major caused
of global public health problem (Sharma & Mohan, 2013). Based on Centers
for Disease Control and Prevention (CDC), in 2016, 10.4 million people around
the world became sick with TB disease and 1.7 million TB-related deaths
worldwide. TB was a leading killer of people who were HIV infected as the virus
weakens a person’s immune system against TB germs.

The
epidemiology of TB had become more serious due to the emergence of drug resistance among M.
tuberculosis isolates and long-term
therapy
using combination of drugs for its treatment (Manikkam et al., 2014). Hence, new antitubercular
drugs to fight against drug resistant M.tuberculosis strains were urgently
needed (Ginsberg, 2010). Less side
effects and improved pharmacokinetic properties were expected from new anti-TB
drugs with extensive and potent activity against drug resistant strains and
ability to reduce the total duration of treatment (De Souza, 2013).

      Actinomycetes were
common soil inhabitant with excellent producers of novel antimicrobial agents
which had the ability to produce exuberant secondary metabolites with
biological significance (Abd-Elnaby,
Abo-Elala, Abdel-Raouf, Abd-elwahab, & Hamed, 2016; Rajan & Kannabiran,
2014; Xu, Ye, Han, Deng, & Hong, 2014). Out of 50% of the total microbial
bioactive metabolites were reported from the members of actinomycetes (Bibb,
2013). In addition, the discovery of
streptomycin shown that the first antibiotic used for antitubercular therapy
was from Streptomyces griseus, and numerous antitubercular antibiotics
such as kanamycin and rifampicin were produced from actinomycetes of
terrestrial origin. However, the survey of Streptomyces
and other common terrestrial actinomycetes were nearly exhausted.

Recently, isolation of
known actinomycetes and antibiotic were frequently reported due to the exploration
of actinomycetes from routine ecosystems but bioprospecting of less explored
ecosystem such as mangrove had been proved consist a high number of bioactive
compound from novel bioactive actinomycetes (Bibb, 2013) including antitubercular
metabolites (Wang et al., 2013).

CHAPTER 3: LITERATURE REVIEW

3.1
TUBERCULOSIS

3.1.1 Overview

Tuberculosis
(TB) was an infectious respiratory disease which was one of the oldest known
human diseases that was still becoming the major causes of mortality caused by M.
tuberculosis (Smith, 2003). This causal pathogen was spread
through airborne droplets and easily infected individuals based on the frequency
of contact with infected person, living in crowd population or unhygienic environments,
and being an immunocompromised person.

There
were two types of TB disease that occurred in two different sites which are at pulmonary
and extrapulmonary organs. TB disease that infected at the pulmonary site known
as Pulmonary TB (PTB) refers to TB disease that affected the lungs with common
signs such as cough, chest radiograph abnormality and may be infectious. While,
TB disease that occurred in other parts of the body such as the brain, kidneys,
larynx, lymph node, bones, or pleura known as Extrapulmonary TB (EPTB). However,
in human immunodeficiency virus (HIV)-infected person, both EPTB and PTB diseases
can occurred. As long as the person was not having PTB, EPTB was not infectious.
Centers for Disease Control and Prevention (CDC) stated that EPTB can also
occurred in the oral cavity, or involved an open lesion with high concentration
of organisms.

Miliary
TB, a rare type of TB was a widespread lymphohematogenous dissemination of M.
tuberculosis, which was a lethal disease if not treated early (Ray, Talukdar, Kundu, Khanra, & Sonthalia, 2013). Tuberculous
meningitis was another form of TB disease that occurred when the tissues around
the brain and spinal cord were infected with TB.

 

3.1.2 Current Statistics of
Tuberculosis Infection

Reported
Tuberculosis in the United States, 2016. Atlanta, by US
Department of Health and Human Services, Centers for Disease Control and
Prevention, CDC stated that a total of 9,272 TB cases (a rate of 2.9 cases per
100,000 persons) are reported in the United States in 2016. This is a decrease
from the number of cases reported in 2015 and the lowest case count on record
in the United States. The case rate of 2.9 per 100,000 persons is a 3.6%
decrease from 2015. Even though the United States continues to make slow
progress, current strategies are not enough to reach the goal of TB elimination
in this century.

According
to World Health Organization, 2015, about 9.6 million new cases of TB were
recorded and 1.5 million patients died, in which, 1.1 million and 0.4 million
were HIV-negative and HIV-positive patients, respectively in 2014. For
multidrug-resistant TB (MDR-TB) cases, 480 000 cases reported, and 190 000 peoples
were estimated died from MDR-TB. India, Indonesia and China accounted the
largest number of cases of the global total with 23 %, 10 %, and 10 %,
respectively.

 

3.1.3 Current Treatment of Tuberculosis

Tuberculosis
disease was when the TB bacteria became actively multiplying in the body and disrupted
the immune system function to stop the bacteria from continuously growing.  It was very important that people who had infected
with TB disease to be treated, finished the medicine, and took the drugs
exactly as prescribed, otherwise they became sick again and more crucial if the
TB bacteria started to develop resistant against those drugs which will be
harder and more expensive to be treated.

By
taking several drugs for 6 to 9 months, probability of the TB disease to be
treated was high.  There were 10 drugs currently approved by the U.S. Food
and Drug Administration (FDA) for treating TB. Isoniazid (INH), rifampin (RIF),
ethambutol (EMB), and pyrazinamide (PZA) were the approved drugs that acted as
the first-line antitubercular agents that form the core of treatment regimens.

 

3.1.4 Drug- Resistant Tuberculosis

Incidence
of TB becomes worst with the emergence of multi-drug resistant (MDR) and
extensively-drug resistant (XDR) strains of M. tuberculosis worldwide
make the incidence of TB became worst as the M. tuberculosis started to develop resistance against
both the first-line and second-line anti-TB drugs (Gupta
et al., 2010; Singh, 2007). Strains that resistant to at least one
of the first-line drugs of isoniazid or rifampicin were known as MDR strains, while
the strains that resistant to both isoniazid and rifampicin, fluoroquinolone
and to at least one of the three injectable second-line drugs such as amikacin,
capreomycin or kanamycin were known as XDR strains. (Galagan,
2014).
The drug-resistant TB was very complicated to treat and
cure, and may resulted into inappropriate management that can led to life-threatening.

 

3.2  ACTINOMYCETES AS A SOURCES OF
ANTI-TUBERCULAR AGENTS

3.2.1
Properties of Actinomycetes

Actinomycetes
were Gram positive filamentous bacteria consisted of high guanosine-cytosine
(GC) and classified to the phylum Actinobacteria (M Goodfellow & Williams, 1983). Most
actinomycetes are slow-growing bacteria and they form well-developed radial
mycelium, which can be divided into aerial mycelium and substrate mycelium on
isolation plates based on morphology and function (Chen, 2015; M Goodfellow & Williams, 1983). The mycelium may
break apart to form rod or coccoid shaped forms in some species. Many genera
also form spores where the sporangia, or spore cases, may be found on aerial
hyphae, on the colony surface, or free within the environment.

Actinomycetes
were the intermediate group between bacteria and fungi due to the present of a
filamentous degree of organization like filamentous fungi (Chen, 2015). Their hyphae were
similar to fungi, and their widths of the mycelium are quite similar to the
width of bacterial cell. However, actinomycetes had been classified as bacteria
since they did not have chitin and cellulose in the cell walls which were often
present in fungi.

 

3.2.2 Antitubercular Potential 

Actinomycetes
hold the important position in production of bioactive metabolites which were responsible
for the production of almost half of the discovered bioactive secondary metabolites
(Abd-Elnaby et al., 2016; Bérdy, 2005; Rajan &
Kannabiran, 2014; Xu et al., 2014). More than 10,000 antibiotics had
been isolated from actinomycetes until today (Kekuda, Shobha, & Onkarappa, 2010).

Further
characterization of these actinomycetes had led to isolation and identification
of novel bioactive compounds with significant therapeutic potential. So far, only
3 % of all antibacterial agents had been reported synthesized by Streptomyces (Watve, Tickoo, Jog, & Bhole, 2001). Actinomycetes
regained its position as the center of antibiotic interest due to the in
concomitant of the information and emerging problem of multi drug resistance (MDR) and new pathogens development
which inactivated the antibiotics and rendered an urgent need for new
antibiotics that would target the emerging multidrug resistance.

 

CHAPTER 4: DISCUSSION

Sediment
samples collected from Pulau Betong, Penang mangrove forest were divided into
two parts as wet samples and dry samples. Wet samples were directly diluted by
using serial dilution method while dry samples were allowed to air-dry at room
temperature for a week before serially diluted. Results showed the number of
isolates from the dry samples were higher than wet samples.

All
31 isolates of actinomycetes were screened for their bioactive compound
production ability and 13 isolates was showed wide range of zone of inhibition
against surrogate TB organisms in the primary screening. Primary screening
methods like cross streak or cross spot method were not suitable for highly
biohazardous organism like M.
tuberculosis thus surrogate TB organisms were used.

 During fermentation process, more yield of
crude extract was produced through solid state fermentation method compared to
submerged state fermentation method when extracted with methanol and ethyl
acetate solvents. The production of higher crude extract in solid state
fermentation was due to the lack of water and completely miscible in organic
solvent (ethyl acetate and methanol) with the fermented biomass. These results
were similar as previous reports (El-Naggar, El-Assar, & Abdul-Gawad, 2009; Mahdi,
Termeh, Ehsan, Bahman, & Elnaz, 2012). The low yield production resulted from
the submerged state fermentation method was due to the use of water immiscible
solvent such as ethyl acetate during extraction.

The
MIC of the crude extracted from the actinomycetes were determined by using TEMA
method against the surrogate TB organisms by observing the color change of MTT
tetrazolium from yellow to purple due to the oxidation of the reagent. The
viable cells with active metabolism converted MTT into a purple color formazan
product. The color remained unchanged when the cells were died as they were unable
to convert MTT into formazan, thus, color formation serves as a marker for the
viable cells. In this study, the crude extract showed a wide range of activity
towards the test organisms with MIC values in the range of 200 – 3200 ?g/mL.
The highest activity was shown by crude extract against M. kansasii with
MIC of 200 ?g/mL. Therefore, the crude extract could be potent sources of
antitubercular drug production, which will lead to the development of novel
drugs for the tuberculosis treatments.

Regarding
the tested surrogate TB organisms strain, the results showed that the
slow-growing M. kansasii was more susceptible than the fast-growing M.
fortuitum and M. smegmatis. These results indicated that the
cellular growth rate could influenced the inhibition whereby the slow grower
had a longer doubling time compared to the fast grower. Fast grower organisms had
a weaker defense ability, thus when the cells were inoculated into a new
medium, they may be lacked in essential enzymes or other components involved in
defense mechanism, so as they took time to synthesize these components, they
were more susceptible to chemical attack. Besides that, the ultra-structure of
these organisms may differ from each other especially on their unique cell
walls, which could had contributed to their defense ability. A previous study
had reported that cell wall thickness was significantly different between
resistant and non-resistant strains of Mycobacterium (Velayati et al., 2009).

CHAPTER 5: CONCLUSION

This study concluded that actinomycetes isolated from
mangrove sediment were priceless natural resources, having immerse
antitubercular potential to treat tuberculosis as they exhibited inhibitory
activity against test Mycobacterium species, M. smegmatis, M.
fortuitum and M. kansasii with MICs of 200 – 3200 ?g/mL. The highest
activity was exhibited by crude extract against M. kansasii with MIC of
200 ?g/mL. In general, crude extract were complex mixture of compound. However,
in this study, only the crude extracts were evaluated and found to be active
against surrogate TB organisms, so additional research like bioassay guided
fractionation and characterization were needed to validate whether a single
useful compound can be found, and it was also needed to determine the MIC and
meaningful toxicity and specificity studies.