Determination of in Vitro Inhibitory Activity of Mycolic Acid Inhibitors To Clinical Isolates of Bacteria and Mycobacterium Tuberculosis

LIFE: International Journal of Health and Life-Sciences
ISSN 2454-5872
Shashikant Vaidya et al.

Special Issue, 2015, pp. 37-54
DETERMINATION OF IN VITRO INHIBITORY ACTIVITY OF

MYCOLIC ACID INHIBITORS TO CLINICAL ISOLATES OF
BACTERIA AND MYCOBACTERIUM TUBERCULOSIS
Shashikant Vaidya
Department of Clinical Pathology, Haffkine Institute for Training, Research and
Testing, Mumbai, India,shashikantvaidya@hotmail.com
Kapil Punjabi
Department of Clinical Pathology, Haffkine Institute for Training, Research and Testing,
Mumbai, India,kapil9372@gmail.com
Shreyasi Mulye
Haffkine Bio-pharmaceutical Corporation Ltd. Mumbai, Indiashreyasi.mulye@gmail.com
Geeta Koppikar
Breach Candy Hospital Trust, Mumbai, Indiainfo@breachcandyhospital.org
Mohan Kulkarni
T.N. Medical College and B.Y.L. Nair Charitable Hospital, Mumbai, India
Abhay Chowdhary
Grant Government Medical College and Sir J.J.Hospital, Mumbai,
Indiaabhaychowdhary@yahoo.com
Abstract
Today tuberculosis (TB) has become the most important communicable disease and a leading
cause of death in the world. The emergence of drug resistance has become the main threat to TB
treatment and control programs. The presence of mycolic acids in cell wall gives Mycobacterium
tuberculosis (M. tuberculosis) many characteristics that defy medical treatment, increased
resistance to chemical damage and dehydration, and prevent the effective activity
of hydrophobic antibiotics. In addition, the mycolic acids allow the bacterium to grow readily
inside macrophages, effectively hiding it from the host's immune system.In this study, in vitro
2015 The author and GRDS Publishing. All rights reserved.
Available Online at: http://grdspublishing.org/LIFE/life.html
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ISSN 2454-5872
inhibitory activity of mycolic acid inhibitors namely Triclosan (TCN), Thiolactomycin (TLM) and
Isoxyl (ISO) was determined against of 87 bacterial clinical isolates and 20 clinical isolates of
M.tuberculosis by agar dilution method. Significant difference was observed in the inhibitory
activity of TCN, TLM and ISO to allbacterial strains. Clinical isolates of Pseudomonas species
were found to be more resistant to TCN than other bacterial strains. While clinical isolates of
Staphyllococcusaureus andKlebsiella species were found to be most susceptible to TCN.
Inhibitory activity of ISO against clinical isolates of M. tuberculosis in both, susceptible and
MDR strains were seen. For susceptible strains, the MIC of ISO ranged from 0.3 - 2.5 mcg/ml
and for MDR strains it was found to in the range of 1.2 20 mcg/ml.TCN was found to be better
mycolic acid inhibitor compound in inhibiting test organisms amongst three compounds. ISO
was found to be least active against bacterial isolates, but was effective against susceptible as
well as MDR strains of M. tuberculosis
Keywords
Drug susceptibility, mycobacterium tuberculosis, ISO
1. Introduction
Tuberculosis (TB), an old, highly infectious disease, declared a global health
emergency by the World Health Organization (WHO) in 1993, is still the second leading
killer in the world, with an approximate 2 billion people being latently infected (World Health
Organization, WHO Global Tuberculosis Report 2014).
Developing new antituberculosis drugs is an expensive exercise and TB is not a disease
of rich nations. Some development projects are underway, but more are needed. TB still remains
a neglected disease in relation to drug development (Chopra et al., 2003).
A number of approaches are considered to identify targets for novel antimycobacterial
agents. They range from biochemical studies of essential pathways to the use of genome scale
tools such as transposon mutagenesis, proteomics and transcript mapping on micro arrays. In
combination with modern approaches, such as structure based drug design and combinatorial
chemistry will lead to the development of new drugs that are not only active against drug
resistant TB, but shorten the chemotherapy schedule. Several targets have been identified and
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ISSN 2454-5872
combination of high throughput screening and rational structure based design is being used to
identify lead molecule (Chopra et al., 2003).
Current chemotherapy for TB relies on Mycobacteria specific drugs that inhibit bacterial
metabolism with a heavy emphasis on inhibitors of the cell wall polymer. Mycobacteria have
conventionally been considered to be surrounded by a thick waxy coat of lipid. Such a coat
around Mycobacterial cells could explain limited permeability, their physical toughness and their
rather general insusceptibility to toxic substances. The Mycobacterial cell wall has a unique
structure comprised of three covalently attached macromolecular structures: arabinogalactan,
peptidoglycan and mycolic acids (Besra et al., 1995). These critical components of the cell
envelope have been the subject of intense research for a number of years because of the fact that
enzymes involved in their biosynthetic pathways including mycolic acids, offer attractive and
selective targets for the development of novel antimycobacterial agents (Brennan, 2002).
Mycolic acids are high molecular weight, alfa-alkyl and beta-hydroxy fatty acids. In
Mycobacterial cell envelope, they are present in free lipids, they are trehalosedimycolate and
trehalosemonomycolate and for the most part esterified to the terminal pentaarabinofuranosyl
units of arabinogalatan and hence part of the backbone of Mycobacterium cell wall (Minnikin,
1982).
They are the most characteristic & essential component of the cell walls of Mycobacteria
and related genera (Besra et al., 1995).
The drugs shown to inhibit mycolic acid biosynthesis are Isoniazid (INH), Ethionamide
(ETH), Isoxyl (ISO) (1,3-bis[4-(3-methylbutoxy)phenyl]theorem), Thiolactomycin (TLM)
((4R)(2E,5E)-2,4,6-trimethyl-3-hydroxy- 2,5,7-octatriene-4-thiolide), and Triclosan ( TCN) (5chloro-2-(2,4-dichlorophenoxy)phenol). In addition, Pyrazinamide (PZ) has been shown to
inhibit fatty acid synthase type I which, in turn, provides precursors for fatty acid elongation to
long-chain mycolic acids by fatty acid synthase II (Schroeder, de Souza, Santos, Blanchard, &
Basso, 2002).
Recently, mycolic acid inhibitors like TLM, TCN and ISO have been reported to have
potent activity against MDR strains of M.tuberculosis (Kremer et al., 2000; Phetsuksiri et al.,
1999; Slayden, et al., 2000).TLM is a unique thiolactone that has been shown to exhibit
antimycobacterial
activity
by
specifically
inhibiting

fatty
acid
and
mycolic
acid
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ISSN 2454-5872
biosynthesis(Kremer et al., 2000).TCN is a biphenyl ether derivative known as 2, 4, 4-Trichloro2-hydroxydiphenyl ether is active against wide range of gram positive and gram-negative
bacteria(Suller & Russell, 2000). It inhibits mycolic acid synthesis and is active against M.
tuberculosis
(Slayden
et
diisoamyloxythiocarbanilide)
al.,
2000).ISO;
demonstrated
potent
thiourea
activity against
(thiocarlide-4,
standard
of
strain
M.tuberculosis (Phetsuksiri et al., 1999).It had been noted that it strongly inhibited mycolic acid
synthesis in M.bovis (Winder, 1982).
There is a need to explore in vitro activities of these compoundsfurther. Hence, in vitro
inhibitory activities of these compounds were determined in the present study against clinical
isolates of bacteria like Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Klebsiella
species, Pseudomonas species and Mycobacterium tuberculosis (M. tuberculosis).
2. Material and Methods

MIC pattern of mycolic acid inhibitors namely TCN(IPCA laboratories Ltd, India), TLM
(National Institute of Health., U.S.A.) and ISO (Cayman Chemical Co., U.S. A)To clinical
isolates of bacteria was determined by agar dilution method (Hawkins, et ak., 1991; Tomioka, et
al., 1993). Total 87 Clinical isolates of bacteria, pre-identified by various biochemical
tests(Thierry et al., 1990)were included in the study.
Out of which 21 strains were of
Klebsiellaspp, 24 strains of E. coli, 20 strains of S.aureus and 22 strains of Pseudomonas spp.

These strains were collected from Department of Microbiology, T.N. Medical College and
B.Y.L. Nair Charitable Hospital, Mumbai. Two fold dilutions of individual drug namely TCN,
TLM and ISO were prepared in sterile water for injection after dissolving it in suitable diluents.
1ml of drug solution (20x) was mixed with 19 ml of sterile molten antibiotic testing medium
(HiMedia Pvt Ltd., India) and poured in sterile petri plates. Bacterial suspension
(105organisms/ml) in 5-microlitre quantities was spotted on agar plates containing various drug
concentrations. The control plates, with no drug was also inoculated all plates were incubated at
37C for 24 hours. The MICs were defined as the minimum concentration of drug that
completely inhibited the growth of the test organism or allowed growth of not more than five
colonies.
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Further MIC pattern of TCN, TLM and ISO to clinical isolates of. Tuberculosis was
determined by agar dilution method. (Hawkins et al., 1991).Total 10 MDR strains, 10 susceptible
strains of M. tuberculosis and standard strain of M. tuberculosis H37RV were included in the
study. These strains were collected from Department of Microbiology of P.D. Hinduja Hospital
and Medical Research Centre, Mumbai. These clinical isolates were tested for drug susceptibility
testing by Bactec460 TBsystem(Siddiqui, et al., 1981)and their drug resistance patterns were
ascertained. Two fold dilutions of individual drugs namely TCN, TLM and ISO were prepared in
sterile water for injection after dissolving it in suitable diluents.1ml of test drug solution (20X)
was mixed with 19 ml of sterile molten Middlebrook 7H11 medium
with OADC
supplements(HiMediaLaboratories Pvt Ltd., India) and poured in sterile Petri plates. Drugs
concentrations (mcg/ml) used were as follows:
ISO: 0.07, 0.15, 0.3, 0.6, 1.2, 2.5, 5, 10, 20 and 40
TCN: 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4
TLM: 0.15, 0.3, 0.6, 1.2, 2.5, 5, 10, 20, 40 and 80

Test strains were inoculated in sterile Dubos broth with glucose and albumin supplement
with 0.05% Tween 80 (HiMediaLaboratories Pvt Ltd., India) and incubated at 370c for 7 to 10
days. The cultures were adjusted to an optical density of 0.1 at 540 nm and then diluted 10 fold
in 0.1% Tween 80 containing normal saline. Bacterial suspension in 5-microlitre quantities was
spotted on agar plates containing various drug concentrations.
The control plates with no drug were also inoculated along with it. Plates were incubated
at 370c for 14 days. The MICS were defined as the minimum concentrations of drugs that
completely inhibit the growth of the test organism or allowed growth of not more than five
colonies(Hawkins et al., 1991).

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3. Results
MIC pattern of S. aureus strains
MIC value (mcg/ml)
45
40
35
30
25
20
15
10
5
0
TCN
TLM
ISO
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Number of strains
Figure 1: MIC pattern of S. aureus strains
The results indicate that MIC of TCN for all strains of S. aureuswas 0.07mcg/ml,
whereas for TLM, the MIC values ranged from 0.07-10 mcg/ml, and for ISO, it ranged from
0.07-40mcg/ml.[Figure 1]
MIC pattern of E.coli strains
MIC value ( mcg/ml)
12
10
10
10
10
10
10
10
10
10
10
10
10
TCN
8
TLM
6
5
ISO
4
2.5
2
0
0.3 0.15 0.07 0.07

0.15 0.15 0.15 0.15 0.07
0.15 0.07
0
2.5
0.3 0.15 0.07 0.07

0.3
0.15 0.15 0.15 0.15 0.15 0.15 0.07
0.15 0.07
0
2.5
2.5
0.15 0.15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Number of strains
Figure 1: MIC pattern of E. coli strains
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ISSN 2454-5872
The results indicate, MIC of strains of E. coli for TCN ranged from 0.07-10mcg/ml,
whereas for TLM, MIC values ranged from 0.07-10 mcg/ml, and for ISO, it ranged from 0.075mcg/ml.[Figure 3.2]
MIC value(mcg/ml)
MIC pattern of Klebsiella strains

45
40
35
30
25
20
15
10
5
0
40
40
40
TCN
TLM
10
5
10
5
ISO
10
5
0.15
0.15
0.15
0.07 0.15 0.15
0.07 0.3
0.07 0.07 0.3
0.07 0.15 0.07 0.15 0.15
0.07 0.3
0.07 0.07 0.3
0.07 0.15 0.07 0.15 0.15
0.07 0.3
0.07 0.07 0.3
0.07 0.15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Number of strains
Figure 2: MIC pattern of Klebsiella strains
The results indicate that, MIC for TCN for all strains of Klebsiellasppswas in range of
0.07mcg/ml, whereas for TLM the MIC values ranged from 0.07-10 mcg/ml, and for ISO,
ranged from 0.07-40mcg/ml.[Figure 3]

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ISSN 2454-5872
MIC value (mcg/ml)
MIC pattern of Pseudomonas strains

45
40
35
30
25
20
15
10
5
0
40
40
40
TCN
TLM
20
20
20
20
ISO
10
10
10
10
5
0.5 0.15 0.15

0.15 0.07
10
10
10
10
10
0.3 0.07 0.15 0.15 0.15

0.3
0.15
0.07
10
10
0.07 0.15 0.07
5
0.3 0.15 0.15
0.3 0.15
0.07
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Number of strains
Figure 3: MIC pattern of Pseudomonas strains
The results indicate that, MIC of strains of Pseudomonas sppfor TCN ranged from 0.0710mcg/ml, whereas for TLM, the MIC values ranged from 0.07-10 mcg/ml, and for ISO, ranged
from 0.07-40mcg/ml.[Figure 4]
Table 3.1: MIC pattern of Mycolic acid inhibitors to all bacterial isolates
Sr.no.
1
2
3
Mycolic acid inhibitor

TCN
TLM
ISO
Geometric Mean MIC ( mcg/ml)

0.09
4.55
7.40
TCN showed lowest MIC followed by TLM. ISO showed highest value of MIC against bacterial
isolates.[Table 3.1]
Table 3.2: MIC pattern of Mycolic acid inhibitors to all bacterial isolates in combination
Group
I
II
III
Mycolic acid
inhibitor
TCN
TLM
TCN
ISO
TLM
Geometric
Mean MIC
( mcg/ml)
0.09
4.55
0.09
7.40
4.55
(+/-)S.D.
2.59604
3.90776
2.59604
11.24982
3.90776

P Value
1.42E-11
4.50E-07
0.027
Remark
Difference
significant
Difference
significant
Difference
is
is
is
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ISSN 2454-5872
ISO
7.40
11.24982
significant
Difference between MIC values between TCN and TLM; TCN and ISO; TLM and ISO to all
bacterial isolates was statistically significant .[Table 3.2]
Distribution of MICs of mycolic acid inhibitors to M.

tuberculosis strains.
60
MIC values
50
50
50
50
50
50
50
50
40
30
25
25
ISO
TLM
TCN
25
20
12.5
10
12.5
12.5
10
6.2
5
2.5
1.2
1.2
1.2
2.5
5
3.1
5
3.1
2.5
5
3.1
0.3
1.2
1.2
0.3
2.5
0.6
1.2
0.3
0.6
1.2
0.6
2.5
0.15
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
No. of strains
Figure 4: MIC pattern of M. tuberculosis strains
The results indicate MIC of strains of M. tuberculosis for TCN ranged from 0.1510mcg/ml, whereas for TLM the MIC values ranged from 3.1-50 mcg/ml, and for ISO, ranged
from 0.3-10mcg/ml.[Figure 5]
Table 3.3: MIC pattern of Mycolic acid inhibitors to M. tuberculosis isolates

Sr.no.
Mycolic acid inhibitor
1
2
3
TCN showed
Geometric Mean(G.M.)MIC ( mcg/ml)
TCN
2.43
TLM
40.08
ISO
2.78
lowest MIC followed by ISO. TLMshowed highest value of MIC against M.
tuberculosis isolates. [Table 3.3]

Table 3.4: MIC pattern of Mycolic acid inhibitors to all M. tuberculosis isolates in combination
Group
Mycolic acid
inhibitor
G.M.Mean MIC
( mcg/ml)
(+/-)S.D.

p value
Remark
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ISSN 2454-5872
TCN
TLM
II
TCN
ISO
III
TLM
ISO
Difference between MIC
40.08
2.43
2.78
2.43
2.78
40.08
values between TCN
51.83
0.004
Difference
2.50
significant
2.73
0.325
Difference
2.50
significant
2.73
0.005
Difference
51.83
significant
and TLM ; TCN and ISO; TLM and ISO to
is
is
is
M.
tuberculosis isolates was statistically significant . [Table 3.4]
4. Discussion
The MICs of TCN, TLM and ISO and antibiotic resistance profile for a range of clinical
isolates of Klebsiella species. E.coli, S.aureus and Pseudomonas species were determined in the
present study.TCN MICs ranged between 0.07 to 10.0 mcg/ml. A 128-fold difference in MIC
between the most sensitive and most resistant strain was observed. Pseudomonas species clinical
isolates were found to be more resistant to TCN than other bacterial strains. Klebsiella species
were found to be most susceptible to TCN. Klebsiella species and E. coli clinical isolates that
were resistant to wide range of antibiotics were susceptible to TCN.MIC was less than 0.3
mcg/ml. Difference between MICs of TCN, TLM and ISO was found to be significant against all
the bacterial strains.
TCN MICs ranged between 0.025 and 1 mcg/ml. A 40-fold difference between the most
sensitive and most resistant strains of S.aureus was observed. Several strains showed resistance
to a wide range of antibiotics and also exhibited low-level resistance to TCN. However other
strains
which
were
resistant
to
several
antibiotics
were
more
susceptible
to
TCN.MICwaslessthan0.1mcg/ml.The clinical significance of the low level TCN resistance seen

in the majority of the strains is unknown (Suller & Russell, 2000). It is thought that strains with
decreased susceptibility to disinfectants may also be susceptible to antibiotics, possibly because
of common resistance mechanism. Some authors demonstrated that low level TCN resistance
amongst S. aureus clinical isolates was observed in equal frequencies whether the organism was
sensitive or resistant to Methicillin(Bamber & Neal, 1999).
Though in our study, several strains showed resistance to a wide range of antibiotics and
also exhibited low-level resistance to TCN, sample size is less in order to draw any positive
conclusions as to the potential cross-resistance between TCN and other antibiotics. Large scale
46

ISSN 2454-5872
testing of isolates from various sources is required. Significant difference was observed in the
inhibitory activity of TCN, TLM and ISO to the all bacterial strains. It was suggested that the
observed perturbation to the cytoplasmic membrane arises indirectly as a consequence of the
action of TCN on this enzyme involved in lipid synthesis, but exponentially growing organisms
be more susceptible or not is not known (Heath, 1998).This was not observed in some studies,
that S. aureus was equally sensitive to TCN whether in the exponential or stationery phase of
growth(Suller & Russell, 2000). Furthermore, non-growing cells were inactivated to the same
degree as the cells in a growth medium.
In our study we could not differentiate the action of TCN, as we have used culture in
exponential phase of growth. Alternatively, it was argued that, although enoylreductase may be a
major target, TCN also has multiple target sites, primarily within the cytoplasmic membrane, that
inhibit lipid RNA and protein synthesis and result in direct membrane damage and cell
death(McDonnell & Pretzer, 1998).The lack of correlation between MICs and lethal effects has
been demonstrated in some studies (Suller & Russell, 2000). Generally antibiotics have single
target sites and consequently increased MICs and reduced bactericidal effectiveness are linked.
In contrast biocides have multiple targets and increased MICs often do not correlate with
decreased bactericidal activities of that compound. It appears that additional, TCN induced
cellular changes are required to produce a bactericidal effect whether the Staphylococcal strain is
resistant or sensitive to TCN as judged purely by MICs (Suller& Russell, 2000).
The presence of proton dependent efflux pumps has been proposed as a possible
mechanism of decreased susceptibility to biocides in E.coli and P.aeuroginosa (Mcmurry, et al.,
1998; Schweizer, 1998).Another proposed mechanism of resistance is cell wall permeability
changes that prevent TCN reaching its target site. P. aeruginosa has a complex cell wall and
exhibit intrinsic resistance to TCN. S. aureus on the other hand has a less complex cell wall and
is more sensitive(Melencke, et al., 1980).Until recently TCN has been regarded as a biocide
with a range of cytoplasmic membrane and intracellular target sites, one such target is the
enzyme enoylreductase, encoded by the Fab 1 gene in E.coli. Enoylreductase uses NADH to
reduce double bonds during fatty acid elongation and thus is a major component of lipid
synthesis. Backed by mutational and some biochemical analysis in E.coli and M.smegmitis
claims have been noted that this is the sole target of TCN in those organisms(Melencke et al.,
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ISSN 2454-5872
1980).TCN has been shown to inhibit the growth of M. tuberculosis isolates(McDonnell &
Pretzer, 1998).
In this study, the MICs of TCN to clinical isolates of M. tuberculosis were determined.
MICs ranged from 0.3 to 10 mcg/ml. A 32-fold difference between the most susceptible and
most resistant strain was observed. After INH, TCN was found to be most active compound
against strains of M.tuberculosis. Difference between MICs of TCN and other compounds was
found to be significant. Genetic and biochemical evidence has indicated two different targets
enzymes for INH within the unique type II fatty acid synthase (FAS) system involved in the
production of mycolic acid. These two components are an enoyl acyl carrier protein (ACP)
reductase, InhA, and a beta ketoacyl-ACP synthase, KasA.TCN inhibits InhA. Overexpression
of InhA conferred more resistance to TCN than INH. Co-expression of both InhA and KasA
resulted in strongly enhanced levels of INH resistance in addition to cross-resistance to both
TLM and TCN.(Slayden et al., 2000). The uses of TCN as biocide still remain controversial until
the mechanism of resistance and relative importance of low-level resistance in the environment
are better understood.
TLM MICs ranged between 0.15 and 10.0 mcg/ml for clinical isolates. A 64-fold
difference in MIC between the most sensitive and most resistant strain of bacteria was observed.
Pseudomonas species and E.coli clinical isolates were found to be more resistant to TLM
followed by S.aureus. Klebsiella species clinical isolates were the most susceptible to TLM.
Difference between MICs of TCN, TLM and ISO was found to be significant against all the
bacterial strains.Strains which showed, resistance to wide range of antibiotics, also exhibited
low-level resistance to Temin order to draw any positive conclusions as to the potential crossresistance between TLM and other antibiotics, large scale testing of isolates from various sources
is required. Amongst 3 compounds, TLM is at middle position in its inhibitory activity.
TLM belongs to a small group of antibacterial compounds that have recently been
collectively termed the thiotetronic acids. TLM exhibits potent in vivo activity against many
pathogenic bacteria is reported(Hamada et al., 1990).It is 4R-2E, 5E-2,4,6-trimethyl-3-hydroxy2,5, 7-octatriene-4-thiolide, purified from a culture filtrate of a strain of the Nocardia species,
was examined for antimicrobial activities against more than 100 strains of oral and periodontally
associated bacteria. It was found that TLM exhibited strong and selective antimicrobial activities
48

ISSN 2454-5872
against Bacteroidesgingivalis and other oral black-pigmented Bacteroidesspecies that may be

etiologically associated with adult periodontitis. TLM also inhibited the growth of
Actinobacillusactinomycetemcomitans, but did not affect the growth of oral Streptococcal
species and Eubacteriumspecies. Strains of Eikenellacorrodens were moderately susceptible to
TLM, while Actinomycesviscosus strains were only slightly susceptible to it. In conclusion, TLM
exhibited highly selective antimicrobial activities to black-pigmented Bacteroides species and
Actinobacillusactinomycetemcomitans, both of which are implicated in the pathogenesis of
human periodontal disease .It is of relevance in the present context that TLM inhibits bacterial
and plant type II fatty acid synthase (FASII) but not mammalian or yeast type I fatty acid
synthase (FASI)(Hayashi, et ak., 1983).
For instance in E.coli, TLM inhibits both B-ketoacyl-ACP synthase I to III and acetyl
coenzyme A (CoA). Consequently, an understanding of the mode of action of TLM is important
in the development of more effective antibiotics that exhibit selective action against bacterial
FAS II. The effects of TLM on Mycobacterial multifunctional FAS I, monofunctional
Mycobacterial polypeptide FAS II and the largely undefined mycolatesynthase were
investigated, in the search for a new antitubercular drug targets and treatments of drug resistant
M.tuberculosis.
In 1996 some authors determined the susceptibilities of M.smegmitis and M.tuberculosis
to various concentrations of TLM on solid media and compared them to published values of INH
and ETH. Our results are comparable to these studies(Slayden et al., 2000).In our study, the
MICs of TLM to clinical isolates of M.tuberculosis was determined. MICs ranged from 3.1to 50
mcg/ml. An approximately 20-fold difference between the most susceptible and most resistant
strain was observed. Amongst 3 compounds tested, TLM is shows lowest inhibitory activity
against M. tuberculosis isolates. Mean MIC was 40.08 mcg/ml. Difference between MICs of
TLM and other compounds were found to be significant. Some studies showed evidence that
TLM targets two B Ketoacyl-carrier protein syntheses, KasA and KasB, consistent with the
fact that both enzymes belong to the fatty acid synthase type II system involved in fatty acid and
mycolic acid biosynthesis(Kremer et al., 2000).
Overexpression of KasA, kas B and kasAB in M.bovis BCG increased in vitro and in
vivo resistance against TLM. In addition MDR clinical isolates were also found to be highly
49

ISSN 2454-5872
sensitive to TLM, indicating promise in counteracting MDR strains of M.tuberculosis.TLM

inhibits KasA in M.tuberculosis.INH and TLM upregulates the expression of an operon
containing five FAS II components. They inhibit mycolic acid synthesis and thisresults in the
accumulation of ACP-bound lipid precursors to mycolic acid. TLM resistant mutants of
M.tuberculosis were more resistant to TLM and INH.Co-over expression of both InhA and KasA
resulted in strongly enhanced levels of INH resistance, in addition to cross-resistance to TLM.
INH appeared to resemble TLM more closely in overall mode of action and kasA levels
appeared to be highly correlated with INH sensitivity.(Kremer et al., 2000).
ISO MICs ranged between 0.15 to 40.0 mcg/ml for test bacterial clinical isolates. A 256fold difference in MIC between the most sensitive and most resistant strain of bacteria was
observed. Pseudomonas spp clinical isolates were found to be more resistant to ISO followed by
KlebsiellasppandS.Aureus clinical isolates. E.coli isolates were more sensitive to ISO.Amongst 3
compounds, ISO showed lowest inhibitory activity against all bacterial clinical isolates.
Difference between MICs of TCN, TLM and ISO was found to be significant against all the
bacterial strains.We could not find any correlation between any other tested antibiotic resistance
and ISO resistance. Several strains showed resistance to wide range antibiotics and also exhibited
low-level resistance to ISO. In order to draw any positive conclusions as to the potential crossresistance between ISO and other antibiotics, large scale testing of isolates from various sources
is required.There is scarce literature available on the antibacterial activity of ISO. ISO inhibits
themy colic acid synthesis in M.tuberculosis (Winder, 1982).
Amongst 3 compounds tested, ISO is shows similar inhibitory activity like TCN against
M. tuberculosisisolates. Difference between MICs of ISO and other compounds was found to be
significant. The inhibitory activity of ISO against M. tuberculosis clinical isolates in both
susceptible and MDR strains are evident. ISO inhibits the mycolic acid synthesis in
M.tuberculosis(Lucchesi, 1963). But previous studies also suggestISO is unique in its ability to
inhibit the synthesis of oleic acid and tuberculo-stearic acid(Phetsuksiri et al., 1999). Oleic acid
is the most abundant unsaturated fatty acid in Mycobacterium species and is a vital constituent of
mycobacterial membrane phospholipids, where it apparently plays an essential role in membrane
physiology. At physiological temperatures, phospholipids containing only saturated fatty acids
cannot form a lipid bilayer, but the introduction of the appropriate unsaturated fatty acids
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ISSN 2454-5872
decreases the transition temperature from gel to the liquid crystalline phases and provides
membranes with the necessary fluidity for physiological function. The vital functions of oleic
acid lead to the conclusion that inhibition of its synthesis results in cell death, and thus, the
enzymes involved in oleic acid synthesis are probably lethal targets for drug therapy.
Mycobacteria contain a large amount of methyl-branched fatty acids, mainly tuberculostearic
acid and multimethyl-branched acids. The inhibition of tuberculostearic acid by ISO is a
consequence of its effect on oleic acid synthesis, since tuberculostearic acid arises by direct
methylation of oleic acid. The mechanism by which ISO inhibits mycolic acid synthesis has yet
to be determined, but clearly it is not merely an effect on oleic acid synthesis(Lucchesi, 1963).
5. Conclusion
To conclude the present study, it can be said that mechanism of action for all three drugs
varies extensively as reported at high concentrations. TCN acts as a biocide with
multiple cytoplasmic and membrane targets. However,
at
the
lower
concentrations
it
appears bacteriostatic, and targets bacteria primarily by inhibiting fatty acid synthesis. ISO is
a thiourea used
in
the
treatmentof tuberculosis,
inhibiting
synthesis
of oleic
acid and tuberculostearic acid. It has considerable antimycobacterial activity in vitro and is
effective against multi-drug resistant strains of M.tuberculosis. However, ISO inhibits the
synthesis of fatty acids partially. The mechanism of TLM resistance in E.coli suggest that two
distinct TLM targets exist in Mycobacteria. In this study, TCN was found to be better compound
in inhibiting test organisms amongst three drugs as low MIC was observed to TCN. ISO was
found to least active against bacterial isolates. ISO was effective against susceptible as well as
MDR strains of M.tuberculosiswith low MIC values with more widespread inhibitory effect.
6. Acknowledgement
The authors were grateful to Dr Clifton Barry, Chief, Tuberculosis Research Section,
Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases,
U.S.Afor providing Thiolactomycinfor research work.

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REFERENCES
Bamber , A. I., & Neal, T. J. (1999). An assessment of triclosan susceptibility in methicillinresistant and methicilin sensitive Staphylococcus aureus. J. of Hosp. Infections, 41, 107109.
Besra, G. S., Khoo, K.-H., McNeil, M. R., Dell, A., Morris, H. R., & Brennan, P. J. (1995). A
new interpretation of the structure of the mycolyl-arabinogalactan complex of
Mycobacterium tuberculosis as revealed through characterization of oligoglycosylalditol
fragments by fast-atom bombardment mass spectrometry and 1H nuclear magnetic
resonance spectroscopy. Biochemistry, 34(13), 4257-4266.
Brennan, P. J. (2002). Plenary lecture, Current developments in drug Discovery for Tuberculosis.
Targeting Cell wall Biogenesis. Introduction symposium on current developments in drug
discovery for tuberculosis, 5-6.
Chopra, P., Meena, L., & Singh, Y. (2003). New drug targets for Mycobacterium tuberculosis.
Indian Journal of Medical Research, 117, 1-9.
Hamada, S., Fujiwara, T., Shimauchi, H., Ogawa, T., Nishihara, T., Koga, T.,. Matsuno, T.
(1990). Antimicrobial activities of thiolactomycin against gramnegative anaerobes
associated with periodontal disease. Oral microbiology and immunology, 5(6), 340-345.
Hawkins, J. E., Wallace Jr, R. J., & Brown, B. A. (1991). Antibacterial susceptibility tests:
Mycobacteria.J. Clin. Microbiol. 23:934-937.
Hayashi, T., Yamamoto, O., Sasaki., H., Kawaguchi, A., & Okazaki, H. (1983). Mechanism of
action of the antibiotic thiolactomycin inhibition of fatty acid synthesis of E. coli.
Biochem. Biophys. Res. Commun, 115, 1108-1113.
Heath, R. J., Yu, Y. T., Snapiro, M. A., Olson, E. and Rock, C.O. (1998). Broad spectrum
antimicrobial biocides target the Fabl component of fatty acid synthesis. J. of Bio. Chem,
273(30), 316-320.
Kremer, L., Douglas, J. D., Baulard, A. R., Morehouse, C., Guy, M. R., Alland, D.,. Brennan, P.
J. (2000). Thiolactomycin and Related Analogues as Novel Anti-mycobacterial Agents
Targeting KasA and KasB Condensing Enzymes inMycobacterium tuberculosis. Journal
of Biological Chemistry, 275(22), 16857-16864.
52

ISSN 2454-5872
Lucchesi, M. (1963). Recherches experimentales sur le 4-4-diisoamyloxythiocarbanilide

(ISOXYL). Acta Tuberc Belg, 54, 42.
McDonnell, G., & Pretzer, D. (1998). Action and targets of triclosan. ASM News, 64(12), 670674.
Mcmurry, L. M., Oethinger, M., & Levy, S. B. (1998). Overexpression of marA, soxS, or acrAB
produces resistance to triclosan in laboratory and clinical strains of Escherichia coli.
FEMS microbiology letters, 166(2), 305-309.
Melencke, B. E., Kranz, R. G., & Lynch, D. L. (1980). Effect of irgasan on bacterial growth and
its adsorption into the cell wall. Microbios, 28, 133-147.
Minnikin, D. E. (1982). Lipids: complex lipids, their chemistry, biosynthesis and roles. The
biology of the mycobacteria, 1, 95-184.
Phetsuksiri, B., Baulard, A. R., Cooper, A. M., Minnikin, D. E., Douglas, J. D., Besra, G. S., &
Brennan, P. J. (1999). Antimycobacterial activities of isoxyl and new derivatives through
the inhibition of mycolic acid synthesis. Antimicrobial agents and chemotherapy, 43(5),
1042-1051.
Schroeder, E., de Souza, O. N., Santos, D., Blanchard, J., & Basso, L. (2002). Drugs that inhibit
mycolic acid biosynthesis in Mycobacterium tuberculosis. Current pharmaceutical
biotechnology, 3(3), 197-225.
Schweizer, H. P. (1998). Intrinsic resistance to inhibitors of fatty acid biosynthesis in
Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation
of unmarked chromosomal mutations for the study of efflux systems. Antimicrobial
agents and chemotherapy, 42(2), 394-398.
Siddiqui, S.H., Libonati, J.P. and Middlebrook, G. (1981): Evaluation of a rapid radiometric
method
for
drug
susceptibility
testing
of
Mycobacterium
tuberculosis.Clin.Microbiol.13:908-912.
Slayden, R. A., Lee, R. E., & Barry, C. E. (2000). Isoniazid affects multiple components of the
type II fatty acid synthase system of Mycobacterium tuberculosis. Molecular
Microbiology, 38(3), 514-525.

53

ISSN 2454-5872
Suller, M., & Russell, A. (2000). Triclosan and antibiotic resistance in Staphylococcus aureus.
Journal of Antimicrobial Chemotherapy, 46(1), 11-18.
Thierry, D., Brisson-Nol, A., Vincent-Levy-Frebault, V., Nguyen, S., Guesdon, J., & Gicquel,
B. (1990). Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110,
and its application in diagnosis. Journal of Clinical Microbiology, 28(12), 2668-2673.
Tomioka, H., Saito, H., Fujii, K., Sato, K., & Hidaka, T. (1993). In vitro antimicrobial activity of
benzoxazinorifamycin, KRM-1648, against Mycobacterium avium complex, determined
by the radiometric method. Antimicrobial agents and chemotherapy, 37(1), 67-70.
Winder, F. G. (1982). Mode of action of the antimycobacterial agents and associated aspects of
the molecular biology of the mycobacteria. The biology of the mycobacteria, 1, 353-438.
World Health Organization, ITS Global Tuberculosis Report 2014,
http://www.who.int/ tdr/news/2014/global-TB-report/en, 2014.

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