Determination of in Vitro Inhibitory Activity of Mycolic Acid Inhibitors to Clinical Isolates of...

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LIFE: International Journal of Health and Life-Sciences

ISSN 2454-5872

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37

Shashikant Vaidya et al.

Special Issue, 2015, pp. 37-54

DETERMINATION OF IN VITRO INHIBITORY ACTIVITY OFMYCOLIC 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 ,[email protected]

Kapil Punjabi Department of Clinical Pathology, Haffkine Institute for Training, Research and Testing,

Mumbai, India ,[email protected]

Shreyasi Mulye Haffkine Bio-pharmaceutical Corporation Ltd. Mumbai, India [email protected]

Geeta Koppikar Breach Candy Hospital Trust, Mumbai, [email protected]

Mohan KulkarniT.N. Medical College and B.Y.L. Nair Charitable Hospital, Mumbai, India

Abhay Chowdhary

Grant Government Medical College and Sir J.J.Hospital, Mumbai,

[email protected]

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 Mycobacteriumtuberculosis (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

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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 aswell 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 leadingkiller 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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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 andselective 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) (5-

chloro-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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biosynthesis(Kremer et al., 2000).TCN is a biphenyl ether derivative known as 2, 4, 4-Trichloro-

2-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 al., 2000).ISO; a thiourea (thiocarlide-4, 4 –

diisoamyloxythiocarbanilide) demonstrated potent activity against standard strain of

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 at37ºC 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

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]

Figure 1: MIC pattern of E. coli strains

0

5

10

15

20

25

30

35

4045

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

M I C v a l u e ( m c g / m l )

Number of strains

MIC pattern of S. aureus strains

TCN

TLM

ISO

0.15 0.15 0.15 0.15 0.07 0.15 0.07 0.07 0.07

10

0.15 0.15 0.15 0.15 0.15 0.15 0.07 0.15 0.07 0.07 0.070.3 0.15 0.150.15

10 10

5

0.3

10 10 10

0

5

2.5 2.5

0.15

10 10

5

0.3

10 10 10

0

5

2.5 2.5

5 5 5

0.15

5

0.15

5

0.15

5 5

0.15

2.5

5 5 5

0.15

5

0.15

5

0.15

5 5

0.15

2.5

0

2

4

6

8

10

12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

M I

C v a l u e ( m c g / m l )

Number of strains

MIC pattern of E.coli strains

TCN

TLM

ISO



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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 0.07-

5mcg/ml.[Figure 3.2]

Figure 2: MIC pattern of Klebsiella strains

The results indicate that, MIC for TCN for all strains of Klebsiellasppswas in range of0.07mcg/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]

0.07 0.15 0.07 0.07 0.07 0.07 0.15 0.07 0.15 0.07 0.07 0.07 0.07 0.15 0.07 0.15 0.07 0.07 0.07 0.07 0.15

10

0.15 0.15 0.3

5

0.3

5

10

0.15 0.15 0.3

5

0.3

5

10

0.15 0.15 0.3

5

0.3

55 5

40

0.15

5 5

0.15

5 5

40

0.15

5 5

0.15

5 5

40

0.15

5 5

0.150

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


Number of strains

MIC pattern of Klebsiella strains

TCN

TLMISO



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Figure 3: MIC pattern of Pseudomonas strains

The results indicate that, MIC of strains of Pseudomonas sppfor TCN ranged from 0.07-

10mcg/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. Mycolic acid inhibitor Geometric Mean MIC ( mcg/ml)

1 TCN 0.09

2 TLM 4.55

3 ISO 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 Mycolic acid

inhibitor

Geometric

Mean MIC( mcg/ml)

(+/-)S.D. P Value Remark

I TCN

TLM

0.09

4.55

2.59604

3.90776

1.42E-11 Difference is

significant

II TCNISO

0.097.40

2.5960411.24982

4.50E-07 Difference issignificant

III TLM 4.55 3.90776 0.027 Difference is

0.15 0.07 0.15 0.15

5

0.07 0.07 0.15 0.15 0.15

10 10 10

5

0.07 0.15 0.07

10

0.07 0.15 0.15 0.150.15

10 10 10

5

0.15

10

5 5

0.3

5 5

10

5

10

5

10 10

0.3

5

0.3 0.150.15 0.5

10

20 20

0.3

20

5 5 5

40

5 5

10

5 5 5 5

40

5

20

40

0

5

10

15

20

25

30

35

40

45

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

MIC pattern of Pseudomonas strains

TCN

TLM

ISO



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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]

Figure 4: MIC pattern of M. tuberculosis strains

The results indicate MIC of strains of M. tuberculosis for TCN ranged from 0.15-

10mcg/ml, whereas for TLM the MIC values ranged from 3.1-50 mcg/ml, and for ISO, rangedfrom 0.3-10mcg/ml.[Figure 5]

Table 3.3: MIC pattern of Mycolic acid inhibitors to M. tuberculosis isolates

Sr.no. Mycolic acid inhibitor Geometric Mean(G.M.)MIC ( mcg/ml)

1 TCN 2.43

2 TLM 40.08

3 ISO 2.78

TCN showed 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

2.5

12.5

25

12.5

6.2

2.5 3.1 3.1

12.5

3.1

25

50 50 50

2.5

50 50 50

25

50

2.5

10

5

1.2 1.2 1.22.5

5 5

2.5

5

0.31.2 1.2

0.3 0.61.2

0.3 0.61.2 0.6 0.150

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

M I C v a l u e s

No. of strains

Distribution of MICs of mycolic acid inhibitors to M.

tuberculosis strains.

ISO

TLM

TCN



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I TCN

TLM

40.08

2.43

51.83

2.50

0.004 Difference is

significant

II TCN

ISO

2.78

2.43

2.73

2.50

0.325 Difference is

significant

III TLMISO

2.7840.08

2.7351.83

0.005 Difference issignificant

Difference between MIC values between TCN and TLM ; TCN and ISO; TLM and ISO to 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



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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, thatinhibit 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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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 KasAresulted 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 cross-

resistance 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-hydroxy-

2,5, 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



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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 acetylcoenzyme 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



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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 256-

fold 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 cross-

resistance 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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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.

ConclusionTo 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 “Thiolactomycin”for research work.



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