In Vitro microbial time-killing curve for newly synthesized aminoacetylenic-2-mercaptobenzothiazole compound

Article abstract of DOI:10.22159/ijpps.2017v9i11.21245

Objective: To determine the time needed for killing different types of microorganisms by a newly synthesized 2-mercapto-1,3-benzothiazole derivative in comparison to ciprofloxacin and fluconazole. Methods: The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) for 2-{[4-(2,6-dimethylPiperidin-1-yl)but-2-yn-1-yl]Sulfanyl}-1,3-benzothiazole(AZ3) compound were determined, using the broth dilution method. The MBC and MFC dilutions were prepared. Broth cultures of Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) were incubated at 37 °C for 24 h, and Candida albicans (C. albicans) was incubated at 25 °C for 48 h. 0.1 ml of each broth culture represent 1.5 x 10⁶CFU/ml was challenged with 9.9 ml broth containing the MBC or MFC concentrations of the AZ3 compound. From each sample at different time intervals, 1 ml was taken and added to 9 ml of sterile distilled water, in order to neutralize the effect of AZ3. Serial dilution was done and a viable count was determined from the appropriate dilutions.Results: The viability of the P. aeruginosa, E. coli, S. aureus, B. subtilis and C. albicans were killed within 3.5 h, 5 h, 24 h, 3 h and 5 h respectively. The time killing curves showed that AZ3 needed longer time for killing S. aureus than the time needed to kill B. subtilis. On the other hand, AZ3 needed a shorter time to kill P. aeruginosa, than the time needed to kill E. coli. In comparison with ciprofloxacin, AZ3 needed a shorter time to kill P. aeruginosa and E. coli, and the same time to kill B. subtilis, while it needed longer time than ciprofloxacin to kill S. aureus. In comparison with fluconazole, AZ3 with lower MFC than fluconazole needed longer time to kill C. albicans.Conclusion: AZ3 showed promising antimicrobial killing activities, in compared with ciprofloxacin and fluconazole, which promoted our interest to investigate the time of killing needed for other 2-mercaptobenzothiazole derivatives against different types of microorganisms.

Full text of DOI:10.22159/ijpps.2017v9i11.21245

International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491
Vol 9, Issue 11, 2017
Original Article
IN VITRO MICROBIAL TIME-KILLING CURVE FOR NEWLY SYNTHESIZED AMINOACETYLENIC-
2-MERCAPTOBENZOTHIAZOLE COMPOUND
ASEEL ALSARAHNI1, ZUHAIR MUHI-ELDEEN2*, ELHAM AL-KAISSI1, HIBA AL-MALLITI1
1Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, University of Petra, Amman, Jordan, 2Department of
Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, University of Petra, Amman, Jordan
Email: zeldeen@uop.edu.jo
Received: 10 Jul 2017 Revised and Accepted: 21 Sep 2017
ABSTRACT
Objective: To determine the time needed for killing different types of microorganisms by a newly synthesized 2-mercapto-1,3-benzothiazole
derivative in comparison to ciprofloxacin and fluconazole.
Methods: The minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) for 2-{[4-(2,6-dimethylPiperidin-1-yl)but-
2-yn-1-yl]Sulfanyl}-1,3-benzothiazole(AZ3) compound were determined, using the broth dilution method. The MBC and MFC dilutions were
prepared. Broth cultures of Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P.
aeruginosa) were incubated at 37 °C for 24 h, and Candida albicans (C. albicans) was incubated at 25 °C for 48 h. 0.1 ml of each broth culture
represent 1.5 x 106 CFU/ml was challenged with 9.9 ml broth containing the MBC or MFC concentrations of the AZ3 compound. From each sample at
different time intervals, 1 ml was taken and added to 9 ml of sterile distilled water, in order to neutralize the effect of AZ3. Serial dilution was done
and a viable count was determined from the appropriate dilutions.
Results: The viability of the P. aeruginosa, E. coli, S. aureus, B. subtilis and C. albicans were killed within 3.5 h, 5 h, 24 h, 3 h and 5 h respectively. The
time killing curves showed that AZ3 needed longer time for killing S. aureus than the time needed to kill B. subtilis. On the other hand, AZ3 needed a
shorter time to kill P. aeruginosa, than the time needed to kill E. coli. In comparison with ciprofloxacin, AZ3 needed a shorter time to kill P.
aeruginosa and E. coli, and the same time to kill B. subtilis, while it needed longer time than ciprofloxacin to kill S. aureus. In comparison with
fluconazole, AZ3 with lower MFC than fluconazole needed longer time to kill C. albicans.
Conclusion: AZ3 showed promising antimicrobial killing activities, in compared with ciprofloxacin and fluconazole, which promoted our interest to
investigate the time of killing needed for other 2-mercaptobenzothiazole derivatives against different types of microorganisms.
Keywords: 2-mercaptobenzothiazole derivatives, Alkylation, Mannich reaction, Time-kill curve
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i11.21245
INTRODUCTION
Kuchta et al. in 1989 carried out a research which involved 22
derivatives of 2-alkylthio-6-amino and 2-alkyl thio-6-
Molecules with benzothiazole (BTA) moiety have different biological
activities [1]. Accordingly, 2-mercaptobenzothiazole (2-MBT)
derivatives have a variety of applications [2]. 2-MBT was isolated as a
natural product, from fermentation cultures of micrococcus species
bacterial symbiont of the marine sponge Tedaniaignis [3]. 2-MBT
consists of benzothiazole which is a heterocyclic compound includes
benzene ring fused with 4,5-positions of the thiazole ring [4, 5], and
the mercapto (thiol group) substituent at position 2 of thiazole ring
that gives the compound antibacterial and anti-inflammatory activity
[6]. 2MBT is prepared by reaction between 2-amino-thiophenol with
refluxing carbon disulfide [6], in the presence of acetic anhydride [7].
formamidobenzothiazole [9]. They were tested against 15 candida
yeast strains, and against A. niger +with a suspension of spore free
mycelium homogenate as inoculums. They found that no significant
effect was observed against A. niger, The pathogenic species C.
albicans and C. gnilliemondii. In another study, 3-(2-alkylsulfanyl-6-
benzothiazolylaminomethyl)-2-benzothiazolethiones
were
synthesized, by the reaction of 2-alkylsulfonyl-6-
aminobenzothiazoles with 3-hydroxy–2-benzoxazolethione in
ethanol (fig. 2). New derivatives were tested against candida,
researchers found that derivatives having the benzyl group at the
second position of MBT ring demonstrated maximum inhibition of
the oxygen evolution rate in spinach chloroplasts [10].
Novel
4-substituted
phenyl–3-chloro-1-[(benzothiazolythio)
accetamidyl]-2-azetidinone derivatives were synthesized (fig. 1).
These novel compounds were screened for their antibacterial and
antifungal activities against S. aureus, A. niger, and C. albicans. The
study showed that compound derivatives having 3-OH–C6H4 and Cl-
C6H4 groups, promoted the activity against candida while compounds
derivatives having 4-OH-C6H4 and 2-Cl-C6H4, at position 4 in the
azetidinone nucleus are very effective against S. aureus and A. niger [8].
.
R=CH3/C2H5/allyl/(CH2)CH3/CH(CH3)C2H5/(CH2)4CH3(CH2)5CH3/(
CH2)6CH3/(CH2)7CH3/(CH2)8CH3/CH2C6H5
Fig. 2: 3-(2-alkylsulfanyl-6-benzothiazolylaminomethyl)-2-
benzothiazolethiones [9]
R= 4-NO2/3,4,5-Tri-OCH3/2-OH/3-OH/4-OH/2-OCH3/4-OCH3/2-
Cl/3-Cl/4-Cl
Fig. 1: 4-substituted phenyl–3-chloro-1-[(benzothiazolythio)
accetamidyl]-2-azetidinone derivatives [8]
In this study, in vitro time exposure needed for killing different types
of microorganisms by AZ3 was determined, and were compared to

Eldeen et al.
Int J Pharm Pharm Sci, Vol 9, Issue 11, 125-130
the time killing curves for ciprofloxacin and fluconazole against the
same microorganisms. The main aim of this study is finding new
promising 2-mercaptobenzothiazole derivatives, which have the
ability to kill different microorganisms in competitive time
compared to other drugs in the market.
in 1,4-dioxane (25 ml) was stirred at room temperature, for 10 min
then was heated and stirred under reflux at 70-75 °C for three h. The
reaction mixture was filtrated and concentrated under reduced
pressure, to give a brown syrup which was dissolved in diethyl
ether, filtrated and concentrated under reduced pressure. The final
products
was
2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-1-yl]
MATERIALS AND METHODS
Experimental
sulfanyl}-1,3-benzothiazole (AZ3). The title compound AZ3 was
synthesized by Mannich reaction in 2.5 g, yield 76.22%, mp: 48.62
°C, UV-HPLC retention time: 2.167 min, FT-IR (KBr cm-1): C-H
stretching aromatic (3080.477 cm-1, 2923.556 cm-1), C-H stretching
tertiary amine (2850.274 cm-1, 2788.563 cm-1, C=C stretching aromatic
(1673.909 cm-1), C=N stretching thiazole (1590.985 cm-1, 1432.851 cm-
1), C-N stretching 3 ° aromatic (1373.168 cm-1), C-N stretching tertiary
cyclic amine (12226.433 cm-1), C-H out of plane bending aromatic
(865.882 cm-1), C-S stretching (717.39 cm-1, 624.823 cm-1), 1H-NMR
(DMSO d6): δ; four types of cyclic amine protons: 0.75 ppm (doublet,
type A, 6H), 0.95 ppm (sextet, type B, 2H), 1.2–1.35 ppm (quartet, type C,
4H), 2 ppm (quintet, type D, 2H), 3.4 ppm (singlet, C-CH2-N, 2H), 4.2 ppm
(singlet, S-CH2C, 2H), four types of aromatic protons: 7.35 ppm (triplet,
type A, 2H), 7.45 ppm (triplet, type B, 2H), 7.85 ppm (doublet, type C,
1H), 8 ppm (doublet, type D, 1H).
Chemicals
2-mercaptobenzothiazole 97%, propargyl bromide, cis-2,6-
dimethylpiperidine 98%, all of them were purchased from (Sigma
Aldrich, USA), magnesium sulphate anhydrous (Lonover, UK),
potassium bromide (KBr) (Scharlau, Spain), potassium carbonate
anhydrous (K2CO3) (Gainland Chemical Company (GCC, UK),
paraformaldehyde polymer (BDH chemicals Ltd Poole, England),
cuprous chloride LRG (east Anglia Chemicals Hadleighlpswich),
acetonitrile 99.7% (PanReAcSintesis)/(PanReAcQuimca SA, EU), 1,4-
dioxane HPLC/ACS (FULL Time, China), chloroform (stabilized with
0.5-1% ethanol) (TEDIA, USA), dimethyl sulfoxide (DMSO) (BBC
Chemicals for lab, EU), diethyl ether (Lonover, England)/(RCL Labs
can, Thailand), absolute ethanol 99.9% (Super Chem, USA), acetone
99% (Scharlau, Spain), distilled water (Ultra, Jordan).
Instrumentation
analytical balance with a precision 0.01 mg (Phoenix instrument,
USA), hot plate with magnetic stirrer (Dragon, China), rotary
evaporator 0-100Kpa/0-700 mmHg (Rocker 600, Germany),
Buchner funnel pump (Vacuubrand, Germany), melting point
apparatus (Gallenkamp, USA), FT-IR spectrophotometer 7800 to 400
cm-1 (Evisa, Poland), DSC (Mettler Toledo, Int Co), UV-VIS
(Evolution 160, USA), HPLC-UV (Finnigan Surveyor, USA), NMR 300
MHZ (Varian 300 MHZ, USA), NMR 500 MHZ (Varian 500 MHZ, USA),
elemental analyzer with variation range (±4) (Euro Vector, Italy),
autoclave machine (Rypa, Spain), incubator (EuroStar, EU), vortex
mixer (Labinco, India), hot plate magnetic stirrer (Dragon, China),
sterile tubes, sterile swabs (mWe, UK), micropipette (Oxford, USA).
Fig. 4: Synthesis of 2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-
1-yl]sulfanyl}-1,3-benzothiazole (AZ3)
Culture media
Muller hinton agar (MHA) (Mastgrp Ltd, UK)/(Himedia, India),
muller hinton broth (MHB) (Mastgrp Ltd, UK), sabourauds dextrose
agar (SDA) (Mastgrp Ltd, UK), sabourauds dextrose broth (SDB)
(Himedia, India), dimethyl sulfoxide (DMSO) (BBC Chemicals for lab,
EU), distilled water (Ultra, Jordan).
Synthesis
Synthesis of 2-(prop-2-yn-1-ylsulfanyl)-1,3-benzothiazole (AZ0)
After the mixture of 2-MBT (5.01 g,0.03 mol), potassium carbonate
anhydrous (3 g, 0.03 mol) and 20-40 ml acetonitrile (ACN) has been
heated and stirred under reflux for 30 min, the propargyl bromide (5
ml, 0.03 mol) was added drop wise. The reaction mixture was heated
and stirred under reflux for 2 h. Then the mixture was filtrated and
concentrated under reduced pressure to give a brown syrup. The
crude product was extracted with 50 ml chloroform and 50 ml
distilled water; chloroform layers were collected, dried over
magnesium sulfate and evaporated under reduced pressure. The
solid brownish crystals were weighed and characterized through
DSC, UV-HPLC, FT-IR, 1H-NMR (fig. 3).
Microorganisms
Staphylococcus aureus (S. aureus ATCC 6538), Bacillus subtilis (B.
subtilis ATCC 6633), Pseudomonas aeruginosa (P. aeruginosa ATCC
9027), Escherichia coli (E. coli ATCC 8739), Candida albicans (C.
albicans ATCC 10231). All these bacterial strains were obtained from
Dar Al Dawa (Na'ur, Jordan).
Determination of microbial time kill curves for AZ3
The minimum inhibitory concentration (MIC) and MBC for AZ3 were
determined firstly. The synthesized compound (AZ3) was diluted
out in the series of twofold dilutions, in a liquid growth medium
dispensed in test tubes [11, 12]. The AZ3 stock solution has to be at
least 1000 µg/ml [12]. Accordingly, AZ3 stoke solution was prepared
in 1000 µg/ml in 10 ml DMSO, this solution was diluted serially in
MHB for bacteria and SDB for fungi, to give concentrations ranging
from 500-7.81 µg/ml. The final volume of the compound dilutions in
each tube was 5 ml. To these tubes, 0.1 ml of the overnight culture
was added. Positive control tube contained 5 ml of sterile broth and
0.1 ml of microbial culture, while the negative control tube
contained 5 ml of the tested compound dilution in sterile broth.
Fig. 3: 2-(prop-2-yn-1-ylsulfanyl)-1,3-benzothiazole (AZ0)
The tubes were incubated at 37 °C for 24 h for bacteria, and at 25 °C
for 48 h for fungi. The MIC was determined by comparison the
turbidity of each concentration tube with the turbidity of the
positive control tube; MIC tube is the lowest concentration of the
compound in which no turbidity was observed [13]. To determine
MBC and MFC, the MIC tube and the tubes with dilutions preceded
were sub-cultured onto agar plates, the plates were incubated at 37
°C for 24 h for bacteria, and at 25 °C for 48 h for fungi. The lowest
concentration tube that gave no growth was the MBC or MFC. Broth
Synthesis of 2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-1-yl]
sulfanyl}-1,3-
benzothiazole (AZ3)
A mixture of 2-(prop-2-yn-1-yl sulfanyl)-1,3-benzothiazole (AZ0)
(2.062 g, 0.01 mol), paraformaldehyde (0.5 g in excess), cyclic amine
(2,6-dimethyl piperidine) and a catalytic amount of cuprous chloride
126

Eldeen et al.
Int J Pharm Pharm Sci, Vol 9, Issue 11, 125-130
dilution test was designed to allow for statistical analysis and was
performed in triplicates.
culture with definite inoculum sizes were challenged with 9.9 ml of
MBC/MFC concentrations for the AZ3 compound.
Time exposure viability test for AZ3 was carried out, to assess the
killing ability rate of AZ3 within a given contact time. The MBC and
MFC concentrations were prepared for compound AZ3 using sterile
broth. The microbial cultures were prepared and incubated at 37 °C
for 24 h for bacteria and at 25 °C for 48 h for fungi. 0.1 ml of each
Then 1 ml was taken from the sample, at different time intervals as
shown in (table 1), and added to 9 ml of sterile distilled water, to
neutralize the AZ3 activity (this step was enough to neutralize as
primarily detected). Serial dilution using sterile distilled water was done,
and the viable count was determined from the appropriate dilutions.
Table 1: Time intervals used for bacterial killing curve
Time intervals (h)
Microorganism
S. aureus
1.5 2.5
3.5
4.5
20
4
24
5
-
-
5
B. subtilis
0.5
1
2
3
E. coli
1
1.5
2.5
1
2
5
-
P. aeruginosa
C. albicans
1.5
0.5
3.5
2
4.5
2.5
5.5
3
AZ3 at 250 µg/ml dilution was demonstrated in fig. 5. The 5 log cycle
reductions were detected, the curve shows that the bacterial count
decreased rapidly over 1 h. Then over 1.5 h, the count of bacteria
decreased slowly. The viability of the P. aeruginosa abolished within
3.5 h. accordingly, the LT 90 % value (time at which 90% of the
original population were killed by the antimicrobial agent) for this
experiment was approximately in the period between 2.5 and 3.5 h.
In the time-killing test for ciprofloxacin against P. aeruginosa, the 7
log cycle reductions were detected, the curve shows that the count of
bacteria decreased rapidly within the first 0.5 h, then the count
decreased slowly within the 4 h. The viability of the P. aeruginosa
was abolished within 5 h.
RESULTS
Minimum inhibitory concentrations results
The newly synthesized compound AZ3 showed activity against all
types of microorganisms tested (table 2), the minimum inhibitory
concentrations (MIC), and minimum bactericidal/fungicidal
concentrations (MBC/MFC) were measured (table 2).
Microbial time-killing curve results
The time exposure viability curve for the addition of 0.1 ml P.
aeruginosa ATCC 9027 culture, with a viable count of (2.47 X 107) to
Table 2: MIC and MBC of AZ3
Microorganisms
MIC(µg/ml)
MBC (µg/ml)
P. aeruginosa
125
250
E. coli
250
500
S. aureus
125
500
B. subtilis
62.5
125
C. albicans
125
250
Values are the mean (n=3), AZ3: 2-{[4-(2, 6-dimethylpiperidin-1-yl) but-2-yn-1-yl] sulfanyl}-1,3-benzothiazole.
Table 3: Bacterial time-killing of AZ3 against P. aeruginosa ATCC 9027
Microorganism
Time (h)
Count (CFU/ml)
Log (CFU/ml)*
P. aeruginosa
0
1.5
2.5
3.5
0
0
4.5
0
0
5.5
0
0
2.47 X107
7.39±0.61
6.8x 103
3.83±0.17
1.4x 103
3.15±0.85
*Values are the mean±SD (n=3), SD: standard deviation, CFU: colony forming unit, AZ3: 2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-1-yl]sulfanyl}-
1,3-benzothiazole.
Fig. 5: Time exposure viability curve for addition of compound AZ3 and ciprofloxacin, at MBC (250 µg/ml) to culture of P. aeruginosa
ATCC 9027
127

Eldeen et al.
Int J Pharm Pharm Sci, Vol 9, Issue 11, 125-130
Values are the mean±SD (n=3), AZ3: 2-{[4-(2,6-dimethylpiperidin-1-
yl)but-2-yn-1-yl]sulfanyl}-1,3-benzothiazole, CFU: colony forming
unit, time (X axis): time intervals needed for killing P. aeruginosa,
Log (CFU/ml) (Y axis): viability of P. aeruginosa at different times.
quickly over 1h, then the count of bacteria decreased slowly over the
next 1h. The viability of E. coli was abolished within 5 h. accordingly,
the LT 90% value for this experiment was approximately in the
period between 2-5 h.
The time exposure viability curve for the addition of 0.1 ml of E. coli
ATCC 8739 culture, with a viable count of (1.42 X107) to AZ3 at 500
µg/ml dilution was demonstrated in fig. 6. The 4 log cycle reductions
were obtained in approximately 5h. The count of bacteria decreased
In The time-killing test for ciprofloxacin against E. coli, The 5 log cycle
reductions were detected. The curve shows that the count of bacteria
decreased within the first 0.5 h, then the count decreased slowly
within the 4 h. The viability of E. coli was abolished within 5 h.
Table 4: Bacterial time-killing of AZ3 against E. coli 8739
E. coli
Microorganism
Time (h)
Count (CFU/ml)
0
1
1.5
2
5
0
0
1.42 *107
7.15±0.85
3.4x103
3.53±0.47
1.63x103
3.21±0.79
3.0x102
2.48±0.52
Log (CFU/ml)*
*Values are the mean±SD (n=3), SD: standard deviation, CFU: colony forming unit, AZ3: 2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-1-yl]sulfanyl}-
1,3-benzothiazole.
Fig. 6: Time exposure viability curve for addition of compound AZ3 and ciprofloxacin, at MBC (500 µg/ml) and (125 µg/ml) respectively to
culture of E. coli ATCC 8739
Values are the mean±SD (n=3), AZ3: 2-{[4-(2, 6-dimethylpiperidin-1-
yl) but-2-yn-1-yl] sulfanyl}-1,3-benzothiazole, CFU: colony forming
unit, time (X-axis): time intervals needed for killing E. coli, Log
(CFU/ml) (Y-axis): viability of microorganisms at different times.
decreased slowly over 18.7 h. The viability of S. aureus was
abolished within 24 h. Accordingly, the LT 90% value for this
experiment was approximately in the period between 20 h and 24 h.
AZ3 needed the longer time of exposure to kill all of the original S.
aureus population.
The time exposure viability curve for the addition of 0.1 ml of S.
aureus ATCC 6538 culture, with a viable count of (1.76x108) to 500
µg/mlAZ3 dilution was demonstrated in fig. 7. The 6 log cycle
reductions were obtained in approximately 24 h. The count of
bacteria decreased quickly after 1.5 h, then the count of bacteria
In the time-killing test for ciprofloxacin against S. aureus, the 7 log
cycle reductions were detected, the curve shows that the count of
bacteria decreased slowly within the first 2 h, then it decreases rapidly
between 2 to 3 h. The viability of S. aureu was abolished within 5 h.
Table 5: Bacterial time-killing of AZ3 against S. aureus 6539
Microorganism
Time (h)
Count (CFU/ml)
(Log CFU/ml)*
S. aureus
Zero
1.5
2.5
3.5
4.5
20
24
0
0
1.76x108
8.25±0.75
1.34x106
6.13±0.87
1.29x106
6.11±0.89
3.4x105
5.53±0.47
5.9x104
4.77±0.23
2.8x104
4.45±0.55
*Values are the mean±SD (n=3), SD: standard deviation, CFU: colony forming unit, AZ3: 2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-1-yl]sulfanyl}-
1,3-benzothiazole.
Fig. 7: Time exposure viability curve for addition of compound AZ3 and ciprofloxacin at MBC (250 µg/ml) to culture of S. aureus ATCC 6538
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Eldeen et al.
Int J Pharm Pharm Sci, Vol 9, Issue 11, 125-130
Values are the mean±SD (n=3), AZ3: 2-{[4-(2,6-dimethyl piperidin-1-
yl) but-2-yn-1-yl]sulfanyl}-1,3-benzothiazole, CFU: colony forming
unit, time (X-axis): time intervals needed for killing S. aureus, Log
(CFU/ml) (Y-axis): viability of S. aureus at different times.
count of bacteria increased over 2 h, then the count of bacteria
decreased sharply over the next 1 h. The viability of B. subtilis was
abolished within 3 h. The LT 90% value for this experiment was
approximately in the period between 2 to 3 h. In
The time exposure viability curve for the addition of 0.1 ml of B.
subtilis ATCC 6633 culture, with a viable count 1.08x103CFU/ml to
AZ3 at 125 µg/ml dilution was demonstrated in fig. 8. The 6 log cycle
reductions were obtained in approximately 5h. Surprisingly, the
The time-killing test for ciprofloxacin against B. subtilis, the 7 log
cycle reductions were detected, the curve shows that the count of
bacteria decreased slowly within the first 2 h. The viability of B.
subtilis was abolished within 3 h.
Table 6: Bacterial time-killing of AZ3 against B. subtilis 6633
B. subtilis
Microorganism
Time
Count (CFU/ml)
Log (CFU/ml)*
0
0.5
2.44 x 103
3.39±0.61
1
2
3
0
0
4
0
0
5
0
0
1.08x103
3.03±0.97
4.6x104
4.66±0.34
6.00x104
4.78±0.22
*Values are the mean±SD (n=3), SD: standard deviation, CFU: colony forming unit, AZ3: 2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-1-yl]sulfanyl}-
1,3-benzothiazole.
Fig. 8: Time exposure viability curve for addition of compound AZ3 and ciprofloxacin, at MBC (125 µg/ml) and (250 µg/ml) respectively to
culture of B. subtilis ATCC 6633
Values are the mean±SD (n=3), AZ3: 2-{[4-(2,6-dimethylpiperidin-1-
yl)but-2-yn-1-yl]sulfanyl}-1,3-benzothiazole, CFU: colony forming
unit, time (X-axis): time intervals needed for killing B. subtilis, Log
(CFU/ml) (Y): viability B. subtilis at different times.
30 min, then the count of fungi increased slowly over the next 30 min.
After 2.5 h, the count of fungi decreased slowly. The viability of
microorganism decreased rapidly within the next 2 h. Accordingly, C.
albicans was totally killed within 5 h. The LT 90% value for this
experiment was approximately in the period between 3-5 h. In the time-
killing test for fluconazole against C. albicans, the 6 log cycle reductions
were detected, the curve shows that the count of bacteria decreased
slowly within the first 45 min, then it decreased rapidly between 45-60
min. The viability of C. albicans was abolished within 3 h.
The time exposure viability curve for the addition of 0.2 ml culture of C.
albicans ATCC 10231, with a viable count 3.0x106 CFU/ml to AZ3 at 250
µg/ml dilution was demonstrated in fig. 9. The 6 log cycle reductions
were obtained in approximately 5 h. The count of fungi decreased over
Table 7: Bacterial time-killing of AZ3 against C. albicans ATCC 10231
C. albicans
microorganism
Time (h)
Count (CFU/ml)
0
0.5
1.98x104
4.30±0.7
1
2
2.5
1.21x105
5.10±0.9
3
5
0
0
3.0x106
6.48±0.52
1.7x105
5.23±0.77
1.32x105
5.12±0.88
8.6x104
4.93±0.07
Log (CFU/ml)*
*Values are the mean±SD (n=3), SD: standard deviation, CFU: colony forming unit, AZ3: 2-{[4-(2,6-dimethylpiperidin-1-yl)but-2-yn-1-yl]sulfanyl}-
1,3-benzothiazole.
Fig. 9: Time exposure viability curve for addition of compound AZ3 and fluconazole, at MFC (250 µg/ml) and (500 µg/ml) respectively to
culture of C. albicans ATCC 10231
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Eldeen et al.
Int J Pharm Pharm Sci, Vol 9, Issue 11, 125-130
Values are the mean±SD (n=3), AZ3: 2-{[4-(2,6-dimethyl-piperidin-
1-yl)but-2-yn-1-yl]sulfanyl}-1,3-benzothiazole, CFU: colony forming
unit, time (X-axis): time intervals needed for killing C. albicans, Log
(CFU/ml) (Y-axis): viability of C. albicans at different times.
Msc. Heba was responsible for part of the laboratory work.
CONFLICT OF INTERESTS
The authors have declared no conflict of interest
REFERENCES
DISCUSSION
The time-killing curve test showed that, 2-{[4-(2,6-dimethyl
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needed much longer time, to kill S. aureus in comparing with the
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needed 20-24 h to kill S. aureus and 2-3 h to kill B. subtilis. The
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resistance and the high virulence nature of S. aureus [14]. On the
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h after exposure to AZ3, then the viable count of bacteria dropped
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AZ3 needed a shorter time to kill P. aeruginosa than the time needed
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CONCLUSION
The newly synthesized 2-{[4-(2, 6-dimethylPiperidin-1-yl) but-2-yn-1-
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activity, against P. aeruginosa, E. coli, and C. albicans. This study may
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ACKNOWLEDGMENT
The authors would like to thank the University of Petra/Faculty of
Pharmacy for providing the necessary facilities to carry out this work.
AUTHORS CONTRIBUTION
Msc. Aseel Alsarahni was responsible for the laboratory work and
writing the paper draft
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Prof. Zuhair Muhi-Eldeen is the chief author, supervised the synthesis
steps
Prof. Elham Al-Kaissi was responsible for writing and supervised the
microbiology part.
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