Adaptation of Staphylococcus aureus to synthetic triazolides

Article abstract of DOI:10.1007/s11094-006-0079-6

The ability of Staphylococcus aureus to adapt to the toxic action of 1,2,4-triazole and its sulfuryl derivates has been studied. A positive correlation is found between the lipophilicity and toxicity of substances. It is established that methylated deriva

Full text of DOI:10.1007/s11094-006-0079-6

Pharmaceutical Chemistry Journal
Vol. 40, No. 3, 2006
MEDICINAL PLANTS
ADAPTATION OF Staphylococcus aureus
TO SYNTHETIC TRIAZOLIDES
E. S. Selezneva,1 Z. P. Belousova,1 A. I. Ivanchina,1 and E. I. Ten’gaev1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 3, pp. 27 – 29, March, 2006.
Original article submitted October 21, 2004.
The ability of Staphylococcus aureus to adapt to the toxic action of 1,2,4-triazole and its sulfuryl derivates has
been studied. A positive correlation is found between the lipophilicity and toxicity of substances. It is estab-
lished that methylated derivates are more toxic than substances without methyl radicals. St. aureus species
grown on a medium containing triazolides in a nontoxic dose become resistant to highly toxic doses of these
substances. The possible mechanisms of St. aureus adaptation to triazolides are discussed.
O
In recent years, the appearance of highly resistant strains
O
S
X
S
N
N
(H3C)3Si
N
N
of Staphylococcus aureus has been observed in cases of
chronic administration of various antibiotics [1, 2]. This situ-
ation leads to the search for new compounds that can serve as
a base for highly effective antistaphylococcal preparations
and to the investigation of mechanisms responsible for the
ability of St. aureus species to adapt to new drugs. In this re-
spect, a promising direction of research is related to the
antistaphylococcal properties of 1,2,4-triazole derivatives,
many of which are used in pharmacology [3] and agriculture
[4 – 6]. Previously, we demonstrated that some triazole de-
rivatives exhibited mutagenicity [7, 8] and antibacterial ac-
tivity [9, 10].
X
Cl
+
O
N
N
;
O
CH3
X =; –CH3;
The IR absorption spectra of the synthesized compounds
were measured using a Spectromom-200 instrument (Hun-
gary) using samples prepared as nujol mulls. The UV spectra
were recorded on a Specord UV-VIS spectrophotometer
(Germany). The melting points were determined on a PTP
device (Khimlaborpribor, Russia). During the synthesis, so-
lutions were evaporated in vacuum at a temperature not ex-
ceeding 40°C.
Despite extensive investigation, relationships between
the structure and activity of various groups of compounds are
for the most part still unclear. This study was aimed at an
analysis of the antibacterial activity of 1,2,4-triazole (I) and
its sulfuryl derivatives (II – IV) (Fig. 1), and the evaluation
of the ability of St. aureus species to adapt to these com-
pounds.
N
N
N
N
N
N
O
N
O
N
O
N
H
N
S
N
S
N
S
EXPERIMENTAL CHEMICAL PART
O
O
O
CH3
Compounds II – IV were synthesized via the interaction
of N-trimethylsilyl-1,2,4-triazole with chloroanhydrides of
methane-, benzene-, and toluenesulfonic acids, respectively,
in anhydrous tetrahydrofuran (THF) according to the follow-
ing scheme:
CH3
IV
III
II
I
Fig. 1. Structures of 1,2,4-triazole (I), methanesulfonic acid N-tria-
zolide (II), benzenesulfonic acid N-triazolide (III), and
toluenesulfonic acid N-triazolide (IV).
1
Samara State University, Samara, Russia.
145
0091-150X/06/4003-0145 © 2006 Springer Science+Business Media, Inc.

146
E. S. Selezneva et al.
10
EXPERIMENTAL BIOLOGICAL PART
9
8
7
6
5
4
3
The biological tests were performed with St. aureus
strain ATCC No. 6587p. The antibacterial activity of com-
pounds I – IV in aqueous solution with concentrations 0.001,
0.01, 0.1, 1, and 10 mg/ml was determined using a conven-
tional method based on the determination of the zone of mi-
crobial growth inhibition as a result of the direct diffusion of
drugs into agar from impregnated paper disks placed onto a
bacterial lawn [13]. There were three series of experiments.
Series 1. Determination of the dependence of the
antistaphylococcal activity of compounds I – IV on the con-
centration (five independent runs for each experimental
point).
I II III IV
Control
I II III IV
Direct
action
I II
Type-1
adaptation
I II
Type-2
adaptation
IV
Fig. 2. A histogram of the ability of St. aureus to develop resistance
with respect to compounds I – IV.
Series 2. Evaluation of the ability of small doses of com-
pounds I – IV to favor the development of adaptation to
highly toxic doses of same drugs. For this purpose, each
compound was introduced into the cultural medium to a con-
centration of 0.001 mg/ml and then St. aureus species were
seeded in these media. The colonies grown on these media
were used to prepare suspensions for seeding on a fresh agar
not containing drugs. The suspensions were diluted with iso-
tonic NaCl solution to a final bacterial load of 5 ´ 109 micro-
bial cells per milliliter (with reference to the turbidity stan-
dard obtained from the Tarasevich Culture Nursery). Then,
test disks impregnated with 10 mg/ml solutions of com-
pounds I – IV were placed onto inoculated bacterial lawn and
the growth inhibition zone size was determined (seven inde-
pendent runs for each experimental point).
The lipophilicity of compounds I – IV was evaluated
from data on the chromatographic mobility, which was deter-
mined by TLC in a toluene – chloroform – methanol
(5 : 2 : 1) solvent system. Some characteristics are summa-
rized in Table 1.
The initial N-trimethylsilyl-1,2,4-triazole was obtained
using a procedure proposed by Birkofer et al. [11].
Methanesulfonic acid N-traizolide of (II) was synthe-
sized as described in [12].
Benzenesulfonic acid N-triazolide (III). A solution of
2 g (0.014 mole) of N-trimethylsilyl-1,2,4-triazole in 10 ml
of dry toluene was cooled down to –10°C. To this solution
was added dropwise with stirring 2 g (0.015 mole) of
benzenesulfonic acid chloroanhydride in 5 ml of toluene and
the mixture was stirred for 3 h at the same temperature.
Then, the reaction mixture (with precipitate) was evaporated
in vacuum to dryness and the residue was recrystallized from
ethanol to obtain 2.5 g (0.012 mole) of product III in the
form of white crystals with a yield of 85%. The purity and
identity of the product was confirmed by the data of elemen-
tal analyses, TLC, and IR spectroscopy; additional evidence
was provided by the melting point determination (Table 1).
Toluenesulfonic acid N-triazolide (IV) was obtained
using a procedure analogous to that described above for
compound III. The target product IV was isolated in the form
of white crystals with a yield of 85%. The melting point and
characteristic IR absorption band positions are given in Ta-
ble 1.
Series 3. Evaluation of the ability of a nontoxic dose
(0.001 mg/ml) of compound I to favor the development of
adaptation to highly toxic (10 mg/ml) doses of compounds
II – IV. For this purpose, St. aureus species were grown in a
medium containing compound I at a concentration of
0.001 mg/ml and then reseeded on a fresh agar not containing
drugs. Then, the experiment was carried out as in series 2.
RESULTS AND DISCUSSION
The results of investigation of the ability of compounds
I – IV to inhibit the growth of St. aureus are presented in Ta-
ble 2. As can be seen from these data, compounds I and II
(but not III and IV) exhibit the concentration-dependent abil-
ity to inhibit the growth of staphylocococcal species. The re-
sults of a two-factor dispersion analysis showed evidence for
reliable ( p < 0.01) dependence of the antistaphylococcal ac-
tivity both on the concentration and on the structure of com-
pounds studies.
TABLE 1. Meting Points and Characteristic IR Absorption Bands
of Compounds III and IV
Characteristic optical
absorption band, cm – 1
Table 3 presents data on the physicochemical parameters
of triazolides I – IV and their ability to inhibit the growth of
St. aureus. A mathematical correlation analysis of these data
confirmed the presence of a positive correlation between
lipophilicity and antistaphylococcal activity (r = 0.66 at
p < 0.01) and between the total energy of a molecule and the
Compound
III
M.p., °C
95
1372
1185
1372
1185
IV
70

Adaptation of Staphylococcus aureus to Synthetic Triazolides
147
TABLE 2. Diameter of the Zone of St. aureus Growth Inhibition by Compounds I – IV
Drug concentrating, mg/ml
Compound
0
0.001
0.01
0.1
1
10
I
6.0 ± 0.00
6.0 ± 0.00
6.0 ± 0.00
6.0 ± 0.00
6.5 ± 0.03
6.8 ± 0.02
6.1 ± 0.04
6.2 ± 0.06
6.5 ± 0.05
7.0 ± 0.07
6.1 ± 0.04
6.3 ± 0.07
6.8 ± 0.04
7.8 ± 0.09
6.2 ± 0.06
6.4 ± 0.10
6.9 ± 0.02
8.0 ± 0.07
6.1 ± 0.03
6.2 ± 0.05
7.2 ± 0.06
9.0 ± 0.11
6.1 ± 0.05
6.2 ± 0.06
II
III
IV
antistaphylococcal activity (r = 0.66 at p < 0.01). It was es-
tablished that methylated derivates are more toxic than sub-
stances without methyl radicals: II > I; IV > III. It should
also be noted that methylation frequently renders compounds
mutagenic [14].
TABLE 3. Comparative Data on Antistaphylococcal Activity and
Physicochemical Characteristics of Compounds I – III
Diameter
Total
dipole
1- R
f
Com-
pound
Total energy
of molecule
of growth
inhibition
zone, mm
R
= log
Rf
m
R
f
In order to elucidate the mechanism of the antibacterial
action of compounds I – IV, we checked for the ability of
these drugs to favor the development of adaptation (type-1
preadaptation) with respect to highly toxic doses of same
compounds (experimental series 2). It was found that the pre-
liminary action of compounds I – II in a concentration of
0.001 mg/ml induces the ability of St. aureus species to
adapt to large (10 mg/ml) doses of these compounds,
whereby the growth inhibition zone size reliably decreases
(Fig. 2). Moreover, the preadaptation induced by compounds
III and IV in a small dose even leads to the so-called “over-
growth” effect, whereby the disks impregnated with these
drugs at a concentration of 10 mg/ml not only fail to inhibit
the growth of St. aureus, but even are overgrown with the
bacterial species.
moment
I
2.73
– 760.21 0.385
– 1277.6 0.538
0.203
7.2 ± 0.06
9.0 ± 0.11
6.1 ± 0.05
6.2 ± 0.06
II
3.867
4.774
5.399
– 0.066
– 0.562
– 0.796
III
IV
– 2209.82 0.785
– 2493.15 0.862
We believe that, being capable of influencing the metab-
olism of purines [15], 1,2,4-triazole is a mutagen for St.
aureus. Mutant clones possess highly active enzymatic sys-
tems, which can both modify the action of triazolides and ac-
tively use triazolides containing benzene rings (compounds
III and IV) in the bacterial cell metabolism. Compounds III
and IV also induce such mutations in St. aureus. Thus, St.
aureus species most frequently maintain the cell homeostasis
using the genetic mechanisms of cell adaptation, which ac-
count for the rapid development of resistance to various
drugs.
In order to refine the mechanisms of adaptive response,
the preadaptation in experimental series 3 was induced by
the initial 1,2,4-triazole (I) in a nontoxic dose and checked
on the highly toxic doses (Fig. 2) of its derivatives (II – IV).
It was established that St. aureus pretreated with compound I
at a concentration of 0.001 mg/ml becomes resistant to a
high dose of its derivatives (II – IV). However, the growth in-
hibition zone size was reliably greater than in series 1 (the
phenomenon of overgrowth was observed only for com-
pound III).
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