The results of all the experiments with antimicrobial effect of tartaric acid 4 new derivatives on Stenotrophomonas maltophilia and Pseudomonas different strains of different species and subspecies are represented in Tables 1–2.
Table 1
The comparison of TA derivatives antimicrobial activity on S. maltophilia different strains (50mkg/ml). TA – tartaric acid, K –K/Na-tartrate, Na – Na-tartrate, CI – cyclohexylimide of TA dissolved in DMSO, BI – benzylimide dissolved in DMSO, BAS – benzylamine complex salt of TA, CAS – Cyclohexylamine complex salt of TA; “+” – growth, “-“ – the absence of growth, С – control on nutrient solid agar media).
Strain
|
BI
|
CI
|
BAS
|
CAS
|
TA
|
PhI
|
PhAS
|
EAS
|
K
|
Na
|
C
|
9303
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
+
|
-
|
-
|
+
|
9306
|
-
|
-
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
9301
|
-
|
-
|
+
|
-
|
+
|
+
|
+
|
-
|
+
|
+
|
+
|
9307
|
-
|
+
|
+
|
+
|
+
|
+
|
-
|
+
|
+
|
+
|
+
|
9310
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
9203
|
+
|
-
|
+
|
-
|
+
|
-
|
-
|
+
|
+
|
+
|
+
|
As it was shown on Table
1, the predominance of strains of
S. maltophilia are non-sensitive to tartaric acid. The strains are more sensitive to imides then to complex salts. For this group of strains, the secondary growth was not registered. Then from all the researched strains was isolated DNA and analyzed. DNA analysis showed the presence of plasmids only in strains:
S. maltophilia 9303, 9301, 9307, 9302.
DNA analysis of target groups of P. putida, P. aeruginosa, P. geniculate, P. chlororaphis, P. syringae, X. vesicatoria, X. beticola and P. fluorescens showed the presence of plasmids only in following strains: 9131, 9068, 9070, 9069, 9091, 9092, 9150, 9114, 9142, 9110, 9106, 9124. On a next stage, there were done transformation of sensitive non-plasmid strains of P. aeruginosa 9056 and E. coli DH5α by the plasmid DNA of these resistant strains. Both used recipients are sensitive to 13 antibiotics of 5 classes, to which were tested target groups of Xanthomonas, Pseudomonas and Stenotrophomonas. That is why the positive control was on antibiotic resistance plasmids of same species, and the negative control on streptomycin genes, which are encoded by bacterial chromosome genes. Thus, during the experiments there were not detected transformants, with the resistance to tartaric acid derivatives or ability to biodegradation of them on mineral media.
Table 2
Tartaric acid natural and synthetic derivatives antimicrobial effect comparison against the various strains of P. fluorescens. (50mkg/ml). N – the number of strain, TA – tartaric acid, K –K/Na- tartrate, Na – Na-tartrate, CI – cyclohexylimide of TA dissolved in DMSO, BI – benzylimide dissolved in DMSO, BAS – benzylamine complex salt of TA, CAS – Cyclohexylamine complex salt of TA; “+” – growth, “-“ – the absence of growth,”+*” - late growth after III day, “+/-“ - late growth after IV day, “+#” - late growth of singular colonies after 5th day, С – control on nutrient solid agar media.
Strain
|
BI
|
CI
|
BAS
|
CAS
|
TA
|
K
|
Na
|
C
|
Strain
|
BI
|
CI
|
BAS
|
CAS
|
TA
|
K
|
Na
|
C
|
9100
|
+*
|
-
|
+#
|
+#
|
+
|
+
|
+
|
+
|
9070
|
-
|
-
|
-
|
-
|
+
|
+
|
-
|
+
|
9205
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
+
|
9072
|
-
|
+#
|
-
|
+#
|
+
|
+
|
+
|
+
|
9106
|
-
|
+#
|
-
|
-
|
+*
|
-
|
-
|
+
|
9077
|
+#
|
+#
|
+#
|
+#
|
-
|
-
|
-
|
+
|
9108
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
+
|
9089
|
-
|
+
|
+
|
-
|
-
|
-
|
-
|
+
|
9095
|
+#
|
-
|
+
|
+
|
+
|
+
|
+
|
+
|
9069
|
-
|
+/-
|
+/-
|
-
|
+
|
-
|
-
|
+
|
9084
|
-
|
+#
|
+#
|
+#
|
+
|
-
|
+
|
+
|
9068
|
-
|
-
|
-
|
-
|
+
|
-
|
+
|
+
|
9123
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
9073
|
+
|
+
|
+
|
+
|
+
|
+
|
-
|
+
|
9096
|
+
|
+#
|
+
|
+/-
|
+
|
-
|
-
|
+
|
9179
|
+
|
+
|
-
|
-
|
+
|
+
|
-
|
+
|
The activity of tartaric acid synthetic derivatives against the range of phytopathogenic microorganisms is presented on table 3.
Table 3
Antimicrobial activity of tartaric acid synthetic derivatives on phytopathogenic Pseudomonas and Xanthomonas different strains. Inhibition zones are presented in mm. The cultures: a – Pseudomonas syringae, b – Xanthomonas vesicatoria; L – complete lysis of growth zone, GС – the positive control on solid nutrient agarised cultural media with 30mm maximal growth zones, “+” – normal growth and the absence of inhibition effect, “+/-“ – less than 10% inhibition; the tested compounds: CAS – cyclohexylamine complex salt of tartaric acid, CI – cyclohexylimide of tartaric acid, BAS – benzylamine complex salt of tartaric acid, BI – benzylimide of tartaric acid, PhAS – Phenylamine complex salt of tartaric acid, PhI – Phenylimide of tartaric acid; the tested compounds concentrations: I- 50mcg/ml, II- 0.001M, III- 0.01M, IV- 0.05M, V- 0,1M, VI- 0,5M.
Bacterial strain
|
CAS
|
CI
|
GC
|
I
|
II
|
III
|
IV
|
V
|
VI
|
II
|
III
|
IV
|
V
|
VI
|
8736
|
a
|
15
|
17.2
|
25
|
25
|
L
|
L
|
23
|
23
|
23
|
23,8
|
L
|
30
|
8740
|
11
|
11
|
12
|
20
|
22
|
L
|
9.1
|
10
|
11.5
|
13
|
17
|
30
|
8744
|
12.1
|
13
|
15
|
14
|
18
|
20
|
14
|
16.2
|
19
|
20.1
|
22
|
30
|
8656
|
8
|
8
|
14.9
|
15.1
|
17
|
L
|
10.3
|
11,4
|
12
|
12.7
|
14
|
30
|
8647
|
b
|
18.2
|
20
|
19.7
|
20.4
|
22
|
L
|
20
|
20.8
|
21
|
21.3
|
22
|
30
|
8651
|
15
|
16.1
|
18.5
|
20.1
|
25
|
L
|
15
|
15,5
|
17,4
|
18,1
|
19
|
30
|
8843
|
L
|
L
|
L
|
L
|
L
|
L
|
L
|
L
|
L
|
L
|
L
|
30
|
Bacterial strain
|
PhI
|
PHAS
|
C
|
I
|
II
|
III
|
IV
|
V
|
VI
|
I
|
II
|
III
|
IV
|
V
|
8736
|
a
|
+
|
+
|
+/-
|
+/-
|
8
|
+
|
+
|
+
|
+
|
+
|
+
|
30
|
8740
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
30
|
8744
|
+
|
+
|
+/-
|
3.5
|
8
|
+
|
+
|
+
|
+
|
+/-
|
6
|
30
|
8656
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+/-
|
3
|
30
|
8647
|
b
|
+
|
+
|
+
|
+/-
|
6
|
+
|
+
|
+
|
+/-
|
3
|
L
|
30
|
8651
|
+
|
+
|
+
|
+/-
|
5
|
+
|
+
|
+
|
+
|
+
|
+
|
30
|
8843
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
30
|
Bacterial strain
|
BAS
|
BI
|
I
|
II
|
III
|
IV
|
V
|
VI
|
I
|
II
|
III
|
IV
|
V
|
VI
|
8736
|
a
|
+
|
+
|
+/-
|
+/-
|
+/-
|
+/-
|
3
|
5
|
L
|
L
|
L
|
30
|
8740
|
+/-
|
5
|
6
|
6,5
|
7
|
8
|
5
|
6
|
6,5
|
7
|
7,2
|
30
|
8744
|
+
|
+
|
+/-
|
4
|
5,5
|
6
|
+
|
+/-
|
+/-
|
3,2
|
4,2
|
30
|
8656
|
+/-
|
2
|
3,8
|
5,5
|
6
|
6,5
|
3,9
|
4
|
4,5
|
5,6
|
L
|
30
|
8653
|
b
|
+.-
|
3
|
4,3
|
5,7
|
6,2
|
L
|
+
|
+
|
+
|
+
|
+
|
30
|
8651
|
|
+
|
+
|
+
|
+/-
|
3,4
|
+
|
+
|
+/-
|
+/-
|
3
|
30
|
8647
|
|
+
|
+
|
+
|
+/-
|
+/-
|
+
|
+
|
+/-
|
+/-
|
3,5
|
30
|
Due to data from Table
3, the maximal activity against the phytopathogenic strains of
Xanthomonas and
Pseudomonas was demonstrated in a majority of cases by cyclohexyl substituted derivatives of tartaric acid. Preheats, it might be defined by the affinity differences in cyclohexyl, phenyl and benzyl groups to enzymes, which are able to degradation of these compounds. The experiments of enzymes activity precipitations in various strains of Pseudomonas and Stenotrophomonas showed the high polyphenol oxidase activity in those strains cells which were resistant to tartaric acid cyclic derivatives effect [Liu at al, 2018; Babayan et al.
2020d]. According to literature data, the genes of 2 types of polyphenol oxidases in researched
Stenotrophomonas maltophilia are encoded by their bacterial chromosome too. Then the resistant strains were cultivated on mineral media for biodegradation tests [Babayan
2020e]. The results showed that biodegradation of tartaric acid complex salts processes more intensively. Probably it is caused by their more hydrophilic properties, in opposite to hydrophobic imides.
Due to the antimicrobial activity tests, the species P. taetrolens, P. chlororaphis and P. geniculate were predominantly resistant to complex salts of tartaric acid, while in case of imides it was noted the inhibition of growth. Thus, for 3 subspecies of P. chlororaphis (P. chlororaphis, subsp. chlororaphis, P. chlororaphis subsp. aureofaciens, P. chlororaphis, subsp. Aurantiaca) and other resistant representatives were done biodegradation tests (table 4).
Table 4
Tartaric acid synthetic derivatives biodegradation by of P. chlororaphis 3 subspecies different strains. The bacterial strains P. chlororaphis subspecies – A – P. chlororaphis, subsp. chlororaphis, B - P. chlororaphis, subsp. aureofaciens, С – P. chlororaphis, subsp. aurantiaca; the tested compounds: CI – cyclohexylimide of tartaric acid (TA), BI – Benzylimide of TA, BAS – benzylamine complex salt of TA, CAS – Cyclohexylamine complex salt of TA; “+” - growth, “-“ - absence of growth, “+*” - secondary growth after 5th day, GС+ – the positive control on nutrient agar cultural media, GC− - the negative control on mineral media.
Strain
|
CI
|
BI
|
BAS
|
CAS
|
GC+
|
GC−
|
Strain
|
CI
|
BI
|
BAS
|
CAS
|
GС+
|
GC−
|
A
|
9189
|
-
|
-
|
+
|
-
|
+
|
-
|
A
|
9165
|
-
|
-
|
+*
|
-
|
+
|
-
|
9171
|
-
|
-
|
+
|
-
|
+
|
-
|
9163
|
-
|
-
|
-
|
-
|
+
|
-
|
9190
|
-
|
-
|
+
|
-
|
+
|
-
|
9157
|
-
|
-
|
-
|
+
|
+
|
-
|
9159
|
-
|
-
|
-
|
-
|
+
|
-
|
9178
|
-
|
-
|
-
|
−
|
+
|
-
|
9177
|
-
|
-
|
+
|
+
|
+
|
-
|
9172
|
-
|
-
|
-
|
-
|
+
|
-
|
9168
|
-
|
-
|
-
|
-
|
+
|
-
|
9174
|
-
|
-
|
-
|
-
|
+
|
-
|
9164
|
-
|
-
|
-
|
-
|
+
|
-
|
9158
|
-
|
-
|
-
|
-
|
+
|
-
|
B
|
9195
|
-
|
-
|
+
|
+
|
+
|
-
|
C
|
9066
|
-
|
-
|
+
|
-
|
+
|
-
|
9200
|
-
|
-
|
-
|
-
|
+
|
-
|
9062
|
-
|
-
|
+
|
+
|
+
|
-
|
9199
|
-
|
-
|
-
|
-
|
+
|
-
|
9064
|
-
|
-
|
-
|
+
|
+
|
-
|
In a majority of considered strains of microorganisms, the effect of tartaric acid synthetic derivatives is being presented by both bacteriostatic (with the prolongation of growth period) and bactericide (the absence of growth even after 7 days of cultivation) activities. It is correlating with well-known antibacterial activity data about the effect oа aldaric acids in general. Moreover, it was noted the forming of singular resistant colonies of some strains of P. fluorescence, after few days of cultivation on nutrient agar cultural media. Probably, this effect is being defined by selection of resistant mutants and is related with some enzymatic activity, for example polyphenol oxidases activity of Pseudomonas [Bagdasaryan et al. 2019; Janusz et al. 2020; Soong et al. 1999].
The results of bacterial growth inhibition by different derivatives of tartaric acid are represented on Fig. 3 and Fig. 4.
Both types of experiments on liquid and solid cultural media showed the maximal inhibitory activity of cyclohexyl substituted derivatives of tartaric acid. Phenyl and ethanolamine derivatives were less active against the various strains of phytopathogenic Pseudomonas syringae, Xanthomonas vesicatoria, Xanthomonas beticola, as well as against the animal and human opportunistic pathogenic Stenotrophomonas maltophilia and Pseudomonas aeruginosa.