Tartaric Acid Synthetic Derivatives Activity Against Pseudomonas, Strenotrophomonas, Xanthomonas Phytopathogens and Oppotunistic Pathogens

The multi-drug resistance is one of the most actual medical and ecological problem. Stenotrophomonas, Xanthomonas, Pseudomonas are common, drug resistant and highly adaptive phytopathogenic and opportunistic pathogenic microbes. Tartaric acid is natural safe antimicrobial agent. In current paper it is discussed the synthesis and antimicrobial activity of tartaric acid new derivatives, which were investigated in our laboratory. There were obtained benzyl, cyclohexyl and phenyl substituted imides as well as complex amine salts of tartaric acid. Their antimicrobial activity was tested on native soil Pseudomonas, Xanthomonas and Stenotrophomonas different strains from The National Microbe Collection of Scientic and (SPC) As a result of cultivation on appropriate compound containing media, as well as genetical analyses of tested microbes and docking analyses, it was found out that tartaric acid carbocyclic and aromatic imides and complex salts are effective as non-selective growth inhibitors. Complex salts of tartaric acid are biodegradable by some soil strains of P. chlororaphis, etc. Thus, all the elaborated compounds can be recommended for further study of their activity as an effective alternative against the multi-drug resistant microorganisms. All the researched strains of microorganisms were cultivated on various selective cultural media with containing the tested synthetic compounds, after the tests of their antibiotic resistance to 13 antibiotics of different classes and generations. The tests were done, due to standard protocols. There selective cultural media were containing 50mcg/ml of following antibiotics: from β-lactamic - Pen/Penicillin, Amp/ampicillin, Amx/Amoxicillin, Amc/Augmentin, Cfx/Cexime and Cro/Ceftriaxone; from aminoglycosides - Gen/Gentamicin, Kan/kanamycin, Str/Streptomycin; from uoroquinolones - Cip/Ciprooxacin; from tetracyclines - Tcn/Tetracycline; from azalides of macrolides -Azm/azithromycin; from amphenicoles - Chl/Chloramphenicol.


Introduction
Tartaric acid (TA) is the most common natural aldaric acid plants. TA and tartrates are well-known compounds, which are broadly used in chemical and food industry, as safe antibacterial agents [Lu et al. 2013; Nagata 2020; Karimi et al. 2015]. Xanthomonas, Stenotrophomonas and Pseudomonas are common inhabitants of wet surfaces of practically every where on Earth. These bacteria are involved in a huge quantity of consumption chains, as reducers and are represented by opportunistic pathogens, pathogens and non-pathogenic strains. Representatives of these genera are very close by their metabolism and wide diversity of adaptation biochemical mechanisms. They are able to biodegradation of wide spectrum of xenobiotics [Park 2016; Gomila 2017]. These bacteria include phytopathogenic (P. syringae), as well as pathogenic and opportunistic pathogens of human and animals (S. maltophilia (former Xanthomonas or later Pseudomonas maltophilia), P. aeruginosa,). Besides, they are able to synthesize various bioactive substances: plant growth stimulators, immune modulatory bioactive compounds, several pests and phytopathogenic microorganism metabolism inhibitors, etc. [Chakhtoura 2018; Choi et al. 2006]. Xanthomonas have many similarities to Pseudomonas and Stenotrophomonas, being a part of 2 close families Pseudomonadaceae and Xanthomonadaceae of Gram-negative bacteria. The majority of Xanthomonas are presented as plant pathogens, with world around spread areal. The mainly important representatives of these microorganisms are Xanthomonas vesicatoria, Xanthomonas beticola (former Curtobacterium acumfaciens pathovar beticola) as well as phytopathogenic Pseudomonas syringae [Anzai et al. 2000].
The multidrug resistance of Pseudomonas, Xanthomonas and Stenotrophomonas has a huge importance because of the ability of these microorganisms to transfer this property to other various microorganisms by intraspeci c gene transfer process [Adesoji et al. 2015]. In current paper there were researched the in uence of synthetic derivatives of tartaric acid on growth of non-pathogenic and opportunistic pathogenic strains of soil microorganisms from genera Stenotrophomonas and Pseudomonas. The New synthetic derivatives of TA, which were synthesized by two stage technology which was elaborated in our laboratory, are presented on Figure 1
All the researched strains of microorganisms were cultivated on various selective cultural media with containing the tested synthetic compounds, after the tests of their antibiotic resistance to 13 antibiotics of different classes and generations. The tests were done, due to standard protocols. There selective cultural media were containing 50mcg/ml of following antibiotics: from β-lactamic -Pen/Penicillin, Amp/ampicillin, Amx/Amoxicillin, Amc/Augmentin, Cfx/Ce xime and Cro/Ceftriaxone; from aminoglycosides -Gen/Gentamicin, Kan/kanamycin, Str/Streptomycin; from uoroquinolones -Cip/Cipro oxacin; from tetracyclines -Tcn/Tetracycline; from azalides of macrolides -Azm/azithromycin; from amphenicoles -Chl/Chloramphenicol. All the used antibiotics were produced by "ASTORIA" The synthetic derivatives of TA ( Fig. 1 and 2), were obtained by the technology which was elaborated in laboratory of NPUA.

Results And Discussion
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 -Natartrate, 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).
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. 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.
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: CAScyclohexylamine 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. CAS  CI  GC   I  II  III  IV  V  VI  II  III  IV  V  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 de ned by the a nity 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.

Bacterial strain
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. uorescence, after few days of cultivation on nutrient agar cultural media. Probably, this effect is being de ned by selection of resistant mutants and is related with some enzymatic activity, for example polyphenol oxidases activity of Pseudomonas [ 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.

Conclusion
During all the experiments with tartaric acid synthetic derivatives in forms of imides and complex salts of it on about 100 native soil strains of Pseudomonas and Stenotrophomonas, the antimicrobial effect of researched 4 compounds was noted. Benzylimide, Cyclohexylimide, as well as benzyl and cyclohexylamine complex salts of TA had demonstrated yourself as effective antibacterial agents against both sensitive and multidrug resistant representatives of both genera Pseudomonas and Stenotrophomonas isolated from soil. Antimicrobial effect of the discussed new semisynthetic compounds in a majority of cases was stronger than in case of well-known tartaric acid derivatives usage. For some strains the prolongation of growth till 1-2 days is detected. In other part of researched representatives of Pseudomonas, Xanthomonas and Stenotrophomonas there were detected single resistant colonies. For plasmid containing ones, the transformations of sensitive Pseudomonas representatives by the plasmids of mutants showed that the property of single colonies is not transmittable. Thus, the mutations are in nucleoid genes and cannot be transmitted to environment, by intraspeci c gene horizontal transfer to other Gram-negative bacteria. Probably, these mutations are related with nucleoid localized polyphenol oxidases genes of Phytopathogenic Xanthomonas and Pseudomonas, as well as opportunistic pathogenic and Stenotrophomonas and Pseudomonas.
During the biodegradation tests, cyclohexyl-derivatives showed themselves as less biodegradative properties than the benzyl-derivatives. Perhaps, it is caused by chemical structure features of cyclohexyl radical and disabilities of appropriate bacterial enzymes to recognize these molecules as substrates, because of low a nity, absence of it or because of some allosteric regulation effects. The opportunistic pathogenic and non-pathogenic strains of Pseudomonas and Stenotrophomonas, isolated form soil can be good models of highly adaptive pathogenic bacteria. Thus, all the tested semisynthetic substances are recommended for further research of their e ciency as antibacterial agents and ecological safeness, as potentially biodegradative compounds.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable" in this section.

Data availability
Data used in this study were retrieved from the Clarivate Analytics PubMed Core Collection.

Competing interests
The authors declare that they have no con ict of interest. The principal Scheme of Tartaric acid derivatization. R = benzyl, phenyl, cyclohexyl, ethanolamine radicals in appropriate derivatives