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Section 1. Biology
https://doi.org/10.29013/AJT-20-5.6-3-7
Bagdasaryan Samvel, senior laboratory assistant, "Armbiotechnology" SPC, NAS RA E-mail: sambagd55@gmail.com Babayan Bella,
PhD student, researcher, "Armbiotechnology" SPC, NAS RA, National Polytechnic University of Armenia (NPUA)
E-mail: bbg.15.04@mail.ru Melkumyan Marina, PhD in Biology, senior researcher, "Armbiotechnology" SPC, NAS RA E-mail: marmelk2109@gmail.com Kinosyan Marina, Researcher, "Armbiotechnology" SPC, NAS RA E-mail: marina.kinosyan@mail.ru
POLYSORBATES BIODEGRADATION POTENTIAL AND PLASMID STABILITY OF SOIL PSEUDOMONAS
Abstract. The main aim of current research was a consideration of xenobiotics biodegradation potential of more than 50 strains of 7 species and 3 subspecies of mentioned bacteria and their antibiotic resistance as interacting parts of one system of adaptation of microorganisms to changing environment. And it has not only theoretical scientific importance, for understanding of bases of Pseudomonas and Stenotrophomonas resistance plasmids stability, but also has a practical significance for solving of modern ecological problems of new multidrug resistant pathogen forming and xeno-biotic biodegradation processes in soil.
Keywords: Pseudomonas, biodegradation, antibiotic-resistance, plasmids, lipases.
Introduction the well-known properties of Pseudomonas, which
The problem of multi-drug resistance of mi- are being isolated from clinics [2]. But the native
crobes, according to WHO reports of recent years, strains of these bacteria, which were isolated from
is one of key importance research directions for up- soil and other wet surfaces of natural and artificial
coming century [1]. The multiple resistance of mi- origin, have the wide spectrum of resistance to anti-
croorganisms to antimicrobial preparations is one of biotics of II and III generations and they are able to
stable transfer this property to other Gram-negative bacteria too. Thus it has not only medical but also ecological and archeological significance, because of very broad life areal of these microorganisms.
According to the research of recent years, Pseudomonas and other cave microorganisms are endangering not only the health visitors and cave researchers, bu also are becoming the new object for resistance research in aspects of both resistance new types discovery and the discovery of new antibiotics [3; 4]. Due to literature data, Pseudomonas are highly adaptive microorganisms. They have a wide diversity of enzymes and biochemical pathways of biodegradation of various native substances and xenobiotics [5; 6]. That is why the study of relation of stabile antibiotic resistance persistence and the transfer of it with xenobiotic biodegradation genes localization in bacterial genome is very significant for understand the mechanisms of this problem.
Materials and Methods
During this research there were used strains from The National Culture Collection of Microorganisms of the MDC of "Armbiotechnology" SPC NAS RA. They were cultivated according to the standard protocols. For the resistance tests here were used 50mkg/
ml concentrations of13 antibiotics of different classes and generations: penicillin/Pen, ampicillin/Amp, amoxicillin/Amx, augmentin/Amc, cefixime/Cfm, ceftriaxone/Cro, kanamycin/Kan, streptomycin/Str, gentamycin/Gen, ciprofloxacin/Cip; tetracycline/ Tet; Chloramphenicol Chl) of amphenicoles, azithromycin (Azm) - of azalides of macrolides [7; 8].
Biodegradation potential was evaluated due to extracellular enzyme activity precipitation. Lipases activities tests were carried out according to standard microbiology and biochemistry protocols on solid cultural media, using substitution of carbon source to appropriate polysorbates -20,-40,-60,-65,-80,-85 with different length of fatty acids [9]. The genetical analyses was done by DNA electrophoresis, transformation and PCR. DNA isolation was done by alkaline extraction for plasmids and with benzyl-chloride method for total DNA. PCR analysis of antibiotic modification enzyme genes was done with the following primers: aph(3')YV, aac(6')II,pCAT639, bla-OXA-10 [10]. The statistical verification of experiments was done by standard methods [11; 12].
Results
The results of polysorbate degradation tests are presented on (table 1).
Table 1. - Polysorbates biodegradation by Pseudomonas
Strain of microorganism Polysorbates C+ C-
20 40 60 65 80 85
G A G A G A G A G A G A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
P. geniculate 9335 3+ — 3+ — 3+ — + — 3+ — 3+ — 5+ —
9336 3+ — 3+ — 3+ — + — 3+ — 3+ — 5+ —
9337 3+ — 3+ 3+ 3+ — 2+ — 3+ — 3+ 3+ 5+ —
9338 2+ — 3+ — 3+ — 2+ — 3+ — 3+ — 5+ —
9339 3+ — 2+ — 3+ — + — 3+ — 3+ — 5+ —
9340 2+ — 3+ — 3+ — 2+ — 3+ — 3+ — 5+ —
9205 3+ — + 2+ 3+ — 3+ — 3+ — 2+ — 5+ —
9203 3+ 2+ + — 2+ + + + 3+ 3+ 3+ — 5+ —
9212 2+ + 2+ - 2+ + + + 2+ — ++ + 5+ —
P. aeruginosa 9059 3+ — + — 3+ — 3+ — 3+ + 3+ 3+ 5+ —
9058 3+ — 3+ — 3+ — 3+ — 3+ — 3+ — 5+ —
9056 - 5+ —
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
9075 3+ + 3+ + 3+ — 2+ — 3+ — 3+ 3+ 5+ —
9077 3+ — 3+ — 3+ — 2+ — 3+ — 3+ — 5+ —
9252 3+ — — — 3+ — + — 3+ 5+ 3+ + 5+ —
P. fluorescens 9089 3+ — 3+ — 3+ — 2+ — 3+ — 3+ — 5+ —
9092 3+ — 3+ — 3+ — 2+ — 3+ — 3+ — 5+ —
9095 3+ 5+ 2+ — 2+ — 2+ — 3+ — 3+ — 5+ —
9096 3+ - 3+ — 3+ — 2+ — 3+ — 3+ — 5+ —
Polysorbates - 20, 40, 65, 80, 85; G - growth (after 5 days), A - activity; "+" - growth/lipase activity presence different intensity;"-" - growth/lipase activity absence; "C+" - positive control on nutrient agar media; "C- negative control on mineral cultural media
Table 2. - Resistance transfer in soil Pseudomonas
Rec Donor R/S of Rec R/S of Donor 1 2 3 4 5 6 7 8 9 10 11 12 13 С2
I pUC18 (C1) S Amp — + +
II S. maltophilia 9302 S Str, Pcn, Amx Amp + + + +
III pUC18 Cip, Cro, Amp — — — — — + — — — — — + — +
IV pUC18 Cip, S Amp + — — — — — — — — — + — — +
Chl, Amc,
V VOG16 Amx, Amp Pcn, Cfm Kan + + + + + + + + +
VI pUC18 Cfm Amp — + — — + — — — — — — +
III S. maltophilia 9285 Cip, Cfm Kan Amc Amx Amp Cfm — — — — + — — — — — — + — +
VI S. maltophilia 9288 Chl, Amc, Amx, Amp, Cfm, R, CroS, TetS, AzmS, CipS — + + + + — + + +
IV S. maltophilia 9277 Cip Amc Amx Amp Cfm + + + + + — +
VI S. maltophilia 9304 Cfm Cro — — — — + — — — — — — — — +
Antibiotics: 1 - penicillin/Pen, 2 - ampicillin/Amp, amoxicillin/Amx, 3 - augmentin/Amc, 4 - cefixime/Cfm, 5 - ceftriaxone/Cro, 6 - kanamycin/Kan, 7 - streptomycin/Str, 8 - gentamycin/Gen, 9 - ciprofloxacin/Cip, 10 -tetracycline/Tet, 11 - Chloramphenicol/Chl, 12 - azithromycin /Azm; Rec. (Recipients): I - P. aeruginosa 9056, II - E. coli DH5a, III - S. maltophilia 9326, IV - P. chlororaphis, subsp. chlororaphis 9329, V - P. fluorescens 9092, VI - P. taetrolens 9243; S - sensitivity, R - resistance, C1 -plasmid transmission control by E. coli DH5a/pUC18 plasmid, C2 - control on nutrient agar, "+" - growth, "-" - the absence oa growth
Then the same procedure of transformation was repeated with other recipient strains. The same results were obtained in 6 used recipients of different species of bacteria. While the tests of stability, the divergence of results was detected. The stability of replication was detected for all the researched donors in case of P. aeruginosa 9056 recipients, except the P. taetrolens recipients which didn't demonstrate the stability after the cultivation of transformants using non selective nutrient agar media. Thus it might
be concluded that the cause of plasmid resistance in general is defined by the genome structure of P. aeruginosa [13]. In case of P. taetrolens, probably there is the system which is blocking the possibility of stable replication of donor DNA in recipient cells. The antibiotic resistance tests of all the obtained transformed strains are presented on table5. The results of antibiotic resistance test were the same for each several donor and all the used recipients, thus in table is represented only one of 6 experiments of each series.
A B
Figure 1. Lipase activity of different stains (A - P. taetrolens 9246 on polysorbate-85; B - P. chlororaphis subsp. aurantiaca 9061)
Conclusions
During the experiments, it was found out that the genes of 5 types of polysorbate degrading lipases are different. They can have both plasmid and nucleoid localization in frames of one species and one subspecies. Probably, one part of these plasmid localized lipases can play a key role for antibiotic resistance plasmid stability in some strains of P. taetrolens, while the stability of plasmids from P. fluorescens, P. chlororaphis and P. aeruginosa, is predominantly caused
by genome of recipient strains. The maximal stability was detected on both selective and non-selective cultural media in recipient cells of P. aeruginosa.
Acknowledgements
This research is supported by the research grant #microbio-5133, from the Armenian National Science and Education Fund (ANSEF) based in New York, USA and The Ministry of Education, Science, Culture and Sports of SCS, in frames of the research project № 18T-2I036.
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