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47
Section 1. Biology
Babayan Bella,
researcher, "Armbiotechnology" SPC NAS RA & National Polytechnic University of Armenia (NPUA) E-mail: bbg.15.04@mail.ru
MULTIPLE ANTIBIOTIC RESISTANCE OF SOIL STRAINS OF PSEUDOMONAS CHLORORAPHIS
Abstract. P. chlororaphis is a very common in nature. It includes few subspecies (P. chlororaphis subsp. chlororaphis, P. chlororaphis subsp. aureofaciens, P. chlororaphis subsp. aurantiaca, P. chlororaphis subsp. taetrolens, etc.) with wide usage as a soil inoculum in agriculture and horticulture. Representatives of P. chlororaphis have a big potential for agrobio-technology for creation of plant bioprotectors against phytopahogenic microorganisms, as an alternative of classical antibiotics. Besides, some strains of P. chlororaphis are being used as growth and fruiting biostimulator producents for horticultural and agricultural crops.
Therefore, according to permanently enlarging of antibiotic wide usage, the research of antibiotic resistance of P. chlororaphis and the ability to spread this property among other microorganisms are very important.
Keywords: Pseudomonas chlororaphis, multidrug resistance, plasmids, PCR.
Introduction
P. chlororaphis is a group of very common Gram-negative bacteria of soil and other wet media. It is often being used as a soil inoculant in agriculture and horticulture. P. chloro-raphis can act as a biocontrol agent against certain fungal plant pathogens via production of phenazine-type antibiotics [1; 2]. They produce phenazine-type antibiotics, which are effective against some phytopahogens. Based on genetic analyses, similar species have been placed in group. P. chlororaphis lends its name to a subgroup within the genus, including P. chlororaphis subsp. chlororaphis, etc. The other members of group are P. aurantiaca, P. aureofaciens, P. fragi, P. lundensis, and P. taetrolens [3]. P. chlororaphis subsp. aurantiaca is an orange soil bacterium, of rhizosphere of potatoes. In P. chlororaphis subsp. aurantiaca produces di-2,4-diacetylfluoroglucylmethan, which is antibiotically active against Gram-positive organisms. P. chlororaphis subsp. aureofaciens is a yellowish, soil bacterium isolated from clay near the River Maas. P. chlororaphis subsp. taetrolens is motile, rod-shaped bacterium that causes musti-ness in eggs [4]. Wide usage of antibiotics in different scopes of human activity often becomes a cause of forming of new pathogenic antibiotic resistant strains, which have inherited their resistance from native Gram-negative soil bacteria [5, 6].
In current paper, the mechanisms of antibiotic resistance of different strains of 4 subspecies of P. chlororaphis were researched.
Materials and Methods
18 strains of 4 subgroups of soil strains of P. chlororaphis from The National Culture Collection of Microorganisms of the MDC of "Armbiotechnology" SPC NAS RA (P. chlororaphis subsp. chlororaphis, P. chlororaphis subsp. aureofaciens, P. chlororaphis subsp. aurantiaca and Pseudomonas chlororaphis subsp taetrolens) were researched. As the control samples there were used non-plasmid sensitive strains P. aeruginosa and E.coli DH5a [7]. All the researched strains were cultivated on liquid and solid agarised cultural media at the temperature 37 °C. Antibiotic resistance was tested according to standard protocols using the following antibiotics, produced by "Astoria": ^-lactamic (Amp/ampicillin, Amx/Amoxicillin, Amc/Aug-mentin, Cfx/Cefixime and Ctx/Ceftriaxone); aminoglycoside (Kan/kanamycin, Stp/Streptomycin); fluoroquinolone (Cip/Ciprofloxacin); Tcn/Tetracycline, Macrolides (Azm/ azithromycin); amphenicol (Cam/Chloramphenicol) [8; 9; 10; 11]. The genetical analysis was done by DNA electrophoresis on 0.8-2.5% agarose gel, transformation by Mandel's method 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')lV, aac(6') II,pCAT639, blaOXA-lO and marker standard fragment mix EcoRI/Hind III [12; 13; 14].
Results and Discussion (table 1), in frame of researched group of strains, the quan-
The research of antibiotic resistance of different strains titative prevalence of multidrug resistant representatives is of P. chlororaphis are presented in (table 1). According to makeable.
Table 1.- Antibiotic resistance of P. chlororaphis different strains. (classes of antibiotics in 50 mkg/ml concentration: 1 - aminoglycosides, 2 - amphenicols, 3 - P-lactams, 3(1) - aminopenicillins, 3(2) - cephalosporins, 4 - tetracyclines, 5 - azalides, 6 - fluorchinolone; "+" - growth, "-" - absence of growth, C - control on solid agarised cultural media)
Strain P. chlororaphis 1 2 3 4 5 6 C
3(1) 3(2)
subsp. aurantiaca Kan Stp Cam Amc Amx Amp CfX Ctx Tcn Azm Cip
9064 - + + - + + + + + + + +
9061 - - + + + + + + + - - +
9062 - - - - - - - - - - - +
subsp. aureofaciens Kan Stp Cam Amc Amx Amp Cfx Ctx Tcn Azm Cip +
9200 + - - - - - - + - - - +
9060 - + + + + + + - + - + +
9192 - - - - - - - - - - - +
9197 - - - - - - - - - - - +
9026 - - - - - - - - - - - +
subsp. chlororaphis Kan Stp Cam Amc Amx Amp Cfx Ctx Tcn Azm Cip +
9329 - - - - - - - - - - + +
9158 - - + - + - + - - - - +
9156 - - + - - + - + - - - +
9167 - - - - - + - + + - + +
9175 - - - + + + + - + - - +
subsp. taetrolens. Kan Stp Cam Amc Amx Amp Cfx Ctx Tcn Azm Cip +
9246 + + + + + + + - - - - +
9243 - - - - - - + - - - - +
9242 - - - - - - + + + - - +
9241 - - - + + - + + - + + +
9248 - - + - - - - + - + + +
The results of comparison of DNA analysis of researched containing and non-plasmid strains. PCR analysis showed sensitive and resistant strains are presented on (table 2 and 3). the presence of 2 types of genes of antibiotic modification The electrophoretic analysis showed the presence of plasmid enzymes.
Table 2.- DNA analysis of P. chlororaphis different strains (p - plasmid, presence ("+")/ absence ("-") of plasmid or gene correspondently)
Strain of Pseudomonas chlororaphis P +/- PCR analysis of antil jiotic modification genes
aph(3')lV aac(6')lI pCAT639/catB7 blaOXA-lO
subsp. chlororaphis
1 2 3 4 5 6
9329 + - - - -
9158 + - - - -
9156 + - - - -
9167 - - - - -
9175 - - - - -
subsp. aureofaciens P +/- aph(3')lV aac(6')lI pCAT639/catB7 bla0XA-10
9200 + - - - -
1 2 3 4 5 6
9060 + - - - -
9192 + - - - -
9197 - - - - 1.6kb
9026 - - - - -
subsp. aurantiaca aph(3')lV aac(6')lI pCAT639/catB7 bla0XA-10
9064 + - - - -
9061 + - - - -
9062 + - - - -
subsp. taetrolens P +/- aph(3')lV aac(6')lI pCAT639/catB7 bla0XA-10
9246 + - - 1.4kb -
9242 + - - - 1.6kb
9243 + - - - -
9241 + - - - -
9248 + - - 1.4kb 1.6kb
In P. chlororaphis subsp. aureofaciens 9197, P. chlororaphis subsp. taetrolens 9248, P. chlororaphis subsp. taetrolens 9242 the gene blaOXA-lO of ^-lactamase OXA-IO, defining the resistance to ^-lactams was detected by PCR analysis. In P. chlororaphis subsp. taetrolens 9246 and P. chlororaphis subsp. taetrolens 9248 the gene catB7 of chloramphenicol acetyltransferase was detected by PCR analysis with primer pCAT639. Genes of
According to (table 3), resistance to both aminoglycosides and ^-lactam antibiotics is caused by plasmids in 28% of researched strains, which belong to subgroups P. aureofaciens and P. aurantiaca. The phenotype of transformants was same in both cases of P. aeruginosa 9056 and E.coli DH5a.
aminoglycoside modification aac(6')lI of N-aminotransferase and aph(3')lV of O-phosphotransferase were not detected. For the definition of detected genes localization and the evaluation of resistance transfer potential of strains, there were done transformation of sensitive strains of microorganisms from different taxonomic groups [15; 16; 17]. The results of transformation are presented in (table 3).
Conclusion
The strains with wide diapason of resistance were observed. The sensitive strains are presented by the minority. In a majority of cases they are resistant to more than 1 antibiotics. Plasmid containing, non-plasmid resistant and sensitive
Table 3.- Analysis of transformation. ("-") absence of transformation)
Plasmid Donor Strain of Pseudomonas Transmission of antibiotic resis- Transmission of antibiotic resis-
Chlororaphis tance to P. aeruginosa 9056 tance to E.coli DH5a
P. chlororaphis. subsp. chlororaphis 9158 - -
P. chlororaphis subsp. chlororaphis 9156 - -
P. chlororaphis subsp. chlororaphis 9329 - -
P. chlororaphis subsp. aurantiaca 9064 Resistance to all ^-lactams Resistance to all ^-lactams
P. chlororaphis subsp. aurantiaca 9061 - -
P. chlororaphis subsp. aurantiaca 9062 - -
P. chlororaphis subsp. aureofaciens 9200 Resistance to Ctx Resistance to Ctx
P. chlororaphis subsp. aureofaciens 9060 Resistance to Amp, Amx, Amc, Cfx Resistance to Amp, Amx, Amc, Cfx
P. chlororaphis subsp. aureofaciens 9192 Resistance to Amp, Amx Resistance to Amp, Amx
P. chlororaphis subsp. taetrolens 9246 - -
P. chlororaphis subsp. taetrolens 9242 - -
P. chlororaphis subsp. taetrolens 9243 - -
P. chlororaphis subsp. taetrolens 9241 - -
P. chlororaphis subsp. taetrolens 9248 - -
Control (E. coli DH5a/pUC18) Resistance to Amp Resistance to Amp
strains were detected. Thus, the direct correlation between such as like in case of streptomycin. Kanamycin and ^-lactams
the plasmid presence and resistance was not detected. The resistance is transferred by plasmids for 4 strains of subsp. au-genes catB7 and blaOXA-10 were identified in 5 strains of rantiaca and subsp. aureofaciens of P. chlororaphis. P. chlororaphis subsp. aureofaciens, subsp. aurantiaca and subsp. Acknowledgments. This work was supported by the RA
taetrolens. These genes have both plasmid and nucleoid lo- MES State Committee of Science, in frames of the research
calization. In current group of strains of subsp. taetrolens the project № 18T-2I036 and the research grant from the Arme-
plasmid genes were no detected. Chloramphenicol resistance nian National Science and Education Fund (ANSEF), № mi-
is not transferred by plasmids and is caused by nucleoid genes, crobio-5133.
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