RESISTANCE TO β-LACTAM ANTIBIOTICS IN SOME SOIL STENOTROPHOMONAS AND PSEUDOMONAS Текст научной статьи по специальности «Биологические науки»

РЕЗИСТЕНТНОСТЬ К 0-ЛАКТАМНЫМ АНТИБИОТИКАМ У НЕКОТОРЫХ ПОЧВЕННЫХ

STENOTROPHOMONAS AND PSEUDOMONAS

Бабаян Б.Г.

Научно-производственный центр (НПЦ) «Армбиотехнология» НАН РА; Национальный Политехнический Университет Армении (НПУА), научный сотрудник

(научный сотрудник в лаборатории экологической безопасности и лаборатории создания новых аг-

рохимикатов и контроля качества) Оганесян Н.А.

Научно-производственный центр (НПЦ) «Армбиотехнология» НАН РА, заведующая лабораторией,

кандидат биологических наук, (Заведующая лабораторией экологической безопасности)

Оганесян А.М.

Ереванский Государственный Университет (ЕГУ), Институт Фармации, заведующая кафедрой,

кандидат биологических наук,

(Заведующая кафедрой фармтехнологии и фармэкономики и менеджмента)

Саргсян А.С.

Научно-производственный центр (НПЦ) «Армбиотехнология» НАН РА, старший научный сотрудник, кандидат биологических наук, (Старший научный сотрудник лаборатории экологической безопасности)

Давидян Т.С.

Научно-производственный центр (НПЦ) «Армбиотехнология» НАН РА, научный сотрудник (научный сотрудник лаборатории экологической безопасности).

RESISTANCE TO 0-LACTAM ANTIBIOTICS IN SOME SOIL STENOTROPHOMONAS AND

PSEUDOMONAS.

Babayan B. G.

Scientific and Production Center (SPC) "Armbiotechnology" NAS RA; National Polytechnic University of

Armenia (NPUA), researcher

(researcher at the laboratory of Ecological Safety and laboratory of New Agrarian Chemicals Creation and

The Quality Control) Hovhannisyan N.A.

Scientific and Production Center (SPC) "Armbiotechnology" NAS RA, Head of laboratory, PhD,

(Head of laboratory of Ecological Safety) Hovhannisyan A.M.

Yerevan State University (YSU), Institute of Pharmacy, Head of Chair, PhD, (Chair of Pharmtechnology and Pharmacy Economics and Management)

Sargsyan A.S.

Scientific and Production Center (SPC) "Armbiotechnology" NAS RA, senior researcher, PhD,

Senior researcher at the laboratory of Ecological Safety)

Davidyan T.S.

Scientific and Production Center (SPC) "Armbiotechnology" NAS RA, researcher

(researcher at the laboratory of Ecological Safety).

Аннотация

Pseudomonas и Stenotrophomonas очень близки по своей морфологии, метаболическим особенностям и механизмам выживания в условиях изменении окружающей среды. Они хорошо известны в качестве патогенных и условно-патогенных микроорганизмов с большим количеством механизмов устойчивости к антибиотикам. Медицинское значение этих штаммов при различных воспалительных патологиях и других оппортунистических заболеваниях, формирующихся в клиниках, растет день ото дня. Кроме того, будучи очень распространенным обитателем влажных поверхностей, представители Pseudomonas и Stenotrophomonas набираются в различных биогеоценозах, будучи вовлеченными в большое количество цепей биодеградации некоторых природных соединений и синтетических ксенобиотиков (алифатические, циклические и ароматические углеводороды и т. д.). Таким образом, они оказывают огромное экологическое влияние на окружающую среду.

В настоящей работе исследована устойчивость к некоторым р-лактамным антибиотикам разных поколений у более чем 70 почвенных штаммов P. aeruginosa, S. maltophilia, P. chlororaphis (subsp. Mororaphis, subsp. аurantiaca и subsp. aureofaciens), P taetrolens, P. putida, P. fluorescens и P. geniculate. Проведено сопоставление плазмидного состава клеток, в так же PCR анализ.

В результате исследования выявлено большое разнообразие Р-лактамаз и локализации их генов. Способность передавать свойство устойчивости плазмидами была идентифицирована у 26 штаммов. У 14 штаммов был обнаружен ген р-лактамазы blaOXA-W. У 2 штаммов резистентность определяется 2 типами

Р-лактамаз. Устойчивость к цефалоспоринам кодируется хромосомными генами, тогда как устойчивость к другим р-лактамам кодируется плазмидами, которые могут передаваться в процессах межвидового горизонтального переноса генов. Выявлены штаммы с сочетанием хромосомной и плазмидной локализации генов резистентности к различным р-лактамам. В некоторых из них присутствуют плазмиды с двумя различными генами устойчивости к антибиотикам, в то время как в другой группе исследованных штаммов в клетках содержатся различные плазмиды, способные к независимому переносу.

Это имеет огромное экологическое и медицинское значение вследствие возможности формирования таким образом новых антибиотико-резистентных патогенных штаммов.

Abstract

Pseudomonas and Stenotrophomonas are very close in their morphology, metabolic features and mechanisms of survival while the changing of environment. They are well known as both pathogenic and opportunistic pathogenic microorganisms with a big quantity of antibiotic resistance mechanisms. The medical significance of these strains in different inflammatory pathologies and other opportunistic diseases forming in clinics growth day by day. Besides, being a very common inhabitant of wet surfaces, the representatives of Pseudomonas and Stenotrophomonas are being recruited in many different biogeocenoses and chains of biodegradation some natural compounds and synthetic xenobiotics. (aliphatic, cyclic, and aromatic hydrocarbons, etc.) Thus they have huge ecological influence on environment.

In current paper, the resistance to some p-lactamic antibiotics of different generations were researched in more than 70 soil strains of P. aeruginosa, S. maltophilia, P. chlororaphis (subsp. Chlororaphis, subsp. aurantiaca and subsp. Aureofaciens), P. taetrolens, P. putida, P. fluorescens and P. geniculate. The plasmid consistence of cells and PCR analysis data were mapped.

As a result, the wide diversity of lactamases and their genes localization was found out. An ability to transfer the property of resistance by plasmids was indicated in 26 strains. In 14 strains was detected p-lactamase gene blaOXA-10. In 2 strains the resistance was defined by 2 types of p-lactamases. The resistance to cephalosporins was encoded by chromosomal genes, while the resistance to other p-lactams was encoded by plasmids, which can be transferred in intraspecific gene transfer processes. There were detected strains with combination of chromosomal and plasmid localization of genes of resistance to different p-lactams. In some of them there are plasmids with two different genes of antibiotic resistance, while in other group of researched strains, there are different plasmids which are able to independent transfer.

It has a huge ecological and medical significance because of their ability to form new antibiotic resistant pathogenic strains.

Ключевые слова: Pseudomonas, Stenotrophomonas, антибиотико-резистентность, плазмиды, p-лактамные антибиотики

Keywords: Pseudomonas, Stenotrophomonas, antibiotic resistance, plasmids, P-lactam antibiotics

Introduction

Stenotrophomonas and Pseudomonas are well-known as pathogenic and opportunistic pathogens with various mechanisms of resistance to different antimicrobial agents. There are more than 140 species of Pseudomonas, including pathogenic and opportunistic pathogens (P. aeruginosa, P. putida etc.). The last ones often can become a cause of different opportunistic inflammatory diseases of human and animals (skin, respirator and urogenital system, etc.), when the immune system of the host organism is suppressed. In immunocompromised patients, S. maltophilia can lead to nosocomial infections too. Among them there are phytopathogenic species too (P. syringae, etc.), which are very common plant pests. Different strains of Pseudomonas and Stenotrophomonas are able to synthesize a wide range of compounds with different biological activity. Some strains produce bactericide compounds and plant hormones (phytohormones), which increase the growth of agricultural plants and block the growth of some phytopathogenic microorganisms [Zowalaty E. Me. et al.2015; Madigan M et al., 2014; Marcelletti R.G. et al., 2014 Baumrin E. et al., 2017; Pompilio A. et al., 2008].

The biochemical mechanisms of adaptation in Pseudomonas are various. They include native antibiotic resistance and acquired properties, which are spec-

ified by the mobile genetic elements (plasmids, transposons, etc.) with the specific genes of multidrug resistance, pandrug-resistance and cross-resistance. Sometimes these properties are related to an ability of microbes to epitomize a biodegradation of different compounds, both native and synthetic xenobiotics. For example, some strains of P. putida, P. geniculata, etc. can utilize cyclic and aliphatic hydrocarbons and their derivatives by the specific enzymes with plasmid and nucleoid localization [Lateef B. et al., 2016]. Despite the huge interest in these microorganisms, which is caused by their large spectrum of metabolic possibilities, not all molecular mechanisms of their cells survival on aggressive media in nature and hospitals are well-researched. Thus the taxonomy of these two families still remains unclear, because of similarities in biochemistry of their cells [Haas D. et al., 2005].

Representatives of Pseudomonas and Stenotroph-omonas, being involved in quorums and various ecosystems, are able to transfer some of their properties by mobile genetic elements (plasmid, transposons) to other microorganisms while the horizontal gene transfer. It is very significant that the antibiotic resistance properties as well as an ability of xenobiotic biodegradation can be spread among the different as non-pathogenic as well as pathogenic microorganisms. Thus, the spread of antibiotic resistant genes from non-pathogenic native to the pathogenic microbes, with formation

of stabile antibiotic resistant strains, presents a real ecological and medical danger for human and animal health [Hauben L, et al., 1999; Felker P, et al., 2005].

In current research the resistance of some native soil strains of Pseudomonas and Stenotrophomonas to P-lactamic antibiotics was researched by microbiological and genetical methods.

Materials and Methods.

Strains and cultural media.

In current research the strains from The National Culture Collection of microorganisms of the Microbial Depository Center of the SPC "Armbiotechnology" NAS RA: P. aeruginosa, Stenotrophomonas malto-philia, P. chlororaphis (subsp. chlororaphis, subsp. au-rantiaca, subsp. aureofaciens) P. taetrolens, P. putida, P. fluorescens and P. geniculata were used. As the control bacteria there were used E. coli DH5a (sensitive strain) and E. coli DH5a/pUC18 (ampicillin resistant strain). All strains were cultivated on solid agarised cultural media and L-broth.

Antibiotic resistance test.

The resistance tests were done by the cultivation on media, containing 50^g/ml of following antibiotics: ampicillin (Amp), amoxicillin (Amx), augmentin (Amc), cefixime (Cfx) and ceftriaxone (Ctx) produced by "Astoria" upon the temperature 37°C in aerobic conditions [Lee J., Zhang L., 2015; Szita G, Biro G., 1990; CLSI 2017].

Resistance genes analysis.

The isolation of plasmid and total DNA was realized by alkaline extraction method and benzyl chloride,

The scientific heritage No 34 (2019) correspondingly [CMI, ESCMID, ECAST 2010; Au-subel F.M, et al., 1997]. Then the isolated DNA was studied by 0.8-2.5% agarose gel electrophoresis [Birn-boim H.C., Doly J.,1979; Heng Zhu, et al., 1993]. The transformation was realized by Mandel's method with CaCl2 usage. PCR analysis of donor, recipient and transformant strains DNA was done with primer blaOXA-10 [Lucotte G., Baneyx F., 1993; Viovy J.-L., 2003; Mandel M., Higa A., 1970; Dagert M., Ehrlich S., 1979; Spilker T., et al., 2004].

Results

The resistance test results of P. aeruginosa, S. maltophilia, P. chlororaphis, P. taetrolens, P. putida, P. fluorescens and P. geniculate to ß-lactam antibiotics of different subgroups and different generations (Amp, Amx, Amc, Cfx and Ctx) are presented in Tables 1-4.

According to presented data, P. aeruginosa 9058 and all researched strains of P. taetrolens cannot transfer the resistance to another microbe. The resistance of some P. aeruginosa is determined by blaOXA-10 chromosomal or plasmid gene, which is encoding a sensitive to clavulanic acid 0XA-10 ß-lactamase. In P. aeruginosa 5249 a mutant version of blaOXA-10, which is not sensitive to clavulanic acid is identified.

The transformants of P. aeruginosa 9057 carry bla0XA-10 and they are resistant to Amp and Amx, while the donor strain is sensitive to Ctx, Amc and Cfx. It can be a result of the presence of other lactamases or efflux system [Moosdeen F., 1997; Subedi D., et al.,2018; Vikram A., et al., 2015].

Strain Resistance to ß-lactams bla0XA-10

Amp Amx Amc Cfx Ctx

P. aeruginosa

9059 +/t +/t +/t +/t - -

9058 +/- +/- - - +/- +

9057 +/t +/t - - - -

9056 - - - - - -

5249 +/t +/t +/t +/t +/t +

P. taetrolens

9214 - - - + - -

9246 + + + + - -

9242 - - - - - +

9243 + - - + + -

9241 + + - + - -

9248 +/- +/- - +/- +/- +

P. putida

9222 +/t +/t +/t - - -

9235 +/t +/t +/t - - -

9254 +/t +/t - - - -

9249 +/t +/t +/t - - +

Table 1.

The correlation of resistance and the presence of gene bla0XA-10 in P. aeruginosa, P. taetrolens and P. putida. (+ resistance; - sensitivity; /t - transfer of resistance; /- - absence of transfer)

Table 2.

The correlation of resistance and the presence of gene blaOXA-10 in S. maltophilia and P. geniculate. (+ re-

sistance; - sensitivity; /t - transfer of resistance; /- - absence of transfer)

Strain Resistance to ß-lactams bla0XA-10

Amp Amx Amc Cfx Ctx

S. maltophilia 9300,

9288 +/t +/t - - - -

9289 +/t +/t +/t - - +

306d2 +/t +/t +/t + - +

9308 + + - - -

9300 +/t - - +/t +/t -

9302 +/t +/t - - - +

9286 + + + - - -

9290 + +/t +/t +/t +/t -

9293 +/t - - +/- +/- -

9294 +/t - - +/- - -

9305 +/t +/t - +/t - -

9326 - - - - + -

P. geniculata

9202 - + + + - -

9211 + + + - - -

9212 + + + + - -

9341 + + + - - -

9290 +/t +/t +/t +/t +/t -

9205 + + + - - -

P. chlororaphis subsp. aureofaciens differ in their resistance, content of plasmids and genes of resistance enzymes. The gene blaOXA-10 is identified both in resistant P. chlororaphis subsp. aureofaciens 9026 and sensitive P. chlororaphis subsp. aureofaciens 9197, the latter probably containing a mutant enzyme. The re-

in P. geniculate the gene blaOXA-10 is not identified, but P. geniculate 9290 strain is able to transfer resistance property to another microbe by plasmids (Tables 1 - 2). In P. chlororaphis, subsp. chlororaphis and P. chlororaphis subsp. aurantiaca (Table 3) there's no bla0XA-10but the resistance of P. chlororaphis, subsp.

searched strains of P. putida and S. maltophilia predominantly carry plasmids but the gene blaOXA-10 is identified only in P. putida 9249, S. maltophilia 306d2, S. maltophilia 9300 and S. maltophilia 9302. Transformants of S. maltophilia 9293, S. maltophilia 9294 are resistant only to Amp, while the resistance of cephalosporin's is caused by chromosomal genes.

Table 3.

- sensitivity;

chlororaphis 9175, P. chlororaphis, subsp. chlororaphis 9167 and P. chlororaphis subsp. aurantiaca 9064 is caused by plasmids.

Among the strains of P. fluorescens (Table 4) bla0XA-10 gene was identified in P. fluorescens 9087, P. fluorescens 9075, P. fluorescens 9070 and P. fluorescens 9110 on a bacterial chromosome.

The correlation of resistance and the presence of gene bla0XA-10 in P. chlororaphis. (+ resistance; _/t - transfer of resistance; /- - absence of transfer)_

Strain of P. chlororaphis Resistance to ß-lactams bla0XA-10

Amp Amx Amc Cfx Ctx

P. chlororaphis subsp. chlororaphis

9158 - + - + - -

9175 - - - - - -

9156 - - - - - -

9168 + + + + - -

9329 - - - - - -

P. chlororaphis subsp. aurantiaca

9062 - - - - - -

9064 +/t +/t +/t +/t +/t -

9061 - - - - - -

P. chlororaphis subsp. aureofaciens

9197 - - - - - +

9192 +/t +/t - - - -

9060 +/t +/t +/t +/t - -

9200 - - - - +/t -

Table 4.

The correlation of resistance and the presence of gene blaOXA-lO in P. fluorescens. (+ resistance; - sensitivity;

/t - transfer of resistance; /- - absence of transfer)

Strain of P. fluorescens Resistance to ß-lactams bla0XA-10

Amp Amx Amc Cfx Ctx

9131 + + + - - -

9209 + + + - + -

9068 + - - - - -

9070 +/t +/t +/t + - +

9091 + + + + - -

9092 + + + + - -

9150 + + + + - -

9127 +/t - - +/t +/t -

9115 + + + + + -

9075 +/t +/t +/t + - +

9142 + + + + - -

9110 +/- +/- +/- +/- +/- +

9097 + + + + - -

9106 + + + + + -

9109 + + + - + -

9072 + + - + + -

9124 + + + - - -

9149 + - - - - -

9087 +/t + + +/t +/t +

Discussion.

During the experiments, it was found out that the strains differ in their plasmid molecular weighs and consistence. According to the presented data, they can be grouped to sensitive and resistant (to one or more antibiotics) strains. Besides, not all observed representatives of each several species demonstrated correlation between antibiotic resistance and presence of plasmids. Not all of resistant strains contain blaOXA-lO gene, just as not all non-plasmid strains are sensitive. Moreover, this gene was identified in some sensitive strains too. Thus, the absence of resistance in phenotype of microbe can be a result of mutation.

For observation of plasmids function in researched microorganisms and for identification of localization of blaoxA-10 gene, the series of transformations of sensitive strains P. aeruginosa 9056 and E. coli DH5a were carried out by the plasmid DNA of resistant strains. Transformant was selected and cultivated on compatible antibiotic containing media. The results of transformation of 2 sensitive strains is identical and demonstrated in Tables 1-3.

According to data from tables 1-3, the among the strains which are resistant to different subgroups of P-lactams, there are some representatives, which can transmit only a part of property, while some of observed strains can transfer the whole resistance spectrum. Thus the strains P. aeruginosa 5249, S. malto-philia 9300, S. maltophilia 9290, P. geniculate 9290, P. aurantiaca 9064, P. aureofaciens 9200, P. fluorescence 9087, P. fluorescence 9127 demonstrate the resistance to cephalosporins of I and III generations (Cfx and Ctx) and their plasmids are able to transfer this property to sensitive strains while the transformation. The resistance of P. aeruginosa 9059 and S. maltophilia 9305 to Cfx can be transmitted by plasmids too. In case of

strains with divergence of property while the transformation, the resistance to Amp, Amx, Amc and cepha-losporins is defined by different genes, one part of which is localized on bacterial chromosome [Liu Y., et al., 2016].

In P. chlororaphis 9167, S. maltophilia 9293, S. maltophilia 9294, P. fluorescens 9311, the resistance is determined by plasmids. Besides, the resistance in different strains is represented by different genes on plasmids and in bacterial chromosome. Moreover, the resistance can be transmitted by plasmids in frames of one species of Pseudomonas and in the processes of interspecific horizontal gene transfer. Probably, in P. chlororaphis 9167, S. maltophilia 9293, S. maltophilia 9294, etc. in terms of absence of bla0XA-10 the degradation of antibiotics proceeds by other enzymes. The resistance can be caused by any plasmid and chromosomal genes of antibiotic entrance prevention systems or efflux system genes, as it is shown for some strains of P. aeruginosa h P. fluorescens. The molecular weight and functions of plasmids in Pseudomonas are versatile. Thus, according to this fact, it might be concluded that in some strains sensitive to this group of antibiotics, the plasmids carry genes, which are encoding another enzyme, for example, the enzymes of biodegradation of other compounds [Maurya A.P., et al., 2017]

Despite the presence of bla0XA-10 gene on plasmids of P. aeruginosa 5249 and P. fluorescens 9087, these strains are Amc-resistant, probably because of mutation in enzyme gene. Among the sensitive to Amp and other antibiotics strains, both plasmid-containing and non-plasmid representatives were detected. In some of them bla0XA-10 is identified, but ß-lactamic activity is absent, because of the lack of expression of ß-lactamase or synthesis of inactive product. Further research of this mutation can have a huge scientific and

medical significance against the multidrug resistant pathogenic strains of Pseudomonas, in treatment of acute and chronic inflammatory infections, especially in therapies against the opportunistic infections of human and animals. According to the collected data, resistance of P. geniculate 9211 and P. putida 9222, is not transferred by the plasmids, because of absence of corresponding genes on plasmid in these strains. Probably, their functions them are related to other metabolic pathways of biodegradation of different natural substances or xenobiotics.

In plasmid DNA of p-lactams resistant and nonsensitive to clavulanic acid in Amc strains blaOXA-lO is identified and probably this feature is defined by the mutation in lactamase gene [Liu Y., et al., 2014].

A gene blaOXA-lO has been also identified in bacterial chromosome of non-plasmid strain S. maltophilia 9302. Except the described mutant strains, the resistance can be determined by other genes of chemical modification of antibiotics or by efflux system, cell membrane penetrance control, etc.

Conclusion

As a result of research of about 70 soil strains of 7 species from Xanthomonadaceae and Pseudomona-daceae, there were detected plasmid containing and non-plasmid microbes, resistant and sensitive to p-lac-tamic antibiotics. In some cases, there was no correlation between plasmid presence and resistance. blaOXA-10 gene was identified in plasmid and bacterial chromosome. For one part of strains the resistance to different p-lactamic antibiotics was determined by different genes, and in some researched strains the resistance to cephalosporins was defined by chromosomal genes, while the plasmids of these strains carry genes of resistance to Amp, Amx and Amc. Some of them carry mutant versions of genes: non-active enzyme; the resistant to clavulanic acid p-lactamase; enzyme with the differing substrate specificity.

All the described features play a key role in survival of microbes. Besides, in P. Chlororaphis 9167 the efflux system is identified. All the described mechanisms are not species-specific. In one part of strains the resistance can be transferred to different microorganisms by plasmids in horizontal gene transfer processes. It has a huge ecological importance because of possibility of forming new antibiotic resistant strains from the natural soil microbes. It should be noted that the described plasmids stay stable in the absence of contact of microbe with any antibiotics. Probably it is caused by the presence in these plasmids of not only the genes of resistance to xenobiotics, but also the genes with a principal metabolic significance.

Acknowledgments. This work was supported by the RA MES State Committee of Science, in the frames of the research project № 18T-2I036.

Author Disclosure Statement. No competing financial interests exist.

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