Determination of the spectrum of antibiotic resistance genes have phenotypic resistant strains of parietal intestinal microbiota in rats by RT-PCR

Автор(и)

  • Y Bukina Zaporozhye State Medical university,
  • A Kamyshny Zaporozhye State Medical university,
  • N Polishchuk Zaporozhye State Medical university,

Ключові слова:

antibiotic resistance, gene indication, resistance genes, RT-PCR, parietal microflora

Анотація

Introduction. The problem of formation of bacterial resistance to glycopeptides and beta-lactam antibiotics (cephalosporins and carbapenems) are used worldwide for the treatment of severe community acquired and nosocomial infections, especially caused by polymicrobial flora has become global and is a major factor limiting the effectiveness of antibiotic therapy. In this regard, the study of genetic microbial resistance determinants allows not only to carry out an effective antibiotic therapy, but also to identify two main processes leading to the development of epidemiologically significant events: the introduction of the agent in the risk population from the outside and in situ pathogen (spontaneous genetic drift) targeted restructuring of the population. Therefore, the aim of our study was to investigate the resistance genes to carbapenems, cephalosporins, glycopeptides have clinically important phenotype of resistant strains of microorganisms families Enterobacteriaceae, Pseudomonadaceae, Bacteroidaceae, Enterococcaceae, Peptostreptococcaceae. Materials and methods. As a material for PCR studies 712 phenotypically resistant strains of microorganisms isolated from 80 rats "Wistar" line in microbiological study microflora of the wall were used. During the investigation 474 isolates of bacteria of the family Enterobacteriaceae, 39 - Pseudomonadaceae, 71 - Bacteroidaceae, 96 - Enterococcaceae, 32 - Peptostreptococcaceae were studied. Isolation of DNA from bacteria in the study was performed using reagents "DNA-Express" ("Litekh", Russia). For the detection of resistance genes by PCR in real time (RT-PCR) reagent kits "FLUOROPOL-RV" ("Litekh", Russia) were used. During the experiment, the VIM genes, OXA-48, NDM, KPC, responsible for the resistance of microorganisms to carbapenems, CTX-M - resistance to cephalosporins, as well as genes Van A and van B, the development of resistance to glycopeptides (vancomycin and teicoplanin) were determined. Analysis of the results of amplification was performed using the program Bio-Rad CFX Manager 3.0 under the "Guidelines on the application of Fluoropol format sets - PB." Results and discussion. During the study of 474 cultures of microorganisms - representatives of the Enterobacteriaceae family, KPC - in 7,81% and OXA-48 - in 8,44%, VIM detected in 14,14% of the strains, NDM – 8,23% in the studied crops. These genes were detected in E.coli strains (6,74%, 7,87%, 14,61%, 4,49%, respectively), from microorganisms of the genus Klebsiella (13,85%, 1,54%, 15,38% , 12,31%), Salmonella (7,03%, 10,16%, 13,28%, 8,59%), Enterobacter (4,76%, 14,29%, 15,87%, 4,76 %), Proteus (7,89%, 10,53%, 14,47%, 6,58%) and Shigella (7,55%, 3,77%, 11,32%, 15,09%). In the study of Bacteroides (Bacteroides spp.) Genes KPC, OXA-48, the VIM and NDM were identified in 9,86 %, 4,23%, 9,86 % and 12,68% of the strains, respectively. PCR study of 39 isolates of P.aeruginosae showed the presence of only VIM gene and only 15,38% of the cultures. In the family Enterococaceae and Peptostreptococaceae these genes were not found. According to Russian researchers have identified strains of Enterobacteriaceae only genes OXA-48 (43,7%) and VIM (17,6%), and VIM gene detection rate in P.aeruginosae was 62.9%. CTX-M gene was detected in 10,97% of the strains of the family Enterobacteriaceae (Klebsiella spp. - 13,85%, Salmonella spp. - 14,6%, Enterobacter spp. - 7,93%, Proteus spp. - 6,58% , Shigella spp. - 13,21%), Bacteroidaceae - 15,49%, Peptostreptococcaceae - 6,25%. In Pseudomonas and Enterococcus CTX-M is not revealed. At the same time, according to the literature, the frequency of detection of gene CTX-M in the family Enterobacteriaceae strains circulating in the Russian Federation, in some regions reaches 100%, thus, the gene is not detected in enterococci and Pseudomonas. In Enterococcaceae phenotypically resistant strains of microorganisms of the family genes in identifying Van A and Van B 11,46% and 6,25% causing resistance to glycopeptides, particularly vancomycin. Thus, of the 46 strains studied E.faecalis, 10,87% of isolates possessed Van A genes and 8,7% - Van B. Out of 50 studied cultures E.faecium 12% were in their genotype Van A and 4% - Van B. However, the frequency of detection of genes Van A and Van B in E.faecalis strains circulating in Russia, 1,7% and 2,2%, while E.faecium - 9,3% and 11,5% respectively. Bacteria of the family Peptostreptococcaceae these genes were not identified. During the molecular genetic studies we have not detected phenotypically resistant strains that have combined carbapenemases activity. Conclusions. The findings of the research results indicate the presence of carbapenem resistance genes in strains of microorganisms families Enterobacteriaceae, Bacteroidaceae and Pseudomonadaceae (P. aeruginosae), to cephalosporins - Enterobacteriaceae, Bacteroidaceae and Peptostreptococcaceae, vancomycin - from family Enterococcaceae bacteria. Phenotypically resistant strains that have combined carbapenemases activity, not revealed. Despite the wide range of activity of appointment carbapenems, cephalosporins and vancomycin should take into account the results of determination of resistance genes causing resistance to these drugs. It is advisable to creating circulation monitoring program of antibiotic-resistant strains, including those with сarbapenemases activity in humans and in the environment.

Посилання

The world health report 2014 – World health statistics 2014 [Electronic resource] / World Health Organization.:http://apps.who.int/iris/bitstream/10665/112738/1/9789240692671_eng.pdf.

Finley, R. L. The scourge of antibiotic resistance: the important role of the environment [Text] / R. L. Finley, P. Collignon, D. G. Larsson [et al.] // Clinical Infection Diseases – 2013. – Vol. 57. – P. 704-710.

Yan, J. J. Metallo-β-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM 3, a novel variant of the VIM 2 enzyme [Text] / J. J. Yan, P. R. Hsueh, W. C. Ko [et al.] // Antimicrobial Agents and Chemotherapy. – 2001. – Vol.45. – P. 2224-2228.

Woodford, N. Arrival of Klebsiella pneumoniae producing KPC carbapenemase in the United Kingdom [Text] / N. Woodford, J. Zhang, M. Warner [et al.] // Journal of Antimicrobial Chemotherapy. – 2008. – Vol. 62. – P. 1261-1264.

Pournaras, S. Clonal spread of KPC 2 carbapenemase producing Klebsiella pneumoniae strains in Greece [Text] / S. Pournaras, E. Protonotariou, E. Voulgari[et al.] // Journal of Antimicrobial Chemotherapy.- 2009. – P. 348-352.

Van Duin, D. Carbapenem-resistant Enterobacteriaceae: a review of trеatment and outcomes [Text] / D. Van Duin, K. S. Kaye, E. A. Neuner, R. A. Bonomo // Diagnostics Microbiology Infections Diseases. – 2013. – Vol. 75. – Р. 115-120.

Courvalin, P. Vancomycin resistance in grampositive cocci [Text] / P. Courvalin // Clinical Infections Diseases. – 2006. – Vol. 42. – P. 25-34.

Davies, J. Inactivation of antibiotics and the dissemination of resistance genes [Text] / J. Davies // Science. – 1994. – P. 375-382.

Evers, S. Genetics of glycopeptide resistance in enterococci [Text] / S. Evers, R. Quintiliani, P. Courvalin // Microbiology Drug Resistance. – 2011. – Vol. 2. – P. 219-23.

Тhe Scientific Center for Medical Research 2012 - Scientific Center statistics 2012 [Electronic resource] / Scientific Center Organization. – Mode of access : http://www.pharmateca.ru/ru/archive/article/31922.

Walsh, T. R. Clinically signicant carbapenemases: an update [Text] / T. R. Walsh [et al.] // Current Opinion in Infections Diseases. – 2008. – Vol. 21. – P. 367-371.

Queenan, A. M. Carbapenemases: the versatile betalactamases [Text] / A. M. Queenan, K. Bush // Clinical Microbiology Reviews. – 2007. – Vol. 20. – P. 440-458.

Lutgring, J. D. The Problem of Carbapenemase-Producing-Carbapenem-Resistant-Enterobacteriaceae Detection [Теxt] / J. D. Lutgring, B. M. Limbago // Clinical Microbiolodgy – 2016. – № 54 (3). – Р. 529-34.

Nordmann, P. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae world wide [Text] / P. Nordmann, L. Poirel // Clinical Microbiology Infections – 2014. – № 20 (9). – Р. 821-830.

Bush, K. A. Functional classification scheme for betalactamases and its correlation with molecular structure [Text] / K. Bush, G. A. Jacoby, A. A. Medeiros // Antimicrobial Agents and Chemotherapy. – 1995. – Vol. 39. – P. 1211-1233.

Queenan, A. M. Carbapenemases: the versatile betalactamases [Text] / A. M. Queenan, K. Bush // Clinical Microbiology Reviews. – 2007. – Vol. 20. – P. 440-458.

Woodford, N. Multiresistant Gramnegative bacteria: the role of high risk clones in the dissemination of antibiotic resistance [Text] / N. Woodford, J. F. Turton, D. M. Livermore // FEMS Microbiology Reviews. – 2011. – Vol. 35. – P. 736-755.

Miriagou, V. Acquired carbapenemases in gramnegative bacterial pathogens: detection and surveillance issues [Text] / V. Miriagou, G. Cornaglia, M. Edelstein [et al.] // Clinical Microbiology and Infection. - 2010. – Vol. 16. – P 112-122.

Hlumcher, F. S. Multiresistant infections: relevance, definition, mechanisms, prevailing pathology, treatment, prevention [Тext] / F. S. Hlumcher, S. O. Dubrov, Y. L. Kuchyn // Interdepartmental Medical Journal «Science & Practice». – 2014. – № 1 (2). – P. 129-149.

Finley, R. L. The scourge of antibiotic resistance: the important role of the environment [Text] / R. L. Finley, P. Collignon, D. G. Larsson [et al.] // Clinical Infection Diseases – 2013. – Vol. 57. – P. 704-710.

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Як цитувати

Bukina, Y., Kamyshny, A., & Polishchuk, N. (2020). Determination of the spectrum of antibiotic resistance genes have phenotypic resistant strains of parietal intestinal microbiota in rats by RT-PCR. Анали Мечниковського Інституту, (2), 21–27. вилучено із https://journals.uran.ua/ami/article/view/191018

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