Genotipizacija, rezistencija na antibiotike i ispitivanje faktora virulencije kliničkih izolata Pseudomonas aeruginosa

Abstract

Utvrđivanje potencijalnih korelacija, između fenotipskih karakteristika (osetljivost na različite klase antibiotika, ekspresija različitih klasa β-laktamaza, učestalost serotipova, proizvodnja pigmenata i stvaranje biofilma) i genotipskih obrazaca kliničkih izolata Pseudomonas aeruginosa različitog porekla, predstavljalo je glavni cilj ovog rada. Tokom dve faze istraživanja, ispitano je 147 kliničkih izolata, od kojih je najveći procenat dobijen iz briseva rana (66%) i urino-kultura (10,9%). Polivalentne serogrupe PMA i PMF, kao i monovalentni serotipovi P1, P6 i P11 bili su najzastupljeniji, dok je najveći procenat izolata bio očekivano netipabilan. Istovremena produkcija oba pigmenta karakterisala je najveći broj izolata, dok su oni sa serotipovima P1, P6 i P11 produkovali više pioverdina nego piocijanina. Sposobnost formiranja biofilma imalo je 77,66% kliničkih izolata, a najizraženija je utvrđena za izolate iz urinokultura i sputuma. Najveći stepen osetljivosti utvrđen je za kolistin, aztreonam, karbapeneme i piperacilin/tazobaktam, dok je izraženija otpornost prikazana prema aminoglikozidima i fluorohinolonima. Višestruku otpornost na tri ili više klasa antibiotika je pokazalo 30,9% izolata iz druge faze istraživanjaje, a skoro polovina je eksprimirala β-laktamaze proširenog spektra, karbapenemaze i metalo-β-laktamaze. RAPD272 PCR analiza pokazala se kao najbolja tehnika koja je izdvojila najveći broj klastera sa ujednačenim obrascima, ističući izraženu heterogenost izolata iste vrste. Korelacija između genetičkih obrazaca i fenotipskih karakteristika nije ustanovljena, ukazujući na potrebu za novim molekularnim metodama u ovom delu Srbije u cilju otkrivanja potencijalnih veza među njima i na izraženu rezistenciju koja se poslednjih godina uvećava.

Determining potential correlations between phenotypic characteristics (sensitivity to different classes of antibiotics, expression of different β-lactamases classes, frequency of serotypes, pigment and biofilm production) and genotypic patterns of clinical isolates of Pseudomonas aeruginosa from different origins was the main goal of this study. Study itself was splitted into two phases, a total of 147 clinical isolates were examined, out of which the largest percentage were from wound swabs (66%) and urine cultures (10.9%). Polyvalent serogroups PMA and PMF, as well as monovalent serotypes P1, P6 and P11 were the most represented, while the highest percentage of isolates was atypical. Simultaneous production of both pigments characterized the most of isolates, while those with serotypes P1, P6 and P11 produced more pyoverdine than pyocyanin. Even 77.66% of clinical isolates had the ability to form a biofilm, and the most pronounced was found for isolates from urine cultures and sputum. The highest susceptibility was found for colistin, aztreonam, carbapenems and piperacillin/tazobactam, while more pronounced resistance was shown onto aminoglycosides and fluoroquinolones. Multiple resistances, up to three or more antibiotics classes, were shown by 30.9% of isolates from the second study phase, and almost half of them expressed broad-spectrum β-lactamases, carbapenemases and metallo-β-lactamases. RAPD272 PCR analysis proved to be the best technique that singled out the largest number of clusters with uniform patterns, emphasizing the pronounced heterogeneity of isolates of the same species. The correlation between genetic patterns and phenotypic characteristics has not been established, indicating the need for new molecular methods in this part of Serbia to discover potential links between them and on the pronounced resistance, which has increased in recent years.