Fenotipske karakteristike kliničkih izolata Pseudomonas aeruginosa
AuthorStanković Nedeljković, Nataša S.
Committee membersDinić, Marina
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Introduction. Pseudomonas aeruginosa (P. aeruginosa) is one of the most common bacteria colonizing the hospital environment. High genetic variability, flexible physiology, adaptability, metabolic potential, production of broad capsules, biofilm forming, control of external membrane permeability and resistance to antibiotics and disinfectants allow the bacillus to be widely dispersed. P. aeruginosa is a common cause of inflammation, if there is a disruption of the body's defense forces for any reason: malignant disease, chemotherapy, neutropenia, diabetes mellitus, cardiovascular diseases, alcoholism, smoking and obesity. The aim. The goals of testing are the analysis of the presence of P. aeruginosa in hospitalized patients' and outpatients` materials, isolates serotyping, testing of fluorescin and pyocyanin production, adhesive ability, biofilm formation, twitching motility, swarming, resistance to oxidative stress, susceptibility testing to imipenem, meropenem, piperacillin-tazobacta...m, colistin, aztreonam, ceftazidime, cefepime, amikacin, gentamicin, netilmicin, tobramycin, ofloxacin and ciprofloxacin, determination of the minimum inhibitory concentrations (MICs) of the piperacillin-tazobactam, ciprofloxacin and amikacin, and testing of the of extended spectrum beta lactamases (ESBL), carbapenemases and mettalo beta-lactamases (MBL) productions. Methods. The study included 100 isolates of P. aeruginosa, 50 from inpatients and 50 outpatients materials. Serotyping of isolates was performed by agglutination reaction per the manufacturer's instructions serum (Bio-Rad, France). Production of pigments was read on specialized substrates for pigments production: Pseudomonas basis for differentiation of fluorescein and Pseudomonas basis for differentiation of piocyanin (HiMedia, India). Testing of adhesive ability was performed in microtiter plates in Lauryl Bertoni broth (Liofilchem, Italy), after incubation of 60 min. The color intensity was read at 610 nm (Biokit, Microwell EL 301, USA). Investigation of biofilm production was performed in microtiter plates in Lauryl Bertoni broth after incubation for 24 hours, and the color intensity was read at 610 nm (Bio-kit, Microwell EL 301, USA). Testing of the twitching movements was performed on 1% Lauryl Bertoni agar, a swarming at 0.5% Lauryl Bertoni agar. The sensitivity to oxidative stress was studied on 2% trypticase soy agar with a sterile filter paper disk diameter of 7 mm, on which there was poured 10 μl of 30% H2O2. The sensitivity to antibiotics was evaluated by disk diffusion method according to CLSI standards (2013, M-100-S-21). MICs of piperacillin-tazobactam, amikacin and ciprofloxacin were determined according to the manufacturers tapes with an antibiotic instructions (Liofilchem, Italy). ESBL production of P. aeruginosa was performed by using cefotaxime, cefotaxime with clavulanic acid, amoxicillin with clavulanic acid, ceftazidime, and ceftazidime with clavulanic acid discs. Production carbapenemases is performed so that the surface of Mueller-Hintovog agar is inoculated culture reference strain E. coli ATCC 2522. In the middle of the board was placed meropenem disk, and lines of tested culture of P. aeruginosa were dragged to disk. MBL production was determined by Hodge test. Results. P. aeruginosa was the most commonly cultured from wound swabs (54%) and urine (21%), while the other patient materials underrepresented. Following serotypes of P. aeruginosa were serologicaly identified: P1, P3, P4, P5, P6, P10, P11, and P12. The most common serotypes were: P11 (20%), P6 (16%) and P1 (12%), and 29% of the isolates were untypibile. P. aeruginosa produces more fluorescein (86%) than pyocyanin (67%). Most of our isolates (93%) had the ability of adherence to polyvinyl chloride, after incubation of 60 min. Almost all isolates of P. aeruginosa (99%) had the ability to form biofilm. All isolates had the abilities of twicing movement and swarming. A significant number of isolates had absolute resistance to toxic effect of 30% H2O2 solution. The isolates showed the greatest degree of susceptibility to colistin and aztreonam (100%), meropenem (92% of isolates of hospital origin and 88% of outpatient), imipenem (86%) and piperacillin-tazobactam (84% of nosocomial isolates and 74% of outpatient). The largest number of isolates had an MIC value for piperacillin-tazobactam 12 mg/ml (37.5%), ciprofloxacin 0.125 mg/ml (43.75%). For amikacin 24.19% of isolates had MIC value of 8 μg/ml. Only one isolate produced a ESBL, 7% carbapenemase and 10% MBL. Conclusion. P. aeruginosa is the most commonly cultured from wound swabs and urine, and much less from other materials. The most serotypes were P11, P6 and P1. Bacillus produces fluorescein more than pyocyanin. Most of isolates have the abilities adhesion and biofilm formation, gliding movement, swarming, and resistance to oxidative stress. All isolates were most susceptible to colistin and aztreonam, followed by meropenem, imipenem and piperacillin-tazobactam.