| Abstract | Infekcije mokraćnoga sustava se ubrajaju među najčešće infekcije kod ljudi, a bakterija Escherichia coli je njihov glavni uzročnik. Pretjerana i nekontrolirana uporaba kinolona dovodi do sve veće otpornosti bakterija na tu skupinu antibiotika. Mehanizmi otpornosti bakterija na kinolone uključuju promjenu ciljne molekule za lijek, pojačanu aktivnost efluksne pumpe i promjenu propusnosti stanične membrane zbog smanjene aktivnosti porina.
Cilj istraživanja je bilo steći uvid u karakteristike sojeva uropatogene E. coli otporne na kinolone s obzirom na molekularne mehanizme otpornosti, prisutnost činitelja virulencije i otpornost na ostale antibiotike, te utvrditi razliku u odnosu na karakteristike sojeva koji su osjetljivi na kinolone. Također je bio cilj utvrditi koliko laboratorijski inducirana otpornost na kinolone kod prethodno osjetljivih sojeva mijenja ostale karakteristike sojeva.
Tijekom jednogodišnjeg razdoblja su se iz uzoraka urina, dobivenih od izvanbolničkih bolesnika sa simptomima infekcije mokraćnog sustava sa područja Splitsko-dalmatinske županije, izolirali svi sojevi E. coli otporni na kinolone. Kontrolna se skupina formirala tako da se za svaki soj otporan na kinolone uzeo slijedeći izolirani bolesnički soj E. coli koji je bio osjetljiv na kinolone.
U skupini kod koje su izolirani sojevi E. coli otporni na kinolone bilo je više muškaraca nego u kontrolnoj skupini, no ta se razlika nije pokazala statistički značajnom. Zapaženo je da stopa otpornosti E. coli na ciprofloksacin raste s povećanjem životne dobi bolesnika, pa su tako otporni sojevi češće izolirani kod starijih bolesnika. Sojevi otporni na kinolone pokazuju veću otpornost na ostale antibiotike. Tako je među sojevima otpornima na kinolone stopa otpornosti na amoksicilin bila 96,55%, na kotrimoksazol 67,82%, te na gentamicin 29,89%, za razliku od osjetljivih sojeva kod kojih je stopa otpornosti na amoksicilin bila 54,02%, na kotrimoksazol 28,74%, te na gentamicin 24,14%. Činitelji virulencije su znatno rjeđe pronađeni kod sojeva otpornih na kinolone (gen fim 21,84%, gen pap 20,69%, gen cnf1 1,15%, hemoliza 2,30%) nego kod sojeva osjetljivih na kinolone (gen fim 90,80%, gen pap 50,57%, gen cnf1 12,64%, hemoliza 52,87%). Među izolatima E. coli otpornima na kinolone nije bilo sojeva koji su posjedovali plazmidne gene otpornosti na kinolone (qnrA, qnrB i qnrS). Mutacije na genu gyrA na pozicijama 83 i 87 bile su prisutne kod 90,80% sojeva E. coli otpornih na kinolone. Sekvencioniranjem gena gyrA kod sojeva E. coli otpornih na kinolone određeno je da su najčešće mutacije na položaju 83 (serin → leucin) i na položaju 87 (asparaginska kiselina → asparagin). Kod jednog testiranog soja otkrivena je izuzetno rijetka mutacija na položaju 106 (glicin → arginin), za koju se dugo smatralo da može nastati jedino nakon indukcije otpornosti na kinolone u eksperimentalnim uvjetima.
Indukcija otpornosti na kinolone uspjela na dva soja E. coli. Sojevi su nakon indukcije zadržali svojstvo izlučivanja hemolizina, kod jednog soja je gen fim ostao prisutan i nakon indukcije, dok je kod drugoga njegova ekspresija bila smanjena. Sojevi nisu posjedovali gene pap i cnf1 ni prije, a ni nakon indukcije otpornosti na kinolone. Sojevi s induciranom otpornošću na kinolone stekli su povećanu otpornost na cefalosporine treće generacije ceftazidim, cefotaksim i ceftriakson, te na antibiotik gentamicin, a jedan je soj stekao otpornost i na antibiotik cefipim. Oba soja kojima je inducirana otpornost na kinolone stekli su dvije točkaste mutacije na genu gyrA (zamjena serina na položaju 83 s leucinom i zamjena asparaginske kiseline na položaju 87 s asparaginom).
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| Abstract (english) | Urinary tract infecions are among most frequent human infections and Escherichia coli is their main cause. Uncontrolled use of quinolones leads to resistance to this group of antibiotic. Quinolone-resistance mechanisms include modification of drug target molecule, increased activity of the eflux pump and changing of cell membrane permeability due to decreased activity of porins.
Aim of this study was to get an insight of characteristics of quinolone-resistant E. coli, taking into cosnsideration the molecular mechanisms of resistance, presence of virulence factors and resistance to other antibiotics, as well as to a determine difference in characteristics of strains that are quinolone-susceptible. Another aim was to determine how much the laboratory-induced quinolone-resistance in previously susceptible strains was changing the other strain characteristics.
During one year period all quinolone-resistant E. coli strains were isolated from urine samples obtained from out-patients with symptoms of urinary tract infections in the Split and Dalmatia County. Control group was formed by taking the next isolated quinolone-susceptible strain of E. coli for each quinolone-resistant strain.
In the group where the strains of quinolone resistant E. coli were isolated, there wasa higher percentageof males than in control group, but this difference was not significant. It was observed that resistance rate of E. coli to ciprofloxacin increases with age of patients and resistant strains were more frequently isolated in elder patients. Strains resistant to quinolones show higher resistance to other antibiotics. Among the quinolone-resistant strains the resistance rate to amoxicillin was 96.55%, to cotrimoxazole 67.82%, and to gentamicin 29.89%, in contrast to sensitive strains where the resistance rate to amoxicillin was 54.02%, to cotrimoxazole 28.74% and to gentamicin 24.14%. Virulence factors were found significantly less frequently in quinolone-resistant strains (fim gene in 21.84%, pap gene in 20.69%, cnf1 gene in 1.15% and hemolysis in 2.30%) than in quinolone-sensitive strains (fim gene in 90,80%, pap gene in 50.57%, cnf1 gene in 12.64% and hemolysis in 52.87%). Among quinolone-resistant E. coli isolates, there were no strains with plasmid quinolone-resistance genes (qnrA, qnrB and qnrS). Mutations in the gyrA gene at positions 83 and 87 were present in 90.80% strains of quinolones resistant E. coli. Sequencing of gyrA gen in quinolones resistant E. coli strains showed that the most common mutation were at position 83 (serine → leucine) and at position 87 (aspartic acid → asparagine). In one tested strain extremely rare mutation at position 106 (glycine → arginine) was detected, which has long been thought to be formed only after the induction of quinolone-resistance in experimental conditions.
Induction of quinolone-resistance was successful in two E. coli strains. After the induction, strains kept the secretion of hemolysin. In one strain, the fim gene remained present after induction, while in the other its expression was reduced. The strains have no pap and cnf1 genes neither before nor after the induction of quinolone-resistance. Strains with induced resistance to quinolones have gained increased resistance to third generation cephalosporins ceftazidime, cefotaxime and ceftriaxone, as well as to gentamicin, while one strain acquired resistance to cefipime. Both strains with induced quinolone-resistance have gained two points mutation in the gyrA gene (replacement of serine at position 83 with leucine and replacement of aspartic acid at position 87 with asparagine).
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