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ID naloge: 98 Letnik: 2001 Predmet: mikrobiologija in imunologija
Primerjava obcutljivosti bakterije Helicobacter pylori za antibiotike z agar dilucijsko metodo in E-testom.
Avtor: Miha Skvarc
Mentor: prof. dr. Marija Gubina, dr.med
Somentor: doc. dr. Bojan Tepeš, dr.med
Izhodišce: Helicobacter pylori je po Gramu negativna, spiralna bakterija, ki naseljuje obmocje želodcne sluznice. Prvic so jo izolirali leta 1982 v Perthu v Avstraliji. Ocenjujejo, da je z njo okuženih prek 50 % ljudi v razvitem svetu. Danes jo povezujejo predvsem z razjedo na dvanajstniku in želodcu, z adenokarcinomom želodca ter z limfomom MALT. Odstranitev te bakterije z antibioticnimi sredstvi z mesta okužbe pripomore k ozdravitvi ulkusne bolezni. Zdravljenje je zahtevno in vkljucuje vec antibiotikov. Pojavljajo se odporne bakterije proti protimikrobnim snovem, še posebno proti metronidazolu in klaritromicinu, kar ogroža uspešnost zdravljenja.
Namen: Za ucinkovito zdravljenje je treba dolociti obcutljivost bakterije za antibiotike, kar se naredi z izdelavo antibiograma. Vecinoma se uporablja enostavnejši E-test. Referencna metoda je agar dilucijska metoda. V raziskavi smo skušali oceniti, v kolikšni meri so rezultati, dobljeni z E-testom, primerljivi z agar dilucijsko metodo.
Hipoteza: Primerjali smo podatke, dobljene z E-testom ter agar dilucijsko metodo za ugotavljanje obcutljivosti bakterije H. pylori za antibiotike. Trdimo, da sta E-test in agar dilucijska metoda enakovredni metodi za dolocanje obcutljivosti za antibiotike pri bakteriji H. pylori.
Metode: V poizkusu smo testirali 41 sevov z agar dilucijsko metodo. Testiranja z E-testom nismo ponavljali, uporabili smo že prej dobljene rezultate. Seve, ki so bili zamrznjeni na _80 °C, smo odmrznili. Nato smo bakterije namnožili in nanesli na plošce, ki so vsebovale Mueller-Hintonov agar s 5 % ovcje krvi, ki smo mu dodali ustrezne koncentracije antibiotika, in sicer od 0,004 do 256 mg/ml. Obenem smo testirali še sev H. pylori ATCC 43504, ki ima znane vrednosti MIK. V raziskavi smo primerjali ucinkovitost šestih antibiotikov. Rezultate dveh metod smo statisticno obdelali s Studentovim t-testom za analizo parnih podatkov.
Rezultati: Rezultati E-testa so se ujemali z agar dilucijsko metodo pri amoksicilinu, klaritromicinu in roksitromicinu. Pri azitromicinu se delovna hipoteza ni potrdila, toda testirali smo samo 23 sevov. Pri testiranju metronidazola smo odkrili 7 sevov, kjer je bila vrednost MIK pri E-testu pod 8 mg/ml, pri agar dilucijski metodi pa nad to mejno koncentracijo, ki služi kot vrednost za dolocitev odpornosti. Pri ciprofloksacinu se je to primerilo pri 6 sevih, pri cemer je mejna koncentracija za ciprofloksacin 1 mg/ml. Ce upoštevamo kriticno vrednost t pri stopnji tveganja p=0,05 ter 40 stopinjah prostosti, je dobljena vrednost t v obeh primerih vecja od kriticne vrednosti t, kar pomeni, da primerljivost rezultatov teh dveh metod ne velja in je med rezultati za metronidazol in ciprofloksacin razlika signifikantna.
Zakljucki: Za izdelavo antibiograma pri bakteriji H. pylori je E-test primeren, kadar testiramo amoksicilin, roksitromicin, klaritromicin in azitromicin. V primeru metronidazola in ciprofloksacina smo zavrgli delovno hipotezo. E-test ni ustrezna metoda za dolocanje obcutljivosti za metronidazol in ciprofloksacin pri bakteriji H. pylori. Priporocamo agar dilucijsko metodo.
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[Abstract / English version]
Comparative evaluation of the agar dilution method and E-test for testing antibiotic susceptibility of Helicobacter pylori strains to six antibiotics.
Author: Miha Skvarc
Mentor: prof. dr. Marija Gubina, dr.med
Co-mentor: doc. dr. Bojan Tepeš, dr.med
Background: Helicobacter pylori is a gram-negative, spiral-shaped bacterium, primarily found in the gastric mucosa. The first isolation from the human stomach was made in 1982 in Australia. It is estimated that over 50 % of the population in the developed countries is infected. The infection with H. pylori is associated with the peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. The treatment with antimicrobial agents successfully eradicates H. pylori from the gastric mucosa, producing favorable clinical response especially for the patients with the peptic ulcer disease. The resistance against antibiotics in particular the metronidazole and the clarithromycin is rising and determents clinical outcome of the antibiotic treatment.
Aim: The determination of the in vitro susceptibility of H. pylori to antimicrobial agents is of vital importance for the successful treatment of infection. The susceptibility testing is mainly performed with the E-test. The reference method is agar dilution method. The aim of this study is to compare the results obtained by the E-test and agar dilution method.
Hypothesis: We compared the results obtained by the E-test with results got with the agar dilution method. We claim that the two methods for determination of the susceptibility of H. pylori to antibiotics are equally reliable.
Methods: 41 H. pylori strains isolated from patients were tested against 6 antibiotics with the agar dilution method. The E-test results and strains were obtained from the previous study. The susceptibility testing was carried out on Mueller-Hinton agar containing 5 % of sheep blood with incorporated antibiotics. The concentration range was from 0.004 to 256 mg/ml. We also tested the ATCC 43504 control strain with the known MIC values. The results got with two methods were statistically compared with paired t test.
Results: The E-test results showed excellent agreement with the agar dilution results for amoxicillin, clarithromycin and roxithromycin. The working hypothesis was wrong in the case of azithromycin but only 23 strains of H. pylori were tested. There were 7 cases of resistance to metronidazole in which MIC values were over 8 mg/ml only by agar dilution method. In 6 cases such incidence happened for ciprofloxacin where the cut off value for determining the resistance is 1 mg/ml. The results compared with paired t test for these two antibiotics have shown that previously mentioned methods have not been equal.
Conclusion: Our results confirm that the E-test is comparable to agar dilution method for susceptibility testing for amoxicillin, azithromycin, clarithromycin and roxithromycin. In the case of metronidazole and ciprofloxacin the hypothesis has not proven to be appropriate. We recommend the agar dilution method for susceptibility testing of H. pylori to metronidazole and ciprofloxacin not the E-test.
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