engleski

A NEW MICRO-AGAR DILUTION METHOD FOR DETERMING AMOXYCILLIN, AZITHROMYCIN AND METRONIDAZOLE MICs FOR HELICOBACTER PYLORI ISOLATES

Objectives: Fast and simple in vitro methods accurately assaying anti - Helicobacter pylori (Hp) activity are needed to facilitate the sensitivity testing. We developed an agar micro dilution technique that uses a 96 - well flat bottom microplate. Methods: 100 clinical isolates used in this study were obtained from gastric biopsies of patients visiting Clinical hospital Merkur, Zagreb. Hp ATCC 43504 was also tested. Micro plates and plates for standard agar dilution method, Hp suspensions and incubation time were done according to NCCLS protocol M7- A4. Mueller - Hinton agar was supplemented with 5% sheep blood. Suspensions were prepared in brucella broth and the turbidity was adjusted to match that of a 2 McFarland standard. Additionally in micro plates was added 0.01% tetrazolium chloride (TTC, Sigma). Adding of TTC gave gold or black appearance of colonies. Volume of agar in each microwell was 400 ľl and inoculum of 2 ľl of bacterial suspension was added in each microwell (including antibiotic -free). After 3 days all plates were examined for visible growth. All tests have been done in duplicate. Results: All strains had identical MICs for amoxicillin in both methods. MICs for azitromycin were different in 15 strains (one dilution difference only and always lower in microplate than in macroplate). MICs for metronidazole differed in 9 strains in the same way. Correlation between MICs by the agar dilution and micro agar dilution was good, with 100% (for amoxicillin), 85% (for azithromycin) and 91% (for metronidazole) of results being within 1 log 2 dilution steps. Appearance of growth on micro plates was easy detectable by gold or black appearance due to TTC added. Discussion: It can be concluded that micro agar dilution method is a practical, easily applicable and cheap alternative of susceptibility testing for clinical microbiology laboratory in determing MICs of H. pylori isolates.

Helicobacter pylori; cultura; sensitivity; MIC

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