Real-time PCR detection of 16S rRNA novel mutations associated with Helicobacter pylori tetracycline resistance in Iran.
BACKGROUND
Tetracycline is an antibiotic widely used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing due to increasing bacterial resistance. The aim of this study was to investigate the occurrence of 16S rRNA mutations associated with resistance or reduced susceptibility to tetracycline of Helicobacter pylori by real-time PCR (RT-PCR) assays from culture.


METHODS
Tetracycline susceptibility and minimal inhibition concentration (MIC) was determined by the Epsilometer test (Etest) method. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture. The 16S rRNA of these isolates was sequenced and resistance-associated mutations were identified. From 104 isolates of H. pylori examined, 11 showed resistance to tetracycline.


RESULTS
LightCycler assay was applied to DNA extracted from 11 tetracycline-susceptible and 11 tetracycline resistance H. pylori isolates. In our study the sequencing of the H. pylori wild types in 16 s rRNA gene were AGA 926-928 with MIC (0.016 to 0.5 μg/ml), while the sequencing and MIC for resistant were GGA and AGC, (0.75 to 1.5 μg/ml), respectively. Also we found a novel mutation in 2 strains with 84° as their melting temperatures and exhibition of an A939C mutation.


CONCLUSIONS
We conclude that real-time PCR is an excellent method for determination of H. pylori tetracycline resistance related mutations that could be used directly on biopsy specimens.
Department of Laboratory Sciences, Marand Branch, Islamic Azad University, Marand, Iran E-mail : mohammadmilano@gmail.com.
Kianoosh
K
Dadashzadeh
Morteza
M
Milani
Mohammad
M
Rahmati
Abolfazl
A
Akbarzadeh
D016481:Helicobacter Infections / Q000188:drug therapy / Q000453:epidemiology / Q000382:microbiology*; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007492:Iran / Q000453:epidemiology; D009154:Mutation / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D013753:Tetracycline Resistance / Q000235:genetics*
D012336:RNA, Ribosomal, 16S