Characterization of Domain V Mutations in Clinical Isolates of in Pakistan and Their Effect on Clarithromycin MIC.

PURPOSE

Clarithromycin is commonly prescribed for infection. Domain V mutations are responsible for clarithromycin resistance. This study was aimed to characterize the clarithromycin resistance and its associated mutations in clinical isolates of in Pakistan.

MATERIALS AND METHODS

Infection was diagnosed in 93 patients' biopsies using culture, rapid urease test, , and gene multiplex PCR. Clarithromycin resistance was assessed by the agar dilution method. Mutations were detected by PCR-RFLP using 46 (1.4 kb) domain V fragments. Sequencing was executed for 13 domain V fragments, of which 12 showed unusual amplicon size (1.2 kb) and 01 had a new II RFLP pattern.

RESULTS

A total of 48 (83%) strains were obtained from 58 (62.3%) PCR -positive samples. Resistance (MIC ≥ 0.001 mg/mL) and intermediate resistance phenotype (MIC = 0.0005 mg/mL) was observed in 22 (46%), and 10 (21%) isolates, respectively. The primary resistance was found in 23 (39.6%) samples. PCR-RFLP detected A2142G, A2143G, and double mutations in 19, 04, and 01 resistant strain, respectively. Sequencing of 10 amplicons obtained from intermediated resistant strains and 03 amplicons from resistant strains showed 138 new mutations. Among them, T2182C was also seen in 04 intermediated resistant isolates, whereas A2142G, A2143G, and A2143C were observed in resistant isolates. The new II RFLP pattern in an intermediated resistant strains was due to A1761G mutation.

CONCLUSION

domain V mutations showed extensive diversity. Multiple mutations in domain V may give endurance to against clarithromycin. Further investigations on the molecular mechanism of antibiotic resistance in seem crucial at this stage.