T2182C Mutation Is Not Associated with Clarithromycin Resistance in Helicobacter pylori

T2182C Mutation Is Not Associated with Clarithromycin Resistance in Helicobacter pyloriIn a recent article, Khan et al. described a new mutation in the 23S rRNA gene, T2182C, conferring clarithromycin resistance to Helicobacter pylori (1).An A-to-G mutation at either position 2142 or position 2143 and an A-to-C mutation at position 2142 in the 23S rRNA gene have been shown to confer resistance to clarithromycin in H. pylori (5).In 2000, we isolated a clarithromycin-resistant (Cla r ) strain from a French patient (MIC Ͼ 256 g/ml).Sequencing the 23S rRNA gene revealed a single T2182C mutation.To confirm the role of the T2182C mutation, we used the PCR fragment containing this mutation to transform a susceptible receptor strain.We obtained Cla r transformants.These results were accepted for oral presentation at the 11th International Workshop on Campylobacter, Helicobacter and Related Organisms held in 2001 (3), but a final checking of the MIC determined by E-test revealed a mixed set of colonies.We observed small colonies in the susceptible area of the plate and big colonies in the resistant area.Subcultivation of isolated colonies provided two different clones.A Cla s clone (MIC, 0.016 g/ml) harbors the T2182C mutation and a wild-type sequence at positions 2142 and 2143.A Cla r clone (MIC, 256 g/ml) harbors both the T2182C mutation and an A2143G mutation.Randomly amplified polymorphic DNA patterns from the Cla s and Cla r isolates demonstrated that they are genetically identical.Coming back to the frozen gastric biopsy sample, we isolated 53 single colonies of H. pylori.Fifty-two colonies were from the Cla s clone, and only one was from the Cla r clone.It is possible that Khan et al. were misled, like we were, by a mixed population of a Cla s clone and a Cla r clone.It is surprising that they mention in their article the use of "pure culture from a single colony [for] further study" (1).T2182C is not a new mutation, and it has been already described in previous reports (2, 4, 6).In 1998, Wang et al. (6) first reported a Cla r strain with A2143G and T2182C mutations.In vitro site-directed mutagenesis experiments suggested that this additional mutation is not associated with clarithromycin resistance (6).In 1999, Matsuoka et al. (4) reported the T2182C mutation in both sensitive and resistant colonies.In 2002, Kim et al. (2) described the same T2182C mutation for four Cla r isolates, but they did not indicate that they worked on single-colony isolates.Attention must be paid to experiments used as evidence that a mutation is responsible for resistance in H. pylori.Experiments have to be realized on several isolated colonies, since a mixture of strains can lead to a false determination of sequence.Transformation with PCR fragments could lead to resistant clones growing on antibiotic plates, since the error rate of Taq polymerase spontaneously generates random mutations containing the well-known A2142G or A2143G mutations (5).The existence of a Cla s isolate (20-222S) harboring a T2182C mutation in the 23S rRNA genes led to the conclusion that the T2182C mutation is not associated with clarithromycin resistance in H. pylori.The susceptible clone 20-222S is available on request.