Comment on: Acquired macrolide resistance genes in Haemophilus influenzae?

Sir, We read the recently published paper of Atkinson et al. with interest. The authors have misrepresented and misquoted our study by referencing primers we did not use or advocate [Table 1 and Table S1 (available as Supplementary data at JAC Online)]. The erm(A) and mef(A) primer sets Atkinson et al. reported to have produced false-positive PCR results were not used in our study nor described in our paper (Table 1). The erm(A) primers they attributed to our study were degenerate primers that have never been used by our group. – 7 We found that the primers for erm(A), erm(B) and erm(F) genes listed in Atkinson et al. (Table S1) were actually designed and reported previously by Chen et al. in a study on the use of rapid real-time PCR assays for the quantification of erm genes in manure, compost, lagoons and a bioreactor. Clearly, when used for Haemophilus influenzae they gave false positives and another primer set should have been used for detection of the genes in question. The erm(A) and mef(A) primer sets used for our H. influenzae study (Table 1), and that do not produce false-positive PCR results, have been described previously. – 7 In addition to confirming our positive PCR results by hybridization of the PCR products with internal probes, we verified that the macrolide genes are on mobile elements by selecting 25 H. influenzae isolates as donors for mating experiments with H. influenzae Rd and/or Enterococcus faecalis JH2-2 as recipients. All 25 donors were able to transfer macrolide resistance to recipients at a frequency ranging from 10 to 10/recipient. The resulting transconjugants were resistant to macrolides and had erm genes and/or a mef(A) gene that were originally found in the donor strains. Mutations do not normally transfer by conjugation (M. C. Roberts, unpublished observations). We should emphasize that a study of 172 H. influenzae isolates from Australia should not be considered representative of H. influenzae across the world. A possible reason for differences between the USA and Australian isolates may be the type of patients included in the Australian study. Only 15.7% (27/172) of the Australian H. influenzae isolates came from patients with exposure to azithromycin compared with 106 USA cystic fibrosis isolates that were from patients participating in a clinical study where 50% had extensive azithromycin therapy three times per week. Only 7 Australian isolates were macrolide resistant compared with 27 macrolide-resistant isolates in the USA study. Therefore, based on different populations (azithromycin exposed) and a low number of isolates resistant to macrolides, there could easily be differences in the distribution of erm/mef(A) genes among H. influenzae from different patient populations even within a single city. It would have been valuable had Atkinson et al. used PCR-specific primers with PCR assays and not with real-time PCR assays. Without this being done, there is no scientific evidence for the authors to conclude that ‘It is interesting to speculate as to whether some of the positive results in the Roberts et al. study, particularly for mef(A) and erm(A), could have been similar false positives’, especially since our strains were internal probe positive and the 25 isolates used for mating experiments could move macrolide resistance by conjugation. It would have been interesting if the resistant Australian strains were tested on their ability to transfer macrolide resistance to a recipient, which would have