Detection and characterization of a macrolide 2'-phosphotransferase from a Pseudomonas aeruginosa clinical isolate.

Macrolides, such as erythromycin, clarithromycin, and roxithromycin, have been used as long-term chemotherapy for diffuse Pseudomonas aeruginosa panbronchiolitis in Japan (2). In the United Kingdom, azithromycin has been used for cystic fibrosis patients infected with P. aeruginosa (3). Recently, highly macrolide-resistant strains, producing erythromycin esterase (1, 6) or macrolide 29-phosphotransferase [MPH(29)] (5, 7, 9), have been recovered with increasing frequency in clinical isolates of members of the family Enterobacteriaceae and also staphylococci (8). No reports, however, have yet been published regarding the presence of a macrolide-inactivating enzyme in P. aeruginosa. The appearance of enzymatically mediated high-level macrolide resistance among recent isolates of P. aeruginosa and the genotypes were investigated in this study. A total of 287 clinical isolates were collected as one sample per patient in hospitals across Japan from 1996 to 1998. The MICs of macrolides were determined by the agar dilution method (4). The MICs of erythromycin at which 50 and 90% of the isolates were inhibited were 200 and 400 mg/ml, respectively. MICs of various macrolides for two isolates, M397 and M398, highly resistant to erythromycin are shown in Table 1. These isolates were highly resistant to all 14and 15-membered-ring macrolides, whereas the MICs of 16-membered ring macrolides for them were almost similar to the corresponding MICs for the macrolide-susceptible strain PAO2142Rp. The two isolates showed similar patterns of multiple drug resistance including carbenicillin, tetracycline, chloramphenicol, streptomycin, and kanamycin. Transfer of the macrolide resistance phenotype from these P. aeruginosa isolates could not be demonstrated, as the transfer frequencies to the recipient strain P. aeruginosa PAO2142Rp were less than 10. Enzymatic inactivation of macrolides using crude extracts with or without a cofactor (40 mM ATP, 40 mM GTP, 2 mM acetyl coenzyme A, 40 mM NAD, 40 mM NADP, 40 mM UDPG, or 80 mM GSH) was determined by measuring residual macrolide activity (6). It was demonstrated that the inactivation of oleandomycin using crude extracts from the two isolates was dependent on only ATP or GTP (Table 1). Rf values of inactivated oleandomycin produced by the crude extracts were in agreement with that of standard oleandomycin 29-phosphate by thin-layer chromatography (5). This suggested that isolates M397 and M398 produced an MPH(29) enzyme. The substrate specificities of the enzyme activity with ATP using crude extract from isolate M398 for oleandomycin, triacetyloleandomycin, erythromycin, clarithromycin, roxithromycin, azithromycin, josamycin, leucomycin, midecamycin, miokamycin, spiramycin, and tylosin were 100, 100, 88, 73, 44, 15, ,2, ,2, ,2, ,2, ,2, and ,2%, respectively. Primers for the macrolide-resistance genes ereA, ereB, ermA, ermB, ermC, mphA (8), and mphB (6) were used to generate specific PCR products. PCR products obtained with the mphA primers were detected for both isolates, M397 and M398 (Table 1). However, the identity of the DNA base sequence between the PCR product from isolate M398 and the mphA gene was 53%. These results suggest that a new mph gene has been detected in P. aeruginosa (accession no. AB048591). The appearance of MPH(29)-producing P. aeruginosa may be a warning to avoid the abuse of macrolide antibiotics and a caution for the future use of macrolides in long-term chemotherapy.