Molecular basis of intrinsic macrolide resistance in clinical isolates of Mycobacterium fortuitum

Abstract

Objectives: Some clinical isolates of Mycobacterium fortuitum are naturally resistant to macrolides, e.g. clarithromycin. Thus, the aim of this study was to identify the gene(s) conferring this resistance.

Methods: M. fortuitum ATCC 6841T DNA libraries were screened for plasmids that complemented the macrolide-susceptible phenotype of Mycobacterium smegmatis variant ermKO4 [erm(38)-negative]. Macrolide-resistant M. smegmatis transformants were selected on agar containing 128 mg/L erythromycin.

Results: Genetic complementation identified an M. fortuitum rRNA methylase gene, termed erm(39), 69% identical to erm(38) of M. smegmatis. In addition, erm(39) was found to be in the same chromosomal location as erm(38) in their respective hosts. Like erm(38), erm(39) conferred resistance (MIC >128 mg/L) to macrolide-lincosamide (ML) agents, but not to streptogramin B. Analysis of erm gene expression in M. fortuitum showed that ML agents increased erm(39) RNA levels, reaching a steady state level approximately 20-fold higher than baseline. Screening of 32 M. fortuitum clinical isolates by PCR showed that all were positive for erm(39), irrespective of clarithromycin susceptibility. A majority of clarithromycin-susceptible (MIC < or = 2 mg/L) isolates were postulated to carry a disabled erm(39) gene as they had a GTG-->CTG mutation in the putative initiation codon of the erm(39) gene.

Conclusions: The similarity of the erm genes of M. smegmatis and M. fortuitum suggests that they were inherited from a common ancestor. Although the clinical impact of erm(39) on the therapeutic utility of clarithromycin is unclear, induction of this gene is consistent with the trailing end-points commonly seen during susceptibility testing of M. fortuitum isolates against macrolides.

Figure 1

Summary of the cloned M. fortuitum DNA fragments that conferred high-level erythromycin resistance to M. smegmatis ermKO. The common region that hybridized with an erm (38) (derived probe is indicated by the arrows. The annotated restriction sites are: B, BamHI; D, BspDI; E, EcoRV; G, BglII; M, MscI [plasmids pMVMF3 and pMVMF1 pMVMF10 had at least one additional BglII and MscI site—the locations of these sites relative to the erm (38) (probe binding site was not mapped in detail in this analysis]. The fragments are oriented so that RNA transcription from the hsp65 promote promoter of the cloning vector (pMV261) proceeds from left to right.

Figure 2

ClustalW alignment of the RNA methylases of M. fortuitum [Erm(39); GenBank acc. AY487229] and M. smegmatis [Erm(38); GenBank acc. AAN86837] AAN86837]. Dark-shaded amino acids are identical the two proteins and light-shaded amino acids are functionally similar.

Figure 3

Genetic organization of the M. fortuitum chromosome in the region of erm(39) ( GenBank acc. AY487229). Genes shown in black have a high degree of identity (≥65%) with M. tuberculosis H37Rv (the gene numbering in this figure is equivalent to the Rv gene index), and M. smegmatis. The genes shown with cross-hatching have a high degree of identity only with M. smegmatis. The hypothetical gene (shown in white) has no convincing homology (i.e. <30% identity) to any known sequence. Key: pgaE —putative polyketide oxygenase (65% amino acid identity with the N-terminus of PgaE of M. smegmatis; 36% identity with N-terminus of oxygenase-reductase PgaM of Streptomyces species, GenBank acc. AAK57530.1); TR—putative transcriptional regulator [73% amino acid identity with TR associated with erm(38) of M. smegmatis; 32% identical to CalR1, a calicheamicin synthesis regulator, GenBank acc. AAM94766]; erm(39)—adenine rRNA methylase [71% amino acid identity with Erm(38) of M. smegmatis, GenBank acc. AAN86837]; 3355—hypothetical gene with unknown function (65% identity with Rv3355c of M. tuberculosis, GenBank acc. NP_217872.1);folD —a tetrahydrofolate dehydrogenase/cyclohydrolase (82% amino acid identity with FolD or Rv3356c of M. tuberculosis, GenBank acc. NP_217873.1); 3359—probable NAD(H)-dependent flavin oxidoreductase (74% identity with N-terminus of Rv3359 of M. tuberculosis, GenBank acc. NP_217876.1).

Figure 4

Real-time RT–PCR analysis of erm(39) expression. (a) A time course study following addition of erythromycin (1 mg/L). RNA levels are presented relative to baseline (time 0). (b) The effect of different antimicrobial agents on erm(39) expression after an incubation of 120 min. ND, control (no drug); CLI, clindamycin (8 mg/L); ERY, erythromycin (1 mg/L); SPM, spiramycin (8 mg/L); Q, quinupristin (16 mg/L). All data points represent the mean of three replicates.