Assessment of Clarithromycin Susceptibility in Strains Belonging to the Mycobacterium abscessus Group by erm (41) and rrl Sequencing

Abstract

Clarithromycin was the drug of choice for Mycobacterium abscessus infections until inducible resistance due to erm(41) was described. Because M. abscessus was split into M. abscessus sensu stricto, Mycobacterium massiliense, and Mycobacterium bolletii, we looked for erm(41) in the three species and determined their clarithromycin susceptibility levels. Ninety strains were included: 87 clinical strains from cystic fibrosis patients (61%) and others (39%), representing 43 M. abscessus, 30 M. massiliense, and 14 M. bolletii strains identified on a molecular basis, and 3 reference strains. Clarithromycin and azithromycin MICs were determined by broth microdilution and Etest with a 14-day incubation period. Mutations in rrl (23S rRNA gene) known to confer acquired clarithromycin resistance were also sought. erm(41) was detected in all strains but with two deletions in all M. massiliense strains. These strains were indeed susceptible to clarithromycin (MIC 90 of 1 g/ml) except for four strains with rrl mutations. M. abscessus strains harbored an intact erm(41) but had a T/C polymorphism at the 28th nucleotide: T28 strains (Trp10 codon) demonstrated inducible clarithromycin resistance (MIC 90 of >16 g/ml), while C28 strains (Arg10) were susceptible (MIC 90 of 2 g/ml) except for two strains with rrl mutations. M. bolletii strains had erm(41) sequences similar to the sequence of the T28 M. abscessus group, associated with inducible clarithromycin resistance (MIC 90 of >16 g/ml). erm(41) sequences appeared species specific within the M. abscessus group and were fully concordant with clarithromycin susceptibility when erm(41) sequencing was associated with detection of rrl mutations. Clarithromycin-resistant strains, including the six rrl mutants, were more often isolated in cystic fibrosis patients, but this was not significantly associated with a previous treatment.Respiratory infections due to rapidly growing mycobacteria were first attributed to the Mycobacterium chelonae complex (45). Some years later, it appeared that they were mostly due to Mycobacterium abscessus (16), a newly described species that was distinguished from M. chelonae by DNA-DNA hybridization (23,25). Indeed, the two species were difficult to distinguish by cultural and biochemical features and even by 16S rRNA gene sequencing, which is widely used for bacterial identification (8, 21). Routine M. abscessus identification became possible mostly with the wide use of PCR sequencing of housekeeping genes other than ribosomal genes (12, 13, 18). Recently, the M. abscessus species has been subclassified into three new species on the basis of rpoB sequences: M. abscessus (sensu stricto), Mycobacterium massiliense, and Mycobacterium bolletii (1, 3). Further taxonomic studies showed that differentiation of the three species was not trivial; they still shared ribosomal sequences, and even multilocus sequencing approaches cannot clearly assign clinical strains to one of the three species (24,27,43,49).The M. abscessus group (also called M. abscessus sensu lato...

Investigation of the population structure of Mycobacterium abscessus complex strains using 17-locus variable number tandem repeat typing and the further distinction of Mycobacterium massiliense hsp65 genotypes

“…The PCR cycling conditions were: initial denaturation at 95 uC for 2 min; 35 cycles of denaturation at 95 uC for 1 min, annealing at 62 uC for 1 min and extension at 72 uC for 1.5 min; and a final extension at 72 uC for 10 min. Mutations in the 23S rRNA gene (rrl) were detected by PCR sequencing as described On: Fri, 11 May 2018 17:12:26 previously (Bastian et al, 2011). The M. massiliense isolates were further classified at the subspecies level based on the results of hsp65 sequencing and the HinfI PCR restriction analysis (PRA) method (Kim et al, 2012).…”

“…In this report, we describe M. abscessus complex isolates as three species [M. abscessus sensu stricto (M. abscessus), M. massiliense and M. bolletii] to enable the comparison of macrolide-acquired and inducible resistance between M. abscessus and M. massiliense. Inducible resistance is conferred by erm(41) -one of the few antibiotic resistance genes of the M. abscessus complex to be characterized, the truncated sequence seen in M. massiliense or the point mutation (C28 sequevar) seen in some M. abscessus strains, both of which result in a nonfunctional erm(41) gene (Bastian et al, 2011;Harris & Kenna, 2014).…”

Inhaled therapies, azithromycin andMycobacterium abscessusin cystic fibrosis patients

“…Most of M. abscessus sensu stricto isolates from CF patients are azithromycin resistant in vivo, through induced expression of the ribosomal methyltransferase erm(41) gene [25]. Thus, low-dose azithromycin maintenance treatment cannot prevent colonisation and further infection with MABSC in most cases.…”

Activity of LCB01-0371, a Novel Oxazolidinone, against Mycobacterium abscessus

“…Although the M. abscessus strain is susceptible to clarithromycin after 3 days of in vitro incubation, the strain will become clarithromycin resistant upon the extension of the incubation time to 2 weeks because of the induction of methyltransferase synthesis. The treatment of this M. abscessus infection then becomes almost impossible if there are no alternative drugs (18). Bedaquiline was …”