Lack of association between rrl and erm(41) mutations and clarithromycin resistance in Mycobacterium abscessus complex

BACKGROUND Mycobacterium abscessus complex (MABC) includes species with high resistance rates among mycobacterial pathogens. In fact, MABC infections may not respond to clarithromycin treatment, which has historically been very effective against MABC infection. Molecular markers have been proposed to detect both acquired (rrl polymorphisms) and inducible (erm(41) polymorphisms) clarithromycin resistance in MABC isolates. OBJECTIVES This study aimed to evaluate the susceptibility profile and molecular markers of clarithromycin resistance in MABC. METHODS The clarithromycin susceptibility profile was determined by broth microdilution with reads on days 3, 5, 7 and 14. Mutations in the rrl and erm(41) genes were evaluated by polymerase chain reaction (PCR) using specific primers, followed by sequencing. FINDINGS A total of 14 M. abscessus subsp. abscessus isolates and 28 M. abscessus subsp. massiliense isolates were evaluated, and clarithromycin resistance was observed in all isolates for up to three days of incubation. None of the 42 isolates exhibited a point mutation in the rrl gene, while all the isolates had a T28 polymorphism in the erm(41) gene. Moreover, all 28 M. abscessus subsp. massiliense isolates had a deletion in the erm(41) gene. MAIN CONCLUSIONS While all the MABC isolates exhibited acquired clarithromycin resistance, no isolates exhibited a point mutation in the rrl gene in this study. The M. abscessus subsp. massiliense isolates demonstrated clarithromycin resistance, which is an uncommon phenotype. The molecular data for the rrl and erm(41) genes were not consistent with the phenotypic test results of clarithromycin susceptibility, indicating a lack of correlation between molecular clarithromycin resistance markers for both acquired and inducible resistance.

In fact, clarithromycin has become the drug of choice for infections caused by MABC, and it was the treatment mainstay until induced resistance was described (Griffith et al. 2007, Nash et al. 2009). Inducible resistance is believed to be mainly related to the capacity of some MABC subspecies to encode a functional erythromycin ribosomal methylase gene, erm (41), which modifies the binding site for macrolide antibiotics, resulting in inducible macrolide resistance (Bastian et al. 2011, Maurer et al. 2014. Because resistance to clarithromycin is increasing, its use as a monotherapy for treatment is not recommended, and it should instead be combined with other drugs (Kasperbauer & de Groote 2015). Additionally, sequence analysis of the erm(41) gene allowed for the identification of M. abscessus subspecies (Griffith et al. 2015).
Acquired resistance to clarithromycin is related to point mutations in a region of the rrl gene encoding the peptidyltransferase domain of 23S rRNA (Nash et al. 2009). The main molecular mechanism of clarithromycinacquired resistance reportedly occurs through adenine point mutations at either position 2058 (A2058G) or position A2059 in the 23S rRNA gene (Maurer et al. 2012 (Brown-Elliott et al. 2002a, b). MABC is comprised of three closely related subspecies (M. abscessus subsp. abscessus, M. abscessus subsp. bolletii and M. abscessus subsp. massiliense), which are the most pathogenic and multidrug-resistant (MDR) isolates among all RGMs (Griffith et al. 2015, Lee et al. 2015. Infections due to MABC are difficult to treat (Kasperbauer & de Groote 2015, Kang & Koh 2016 because they are intrinsically resistant to not only classical antituberculosis drugs but also to most antibiotics that are currently available (Ryu et al. 2016, Stout et al. 2016. of incubation of the MABC with clarithromycin, while inducible resistance requires prolonged incubation (14 days of incubation) (Bastian et al. 2011).
The aim of this study was to evaluate correlation between the susceptibility profile and the molecular mechanisms of clarithromycin resistance in MABC.

MATERIALS AND METHODS
A total of 42 isolates from a previous surveillance study (Nunes et al. 2014) of samples from different sources collected between 2007 and 2013 were used in this study. The susceptibility profile was determined by broth microdilution following the guidelines of the Clinical and Laboratory Standards Institute [CLSI document M24-A2 (CLSI 2011)] with readings on days three, five, seven and 14. Point mutations in the rrl gene were evaluated by polymerase chain reaction (PCR) amplification with primers 18 (AGT CGG GAC CTA AGG CGA G) and 21 (TTC CCG CTT AGA TGC TTT CAG) (Meier et al. 1994). Polymorphisms in the erm(41) gene were evaluated with primers ERM1f (CGC CAA CGA GCA GCT CG) (Bastian et al. 2011) and MC823 (GAC TTC CCC GCA CCG ATT CCA C) (Nash et al. 2009) using the following steps: 94ºC for 5 min, 40 cycles of 94ºC for 1 min, 64ºC for 1 min and 72ºC for 1 min, and 72ºC for 10 min. M. abscessus ATCC 19977 was used as quality control.
Genetic profiling of the two genes was performed by partial sequencing using the ABI 3500 Genetic Analyser with 50-cm capillaries and the POP7 polymer (Applied Biosystems, Thermo Fisher Scientific, Califórnia, EUA). PCR products were labelled with 3.2 pmol of specific primers and 1 µL of the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) in a final volume of 10 µL. Sequence alignment was performed using the program Biological Sequence Alignment Editor -BioEdit 7.2-5. Homology analysis was performed by comparing the consensus sequences obtained for each isolate with those deposited in GenBank using the BLAST algorithm (Basic Local Alignment Search Tool, http://www. ncbi.nlm.nih.gov/BLAST). The erm(41) sequences of M. abscessus subsp. Abscessus T28 sequevar ATCC19977 (GenBank accession number HQ127365) and M. abscessus subsp. Massiliense CIP108297 (GenBank HQ127368) were used as references. This study was approved by the Ethics Committee of "Grupo de Pesquisa e Pós-Graduação (GPPG)" of "Hospital de Clínicas de Porto Alegre" (reference number CAAE: 35684114.5.0000.5327).

RESULTS
A total of 14 isolates belonging to M. abscessus subsp. Abscessus and 28 isolates belonging to M. abscessus subsp. Massiliense were identified by analysing the erm(41) gene.
Clarithromycin resistance was observed in all the isolates for up to three days of incubation.
For all the isolates, PCR yielded amplicons of the expected size (1500 bp) for the rrl gene; however, none of the 42 isolates exhibited point mutations at positions 2058 or 2059 in the peptidyltransferase region of 23S rRNA (rrl gene), which is the gene related to acquired clarithromycin resistance. For the erm(41) gene, the PCR reactions generated fragments of 764 bp for M. abscessus subsp. Abscessus and smaller amplicons (~250 bp) for all the M. abscessus subsp. Massiliense isolates. These isolates also presented the T/C polymorphism at the 28th nucleotide of erm(41), corresponding to a tryptophan at the 10th codon. Moreover, all M. abscessus subsp. massiliense isolates had a deletion in erm(41) compared to the reference sequence.
Interestingly, point mutations were identified at other positions in the erm(41), gene but they did not correspond to amino acid alteration (silent mutations) ( Table).

DISCUSSION
Widespread use of macrolides in clinical medical care has created favourable conditions for the selection of resistant RGM isolates (Bailey et al. 2008). The two main mechanisms of macrolide resistance are drug-efflux pumps and modification of ribosomal drug target sites via erm methylase expression (Nessar et al. 2012). This modified erm prevents binding of the antibiotic to the ribosome by mono-or di-methylation, which disturbs the shape and chemical makeup of the drug binding site, thereby reducing the affinity of the macrolide to the ribosome (Douthwaite et al. 2005, Mougari et al. 2016b. Erm expression is highly regulated, as it is induced in the presence of macrolides; however, its expression is not favourable for the cell because it is associated with significant fitness costs (Brierley 2013). Nash et al. (2009) and Kim et al. (2010) investigated several isolates of the M. abscessus complex and found that M. abscessus subsp. massiliense was susceptible to clarithromycin, while the susceptibility profile of M. abscessus subsp. abscessus was variable. Conversely, we found a high percentage of M. abscessus subsp. massiliense isolates that exhibited clarithromycin resistance, which was also reported by Mougari et al. (2016b). This susceptibility profile difference may be related to local or regional characteristics of MABC isolates. It should be noted that most of the isolates used in our study belonged to the same clone (data not shown), although they were originated from several outbreaks in different cities in southern Brazil (Nunes et al. 2014).
Molecular profiles of the MABC isolates established by Nash et al. (2009) and Kim et al. (2010) revealed the T28 polymorphism of erm(41) to be related to resistance and the C28 polymorphism to be related to susceptibility. Therefore, the T28 polymorphism in M. abscessus subsp. abscessus could be considered a clarithromycin resistance marker. In our study, all 14 isolates of M. abscessus subsp. abscessus exhibited a T28 polymorphism. It should be noted that in the study by Kim et al. (2010), no prolonged incubation periods were evaluated (microdilution reads were made on the 5th day of incubation).
Regarding M. abscessus subsp. massiliense, the T28 polymorphism is not necessarily related to clarithromycin inducible resistance, as position 28 is located upstream of the deletion that leads to resistance. However, several M. abscessus subsp. massiliense isolates with a functional erm(41) gene have been reported (Gray et al. 2014). Moreover, two M. abscessus subsp. massiliense isolates with a functional erm(41) gene showing inducible clarithromycin resistance after 14 days have been described (Shallom et al. 2013).
Regarding the molecular basis of acquired resistance, sequencing of the rrl gene showed an absence of mutations (wild type) at positions 2058 and 2059 of 23S rRNA in all the isolates analysed in this study. However, we found isolates that exhibited resistance for up to three days of incubation, indicating that another molecular mechanism may be involved in acquired resistance. Although rrl wild-type isolates resistant to clarithromycin are not an usual finding, Maurer et al. (2012) described the presence of resistant isolates with no mutations in the rrl gene in the MABC. Therefore, mutations solely in 23S rRNA cannot be used as a marker for acquired resistance to clarithromycin.
Because Esteban et al. (2009) demonstrated low minimum inhibitory concentration (MIC) for erm(41)positive isolates and high MIC for erm(41)-negative isolates, presence of the erm(41) gene is not necessarily correlated with clarithromycin resistance (higher MIC).
In this study, point mutations in the erm(41) gene were evidenced, but this exchange of nucleotides did not lead to amino acid alteration. According to Brown-Elliott et al. (2015), only the T28C substitution resulted in loss of erm(41) gene function, and no other nucleotide substitution is known to be associated with macrolide susceptibility. This finding disagrees with that of the Kim et al. (2016) study, which demonstrated that M. abscessus subsp. abscessus has a C-to-T mutation at position 19 (C19 → T), which leads to an Arg → stop codon mutation at codon seven of the erm(41) gene and results in loss of erm(41) gene function.
The main finding of our study was the high number of M. abscessus complex isolates with resistance to clarithromycin that did not correlate with mutations in the rrl gene. Therefore, the results of erm (41) and rrl sequencing should not be considered fully concordant with phenotypic clarithromycin susceptibility tests.