Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in non-tuberculous mycobacteria isolates from Kenya

Introduction

Non-tuberculous Mycobacteria (NTM) are a group of about 170 Mycobacteria species that do not include Mycobacterium tuberculosis and Mycobacterium leprae (Falkinham, 2017; Koh et al., 2006; Peixoto et al., 2020; Sam et al., 2020; Simons et al., 2011). In mycobacterial culture using Lowestein-Jensen (LJ) media which is a solid selective media for mycobacteria, NTM varies in their growth characteristics, with Rapid Growing Mycobacteria (RGM) forming visible colonies within seven days of incubation and Slow-Growing Mycobacteria (SGM) taking up to fourty days. SGM include Mycobacterium avium complex (MAC), Mycobacterium chimaera, Mycobacterium kansasii, Mycobacterium malmoense, and Mycobacterium xenopi, whereas RGM comprise species from the Mycobacterium abscessus and Mycobacterium fortuitum complexes (Alffenaar et al., 2021). In human infections, identifying NTM is crucial for determining clinically relevant species and the best treatment plan (Chalmers et al., 2019; Mwangi et al., 2022a).

NTM treatment consists of a macrolide-based antibiotic regimen which include clarithromycin or azithromycin, combined with other selected antibiotics which are given for 12 to 18 months until culture negativity is achieved. (Falkinham, 2018). In addition to macrolides, the antibiotic of choice is largely determined by the infecting NTM, its growth rate, and the intricacy of the mycolic acid cell wall (Goswami et al., 2016). Rifampicin and ethambutol are also used in SGM treatment, while aminoglycosides, cefoxitin, imipenem, or tigecycline are used in RGM treatment (Brown-Elliott et al., 2012; Saxena et al., 2021). Due to the slow growth of NTM compared to other bacterial infections, antimicrobial combination therapy is strongly recommended in NTM treatment to avoid the development of drug resistance (Falkinham, 2018; Pharmd et al., 2019). The antibiotic regimen of patients with pre-existing lung diseases including asthma, chronic obstructive pulmonary disease, cystic fibrosis, and bronchiectasis or with immunodeficiencies are the same as for patients without these comorbidities (Koh, 2017).

Anti-mycobacterial drugs attach to their binding sites with a high affinity, preventing the target gene product from functioning normally (Alffenaar et al., 2021). Changes in the structure of the target regions caused by mutations, on the other hand, interfere with the medications' ability to attach to them, resulting in antibiotic resistance. As a result, determining the antibiotic resistance profile of NTM is critical for determining an effective treatment strategy for a specific NTM infection (Goswami et al., 2016; Nasiri et al., 2017).

Acquired resistance to anti-NTM drugs develops due to mutations in the NTM drug target regions, subsequently promoting NTM evolution to mutant strains that are insusceptible to anti-NTM drugs (Huh et al., 2019; Munita & Arias, 2016; Nasiri et al., 2017; Pharmd et al., 2019; Saxena et al., 2021). Prolonged exposure to NTM antibiotics, as seen in the lengthy NTM regimen, sub-optimal administration of anti-NTM drugs, as seen in patients who do not adhere to the regimen or who are lost to follow up, and incorrect prescription for NTM infection due to NTM misdiagnosis all promote mutation in the drug target regions (Gopalaswamy et al., 2020; Munita & Arias, 2016; Zhou et al., 2020).

Macrolides inhibit protein synthesis by binding to the peptide exit tunnel of ribosomes, hence preventing the growing peptide chain from exiting the peptidyl transferase center of the ribosome (Hansen et al., 2002). Mutation of the rrl gene at positions A2058 and A2059 accounts for 80-100% of the macrolide resistance in NTM. Macrolide resistance can also be conferred by the erm (41) gene which encodes a ribosomal methyltransferase that methylates the rrl thus blocking the drug-binding site of the macrolide (Bastian et al., 2011; Huh et al., 2019).

Aminoglycosides inhibit protein synthesis by binding the bacterial 30S ribosomal subunit interfering with bacterial protein translation and leading to cell death (Saxena et al., 2021). Drug resistance to aminoglycosides is associated with modification in the rrs gene mainly observed as point mutation at position 1408 (Brown-Elliott et al., 2013). In addition, mutations at positions 1406, 1409, and 1491 have been shown to confer resistance to aminoglycosides in some NTM (Olivier et al., 2017).

Rifampicin is a key drug in treating mycobacterial diseases. It is the recommended first line drug for treatment of M. tuberculosis alongside other drugs (WHO, 2014). It inhibits the synthesis of Ribonucleic acid (RNA) by binding to the ß-subunit of the RNA polymerase that is encoded by rpoB gene. Most rifampicin-resistant mycobacteria have mutations in an 81-bp rifampicin resistance determining region (RRDR) within the rpoB gene. Mutations in this region account for 95% of rifampicin resistance in mycobacteria. The commonly observed mutations within the RRDR of rpoB are often seen at codons 526, and 531 (Li et al., 2016). M. kansasii has also shown mutation conferring resistance to rifampicin at codons 513 and 516, while MAC also shows resistance to rifampicin with mutations outside the RRDR at codon 626 (K626T) (Ramasoota et al., 2006).

Detection of drug resistance in NTM can be carried out by drug susceptibility testing (DST) through broth microdilution, (Litvinov et al., 2018; Park et al., 2020), sequencing for mutations in the rrl, rrs and rpoB genes (Brown-Elliott et al., 2012; Huh et al., 2019; Saxena et al., 2021), or by using GenoType NTM-DR test (Hain, Lifescience, Nehren, Germany) which is a line probe assay (LPA) that detects resistance to macrolides and aminoglycosides (Bouzinbi et al., 2020).

We, therefore, investigated drug resistance in NTM by describing the mutation patterns in rrl, rrs, and rpoB genes for macrolides, aminoglycosides and rifampicin respectively, in NTM isolated from symptomatic TB-negative patients from Kenya.

Methods

We carried out a cross-sectional study that included 122 NTM identified by mycobacterial culture and hsp65 targeted sequencing from the sputum of symptomatic TB-negative patients (Mwangi et al., 2022a). The patients had provided approximately 5 ml of sputum to the National Tuberculosis Reference Laboratory (NTRL) in Kenya between January to November 2020.

In this study, the 122 NTM including 54 RGM and 68 SGM underwent targeted sequencing of the rrl gene. The RGM were also sequenced for rrs, and the SGM were sequenced for rpoB genes using ABI 3730XL analyzer (Applied Biosystems, Foster City, California, USA).

Laboratory procedures

Results

Our study established that 23% (28/122) of NTM harbored mutations associated with resistance to at least one of the antibiotics in the macrolide-based therapy. One isolate (C47) containing M. abscessus had mutations conferring resistance in both rrl and rrs genes.

The bulk of the isolates with mutations associated with drug resistance originated from the Lake Victoria, Coastal, and Nairobi regions, with 5% (6/122) NTM showing mutations in the rrl, rrs, or rpoB genes (p=0.012)) (Table 2). The age group was statistically significant (p=0.012), with isolates from participants aged 21 to 35 years old having the highest (n=10, 35.7%) number of NTM with target gene alterations for rrl, rrs, and rpoB genes. Males accounted for 76.5% (21/28) of the NTM isolates with mutations in the drug target genes and indicated a strong statistical significance (p=0.000). The majority of NTM with these mutations were detected in new (n=8; 28.5%) and TB relapse patients (n=8; 28.5%) patients, which had a significant statistical significance (p=0.001) (Table 2).

Mutations in rrl gene for the NTM were highly significant with a p value of <0.001. Of the NTM with mutations in their drug target genes, 10.4% (12/122) had mutations in the rrl gene (Table 2) with 58.3% (7/12) comprising RGM (Table 3) and 41.7% (5/12) being SGM (Table 3). Point mutation at position 2058 (A2058G, A2058C, A2058T) of the rrl gene was seen for 83.3% (10/12) of NTM including M.intracellulare, M. abscessus subsp abscessus, M. nebraskense, M. massiliense, M. kumamototense, M. heraklionense, and M. bourgelatii. A rrl A2059G mutation was seen for M. abscessus subsp abscessus representing16.6% (2/12) of the NTM (Table 3).

Of the 54 RGM included for rrs characterization, 11.1% (6/54) exhibited mutations at position 1408(A1408G), The NTM species presenting with these mutations include M. abscessus subsp abscessus, M. chelonae, and M. alsense (Table 4). The NTM species had a low likelihood (p=0.06) of developing mutations in rrs gene for aminoglycoside resistance.

A low association (p=0.89) for rpoB mutation in SGM was observed. Mutations within codons between 503-533 of the rpoB were seen for 14.7% (10/68) of SGM. These SGM included M. avium subsp avium, (S531W S531L, S531Y) M. intracellulare (F506L), M. yongonense (E509H) and M. gastri which had multiple mutations at positions D516V, H526D and, S531F (Table 5).

Discussion

Non‐tuberculous mycobacteria (NTM) are an important cause of pulmonary disease worldwide, and are being isolated increasingly (Rivero-Lezcano & Carolina González-Cortés, 2019). They are often mistakenly treated as M. tuberculosis in countries devoid of laboratory competence for mycobacterial species differentiation (Pokam et al., 2022). Recently, there has been a considerable rise in infections caused by NTM (Saxena et al., 2021). These mycobacteria, which comprise a large and diverse range of species, have developed resistance to most conventional antibiotics, rendering their treatments unsatisfactory (Brown-Elliott et al., 2012).

This study established that 23% of NTM harbored mutations associated with resistance to at least one of the antibiotics in the macrolide-based therapy. Regional distribution of NTM with mutations in the target genes had a significant correlation (p=0.012) with the bulk of the isolates originating from the Lake Victoria (n=6, 5%), Coastal (n=6, 5%), and Nairobi (n=6, 5%) regions. The observed regional diversity of NTM harboring drug resistance mutations across Kenya could be attributed to NTM evolution to evade natural antibiotics secreted as secondary metabolites by nearby environmental bacteria in various geographical landscapes (Moore et al., 2019). The 21-35 years age group had the highest number of NTM isolates presenting with drug resistance associated mutations while males comprised the majority (n=21, 76.5%) of cases with NTM showing these mutations. These mutations were associated with a history of previous TB infection as seen in the high number of TB relapse cases recorded in this study (n=8; 28.5%, p=0.001). This is a common observation in sub-Saharan Africa given the high incidence of M. tuberculosis disease and the frequent misdiagnosis of NTM infection with TB seen in this region (Aliyu et al., 2013; Hoza et al., 2016; Pokam et al., 2022; Mwangi et al., 2022a).

Despite NTM demonstrating high levels of resistance to a broad range of antibiotics, macrolides including clarithromycin, erythromycin, and azithromycin remain the most effective antibiotic with >80% of isolates showing susceptibility (Ananta et al., 2018). However, some NTM including MAC and M. abscessus have been associated with increased resistance to macrolides leading to treatment failure (Saxena et al., 2021). The mechanisms of macrolide resistance have been studied at the molecular level and has consistently demonstrated that 80-100% of phenotypic macrolide resistant clinical isolates contained point mutations at positions A2058 and A2059 in the 23S rRNA gene for NTM (Huh et al., 2019). According to 23S rRNA crystallography investigations, A2058 and A2059 serve as important contact sites for macrolide binding since they are conveniently situated on the surface of the peptide exit tunnel of ribosomes. The architecture of the macrolide binding site is changed by a point mutation of A2058/A2059, which creates a smooth, sunken interaction surface that weakens the interaction of macrolides with their binding site hence leading to drug resistance (Poehlsgaard et al., 2005). Our study demonstrated that majority of macrolide-resistant isolates (88.3%) including M. intracellulare (A2058G), M. nebraskense (A2058C), M. massiliense (A2058T), M. kumamototense (A2058T), M. heraklionense (A2058T), and M. bourgelatii (A2058C) had a point mutation at position 2058 in the rrl, while two (16.7%) M. abscessus subsp abscessus isolates had mutation at position A2059G. The increased potential for development of these mutations in MAC species including M. intracellulare, and M. abscessus could be attributed to inherent factors such as a high propensity for genetic mutation in the drug target region, and high drug tolerance levels (Park et al., 2020), environmental factors facilitating the emergence of mutations in the rrl and gene subsequent ease of transmission to humans (Beverley Cherie Millar, 2019). Other mutations that could confer resistance to macrolides include T2419 in M. intracellulare (Huh et al., 2019). However, this mutation was not demonstrated in the Kenyan isolates of this study.

The commonly used aminoglycosides for the treatment of NTM are amikacin, streptomycin, kanamycin, tobramycin, and streptomycin (Krause et al., 2016). In our present study, three M. abscessus subsp abscessus, two M. chelonae, and one M. alsense presented with a A1408G mutation. Genotypic characteristics in rrs that are associated with aminoglycoside resistance usually are in concordance with DST broth microdilution and GenoType NTM-DR assays, implying that mutations within rrs are the molecular basis of aminoglycoside resistance in NTM (Bouzinbi et al., 2020). For instance, a study that selected in-vitro aminoglycoside-resistant M. abscessus and M. chelonae presented an A→G mutation at position 1408 within the rrs upon sequencing. This confirms that a single point mutation at 1408 is adequate to confer high-level aminoglycoside resistance (Nessar et al., 2011). Further, individual mutations at T1406, C1409 and G1491 have also indicated considerable resistance to aminoglycoside in most M. abscessus subspecies (Nessar et al., 2011). Similar to other bacteria, NTM present with low-level aminoglycoside resistance through the production of drug-modifying enzymes including acetyltransferases (Sanz-García et al., 2019), phosphorylases, adenylates, and methylases which act at various points on the aminoglycoside scaffold making it less potent (Krause et al., 2016; Munita & Arias, 2016; Sanz-García et al., 2019; Tarashi et al., 2022; Zaragoza Bastida et al., 2017). Why does mutation at 1408 confer more drug resistance compared to other point mutation.

Similar to M. tuberculosis, resistance to rifampicin in NTM is associated with mutations in the 81 bp RRDR corresponding to codons 503 to 533 of the rpoB gene (Saxena et al., 2021; Zhou et al., 2020). Our study identified 11.6% (10/68) NTM with mutations occurring at codon 531 in M. avium, codon 506 in M. intracellulare, 509 in M. yongonense. M. gastri had amino acid substitutions at positions 516, 526, 531 of the rpoB gene. Our findings did not establish mutations outside of the RRDR. However, low-level rifampicin resistance has previously been demonstrated in M. intracellulare with mutations occurring outside the RRDR at position N494S (Park et al., 2020). Broth microdilution analysis which is the gold standard for rifampicin drug resistance testing presents a high minimum inhibition concentration (MIC) for isolates with mutations in the RRDR, hence confirming the role of these mutations in conferring high-level resistance to rifampicin (Huh et al., 2019). We further demonstrated that M. gastri harbored more than one mutation within the RRDR which is a unique attribute observed for M. kansasii complex species to which M.gastri belongs (Wu et al., 2018). A similar study obtained rifampicin-resistant M. kansasii from clinical isolates and in vitro generated mutant, and demonstrated mutations in codons 513, 526, and 531 of rpoB which is a common pattern in some SGM and M. tuberculosis (Klein et al., 2001).

We found considerable levels of mutations in the drug target genes for macrolides, aminoglycosides and rifampicin in Kenyan NTM. To guide therapy, both species-level identification and drug resistance testing of NTM should be performed before starting treatment for NTM infection.

Conclusion

We demonstrated significant mutations associated with drug resistance for macrolides, aminoglycosides and rifampicin in NTM isolated from symptomatic TB-negative patients in Kenya.

M. abscessus and MAC were the dominant NTM with rrl mutations, and most mutations occurred at position 2058. Majority of RGM had mutation at position 1408 of the rrs gene, while rpoB mutations presented within the RRDR.

Data availability

Figshare. Mutation Patterns of Resistance Genes for Macrolides, Aminoglycosides, and Rifampicin in Non‐tuberculous Mycobacteria Isolates from Kenya. DOI: https://doi.org/10.6084/m9.figshare.20331378.v2 (Mwangi et al., 2022b).

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Author contributions

ZMM - Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

GN - Investigation, Writing – Review & Editing

MWM - Supervision, Writing- Review and Editing

FGO - Conceptualization, Methodology, Supervision, Writing- Review and Editing

WDB - Conceptualization, Methodology, Supervision, Writing- Review and Editing