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

Version Changes

Revised. Amendments from Version 4

Title: Inclusion of a hyphen in Non-tuberculous Abstract: Inclusion of a hyphen in Non-tuberculous Mycobacterium (NTM). Used upper case for the first letters of the acronym SGM and RGM. In the results section, the percentage are outside the brackets while proportions have been indicated in brackets. For mutations, nucleotide change has been indicated for each position. Introduction: Details of Lowenstein Jensen media for growth of mycobacteria has been included. Second paragraph, the duration of treatment of NTM infections has been indicated. indication that Rifampicin is recommended for first-line treatment of MTB alongside other drugs. Laboratory procedures: Sputum sample estimate volume for each participant has been provided.  Details on FM microscopy readers and reviewers is now indicated Results: The table with results indicating that the bulk of the samples were obtained from Lake Victoria, Coastal and Nairobi region has been mentioned. Discussion: rrl significant mutations are critically discussed and their relevance to macrolide drug resistance

Abstract

Background: Non-tuberculous mycobacteria (NTM) treatment constitutes a macrolide-based antibiotic regimen in combination with aminoglycosides for Rapid-Growing Mycobacteria (RGM), and rifampicin for Slow-Growing Mycobacteria (SGM). Mutations in the anti-NTM drug target regions promote NTM evolution to mutant strains that are insusceptible to NTM drugs leading to treatment failure. We, therefore, described the mutation patterns of anti-NTM drug target genes including rrl, rrs, and rpoB in NTM isolates from Kenya. 

Methods: We carried out a cross-sectional study that included 122 NTM obtained from the sputum of symptomatic tuberculosis-negative patients in Kenya. All 122 NTM underwent targeted sequencing of the rrl gene. The 54 RGM were also sequenced for rrs, and the 68 SGM were sequenced for rpoB genes using ABI 3730XL analyzer. The obtained sequences were aligned to their wild-type reference sequences for each gene using Geneious then mutations were identified. Pearson chi-square at a 95% confidence interval tested the association of NTM to mutation patterns for each gene.

Results: NTM harboring mutations associated with resistance to at least one of the antibiotics used in the macrolide-based therapy were 23% (28/122). Of these NTM, 10.4% (12/122) had mutations in the rrl gene with 58.3% (7/12) comprising RGM and 41.7% (5/12) being SGM. Mutation at position 2058 (A2058G, A2058C, A2058T) of the rrl gene was seen for 83.3% (10/12) of NTM, while 16.6% (2/12) harbored a A2059G mutation. Of the 54 RGM included for rrs characterization, 11.1% (6/54) exhibited mutations  at position 1408(A1408G), while 14.7% (10/68) of the SGM had mutations in the rpoB gene at positions S531W, S531L, S531Y, F506L, E509H with M.gastri having multiple mutations at positions D516V, H526D and, S531F.

Conclusion: We demonstrated a significant level of mutations associated with drug resistance for macrolides, aminoglycosides, and rifampicin in NTM isolated from symptomatic TB negative patients in 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

Sample processing, mycobacterial culture and growth identification

The sputum samples were decontaminated using the N-acetyl-L-cysteine 2% NaOH (NALC-NaOH) procedure, then inoculated into Mycobacteria Growth Indicator Tube (MGIT) and Lowestein-Jenseen (LJ) medias, incubated at 37°C and monitored for growth for up to eight weeks respectively. At the same time, sputum smears were prepared, air dried, heat fixed then fluorochrome stained with auramine O where mycobacteria appeared as bright yellow fluorescent rods when viewed under a light emitting diodes (LED) microscope. Fluorescent microscopy was performed by NTRL laboratory technologist then smears were reviewed by the section head before reporting.

The culture growth in MGIT and LJ underwent the Mtb identification testing using the SD Bioline TB Ag MPT64 assay (capilia) (Standard Diagnostics, Yongin-si, Gyeonggi-do, Republic of Korea) and capilia positive samples were excluded from the study. The capilia negative samples underwent ZN microscopy with presence of AFB indicating a possible NTM.

DNA extraction

Mycobacterial DNA was extracted from 500 μL of re-suspended colonies using using GenoLyse ^® ( Hain Lifescience, Nehren, Germany) according to the manufacturer's instructions. Briefly, 100 ul of lysis buffer (A-LYS) buffer was added to each cryovial containing the resuspended colonies and incubated for five minutes at 95 ^oC after which 100 ul neutralization buffer (A-NB) was added and centrifugation done at 5000G for ten minutes. The supernatant was transferred to a newly labelled cryovial awaiting PCR.

Conventional PCR, DNA gel electrophoresis and DNA purification

Conventional PCR targeting rrl, rpoB and rrs genes of NTM were conducted using the Horse-Power™ Taq DNA Polymerase MasterMix (Canvax, Córdoba, Spain) in a final reaction volume for each gene of 13 μl comprising 6.25 μl of 2X Horse-Power™ Taq DNA Polymerase MasterMix, 2.5 μl DNA template, 0.25 μl of each of both forward and reverse primers ( Table 1) at a final concentration of 10 pmoles, and 3.75 μl of nuclease-free water to make up the reaction volume. The PCR assays were carried out with a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA) ( Table 1). Thermal cycling conditions for rrl were as follows: one cycle of 95°C for five minutes, 35 cycles of 95°C for one minute, 55°C for one minute, 72°C for one minute, and a final extension for ten minutes at 72°C. PCR for rrs was conducted as follows 95°C for five minutes, 35 cycles of 95°C for one minute, 60°C for one minute, 72°C for one minute, and a final extension for seven minutes at 72°C. PCR for the rpoB was conducted as follows: 95°C for five minutes, 35 cycles of 95°C for one minute, 58°C for one minute, 72°C for one minute, and a final extension for seven minutes at 72°C. Amplified products were confirmed on a 1% Agarose gel stained with 4.6 μl SYBR safe DNA stain (Invitrogen, Carlsbad, California, USA), and results were visualized with an UltraViolet gel viewer (Terra Universal, S. Raymond Ave, Fullerton, CA, USA). For the corresponding runs, a positive control containing drug-resistant NTM with mutations in the rrl, rpoB and rrs genes was used, as well as a negative control containing drug sensitive NTM without mutations in the drug target genes were included.

Table 1. Nucleotide sequence of primers for rrl, rpoB and rrs genes.

Gene	Primers	Size	Reference
rrl	F (5′-TTTAAGCCCCAGTAAACGGC-3′) R (5′-GTCCAGGTTGAGGGAACCTT-3′)	420 bp	( Park et al., 2020)
rpoB	F (5′-GGCAAGGTCACCCCGAAGGG-3′) R (5′-AGCGGCTGCTGGGTGATCATC-3′)	723 bp	( Adékambi et al., 2003)
rrs	F (5′-AAGGAGGTGATCCAGCCGCA-3′) R (5′-TCCCTTGTGGCCTGTGTGCA-3′)	341 bp	( Kim et al., 2021)

The PCR products were enzymatically purified using ExoSAP IT (Applied Biosystems, Foster City, California, USA). Purification conditions were done at 37°C for fifteen minutes followed by a second incubation at 80°C for fifteen minutes and a final cooling step at 4°C for five minutes.

rrl, rpoB and rrs genes sequencing

The purified amplicons were sequenced in the forward and reverse directions by Sanger sequencing using Big Dye™ Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) and the forward and reverse primers. The sequencing reaction for each gene was a 10 μl reaction comprising 1.25 μl of Big Dye Terminator, 3 μl of 5× Sequencing Buffer, 1 μl of 1 pmol of the sequencing primer, and 1.5 μl of the PCR product. The reaction volume was made up by adding 3.25 μl of nuclease-free water. The reaction proceeded through 96°C for 1 minute then 25 cycles of 96°C for 10 seconds, 50°C for five seconds, and 60°C for four minutes.

Purification of cycle-sequencing products was done using the BigDye X Terminator™ purification kit following the manufacturer’s instructions (Applied Biosystems, Foster City, California, USA) and purified products were loaded onto the ABI 3730 genetic analyzer (Applied Biosystems, Foster City, California, USA) for capillary electrophoresis.

Data analysis

The obtained sequences were first assembled into contigs and the consensus sequences aligned to their wild-type reference sequences for each gene using Geneious version 11.0 (Biomatters Ltd, Auckland, New Zealand). Mutations in the drug resistance genes were identified visually. STATA version 14 (StataCorp, College Station, Texas, USA) was used to test the association of NTM species to mutation patterns using Pearson chi-square at 95% confidence interval.

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).

Table 2. Sociodemographic characteristics of patients with drug-resistant NTM.

Variables	NTM (n, %)	Genes with mutations associated with drug resistance to NTM			P value
		rrl (n, %)	rrs (n, %)	rpoB (n, %)
Regions
Mt. Kenya	23(18.9)	2(16.7)	-	-
North Rift Valley	13(10.7)	1(8.3)	-	-
South Rift valley	9(7.3)	2(16.7)	-	2(20)
Lake Victoria	8(6.6)	-	2 (33.3)	4(40)	0.012
Lower Eastern	8(6.6)	1(8.3)	-	2(20)
Coast	33(27)	2(16.7)	2 (33.3)	2(20)
Northeastern	4(3.3)	-	-	-
Nairobi	24(19.6)	4(33.3)	2 (33.3)	-
Subtotal	122(100)	12 (100)	6 (100)	10(100)
Age group
<21	5(4)	-	-	-
21-35	49(40.1)	4(33.3)	1(16.7)	5(50)
36-50	40(32.8)	5(41.7)	3(50)	1(10)	0.012
51-65	21(17.3)	2(16.7)	2 (33.3)	3(30)
>65	7(5.8)	1(8.3)	0	1(10
Subtotal	122(100)	12(100)	6(100)	10(100)
Gender
Female	32 (27)	4(33.3)	2 (33.3)	1(10)
Male	90(73)	8(66.7)	4(66.7)	9(90)	0.000
Subtotal	122(100)	12(100)	6(100)	10(100)
Type of patient
New	33(27)	3(25)	2 (33.3)	3(30)
Retreatment	21(17)	3(25)	1(16.7)	2(20)
Relapse	30(25)	2(16.7)	2(33.3)	4(40)	0.001
MDR follow-up	38(31)	4(33.3)	1(16.7)	1(10)
Subtotal	122(100)	12(100)	6(100)	10(100)

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).

Table 3. NTM showing mutations associated with macrolide resistance.

Isolate ID	NTM species	Growth rate	Position of mutation in rrl	X 2 , p value
C21, C64	M. intracellulare	SGM	A2058G
C31	M. nebraskense	SGM	A2058C
C109	M. kumamototense	SGM	A2058T
C132	M. heraklionense	SGM	A2058T
C36, 63	M. abscessus subsp abscessus	RGM	A2058C	87, <0.001
C47	M. abscessus subsp abscessus	RGM	A2058G
C88	M. massiliense	RGM	A2058T
C133	M. bourgelatii	RGM	A2058C
C46, C71	M. abscessus subsp abscessus	RGM	A2059G

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.

Table 4. Rapid-growing NTM showing mutations associated with aminoglycoside resistance.

Isolate ID	NTM species	Position of mutation in rrs	X 2, p value
C12, C53	M. chelonae	A1408G
C58	M. alsense	A1408G	25, 0.06
C46, C47, C70	M. abscessus subsp abscessus	A1408G

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).

Table 5. Slow- growing NTM showing mutations associated with rifampicin resistance.

Isolate ID	NTM species	Codons with mutation in rpoB	X 2, p value
C66	M. avium subsp avium	S531W
C78, C94	M. avium subsp avium	S531L
C128	M. avium subsp avium	S531Y	26, 0.89
C10, C57, C64, C166	M. intracellulare	F506L
C74	M. yongonense	E509H
C151	M. gastri	D516V, H526D, S531F

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.

Limitations of the study

This study investigated the acquired mechanisms of drug resistance for NTM. Other intrinsic factors apart from drug target gene mutation could influence the sensitivity of NTM to antibiotics.

Despite this limitation, the study documents drug resistance-associated mutation patterns of Kenyan NTM for the first time, and advocates for drug resistance testing before commencement of treatment for NTM infection.

Ethical clearance

This study was approved by the Kenyatta National Hospital-University of Nairobi Ethics Review Committee (Ref: KNH-ERC/A/38) on 30 ^th January 2020. Waiver for individual informed consent was granted as the study utilized remnant clinical samples and the research posed no greater than minimal risk to the study subjects.

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:

• -Mutation patterns for rrl, rrs, and rpoB genes in NTM

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

Funding Statement

This work was supported by the Meru University of Science and Technology 6th call for proposals awarded to Zakayo M Mwangi. The content is solely the responsibility of the authors and does not represent the views of the funders.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.