Keywords
Nontuberculous mycobacteria, slow-growing mycobacteria, rapid-growing mycobacteria, macrolides, aminoglycosides, rifampicin
This article is included in the Cell & Molecular Biology gateway.
Nontuberculous mycobacteria, slow-growing mycobacteria, rapid-growing mycobacteria, macrolides, aminoglycosides, rifampicin
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, NTM varies in their growth characteristics, with rapid growing NTM (RGM) forming visible colonies within seven days of incubation and slow-growing NTM (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 determining the best treatment plan (Chalmers et al., 2019; Mwangi et al., 2022).
NTM treatment consists of a macrolide-based antibiotic regimen, such as clarithromycin or azithromycin, combined with other selected antibiotics that work synergistically to disrupt NTM metabolic processes and growth (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).
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 mostly 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 including those caused by M. tuberculosis (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 occurring 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.
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., 2022). The samples were obtained from the National Tuberculosis Reference Laboratory (NTRL) in Kenya between January to November 2020. All the 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 (Applied Biosystems, Foster City, California, USA).
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.
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.
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 95oC 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 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).
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 fifteenminutes and a final cooling step at 4°C for five minutes.
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.
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.
Our study established that twenty-eight (23%) of NTM are resistant 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 drug-resistant isolates originated from the Lake Victoria, Coastal, and Nairobi regions, with six (5%) NTM showing mutations in the rrl, rrs, or rpoB genes (p=0.012). 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 twenty one (76.5%) of the 28 drug-resistant NTM identified, indicating a strong statistical significance (p=0.000). The majority of drug-resistant NTM isolates 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. Twelve (10.4%) of NTM were resistant to macrolides (Table 2) with seven (58.3%) of RGM (Table 3) and five (41.7%) of SGM (Table 3) showing mutations within rrl gene. Point mutation at position 2058 was seen in M.intracellulare, M. abscessus subsp abscessus, M. nebraskense, M. massiliense, M. kumamototense, M. heraklionense, and M. bourgelatii. Only for M. abscessus subsp abscessus was a mutation at 2059 observed (Table 3).
Resistance to aminoglycosides was demonstrated by mutation at position A1408G of the rrs gene for six (11.1%) of the 54 RGM. The NTM with aminoglycoside resistance 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.
Isolate ID | NTM species | Position of mutation in rrs | X2, 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 rifampicin resistance in SGM was observed. Mutations within codons between 503-533 of the rpoB were seen for ten (14.7%) SGM. These SGM included M. avium subsp avium, M. intracellulare, M. yongonense and M. gastri with mutations at codons 506, 509, 516, 526, and 531 respectively (Table 5).
Nontuberculous 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 are resistant to at least one of the antibiotics in the macrolide-based therapy. Regional distribution of drug resistant NTM had a significant correlation (p=0.012) with the bulk of drug-resistant isolates originating from the Lake Victoria (n=6, 5%), Coastal (n=6, 5%), and Nairobi (n=6, 5%) regions. The observed regional diversity in drug resistant NTM 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 while males comprised the majority (n=21, 76.5%) of the drug-resistant NTM identified. NTM drug resistance was 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., 2022).
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). Our study demonstrated a similar pattern where M. abscessus subsp abscessus (A2058G/C, A2059G) and M. intracellulare (A2058G) formed the majority of isolates (58.3%) with macrolide resistance. Other NTM presenting with high levels of resistance to macrolides were M. nebraskense (A2058C), M. massiliense (A2058T), M. kumamototense(A2058T), M. heraklionense (A2058T), and M. bourgelatii (A2058C). The increased potential for development of drug resistance 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 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 aminoglycoside resistance with A1408G mutation. Genotypic characteristics in rrs that indicate 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-GarcÃa 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-GarcÃa et al., 2019; Tarashi et al., 2022; Zaragoza Bastida et al., 2017).
Similar to M. tuberculosis, resistance to rifampicin in NTM is mediated by 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 ten (11.6%) rifampicin-resistant 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 drug resistance 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.
We demonstrated a significant level of 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 macrolide resistance, and most macrolide-resistant NTM harbored mutation at position 2058. Majority of RGM had mutation at position 1408 of the rrs gene, while rifampicin resistant SGM had mutations within the RRDR of the rpoB gene.
This study investigated the acquired mechanisms of drug resistance for NTM. Other intrinsic factors apart from drug target gene mutation could influence sensitivity of NTM to antibiotics.
Despite this limitation, the study documents drug resistance mutation patterns of Kenyan NTM for the first time, and advocates for drug resistance testing before commencement of treatment for NTM infection.
This study was approved by Kenyatta National Hospital-University of Nairobi Ethics Review Committee (Ref: KNH-ERC/A/38) on 30th 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.
Figshare. Mutation Patterns of Resistance Genes for Macrolides, Aminoglycosides, and Rifampicin in Nontuberculous Mycobacteria Isolates from Kenya. DOI: https://doi.org/10.6084/m9.figshare.20331378.v2.
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).
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
We are grateful to the management of the National Public Health Laboratories- Kenya for granting us permission to access the National Tuberculosis Reference Laboratory and carry out this research. Much appreciation to the laboratory staff at the National Tuberculosis Reference.
Laboratory for their technical support during the collection and initial analysis of sputum samples for this study.
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Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Infectious diseases with special reference to tuberculosis and leprosy, Cell Biology, Microbiology, Immunology and Biotechnology.
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Are the conclusions drawn adequately supported by the results?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Infectious diseases and antibiotic resistance
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