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

Zakayo Mwangi; Grace Naeku; Marianne Mureithi; Frank Onyambu; Wallace Bulimo

doi:10.12688/f1000research.124002.4

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Research Article

Revised

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

[version 4; peer review: 1 approved, 1 approved with reservations, 1 not approved]

Zakayo Mwangi^1,2, Grace Naeku³, Marianne Mureithi², Frank Onyambu^1,4, Wallace Bulimo⁵

Zakayo Mwangi^1,2, Grace Naeku³, [...] Marianne Mureithi², Frank Onyambu^1,4, Wallace Bulimo⁵

PUBLISHED 20 Feb 2023

Author details Author details

¹ Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, Meru, 972, Kenya
² Department of Medical Microbiology and Immunology, University of Nairobi, Nairobi, Nairobi, 30197, Kenya
³ Molecular and Infectious Disease Research Laboratory, University of Nairobi, Nairobi, Nairobi, 30197, Kenya
⁴ Centre for Molecular Biosciences and Genomics, Centre for Molecular Biosciences and Genomics, Nairobi, Nairobi, 29408, Kenya
⁵ Kenya Medical Research Institute, Kenya Medical Research Institute, Nairobi, Nairobi, 54840, Kenya

Zakayo Mwangi
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Grace Naeku
Roles: Investigation, Writing – Review & Editing

Marianne Mureithi
Roles: Supervision, Writing – Review & Editing

Frank Onyambu
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Wallace Bulimo
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Cell & Molecular Biology gateway.

Abstract

Background: Nontuberculous 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: Twenty-eight (23%) of the NTM harbored mutations associated with resistance to at least one of the macrolide-based therapy antibiotics. Twelve (10.4%) NTM comprising 7(58.3%) of RGM and 5(41.7%) of SGM had mutations in the rrl gene.
For ten (83.3%) NTM, mutations were found at position 2058, while for two (16.6%) NTM, mutations were found at position 2059. Six (11.1%) of the 54 RGM exhibited mutations in the aminoglycoside target gene rrs at location 1408. Ten (14.7%) of the 68 SGM harbored mutations in the rpoB gene with 40 percent having mutations at codon 531.
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.

Keywords

Nontuberculous mycobacteria, slow-growing mycobacteria, rapid-growing mycobacteria, macrolides, aminoglycosides, rifampicin

Corresponding author: Zakayo Mwangi

Competing interests: No competing interests were disclosed.

Grant information: 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.

Copyright: © 2023 Mwangi Z et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Mwangi Z, Naeku G, Mureithi M et al. Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in nontuberculous mycobacteria isolates from Kenya [version 4; peer review: 1 approved, 1 approved with reservations, 1 not approved]. F1000Research 2023, 11:962 (https://doi.org/10.12688/f1000research.124002.4) First published: 19 Aug 2022, 11:962 (https://doi.org/10.12688/f1000research.124002.1) Latest published: 03 Apr 2023, 11:962 (https://doi.org/10.12688/f1000research.124002.5)

Revised Amendments from Version 3

Positive control has been included in the methods section.

See the authors' detailed response to the review by Leena Al-Hassan
See the authors' detailed response to the review by Utpal Sengupta
See the authors' detailed response to the review by Clement Likhovole

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, 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 the best treatment plan (Chalmers et al., 2019; Mwangi et al., 2022a).

NTM treatment consists of a macrolide-based antibiotic regimen, such as clarithromycin or azithromycin, combined with other selected antibiotics that synergise 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 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 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 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 NTM isolates included 54 RGM and 68 SGM. 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).

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.

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 twenty-eight (23%) of NTM harbored mutations associated with resistance to at least one of the macrolide-based therapy antibiotics. 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 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 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
Variables	NTM (n, %)	rrl (n, %)	rrs (n, %)	rpoB (n, %)	P value
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. Twelve (10.4%) of NTM comprising (Table 2) seven (58.3%) of RGM (Table 3) and five (41.7%) of SGM (Table 3) had mutations within the 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).

Table 3. NTM showing mutations associated with macrolide resistance.

Isolate ID	NTM species	Growth rate	Position of mutation in rrl	X², 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

Mutation at position A1408G of the rrs gene was seen for six (11.1%) of the 54 RGM. 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	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 rpoB mutation 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).

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

Isolate ID	NTM species	Codons with mutation in rpoB	X², 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

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 harbored mutations associated with resistance to at least one of the macrolide-based therapy antibiotics. 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 in NTM harboring mutations in drug target genes 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. Drug resistance associated mutations 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., 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 in NTM (Huh et al., 2019). 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 mutations in the rrl gene. Other NTM presenting with mutations associated with macrolides resistance were M. nebraskense (A2058C), M. massiliense (A2058T), M. kumamototense (A2058T), M. heraklionense (A2058T), and M. bourgelatii (A2058C). 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 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).

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 ten (11.6%) 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 Nontuberculous 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

Acknowledgments

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 study's collection and initial analysis of sputum samples.

References

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Version 5

VERSION 5 PUBLISHED 19 Aug 2022

Author details Author details

Zakayo Mwangi
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Grace Naeku
Roles: Investigation, Writing – Review & Editing

Marianne Mureithi
Roles: Supervision, Writing – Review & Editing

Frank Onyambu
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Wallace Bulimo
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

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.

Article Versions (5)

version 5

Revised

Published: 03 Apr 2023, 11:962

https://doi.org/10.12688/f1000research.124002.5

version 4

Revised

Published: 20 Feb 2023, 11:962

https://doi.org/10.12688/f1000research.124002.4

version 3

Revised

Published: 05 Dec 2022, 11:962

https://doi.org/10.12688/f1000research.124002.3

version 2

Revised

Published: 24 Oct 2022, 11:962

https://doi.org/10.12688/f1000research.124002.2

version 1

Published: 19 Aug 2022, 11:962

https://doi.org/10.12688/f1000research.124002.1

© 2023 Mwangi Z et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Mwangi Z, Naeku G, Mureithi M et al. Mutation patterns of resistance genes for macrolides, aminoglycosides, and rifampicin in nontuberculous mycobacteria isolates from Kenya [version 4; peer review: 1 approved, 1 approved with reservations, 1 not approved]. F1000Research 2023, 11:962 (https://doi.org/10.12688/f1000research.124002.4)

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Open Peer Review

Current Reviewer Status: ?

Key to Reviewer Statuses VIEW HIDE

ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 4

VERSION 4

PUBLISHED 20 Feb 2023

Revised

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Reviewer Report 24 Mar 2023

Clement Likhovole, Department of Microbiology, UZIMA University, Kisumu, Kenya

Approved with Reservations

https://doi.org/10.5256/f1000research.144441.r167243

General comments

The manuscript addresses an important area in Tuberculosis research particularly analysis of drug resistant mutations that have been linked to treatment failure.

Abstract:

The authors to include a

General comments

The manuscript addresses an important area in Tuberculosis research particularly analysis of drug resistant mutations that have been linked to treatment failure.

Abstract:

The authors to include a hyphen in Non-tuberculous Mycobacterium (NTM).
Use upper case for the first letters of the following acronyms, SGM and RGM.
In the results section, percentages should not be in brackets. The proportions to indicated in brackets e.g. 23% (23/100). This should be done all the data presented in this section. When reporting on the mutations, also indicate the amino acid or nucleotide change as well. For example, which mutation is present on codon 2058? Change from what to what?

Introduction:

Second sentence, the authors to describe in detail the type of culture used to grow NTMs for 40 days. MGIT?
Second paragraph, authors to indicate the duration of treatment of NTM infections. Also the length of treatment with Rifampicin to be mentioned. Is it the same for patients infected with comorbidities?
The authors have listed several drugs used for the treatment of NTMs but described only one resistance mechanisms for NTMs, do they all have the same type of resistance mechanism? (Macrolides - Clarithromycon and Azithromycin).
Paragraph seven, the authors to indicate that Rifampicin is recommended for first-line treatment of MTB alongside other drugs.

Laboratory procedures:

The authors to estimate the volume of sputum sample collected from each participant.
For the FM microscopy, how many readers were used before reporting the smear results?
What volume of DNA was finally collected after extraction? Was quantification of the DNA material done?

Results:

The authors to mention the table with results indicating that the bulk of the samples were obtained from Lake Victoria, Coastal and Nairobi region.

Discussion:

The authors to refer to my previous comments on Non-tuberculous mycobacterium.
The authors to critically discuss the relevance of the rrl significant mutations reported in the study sites. What are the implications of these finding?

Is the work clearly and accurately presented and does it cite the current literature?

Yes
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: TB molecular biology and immunology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 29 Mar 2023

Zakayo Mwangi, Meru University of Science and Technology, Kenya

29 Mar 2023

Author Response

Thank you for your valuable feedback.
Competing Interests: No competing interests were disclosed.
Thank you for your valuable feedback.
Thank you for your valuable feedback.
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 29 Mar 2023

Zakayo Mwangi, Meru University of Science and Technology, Kenya

29 Mar 2023

Author Response

Thank you for your valuable feedback.
Competing Interests: No competing interests were disclosed.
Thank you for your valuable feedback.
Thank you for your valuable feedback.
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 27 Feb 2023

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

Not Approved

https://doi.org/10.5256/f1000research.144441.r163993

No further comments. Please see previous comments ... Continue reading

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Version 3

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PUBLISHED 05 Dec 2022

Revised

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Reviewer Report 05 Dec 2022

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

Not Approved

https://doi.org/10.5256/f1000research.141647.r157361

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Author Response 15 Dec 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

15 Dec 2022

Author Response

Thank you for the feedback.
Competing Interests: No competing interests were disclosed.
Thank you for the feedback.
Thank you for the feedback.
Competing Interests: No competing interests were disclosed. Close
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Author Response 15 Dec 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

15 Dec 2022

Author Response

Thank you for the feedback.
Competing Interests: No competing interests were disclosed.
Thank you for the feedback.
Thank you for the feedback.
Competing Interests: No competing interests were disclosed. Close
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Version 2

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PUBLISHED 24 Oct 2022

Revised

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Reviewer Report 22 Nov 2022

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

Not Approved

https://doi.org/10.5256/f1000research.139953.r154017

Thank you for revising the manuscript and incorporating the comments into the text. However I would highly suggest that you revise the sections where you correlate the genotype with phenotype, since you have not done the AST (MIC) in this study. If you have not done the AST testing, you cannot state that they are resistant. Your study is primarily genotype based, and you have detected mutations in target resistance determinants, and I therefore suggest you change the wording of the results and discussion to reflect that. These mutations may be silent mutations, and if you do not have phenotype data to support it, you cannot state the fact that they are resistant. You can only say that they harbour the mutations which in previous reports have led to resistance.

These statements "Twenty-eight (23%) of the NTM were resistant to at least one of the antibiotics used in the macrolide-based treatment " and "Ten (14.7%) of the 68 SGM were resistant to rifampicin, with 40 percent having mutations at codon 531 in the rpoB gene" cannot be proven without AST. You should re-word it to say that 23% harboured mutations associated with resistance to macrolide antibiotics.

Same for the conclusion: you have not demonstrated a significant level of drug resistance for macrolides, aminoglycosides and rifampicin in NTM - you have detected mutations in gene targets that have previously been associated with drug resistance.

I hope my point is clear. I am not doubting your results, and I think it's interesting that you have detected these mutations, however it's not scientifically sound to assume resistance based on genotype only, and you must phrase your results and conclusions according to the methods used and data generated. The way it is written now indicates that you have the AST data for the isolates and you are sure that they are rifampicin or aminoglycoside resistant. I would strongly recommend you perform the AST - even for a subset of isolates - to confirm that the observed genotype correlates with the phenotype.

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Infectious diseases and antibiotic resistance

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Author Response 05 Dec 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

05 Dec 2022

Author Response

I appreciate your re-evaluation and the way in which you made the issues that need to be addressed evident.

I revised the paper in accordance with your advice and ... Continue reading I appreciate your re-evaluation and the way in which you made the issues that need to be addressed evident.

I revised the paper in accordance with your advice and resubmitted it for review.
I appreciate your re-evaluation and the way in which you made the issues that need to be addressed evident.

I revised the paper in accordance with your advice and resubmitted it for review.
Competing Interests: No competing interests were disclosed. Close
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Author Response 05 Dec 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

05 Dec 2022

Author Response

I appreciate your re-evaluation and the way in which you made the issues that need to be addressed evident.

I revised the paper in accordance with your advice and ... Continue reading I appreciate your re-evaluation and the way in which you made the issues that need to be addressed evident.

I revised the paper in accordance with your advice and resubmitted it for review.
I appreciate your re-evaluation and the way in which you made the issues that need to be addressed evident.

I revised the paper in accordance with your advice and resubmitted it for review.
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 03 Nov 2022

Utpal Sengupta, Stanley Browne Laboratory, The Leprosy Mission Community Hospital, New Delhi, Delhi, India

Approved

https://doi.org/10.5256/f1000research.139953.r154016

The manuscript can ... Continue reading

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Author Response 05 Dec 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

05 Dec 2022

Author Response

We sincerely appreciate your second reading and approval.
Competing Interests: No competing interests were disclosed.
We sincerely appreciate your second reading and approval.
We sincerely appreciate your second reading and approval.
Competing Interests: No competing interests were disclosed. Close
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Author Response 05 Dec 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

05 Dec 2022

Author Response

We sincerely appreciate your second reading and approval.
Competing Interests: No competing interests were disclosed.
We sincerely appreciate your second reading and approval.
We sincerely appreciate your second reading and approval.
Competing Interests: No competing interests were disclosed. Close
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Version 1

VERSION 1

PUBLISHED 19 Aug 2022

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Reviewer Report 10 Oct 2022

Utpal Sengupta, Stanley Browne Laboratory, The Leprosy Mission Community Hospital, New Delhi, Delhi, India

Approved with Reservations

https://doi.org/10.5256/f1000research.136168.r150334

The manuscript has been written on the findings of antimycobacterial resistance of NTM isolated from 122 sputum M. tuberculosis-negative patients. The authors have investigated their resistance patterns on the isolated strains using gene sequencing of the drug-resistant genes and noted the mutations in the respective codon positions.

Materials and methods have been well described. Data were properly analyzed and presented adequately in the tables. However, there were a lot of discrepancies in Table 2 in the first column where the total sample number was shown to be 135 instead of 122 and therefore the percentage expressions were wrongly mentioned in the table and in the text. The percentages have to be calculated correctly and Table 2 has to be modified and described accordingly in the text.

The discussion has been adequate, citing adequate references. However, the discussion with respect to the results of Table 2 has to be modified and discussed for a correct understanding of the reader.

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.

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Author Response 24 Oct 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

24 Oct 2022

Author Response

I appreciate your review. The issue mentioned above has been addressed.
Competing Interests: No competing interests were disclosed.
I appreciate your review. The issue mentioned above has been addressed.
I appreciate your review. The issue mentioned above has been addressed.
Competing Interests: No competing interests were disclosed. Close
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Author Response 24 Oct 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

24 Oct 2022

Author Response

I appreciate your review. The issue mentioned above has been addressed.
Competing Interests: No competing interests were disclosed.
I appreciate your review. The issue mentioned above has been addressed.
I appreciate your review. The issue mentioned above has been addressed.
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 22 Sep 2022

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

Approved with Reservations

https://doi.org/10.5256/f1000research.136168.r148288

This study characterises mutation patterns for resistance genes in NTM from Kenya.

Although the study is interesting, I think the authors need to give more information in the methodology and results section on:

Identification: 122 isolates were collected, but no details in the results on how many were slow-growing and rapid-growing NTM.
MIC: No mention of methodology for MIC and results.
The PCR: Did you include any positive or negative controls in the experiments?
The discussion on mutations and their role in resistance is quite vague. It's not clear whether the mutations identified can be linked to the observed MIC. Have the researchers checked that these are not silent mutations?

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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Author Response 06 Oct 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

06 Oct 2022

Author Response
I appreciate your review.
- Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.
... Continue reading
I appreciate your review.

Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.

The phenotypic drug sensitivity by MIC analysis was not done in this study. We performed genotypic characterization of the drug target genes by describing the mutations associated with drug resistance through target sequencing of the drug target genes.

Positive and negative controls were included during PCR and subsequent sequencing of respective target genes. The controls were based on Genotype NTM-DR results.

In the discussion, the mutations identified in this study have been compared to those seen in previous studies that had also included MIC in their analysis. The point mutations identified in the drug target genes were seen to be associated with phenotypic drug resistance.
I appreciate your review.

Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.

The phenotypic drug sensitivity by MIC analysis was not done in this study. We performed genotypic characterization of the drug target genes by describing the mutations associated with drug resistance through target sequencing of the drug target genes.

Positive and negative controls were included during PCR and subsequent sequencing of respective target genes. The controls were based on Genotype NTM-DR results.

In the discussion, the mutations identified in this study have been compared to those seen in previous studies that had also included MIC in their analysis. The point mutations identified in the drug target genes were seen to be associated with phenotypic drug resistance.
Competing Interests: No competing interests Close
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Author Response 06 Oct 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

06 Oct 2022

Author Response
I appreciate your review.
- Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.
... Continue reading
I appreciate your review.

Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.

The phenotypic drug sensitivity by MIC analysis was not done in this study. We performed genotypic characterization of the drug target genes by describing the mutations associated with drug resistance through target sequencing of the drug target genes.

Positive and negative controls were included during PCR and subsequent sequencing of respective target genes. The controls were based on Genotype NTM-DR results.

In the discussion, the mutations identified in this study have been compared to those seen in previous studies that had also included MIC in their analysis. The point mutations identified in the drug target genes were seen to be associated with phenotypic drug resistance.
I appreciate your review.

Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.

The phenotypic drug sensitivity by MIC analysis was not done in this study. We performed genotypic characterization of the drug target genes by describing the mutations associated with drug resistance through target sequencing of the drug target genes.

Positive and negative controls were included during PCR and subsequent sequencing of respective target genes. The controls were based on Genotype NTM-DR results.

In the discussion, the mutations identified in this study have been compared to those seen in previous studies that had also included MIC in their analysis. The point mutations identified in the drug target genes were seen to be associated with phenotypic drug resistance.
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Version 5

VERSION 5 PUBLISHED 19 Aug 2022

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Leena Al-Hassan, Brighton and Sussex Medical School, Brighton, UK
Utpal Sengupta, The Leprosy Mission Community Hospital, New Delhi, India
Clement Likhovole, UZIMA University, Kisumu, Kenya

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7 Views

04 Apr 2023 | for Version 5

Clement Likhovole, Department of Microbiology, UZIMA University, Kisumu, Kenya

7 Views Cite this report Responses(1)

Approved

The authors have revised the manuscript as per the recommended changes. The revised manuscript is adequate in form and content

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

TB molecular biology and immunology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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16 Views

24 Mar 2023 | for Version 4

Clement Likhovole, Department of Microbiology, UZIMA University, Kisumu, Kenya

16 Views Cite this report Responses(1)

Approved With Reservations

General comments

The manuscript addresses an important area in Tuberculosis research particularly analysis of drug resistant mutations that have been linked to treatment failure.

Abstract:

The authors to include a hyphen in Non-tuberculous Mycobacterium (NTM).
Use upper case for the first letters of the following acronyms, SGM and RGM.
In the results section, percentages should not be in brackets. The proportions to indicated in brackets e.g. 23% (23/100). This should be done all the data presented in this section. When reporting on the mutations, also indicate the amino acid or nucleotide change as well. For example, which mutation is present on codon 2058? Change from what to what?

Introduction:

Second sentence, the authors to describe in detail the type of culture used to grow NTMs for 40 days. MGIT?
Second paragraph, authors to indicate the duration of treatment of NTM infections. Also the length of treatment with Rifampicin to be mentioned. Is it the same for patients infected with comorbidities?
The authors have listed several drugs used for the treatment of NTMs but described only one resistance mechanisms for NTMs, do they all have the same type of resistance mechanism? (Macrolides - Clarithromycon and Azithromycin).
Paragraph seven, the authors to indicate that Rifampicin is recommended for first-line treatment of MTB alongside other drugs.

Laboratory procedures:

The authors to estimate the volume of sputum sample collected from each participant.
For the FM microscopy, how many readers were used before reporting the smear results?
What volume of DNA was finally collected after extraction? Was quantification of the DNA material done?

Results:

The authors to mention the table with results indicating that the bulk of the samples were obtained from Lake Victoria, Coastal and Nairobi region.

Discussion:

The authors to refer to my previous comments on Non-tuberculous mycobacterium.
The authors to critically discuss the relevance of the rrl significant mutations reported in the study sites. What are the implications of these finding?

Is the work clearly and accurately presented and does it cite the current literature?

Yes
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

TB molecular biology and immunology

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19 Views

27 Feb 2023 | for Version 4

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

19 Views Cite this report Responses(0)

Not Approved

No further comments. Please see previous comments about the need to correlate findings with MIC.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Infectious diseases and antibiotic resistance

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37 Views

05 Dec 2022 | for Version 3

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

37 Views Cite this report Responses(1)

Not Approved

I have read the revised version, but I still see that the manuscript has misleading information that needs to be re-written. The data provided need to associated with phenotypic data on resistance. Identification of mutations in genes is not sufficient to prove resistance, as the researchers have not confirmed any silent mutations.

The positive controls used in the methodology should be isolates that are confirmed resistant and harbour these mutations. This is the definition of a positive control. The negative control is an isolates that is genotypically confirmed having no mutations in the target genes.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Infectious diseases and antibiotic resistance

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29 Views

22 Nov 2022 | for Version 2

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

29 Views Cite this report Responses(1)

Not Approved

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Infectious diseases and antibiotic resistance

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Reviewer Report

24 Views

03 Nov 2022 | for Version 2

Utpal Sengupta, Stanley Browne Laboratory, The Leprosy Mission Community Hospital, New Delhi, Delhi, India

24 Views Cite this report Responses(1)

Approved

The manuscript can be accepted now.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Infectious diseases with special reference to tuberculosis and leprosy, Cell Biology, Microbiology, Immunology and Biotechnology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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18 Views

10 Oct 2022 | for Version 1

Utpal Sengupta, Stanley Browne Laboratory, The Leprosy Mission Community Hospital, New Delhi, Delhi, India

18 Views Cite this report Responses(1)

Approved With Reservations

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.

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Reviewer Report

54 Views

22 Sep 2022 | for Version 1

Leena Al-Hassan, Department of Global Health and Infection, Brighton and Sussex Medical School, Brighton, UK

54 Views Cite this report Responses(1)

Approved With Reservations

Identification: 122 isolates were collected, but no details in the results on how many were slow-growing and rapid-growing NTM.
MIC: No mention of methodology for MIC and results.
The PCR: Did you include any positive or negative controls in the experiments?
The discussion on mutations and their role in resistance is quite vague. It's not clear whether the mutations identified can be linked to the observed MIC. Have the researchers checked that these are not silent mutations?

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

Respond to this report

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Author Response

06 Oct 2022

Zakayo Mwangi, Department of Medical Laboratory Science, Meru University of Science and Technology, Meru, 972, Kenya

I appreciate your review.

Under methods, it has been indicated that of the 122 NTM isolated, 54 were rapid-growing NTM while 68 were slow-growing NTM.
The phenotypic drug sensitivity by MIC analysis was not done in this study. We performed genotypic characterization of the drug target genes by describing the mutations associated with drug resistance through target sequencing of the drug target genes.
Positive and negative controls were included during PCR and subsequent sequencing of respective target genes. The controls were based on Genotype NTM-DR results.
In the discussion, the mutations identified in this study have been compared to those seen in previous studies that had also included MIC in their analysis. The point mutations identified in the drug target genes were seen to be associated with phenotypic drug resistance.

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Competing Interests

No competing interests

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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

Abstract

Keywords

Revised Amendments from Version 3

Introduction

Methods

Laboratory procedures

Sample processing, mycobacterial culture and growth identification

DNA extraction

Conventional PCR, DNA gel electrophoresis and DNA purification

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

rrl, rpoB and rrs genes sequencing

Data analysis

Results

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

Table 3. NTM showing mutations associated with macrolide resistance.

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

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

Discussion

Conclusion

Limitations of the study

Ethical clearance

Data availability

Author contributions

Acknowledgments

References

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