Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia
Abstract
:1. Introduction
2. Results
2.1. Study Samples
2.2. Resistance Profiles
2.3. Genotypes, Phylogeny and Clustering
Phylogenetic Analysis of Global Strains Belonging to Sub-Lineages L4.3.4.1 and L4.3.4.2.1
3. Discussion
4. Materials and Methods
4.1. Sample Collection
4.2. Drug Susceptibility Testing and DNA Extraction
4.3. Targeted Sequencing of Drug-Resistance Associated Genes
4.4. Whole Genome Sequencing
- Library preparation and sequencing
- b.
- Data Analysis: Clustering, phylogenetic analysis and resistance patterns
- c.
- Phylogenetic analysis of global strains belonging to sub-lineages L4.3.4.1 and L4.3.4.2.1
4.5. Drug Susceptibility Testing to Second Line Drugs
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Mutations | Number | Percentage (%) | ||
---|---|---|---|---|
Rifampicin | Asp435Val a | 8 | 13 | |
His445Asp a | 7 | 11 | ||
Ser450Leu a | 34 | 54 | ||
Rifampicin mutation only (His445Tyr a) | 2 | 3 | ||
No mutation | 1 | 2 | ||
Other rifampicin resistance mutations | 11 | 17 | ||
Isoniazid | Ser315Thr b | 60 | 95 | |
15 C>T c | 1 | 2 | ||
No mutation | 2 | 3 | ||
MDR | Asp435Tyr a, Ser315Thr b | 2 | 3 | |
Asp435Val a, Ser315Thr b | 8 | 13 | ||
His445Asp a, Ser315Thr b | 7 | 11 | ||
Ser450Leu a, Ser315Thr b | 30 | 48 | ||
Other MDR mutation combinations | 9 | 14 | ||
Others (mono and polyresistant) | 3 | 5 | ||
Ser450Leu a, Ser315Thr b, Asp94His d | 1 | 2 | ||
Pre-XDR | Ser450Leu a, Ser315Thr b, Asp94Gly d | 1 | 2 | |
His445Leu a, Ser315Thr b, Asp94Gly d | 2 | 3 | ||
Polyresistant | 15 C>T c, Gly88Cys d | 1 | 2 | |
Non polyresistant | 62 | 98 |
Drug Susceptibility Pattern | Gene Mutations | Treatment | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
ID | Age | Sex | I | R | E | S | Bdq | Lfx | Mfx | Cfz | Phenotype | gryA | rpoB | katG | inh_promoter | pncA | embB | rpsL | ethA | folC | Sublineage | |||||
A | 41 | M | X | √ | √ | √ | √ | X | X | √ | poly-resistant | G88C | - | - | 15C>T | R154G | - | - | 15C > T | - | L4.3.4.2.1 | Lfx | I | R | E | Z |
B | 29 | M | X | X | √ | √ | √ | X | X | √ | pre-XDR | D94H | S450L | S315T | - | - | M306I | - | - | - | L4.1.2 | Lfx | Bdq | Lzd | Cs | - |
C | 48 | F | X | X | √ | √ | √ | X | X | √ | pre-XDR | D94G | S450L | S315T | - | R154G | Y319S | K43R | 1412_1413insGG | E40G | L4.3.4.2.1 | - | - | - | - | - |
D | 24 | F | X | X | √ | √ | √ | X | X | √ | pre-XDR | D94G | H445L | S315T | - | - | M306V | - | - | - | L4.3.4.2.1 | Lfx | Km | Eto | Cs | - |
E | - | - | - | - | - | - | - | - | - | - | * pre-XDR | D94G | H445L | S315T | - | - | M306V | - | - | - | L4.3.4.2.1 | - | - | - | - | - |
F | 42 | M | X | X | X | X | - | - | - | - | MDR | - | S450L | S315T | - | H43P | M306V | - | 1215_1224del | - | L4.6.1.2 | Lfx | Km | Eto | Cs | Z |
G | 27 | F | X | X | √ | √ | - | - | - | - | MDR | - | S450L | S315T | - | - | - | - | T61M | - | L4.3.4.2.1 | - | - | - | - | - |
Drug | Mutations | Number | Percentage (%) | |
---|---|---|---|---|
Non-MDR | Polyresistant | Gly88Cys, 15 C>T, Arg154Gly | 1 | 2 |
Pyrazinamide (Z) | Arg154Gly | 1 | 2 | |
Trp119Cys | 1 | 2 | ||
His43Pro | 1 | 2 | ||
Val128Gly | 1 | 2 | ||
MDR plus | Other pyrazinamide mutations | 6 | 12 | |
No mutations | 40 | 80 | ||
Ethambutol (E) | Met306Val | 2 | 4 | |
Met306Val | 4 | 8 | ||
Met306Ile | 3 | 6 | ||
Tyr319Ser | 3 | 6 | ||
Other ethambutol mutations | 8 | 16 | ||
No mutations | 30 | 60 | ||
Streptomycin (S) | Lys88Arg | 6 | 12 | |
Other streptomycin mutations | 9 | 18 | ||
No mutations | 35 | 70 | ||
Ethionamide (Eto) | Thr61Met | 1 | 2 | |
Ser450Leu, Ser315Thr, 15C>T | 1 | 2 | ||
Other ethionamide mutations | 2 | 4 | ||
No mutations | 46 | 92 | ||
Para-amino salicylic acid (PAS) | Glu40Gly | 1 | 2 | |
No mutations | 49 | 98 |
Gene | Primer Set | |
---|---|---|
rpoB | Foward | CAGGACGTGGAGGCGATCAC |
Reverse | GAGCCGATCAGACCGATGTTGG | |
katG | Foward | ATGGCCATGAACGACGTCGAAAC |
Reverse | CGCAGCGAGAGGTCAGTGGCCAG | |
inhA | Foward | TCACACCGACAAACGTCACGAGC |
Reverse | AGCCAGCCGCTGTGCGATCGCCA | |
gyrA | Foward | AGCGCAGCTACATCGACTATGCG |
Reverse | CTTCGGTGTACCTCATCGCCGCC | |
gryB | Foward | CGGCACGTAAGGCACGAGAG |
Reverse | GAACCGGAACAACAACGTCAAC | |
rrs | Foward | CGGATCGGGGTCTGCAACTCGAC |
Reverse | CAAGAACCCCTCACGGCCTACG |
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Chizimu, J.Y.; Solo, E.S.; Bwalya, P.; Kapalamula, T.F.; Mwale, K.K.; Squarre, D.; Shawa, M.; Lungu, P.; Barnes, D.A.; Yamba, K.; et al. Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia. Antibiotics 2023, 12, 1126. https://doi.org/10.3390/antibiotics12071126
Chizimu JY, Solo ES, Bwalya P, Kapalamula TF, Mwale KK, Squarre D, Shawa M, Lungu P, Barnes DA, Yamba K, et al. Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia. Antibiotics. 2023; 12(7):1126. https://doi.org/10.3390/antibiotics12071126
Chicago/Turabian StyleChizimu, Joseph Yamweka, Eddie Samuneti Solo, Precious Bwalya, Thoko Flav Kapalamula, Kaemba Kunkuta Mwale, David Squarre, Misheck Shawa, Patrick Lungu, David Atomanyi Barnes, Kaunda Yamba, and et al. 2023. "Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia" Antibiotics 12, no. 7: 1126. https://doi.org/10.3390/antibiotics12071126
APA StyleChizimu, J. Y., Solo, E. S., Bwalya, P., Kapalamula, T. F., Mwale, K. K., Squarre, D., Shawa, M., Lungu, P., Barnes, D. A., Yamba, K., Mufune, T., Chambaro, H., Kamboyi, H., Munyeme, M., Hang’ombe, B. M., Kapata, N., Mukonka, V., Chilengi, R., Thapa, J., ... Suzuki, Y. (2023). Genomic Analysis of Mycobacterium tuberculosis Strains Resistant to Second-Line Anti-Tuberculosis Drugs in Lusaka, Zambia. Antibiotics, 12(7), 1126. https://doi.org/10.3390/antibiotics12071126