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Emergence of mixed infection of Beijing/Non-Beijing strains among multi-drug resistant Mycobacterium tuberculosis in Pakistan

View Article: PubMed Central - PubMed

ABSTRACT

Tuberculosis (TB) remains as one of the deadliest diseases after HIV globally with 95 % of deaths confined to low-and-middle income countries. Pakistan is fifth among the 22 high-burden TB countries with the incidence rate of 230/100,000 persons, however, studies related to prevalent Mycobacterium tuberculosis strains and their spread, drug resistance pattern and evolutionary genetics are inadequate. The present study was undertaken to highlight the circulation of M. tuberculosis strains causing drug resistant TB in our community by targeting the molecular marker IS6110 and then characterization of these strains as Beijing and Non-Beijing genotypes. Sputum samples from 102 MDR TB suspects from different cities of Punjab were collected and their record was stored in a database. Sputum samples were evaluated by Ziehl Neelson staining and cultured on Lownstein Jensen medium by Modified Petroff’s method. DST was performed for first-line anti-mycobacterial drugs by indirect proportion method. Mycobacterium tuberculosis isolates were investigated for the presence of IS6110 and further identification as Beijing, Non-Beijing or mixed genotype. Percentage of male and female patients was found to be 58.8 and 41.2 % respectively. DST showed resistance of 93 % of isolates to isoniazid and rifampicin. All of the isolates showed positive results for IS6110 amplification. Based on PCR amplification of Beijing and non-Beijing primer sets 4.9 % of the patients showed infection with pure Beijing isolates, 14.7 % with both Beijing and non-Beijing isolates and 80.3 % with pure non-Beijing isolates. Analysis of IS6110 and Beijing sequences showed the presence of putative transposase conserved domain while non-Beijing sequences were epitomized with RAMP_I_III superfamily domain (CRISPR-associated protein family). TB in Pakistan is predominantly caused by Non-Beijing genotypes, but Beijing strains showed incessant circulation in our community as both single and mixed (co-infecting Non-Beijing and Beijing) strains.

No MeSH data available.


Related in: MedlinePlus

Comparison of genetic clusters on the basis of translated protein sequences.  a Aligned Non-Beijingsequences cluster 1 (Non-Beijing 203, 58, 56, 12) is indicated with blue arrows; cluster 2 (Non-Beijing 6, 55) isindicated with red arrows; cluster 3 (Non-Beijing 182, 65, 69, 48) is indicated with orange arrows. b *Phylogram ofthe M. tuberculosis sequences on the basis of non-Beijing specific sequences. *The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 4.40932938 is shown. The percentage of replicate trees in which the associated taxa clustered together inthe bootstrap test (100 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 14 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 137 positions in the final dataset. Evolutionary analyses were conducted in MEGA6
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Fig5: Comparison of genetic clusters on the basis of translated protein sequences. a Aligned Non-Beijingsequences cluster 1 (Non-Beijing 203, 58, 56, 12) is indicated with blue arrows; cluster 2 (Non-Beijing 6, 55) isindicated with red arrows; cluster 3 (Non-Beijing 182, 65, 69, 48) is indicated with orange arrows. b *Phylogram ofthe M. tuberculosis sequences on the basis of non-Beijing specific sequences. *The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 4.40932938 is shown. The percentage of replicate trees in which the associated taxa clustered together inthe bootstrap test (100 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 14 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 137 positions in the final dataset. Evolutionary analyses were conducted in MEGA6

Mentions: BLAST results based on IS6110 showed 100 % sequence similarity with putative transposases of M. tuberculosis isolates. Analysis of Beijing sequences with NCBI BLAST showed 98 % sequence similarity with transposase of M. tuberculosis Beijing-like strain (GenBank: CP010873.1) while non-Beijing sequences showed 99 % sequence similarity with CRISPR-associated protein Csm5 of M. tuberculosis. 3 of the non-Beijing sequences have been submitted to the Genbank and their accession numbers are KR362602 (Non-Beijing 55), KR362601 (Non-Beijing 65) and KR362600 (Non-Beijing 69). Phylogenetic analysis revealed similarity with reference strains as well as among the strains themselves (Figs. 3, 4, 5).Fig. 3


Emergence of mixed infection of Beijing/Non-Beijing strains among multi-drug resistant Mycobacterium tuberculosis in Pakistan
Comparison of genetic clusters on the basis of translated protein sequences.  a Aligned Non-Beijingsequences cluster 1 (Non-Beijing 203, 58, 56, 12) is indicated with blue arrows; cluster 2 (Non-Beijing 6, 55) isindicated with red arrows; cluster 3 (Non-Beijing 182, 65, 69, 48) is indicated with orange arrows. b *Phylogram ofthe M. tuberculosis sequences on the basis of non-Beijing specific sequences. *The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 4.40932938 is shown. The percentage of replicate trees in which the associated taxa clustered together inthe bootstrap test (100 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 14 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 137 positions in the final dataset. Evolutionary analyses were conducted in MEGA6
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4837763&req=5

Fig5: Comparison of genetic clusters on the basis of translated protein sequences. a Aligned Non-Beijingsequences cluster 1 (Non-Beijing 203, 58, 56, 12) is indicated with blue arrows; cluster 2 (Non-Beijing 6, 55) isindicated with red arrows; cluster 3 (Non-Beijing 182, 65, 69, 48) is indicated with orange arrows. b *Phylogram ofthe M. tuberculosis sequences on the basis of non-Beijing specific sequences. *The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 4.40932938 is shown. The percentage of replicate trees in which the associated taxa clustered together inthe bootstrap test (100 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 14 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 137 positions in the final dataset. Evolutionary analyses were conducted in MEGA6
Mentions: BLAST results based on IS6110 showed 100 % sequence similarity with putative transposases of M. tuberculosis isolates. Analysis of Beijing sequences with NCBI BLAST showed 98 % sequence similarity with transposase of M. tuberculosis Beijing-like strain (GenBank: CP010873.1) while non-Beijing sequences showed 99 % sequence similarity with CRISPR-associated protein Csm5 of M. tuberculosis. 3 of the non-Beijing sequences have been submitted to the Genbank and their accession numbers are KR362602 (Non-Beijing 55), KR362601 (Non-Beijing 65) and KR362600 (Non-Beijing 69). Phylogenetic analysis revealed similarity with reference strains as well as among the strains themselves (Figs. 3, 4, 5).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Tuberculosis (TB) remains as one of the deadliest diseases after HIV globally with 95 % of deaths confined to low-and-middle income countries. Pakistan is fifth among the 22 high-burden TB countries with the incidence rate of 230/100,000 persons, however, studies related to prevalent Mycobacterium tuberculosis strains and their spread, drug resistance pattern and evolutionary genetics are inadequate. The present study was undertaken to highlight the circulation of M. tuberculosis strains causing drug resistant TB in our community by targeting the molecular marker IS6110 and then characterization of these strains as Beijing and Non-Beijing genotypes. Sputum samples from 102 MDR TB suspects from different cities of Punjab were collected and their record was stored in a database. Sputum samples were evaluated by Ziehl Neelson staining and cultured on Lownstein Jensen medium by Modified Petroff’s method. DST was performed for first-line anti-mycobacterial drugs by indirect proportion method. Mycobacterium tuberculosis isolates were investigated for the presence of IS6110 and further identification as Beijing, Non-Beijing or mixed genotype. Percentage of male and female patients was found to be 58.8 and 41.2 % respectively. DST showed resistance of 93 % of isolates to isoniazid and rifampicin. All of the isolates showed positive results for IS6110 amplification. Based on PCR amplification of Beijing and non-Beijing primer sets 4.9 % of the patients showed infection with pure Beijing isolates, 14.7 % with both Beijing and non-Beijing isolates and 80.3 % with pure non-Beijing isolates. Analysis of IS6110 and Beijing sequences showed the presence of putative transposase conserved domain while non-Beijing sequences were epitomized with RAMP_I_III superfamily domain (CRISPR-associated protein family). TB in Pakistan is predominantly caused by Non-Beijing genotypes, but Beijing strains showed incessant circulation in our community as both single and mixed (co-infecting Non-Beijing and Beijing) strains.

No MeSH data available.


Related in: MedlinePlus