Genomic analyses of the ancestral Manila family of <i>Mycobacterium tuberculosis</i>

<div><p>With its airborne transmission and prolonged latency period, <i>Mycobacterium tuberculosis</i> spreads worldwide as one of the most successful bacterial pathogens and continues to kill millions of people every year. <i>M</i>. <i>tuberculosis</i> lineage 1 is inferred to originate ancestrally based on the presence of the 52-bp TbD1 sequence and analysis of single nucleotide polymorphisms. Previously, we briefly reported the complete genome sequencing of <i>M</i>. <i>tuberculosis</i> strains 96121 and 96075, which belong to the ancient Manila family and modern Beijing family respectively. Here we present the comprehensive genomic analyses of the Manila family in lineage 1 compared to complete genomes in lineages 2–4. Principal component analysis of the presence and absence of CRISPR spacers suggests that Manila isolate 96121 is distinctly distant from lineages 2–4. We further identify a truncated <i>whiB5</i> gene and a putative operon consisting of genes encoding a putative serine/threonine kinase PknH and a putative ABC transporter, which are only found in the genomes of Manila family isolates. Six single nucleotide polymorphisms are uniquely conserved in 38 Manila strains. Moreover, when compared to <i>M</i>. <i>tuberculosis</i> H37Rv, 59 proteins are under positive selection in Manila family isolate 96121 but not in Beijing family isolate 96075. The unique features further serve as biomarkers for Manila strains and may shed light on the limited transmission of this ancestral lineage outside of its Filipino host population.</p></div

CRISPR spacer rearrangements in Manila family 96121, Beijing family 96075, and H37Rv.

<p>Green highlights spacers conserved with other MTBC species but lost in modern Beijing family isolate 96075 and H37Rv. Red highlights spacers specific in Manila family isolate 96121. Blue highlights spacers present in H37Rv but absent in Manila family isolate 96121 and Beijing family isolate 96075.</p

Strain information list.

<p>Strain information list.</p

List of proteins under positive selection in Manila family 96121 but not Beijing family 96075.

<p>List of proteins under positive selection in Manila family 96121 but not Beijing family 96075.</p

Evolution of CRISPR spacers in complete genomes of 21 <i>M</i>. <i>tuberculosis</i> strains.

<p>(A) Accumulation curve of spacers in complete genomes of 21 <i>M</i>. <i>tuberculosis</i> strains. (B) PCA of presence and absence of CRISPR spacers in complete genomes of 21 <i>M</i>. <i>tuberculosis</i> strains. The blue arrow indicates the position of Manila family 96121, illustrating a dramatic difference in the composition of spacers in Manila family 96121.</p

List of Manila 96121 nonsynonymous SNPs in protein coding genes.

<p>List of Manila 96121 nonsynonymous SNPs in protein coding genes.</p

Dual color fluorescence <i>in situ</i> hybridization (FISH) assays for detecting <i>Mycobacterium tuberculosis</i> and <i>Mycobacterium avium</i> complexes and related pathogens in cultures

<div><p>Two rapid dual color fluorescence <i>in situ</i> hybridization (FISH) assays were evaluated for detecting <i>M</i>. <i>tuberculosis</i> and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the <i>Mycobacterium tuberculosis</i> complex (MTBC) and a green fluorescent probe specific for the <i>Mycobacterium</i> and <i>Nocardia</i> genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and <i>M</i>. <i>avium</i> complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as <i>Mycobacterium</i> species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of <i>Corynebacterium</i> gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as <i>Mycobacterium</i> species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of <i>Nocardia</i>, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for <i>M</i>. <i>tuberculosis</i> was determined to be 5.1x10⁴ cfu per ml and for <i>M</i>. <i>avium</i> 1.5x10⁴ cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.</p></div

Results of the DNA sequence-based characterization and the MN Genus-MTBC and MTBC-MAC FISH assays on clinical mycobacterial cultures from India, Peru and the USA.

<p>Results of the DNA sequence-based characterization and the MN Genus-MTBC and MTBC-MAC FISH assays on clinical mycobacterial cultures from India, Peru and the USA.</p

Results of the MN Genus-MTBC and MTBC-MAC FISH assays on reference cultures of pathogens not belonging to the <i>Mycobacterium</i> or <i>Nocardia</i> genera.

<p>Results of the MN Genus-MTBC and MTBC-MAC FISH assays on reference cultures of pathogens not belonging to the <i>Mycobacterium</i> or <i>Nocardia</i> genera.</p

Estimated sensitivity, specificity, and positive and negative predictive values of the FISH probes on 243 clinical mycobacterial cultures that could be identified by DNA sequencing.

<p>Estimated sensitivity, specificity, and positive and negative predictive values of the FISH probes on 243 clinical mycobacterial cultures that could be identified by DNA sequencing.</p