Genotypic and phenotypic diversity of Mycobacterium tuberculosis complex genotypes prevalent in West Africa

Abstract

Findings from previous comparative genomics studies of the Mycobacterium tuberculosis complex (MTBC) suggest genomic variation among the genotypes may have phenotypic implications. We investigated the diversity in the phenotypic profiles of the main prevalent MTBC genotypes in West Africa. Thirty-six whole genome sequenced drug susceptible MTBC isolates belonging to lineages 4, 5 and 6 were included in this study. The isolates were phenotypically characterized for urease activity, tween hydrolysis, Thiophen-2-Carboxylic Acid Hydrazide (TCH) susceptibility, nitric oxide production, and growth rate in both liquid (7H9) and solid media (7H11 and Löwenstein-Jensen (L-J)). Lineage 4 isolates showed the highest growth rate in both liquid (p = 0.0003) and on solid (L-J) media supplemented with glycerol (p<0.001) or pyruvate (p = 0.005). L6 isolates optimally utilized pyruvate compared to glycerol (p<0.001), whereas L5 isolates grew similarly on both media (p = 0.05). Lineage 4 isolates showed the lowest average time to positivity (TTP) (p = 0.01; Average TTP: L4 = 15days, L5 = 16.7days, L6 = 29.7days) and the highest logCFU/mL (p = 0.04; average logCFU/mL L4 = 5.9, L5 = 5.0, L6 = 4.4) on 7H11 supplemented with glycerol, but there was no significant difference in growth on 7H11 supplemented with pyruvate (p = 0.23). The highest release of nitrite was recorded for L5 isolates, followed by L4 and L6 isolates. However, the reverse was observed in the urease activity for the lineages. All isolates tested were resistant to TCH except for one L6 isolate. Comparative genomic analyses revealed several mutations that might explain the diverse phenotypic profiles of these isolates. Our findings showed significant phenotypic diversity among the MTBC lineages used for this study.

Fig 1. Utilization of different carbon sources on Löwenstein-Jensen media slants.

Growth was compared as degree of positivity (WHO standard for grading Mtb growth on L-J) on both glycerol and pyruvate supplemented media slants. Two-way analysis of variance (ANOVA) considering Time as a factor showed significant difference between the 3 lineages on glycerol (p<0.001) and on pyruvate (p = 0.005) supplemented media.

Fig 2. Time to positivity (TTP) on 7H11 media.

The average time to positivity was measured in terms of the number of days it took for the first colonies to appear for CFU determination and plotted against the various media compositions.

Fig 3. Growth rate on 7H11 media.

The average logCFU/ml was plotted against the different supplemented media.

Fig 4. Growth rate in liquid medium.

Optical density at 600nm was measured at the data points (days) and indicated by the symbols. The data points are the averages of individual strains belonging to the 3 different lineages.

Fig 5. Comparison of nitrite concentration.

The individual isolates are represented by the black points in the boxplot.

Fig 6. Comparison of nitrate concentration.

The individual isolates are represented by the black points in the boxplot.

Fig 7. Comparison of urease activity of the three different lineages.

A negative control depicted by the blue line was only urea broth. H37Rv was included in the experimental set-up as a positive control (black line).

Fig 8. Tween hydrolysis test.

A negative control depicted by the blue line had only the Tween hydrolysis reagent. Mycobacterium aurum represented by the purple line was included in the experimental set-up as a positive control.