Novel Populations of Mycobacterium smegmatis Under Hypoxia and Starvation: Some Insights on Cell Viability and Morphological Changes
<p>Correlation between growth measurement and cell viability methods of <span class="html-italic">M. smegmatis</span>. (<b>A</b>) Growth and cell viability measurements during starvation. (<b>B</b>) Growth and cell viability measurements during hypoxia. White circles represent the optical density (OD), blue squares represent the number of CFU/mL, red diamonds represent total bacteria/mL (t-bact/mL) detected by FCM, and green triangles represent viable bacteria/mL (v-bact/mL) detected by FCM. The time when NRP1 (initial fading of the methylene blue, ▽f) and NRP2 phases (total discolouration of the methylene blue occurred, ▽d) began are indicated. Values are displayed on a logarithmic scale and mean ± SD are plotted. All experiments were carried out in triplicate.</p> "> Figure 2
<p>Multiparametric FCM analysis of <span class="html-italic">M. smegmatis</span> grown under starvation conditions. (<b>A</b>) Dot plots of dead (†), viable (§) and the combination of dead and viable (*) cell populations are displayed; green fluorescence (FL1-H, SYTO 9) vs. red fluorescence (FL2-H, PI) is charted through time (0 h to 120 h). (<b>B</b>) Overlap of frequency histograms of the fluorescence profile of SYTO 9 (FL1-H) at different starvation times. (<b>C</b>) Histograms of cell size. The forward scatter depicts size (FSC-H), and the ordinate (cell counts) denotes single events. (<b>D</b>) Frequency histograms of cell granularity/complexity (SSC-H); the ordinate (cell counts) denotes single events.</p> "> Figure 3
<p>Multiparametric analysis of the results obtained by FCM of M. smegmatis in hypoxia. (<b>A</b>) Dot plots of dead cells defined as SYTO 9lowPIhigh (†), viable cells defined as SYTO 9<sup>high</sup>PI<sup>low</sup> (§) and cells defined as SYTO 9<sup>high</sup>PI<sup>high</sup> (‡) are displayed; fluorescence microspheres were used for calibration (p), red fluorescence (FL2-H, PI) vs. green fluorescence (FL1-H, SYTO 9) is charted through time (0 h to 120 h). (<b>B</b>) Measurements of mycobacterial populations detected by FCM and their correlation with measurements in solid media. The blue squares represent CFU/mL; brown diamonds represent total bacteria per mL (t-bact/mL); black circles represent SYTO 9<sup>high</sup>PI<sup>high</sup> bacteria/mL (SYTO 9<sup>high</sup>PI<sup>high</sup>-bact/mL); green triangles represent viable bacteria per mL (v-bact/mL); red circles represent dead bacteria per mL (d-bact/mL). The times when a noticeable fading (NRP1, ▽f) and the total discolouration (NRP2, ▽d) of the methylene blue occurred are indicated. Values are displayed on a logarithmic scale and mean ± SD are charted. All experiments were carried out in triplicate. (<b>C</b>,<b>D</b>) Frequency histograms of cell size (FSC-H) and cell complexity (SSC-H) of <span class="html-italic">M. smegmatis</span>, respectively. In both cases, the ordinate (cell counts) denotes single events.</p> "> Figure 4
<p>Gene expression of <span class="html-italic">M. smegmatis</span> cultures under in vitro latency conditions over time. Absolute gene expression normalised to 16S rRNA was measured in <span class="html-italic">M. smegmatis</span> either under starvation (<b>A</b>) or hypoxia (<b>C</b>); standard deviations are charted and <span class="html-italic">p</span> < 0.05 was considered significantly different (represented by a star) between expression in time points of in vitro latency and the time 0 h (exponential phase). The relative gene quantification in starvation (at 4, 12, 24 and 120 h) is expressed as the ratio of transcription overtime/transcription at 0 h (<b>B</b>). The relative gene quantification in hypoxia (at 12, 36, 72 and 120 h) is expressed as the ratio of transcription overtime/transcription at 0 h (<b>D</b>).</p> "> Figure 5
<p>TEM images of <span class="html-italic">M. smegmatis</span> under starvation. (<b>A</b>) Representative electron micrographs at 0 h, 12 h, 24 h, and 120 h of incubation; magnification was ×10 K; scale bars represent 2 µm; arrows point to electron-transparent cells without nucleoid and debris are indicated with an asterisk. (<b>B</b>) Ultrastructure of <span class="html-italic">M. smegmatis</span> at 0 h, 24 h, and 120 h of incubation; magnification was ×150 K and scale bars represent 100 nm. Arrow heads point to different cells structure such as a a: cytoplasmatic material, b: cell wall, c: chromatin, d: cellular debris, and e: electron-transparent zone surrounding the nucleoid. The acceleration voltage was 70 kV. These images represent three independent experiments and were chosen from five random fields.</p> "> Figure 6
<p>SEM images of <span class="html-italic">M. smegmatis</span> under starvation. (<b>A</b>) Cells at 0, 24 and 120 h of starvation, at ×10 K magnification. (<b>B</b>) Cells at ×25 K, ×30 K and ×40 K magnification at 24 and 120 h of starvation. Rod cell surfaces with a rough appearance are indicated with an asterisk, and septa are indicated by an arrow. These images represent three independent experiments and were chosen from five random fields. Scale bars represent 500 nm, and the acceleration voltage was 15 kV in all cases.</p> "> Figure 7
<p>SEM images of <span class="html-italic">M. smegmatis</span> cells grown under hypoxic conditions. (<b>A</b>) Representative electron micrographs taken at various times; magnification was ×10 K, and scale bars represent 1 µm. (<b>B</b>) Morphological changes at a higher magnification; times 0 to 36 h were ×20 K, and the scale bar represents 1 µm; times 72 to 120 were ×30 K, and scale bars represent 500 nm. Regular rods (RR), pleomorphic cells (PC), and rough surface cells (RC); the star indicates cellular debris. The acceleration voltage was 15 kV. These images represent three independent experiments and were chosen from five random fields.</p> "> Figure 8
<p>TEM images of <span class="html-italic">M. smegmatis</span> under hypoxia. (<b>A</b>) <span class="html-italic">M. smegmatis</span> cells over time, at ×10K magnification. Scale bars represent 2 µm. (<b>B</b>) <span class="html-italic">M. smegmatis</span> cells at 0 h and 72 h under hypoxia, magnification was ×45 K or ×150 K, respectively. Scale bars represent 100 nm. (<b>C</b>) <span class="html-italic">M. smegmatis</span> cells at 120 h under hypoxia, magnification was ×150 K. Scale bars represent 100 nm. Black arrows point to cells with a low density of electrons. Numbers in panels point to ① Lipid-body-like structures, ② compacted DNA with highly dense electrons, ③ low-density electron zone, ④ loss of rigidity of cell wall, ⑤ cytoplasm, ⑥ star-shaped nucleoid and ⑦ vesicular structures with internal membranes. An acceleration voltage of 70 kV was used. Smaller frames within each panel were magnified ×120 K, and the acceleration voltage was 60 kV; scale bars represent 500 nm. These images represent three independent experiments and were chosen from five random fields.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strain and Growth Conditions
2.2. Starvation Model of Dormancy
2.3. Hypoxic Model of Dormancy
2.4. Growth Measurements and Bacterial Viability Determinations
2.5. Flow Cytometry Analysis
2.6. Transmission Electron Microscopy
2.7. Scanning Electron Microscopy
2.8. RNA Isolation and cDNA Synthesis
2.9. Quantitative Real-Time PCR
2.10. Statistical Analyses
3. Results
3.1. Growth Measurements and Cell Viability of M. smegmatis
3.2. Multiparametric Analysis of Mycobacterial Growth Obtained by FCM
3.3. Gene Expression
3.4. Morphological Changes in Dormant M. smegmatis Examined by Electron Microscopy
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Period of Cell Culture Under Starvation (h) | Cell Length (µm) § | Morphology ‡ |
---|---|---|
* 0 | 3.83 ± 1.29 | Rods |
4 | 2.90 ± 0.79 | Rods |
12 | 2.11 ± 0.28 | Rods |
24 | 1.52 ± 0.34 | Rods with rough appearance |
120 | 1.41 ± 0.34 | All rods have a rough surface |
Period Under Hypoxia | Cell Length (µm) § | Morphology ‡ (% of Fraction of Cells) | Pleomorphic + Rough Cells (%) | ||
---|---|---|---|---|---|
Rod-Shaped | Pleomorphic ▲ | Rough | |||
* 0 h | 3.83 ± 1.29 | 100 | 0 | 0 | 0 |
12 h | 3.04 ± 0.91 | 75 | 25 | 0 | 25 |
36 h | 3.73 ± 1.42 | 16 | 79 | 5 | 84 |
72 h | 3.53 ± 1.76 | 18 | 67 | 15 | 82 |
120 h | 3.38 ± 0.73 | 16 | 56 | 28 | 84 |
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Zaragoza-Contreras, R.; Aguilar-Ayala, D.A.; García-Morales, L.; Ares, M.A.; Helguera-Repetto, A.C.; Cerna-Cortés, J.F.; León-Solis, L.; Suárez-Sánchez, F.; González-Y-Merchand, J.A.; Rivera-Gutiérrez, S. Novel Populations of Mycobacterium smegmatis Under Hypoxia and Starvation: Some Insights on Cell Viability and Morphological Changes. Microorganisms 2024, 12, 2280. https://doi.org/10.3390/microorganisms12112280
Zaragoza-Contreras R, Aguilar-Ayala DA, García-Morales L, Ares MA, Helguera-Repetto AC, Cerna-Cortés JF, León-Solis L, Suárez-Sánchez F, González-Y-Merchand JA, Rivera-Gutiérrez S. Novel Populations of Mycobacterium smegmatis Under Hypoxia and Starvation: Some Insights on Cell Viability and Morphological Changes. Microorganisms. 2024; 12(11):2280. https://doi.org/10.3390/microorganisms12112280
Chicago/Turabian StyleZaragoza-Contreras, Ruben, Diana A. Aguilar-Ayala, Lázaro García-Morales, Miguel A. Ares, Addy Cecilia Helguera-Repetto, Jorge Francisco Cerna-Cortés, Lizbel León-Solis, Fernando Suárez-Sánchez, Jorge A. González-Y-Merchand, and Sandra Rivera-Gutiérrez. 2024. "Novel Populations of Mycobacterium smegmatis Under Hypoxia and Starvation: Some Insights on Cell Viability and Morphological Changes" Microorganisms 12, no. 11: 2280. https://doi.org/10.3390/microorganisms12112280
APA StyleZaragoza-Contreras, R., Aguilar-Ayala, D. A., García-Morales, L., Ares, M. A., Helguera-Repetto, A. C., Cerna-Cortés, J. F., León-Solis, L., Suárez-Sánchez, F., González-Y-Merchand, J. A., & Rivera-Gutiérrez, S. (2024). Novel Populations of Mycobacterium smegmatis Under Hypoxia and Starvation: Some Insights on Cell Viability and Morphological Changes. Microorganisms, 12(11), 2280. https://doi.org/10.3390/microorganisms12112280