Most-Probable-Number-Based Minimum Duration of Killing Assay for Determining the Spectrum of Rifampicin Susceptibility in Clinical Mycobacterium tuberculosis Isolates.

Accurate antibiotic susceptibility testing is essential for successful tuberculosis treatment. Recent studies have highlighted the limitations of MIC-based phenotypic susceptibility methods in detecting other aspects of antibiotic susceptibilities in bacteria. Duration and peak of antibiotic exposure, at or above the MIC required for killing the bacterial population, has emerged as another important factor for determining antibiotic susceptibility. This is broadly defined as antibiotic tolerance. Antibiotic tolerance can further facilitate the emergence of antibiotic resistance. Currently, there are limited methods to quantify antibiotic tolerance among clinical M. tuberculosis isolates. In this study, we develop a most-probable-number (MPN)-based minimum duration of killing (MDK) assay to quantify the spectrum of M. tuberculosis rifampicin susceptibility within subpopulations based on the duration of rifampicin exposure required for killing the bacterial population. MDK90-99 and MDK99.99 were defined as the minimum duration of antibiotic exposure at or above the MIC required for killing 90 to 99% and 99.99% of the initial (pretreatment) bacterial population, respectively. Results from the rifampicin MDK assay applied to 28 laboratory and clinical M. tuberculosis isolates showed that there is variation in rifampicin susceptibility among isolates. The rifampicin MDK99/99.99 time for isolates varied from less than 2 to 10 days. MDK was correlated with larger subpopulations of M. tuberculosis from clinical isolates that were rifampicin tolerant. Our study demonstrates the utility of MDK assays to measure the variation in antibiotic tolerance among clinical M. tuberculosis isolates and further expands clinically important aspects of antibiotic susceptibility testing.

Development of ligand-coated beads to measure macrophage antimicrobial activities.

BACKGROUND INFORMATION: After macrophage recognises and phagocytoses the microorganism, their phagosome undergoes a maturation process, which creates a hostile environment for the bacterium. The lumen is acidified, and proteolysis occurs to kill and degrade pathogen for further antigen presentation. It is important to understand the association between the macrophage intracellular activities and the outcome of infection. Different methods have been developed to measure the phagosome dynamics of macrophages, but there are still limitations. RESULTS: We used Mycobacterium tuberculosis (Mtb) antigens, the causative agent of tuberculosis (TB), as a model of infectious disease. Adopting a fluorescent bead-based assay, we developed beads coated with trehalose 6,6'dimycolate (TDM) from Mtb cell wall and ß-glucan from yeast cell wall to measure the macrophage phagosomal activities using a microplate reader. We examined the consistency of the assay using J774 cells and validated it using human monocyte-derived macrophages (hMDM) from healthy volunteers and TB patients. There was a decreased pH and increased proteolysis in the lumen of J774 cells after phagocytosing the ligand-coated beads. J774 macrophage showed no difference in the acidification and proteolysis in response to control IgG beads, TDM and ß-glucan beads. hMDM from healthy volunteers or TB patients showed heterogeneity in the intracellular activities when treated with ligand-coated beads. CONCLUSIONS AND SIGNIFICANCE: The beads coated with specific ligands from Mtb worked well in both macrophage cell line and human primary macrophages, which can be exploited to further study the phagosomal function of macrophage in TB. Our bead model can be applied to different ligands from other pathogens, which could extend the understanding of the associations between macrophage antimicrobial functions and outcomes of infectious diseases and the possible cellular mechanisms involved.

Asymmetric cell division in Mycobacterium tuberculosis and its unique features.

Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed.

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates from a longitudinal study.

Antibiotic tolerance in Mycobacterium tuberculosis reduces bacterial killing, worsens treatment outcomes, and contributes to resistance. We studied rifampicin tolerance in isolates with or without isoniazid resistance (IR). Using a minimum duration of killing assay, we measured rifampicin survival in isoniazid-susceptible (IS, n=119) and resistant (IR, n=84) isolates, correlating tolerance with bacterial growth, rifampicin minimum inhibitory concentrations (MICs), and isoniazid-resistant mutations. Longitudinal IR isolates were analyzed for changes in rifampicin tolerance and genetic variant emergence. The median time for rifampicin to reduce the bacterial population by 90% (MDK90) increased from 1.23 days (IS) and 1.31 days (IR) to 2.55 days (IS) and 1.98 days (IR) over 15-60 days of incubation, indicating fast and slow-growing tolerant sub-populations. A 6 log10-fold survival fraction classified tolerance as low, medium, or high, showing that IR is linked to increased tolerance and faster growth (OR = 2.68 for low vs. medium, OR = 4.42 for low vs. high, p-trend = 0.0003). High tolerance in IR isolates was associated with rifampicin treatment in patients and genetic microvariants. These findings suggest that IR tuberculosis should be assessed for high rifampicin tolerance to optimize treatment and prevent the development of multi-drug-resistant tuberculosis.

Percoll discontinuous density gradient centrifugation method for the fractionation of the subpopulations of Mycobacterium smegmatis and Mycobacterium tuberculosis from in vitro cultures.

Bacterial populations in the in vitro laboratory cultures, environment, and patients contain metabolically different subpopulations that respond differently to stress agents, including antibiotics, and emerge as stress tolerant or resistant strains. To contain the emergence of such strains, it is important to study the features of the metabolic status and response of the subpopulations to stress agents. For this purpose, an efficient method is required for the fractionation and isolation of the subpopulations from the cultures. Here we describe in detail the manual setting up of a simple, easy-to-do, reproducibly robust Percoll discontinuous density gradient centrifugation for the fractionation of subpopulations of short-sized cells (SCs) and normal/long-sized cells (NCs) from Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, which we had reported earlier. About 90-98% enrichment was obtained respectively for SCs and NCs for M. smegmatis and 69-67% enrichment was obtained respectively for the SCs and NCs for M. tuberculosis.•The Percoll discontinuous density gradient centrifugation helps the fractionation and isolation of mycobacterial subpopulations that differ in density.•The method offers a consistently reproducible high enrichment of the subpopulations of SCs and NCs from the in vitro cultures of M. smegmatis and M. tuberculosis.•Our earlier reports on the consistency in the differential response of the subpopulations, enriched using the method, to oxidative, nitrite, and antibiotic stress proves its validity.

Rifampicin tolerance and growth fitness among isoniazid-resistant clinical Mycobacterium tuberculosis isolates: an in-vitro longitudinal study.

Antibiotic tolerance in Mycobacterium tuberculosis leads to less effective bacterial killing, poor treatment responses and resistant emergence. There is limited understanding of antibiotic tolerance in clinical isolates of M. tuberculosis. Therefore, we investigated the rifampicin tolerance of M. tuberculosis isolates, with or without pre-existing isoniazid-resistance. In-vitro rifampicin survival fractions determined by minimum duration of killing assay in isoniazid susceptible (n=119) and resistant (n=84) M. tuberculosis isolates. Rifampicin tolerance was correlated with bacterial growth, rifampicin minimum inhibitory concentrations (MICs) and isoniazid-resistant mutations. The longitudinal isoniazid-resistant isolates were analyzed for rifampicin tolerance based on collection time from patients and associated emergence of genetic variants. The median duration of rifampicin exposure reducing the M. tuberculosis surviving fraction by 90% (minimum duration of killing-MDK90) increased from 1.23 (95%CI 1.11; 1.37) and 1.31 (95%CI 1.14; 1.48) to 2.55 (95%CI 2.04; 2.97) and 1.98 (95%CI 1.69; 2.56) days, for IS and IR respectively, during 15 to 60 days of incubation respectively. Increase in MDK90 time indicated the presence of fast and slow growing tolerant sub-populations. A range of 6 log10-fold survival fraction enabled classification of tolerance as low, medium or high and revealed isoniazid-resistance association with increased tolerance with faster growth (OR=2.68 for low vs. medium, OR=4.42 for low vs. high, P-trend=0.0003). The high tolerance in longitudinal isoniazid-resistant isolates was specific to those collected during rifampicin treatment in patients and associated with bacterial genetic microvariants. Our study identifies a range of rifampicin tolerance and reveals that isoniazid resistance is associated with higher tolerance with growth fitness. Furthermore, rifampicin treatment may select isoniazid-resistant isolate microvariants with higher rifampicin tolerance, with survival potential similar to multi-drug resistant isolates. These findings suggest that isoniazid-resistant tuberculosis needs to be evaluated for rifampicin tolerance or needs further improvement in treatment regimen. It is made available under a CC-BY 4.0 International license.

Evolution and transmission of antibiotic resistance is driven by Beijing lineage Mycobacterium tuberculosis in Vietnam.

IMPORTANCE: Drug-resistant tuberculosis (TB) infection is a growing and potent concern, and combating it will be necessary to achieve the WHO's goal of a 95% reduction in TB deaths by 2035. While prior studies have explored the evolution and spread of drug resistance, we still lack a clear understanding of the fitness costs (if any) imposed by resistance-conferring mutations and the role that Mtb genetic lineage plays in determining the likelihood of resistance evolution. This study offers insight into these questions by assessing the dynamics of resistance evolution in a high-burden Southeast Asian setting with a diverse lineage composition. It demonstrates that there are clear lineage-specific differences in the dynamics of resistance acquisition and transmission and shows that different lineages evolve resistance via characteristic mutational pathways.

Unveiling unusual features of formation of septal partition and constriction in mycobacteria--an ultrastructural study.

The ultrastructural functions of the electron-dense glycopeptidolipid-containing outermost layer (OL), the arabinogalactan-mycolic acid-containing electron-transparent layer (ETL), and the electron-dense peptidoglycan layer (PGL) of the mycobacterial cell wall in septal growth and constriction are not clear. Therefore, using transmission electron microscopy, we studied the participation of the three layers in septal growth and constriction in the fast-growing saprophytic species Mycobacterium smegmatis and the slow-growing pathogenic species Mycobacterium xenopi and Mycobacterium tuberculosis in order to document the processes in a comprehensive and comparative manner and to find out whether the processes are conserved across different mycobacterial species. A complete septal partition is formed first by the fresh synthesis of the septal PGL (S-PGL) and septal ETL (S-ETL) from the envelope PGL (E-PGL) in M. smegmatis and M. xenopi. The S-ETL is not continuous with the envelope ETL (E-ETL) due to the presence of the E-PGL between them. The E-PGL disappears, and the S-ETL becomes continuous with the E-ETL, when the OL begins to grow and invaginate into the S-ETL for constriction. However, in M. tuberculosis, the S-PGL and S-ETL grow from the E-PGL and E-ETL, respectively, without a separation between the E-ETL and S-ETL by the E-PGL, in contrast to the process in M. smegmatis and M. xenopi. Subsequent growth and invagination of the OL into the S-ETL of the septal partition initiates and completes septal constriction in M. tuberculosis. A model for the conserved sequential process of mycobacterial septation, in which the formation of a complete septal partition is followed by constriction, is presented. The probable physiological significance of the process is discussed. The ultrastructural features of septation and constriction in mycobacteria are unusually different from those in the well-studied organisms Escherichia coli and Bacillus subtilis.

Mycobacterium tuberculosis expresses ftsE gene through multiple transcripts.

Bacterial FtsE gene codes for the ATP-binding protein, FtsE, which in complex with the transmembrane protein, FtsX, participates in diverse cellular processes. Therefore, regulated expression of FtsE and FtsX might be critical to the human pathogen, Mycobacterium tuberculosis, under stress conditions. Although ftsX gene of M. tuberculosis (MtftsX) is known to be transcribed from a promoter inside the upstream gene, ftsE, the transcriptional status of ftsE gene of M. tuberculosis (MtftsE) remains unknown. Therefore, the authors initiated transcriptional analyses of MtftsE, using total RNA from M. tuberculosis cells that were grown under stress conditions, which the pathogen is exposed to, in granuloma in tuberculosis patients. Primer extension experiments showed the presence of putative transcripts, T1, T2, T3, and T4. T1 originated from the intergenic region between the upstream gene, MRA_3135, and MtftsE. T2 and T3 were found initiated from within MRA_3135. T4 was transcribed from a region upstream of MRA_3135. RT-PCR confirmed co-transcription of MRA_3135 and MtftsE. The cloned putative promoter regions for T1, T2, and T3 elicited transcriptional activity in Mycobacterium smegmatis transformants. T1, T2, and T3, but no new transcript, were present in the M. tuberculosis cells that were grown under the stress conditions, which the pathogen is exposed to in granuloma in tuberculosis patients. It showed lack of modulation of MtftsE transcripts under the stress conditions tested, indicating that ftsE may not have a stress response-specific function in M. tuberculosis.

Virulence of Mycobacterium tuberculosis Clinical Isolates Is Associated With Sputum Pre-treatment Bacterial Load, Lineage, Survival in Macrophages, and Cytokine Response.

It is uncertain whether differences in Mycobacterium tuberculosis (Mtb) virulence defined in vitro influence clinical tuberculosis pathogenesis, transmission, and mortality. We primarily used a macrophage lysis model to characterize the virulence of Mtb isolates collected from 153 Vietnamese adults with pulmonary tuberculosis. The virulence phenotypes were then investigated for their relationship with sputum bacterial load, bacterial lineages, bacterial growth, and cytokine responses in macrophages. Over 6 days of infection, 34 isolates (22.2%) showed low virulence (< 5% macrophages lysed), 46 isolates (30.1%) showed high virulence (≥90% lysis of macrophages), and 73 isolates (47.7%) were of intermediate virulence (5-90% macrophages lysed). Highly virulent isolates were associated with an increased bacterial load in patients' sputum before anti-tuberculosis therapy (P = 0.02). Isolate-dependent virulence phenotype was consistent in both THP-1 and human monocyte-derived macrophages. High virulence isolates survived better and replicated in macrophages one hundred fold faster than those with low virulence. Macrophages infected with high virulence isolates produced lower concentrations of TNF-α and IL-6 (P = 0.002 and 0.0005, respectively), but higher concentration of IL-1ß (P = 5.1 × 10-5) compared to those infected with low virulence isolates. High virulence was strongly associated with East Asian/Beijing lineage [P = 0.002, Odd ratio (OR) = 4.32, 95% confident intervals (CI) 1.68-11.13]. The association between virulence phenotypes, bacterial growth, and proinflammatory cytokines in macrophages suggest the suppression of certain proinflammatory cytokines (TNF-α and IL-6) but not IL-1ß allows better intracellular survival of highly virulent Mtb. This could result in rapid macrophage lysis and higher bacterial load in sputum of patients infected with high virulence isolates, which may contribute to the pathogenesis and success of the Beijing lineage.

Mycobacterial Cultures Contain Cell Size and Density Specific Sub-populations of Cells with Significant Differential Susceptibility to Antibiotics, Oxidative and Nitrite Stress.

The present study shows the existence of two specific sub-populations of Mycobacterium smegmatis and Mycobacterium tuberculosis cells differing in size and density, in the mid-log phase (MLP) cultures, with significant differential susceptibility to antibiotic, oxidative, and nitrite stress. One of these sub-populations (~10% of the total population), contained short-sized cells (SCs) generated through highly-deviated asymmetric cell division (ACD) of normal/long-sized mother cells and symmetric cell divisions (SCD) of short-sized mother cells. The other sub-population (~90% of the total population) contained normal/long-sized cells (NCs). The SCs were acid-fast stainable and heat-susceptible, and contained high density of membrane vesicles (MVs, known to be lipid-rich) on their surface, while the NCs possessed negligible density of MVs on the surface, as revealed by scanning and transmission electron microscopy. Percoll density gradient fractionation of MLP cultures showed the SCs-enriched fraction (SCF) at lower density (probably indicating lipid-richness) and the NCs-enriched fraction (NCF) at higher density of percoll fractions. While live cell imaging showed that the SCs and the NCs could grow and divide to form colony on agarose pads, the SCF, and NCF cells could independently regenerate MLP populations in liquid and solid media, indicating their full genomic content and population regeneration potential. CFU based assays showed the SCF cells to be significantly more susceptible than NCF cells to a range of concentrations of rifampicin and isoniazid (antibiotic stress), H2O2 (oxidative stress),and acidified NaNO2 (nitrite stress). Live cell imaging showed significantly higher susceptibility of the SCs of SC-NC sister daughter cell pairs, formed from highly-deviated ACD of normal/long-sized mother cells, to rifampicin and H2O2, as compared to the sister daughter NCs, irrespective of their comparable growth rates. The SC-SC sister daughter cell pairs, formed from the SCDs of short-sized mother cells and having comparable growth rates, always showed comparable stress-susceptibility. These observations and the presence of M. tuberculosis SCs and NCs in pulmonary tuberculosis patients' sputum earlier reported by us imply a physiological role for the SCs and the NCs under the stress conditions. The plausible reasons for the higher stress susceptibility of SCs and lower stress susceptibility of NCs are discussed.

Ultrastructural Analysis of Cell Envelope and Accumulation of Lipid Inclusions in Clinical Mycobacterium tuberculosis Isolates from Sputum, Oxidative Stress, and Iron Deficiency.

Introduction: Mycobacteria have several unique cellular characteristics, such as multiple cell envelope layers, elongation at cell poles, asymmetric cell division, and accumulation of intracytoplasmic lipid inclusions, which contributes to their survival under stress conditions. However, the understanding of these characteristics in clinical Mycobacterium tuberculosis (M. tuberculosis) isolates and under host stress is limited. We previously reported the influence of host stress on the cell length distribution in a large set of clinical M. tuberculosis isolates (n = 158). Here, we investigate the influence of host stress on the cellular ultrastructure of few clinical M. tuberculosis isolates (n = 8) from that study. The purpose of this study is to further understand the influence of host stress on the cellular adaptations of clinical M. tuberculosis isolates. Methods: We selected few M. tuberculosis isolates (n = 8) for analyzing the cellular ultrastructure ex vivo in sputum and under in vitro stress conditions by transmission electron microscopy. The cellular adaptations of M. tuberculosis in sputum were correlated with the ultrastructure of antibiotic sensitive and resistant isolates in liquid culture, under oxidative stress, iron deficiency, and exposure to isoniazid. Results: In sputum, M. tuberculosis accumulated intracytoplasmic lipid inclusions. In liquid culture, clinical M. tuberculosis revealed isolate to isolate variation in the extent of intracytoplasmic lipid inclusions, which were absent in the laboratory strain H37Rv. Oxidative stress, iron deficiency, and exposure to isoniazid increased the accumulation of lipid inclusions and decreased the thickness of the cell envelope electron transparent layer in M. tuberculosis cells. Furthermore, intracytoplasmic compartments were observed in iron deficient cells. Conclusion: Our ultrastructural analysis has revealed significant influence of host stress on the cellular adaptations in clinical M. tuberculosis isolates. These adaptations may contribute to the survival of M. tuberculosis under host and antibiotic stress conditions. Variation in the cellular adaptations among clinical M. tuberculosis isolates may correlate with their ability to persist in tuberculosis patients during antibiotic treatment. These observations indicate the need for further analyzing these cellular adaptations in a large set of clinical M. tuberculosis isolates. This will help to determine the significance of these cellular adaptations in the tuberculosis treatment.

Influence of Stress and Antibiotic Resistance on Cell-Length Distribution in Mycobacterium tuberculosis Clinical Isolates.

Mycobacterial cellular variations in growth and division increase heterogeneity in cell length, possibly contributing to cell-to-cell variation in host and antibiotic stress tolerance. This may be one of the factors influencing Mycobacterium tuberculosis persistence to antibiotics. Tuberculosis (TB) is a major public health problem in developing countries, antibiotic persistence, and emergence of antibiotic resistance further complicates this problem. We wanted to investigate the factors influencing cell-length distribution in clinical M. tuberculosis strains. In parallel we examined M. tuberculosis cell-length distribution in a large set of clinical strains (n = 158) from ex vivo sputum samples, in vitro macrophage models, and in vitro cultures. Our aim was to understand the influence of clinically relevant factors such as host stresses, M. tuberculosis lineages, antibiotic resistance, antibiotic concentrations, and disease severity on the cell size distribution in clinical M. tuberculosis strains. Increased cell size and cell-to-cell variation in cell length were associated with bacteria in sputum and infected macrophages rather than liquid culture. Multidrug-resistant (MDR) strains displayed increased cell length heterogeneity compared to sensitive strains in infected macrophages and also during growth under rifampicin (RIF) treatment. Importantly, increased cell length was also associated with pulmonary TB disease severity. Supporting these findings, individual host stresses, such as oxidative stress and iron deficiency, increased cell-length heterogeneity of M. tuberculosis strains. In addition we also observed synergism between host stress and RIF treatment in increasing cell length in MDR-TB strains. This study has identified some clinical factors contributing to cell-length heterogeneity in clinical M. tuberculosis strains. The role of these cellular adaptations to host and antibiotic tolerance needs further investigation.

Highly Deviated Asymmetric Division in Very Low Proportion of Mycobacterial Mid-log Phase Cells.

In this study, we show that about 20% of the septating Mycobacterium smegmatis and Mycobacterium xenopi cells in the exponential phase populationdivideasymmetrically, with an unusually high deviation (17 ± 4%) in the division site from the median, to generate short cells and long cells, thereby generating population heterogeneity. This mode of division is very different from the symmetric division of themajority (about 80%) of the septating cells in the Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG exponential phase population, with 5-10% deviation in the division site from the mid-cell site, as reported by recent studies. The short cells and the long cells further grew and divided to generate a population. We speculate that the generation of the short cells and the long cells through the highly deviated asymmetric divisionin the low proportions of mycobacterial population may have a role in stress tolerance.

The ftsZ Gene of Mycobacterium smegmatis is expressed Through Multiple Transcripts.

The principal essential bacterial cell division gene ftsZ is differentially expressed through multiple transcripts in diverse genera of bacteria in order to meet cell division requirements in compliance with the physiological niche of the organism under different environmental conditions. We initiated transcriptional analyses of ftsZ gene of the fast growing saprophytic mycobacterium, Mycobacterium smegmatis, as the first step towards understanding the requirements for FtsZ for cell division under different growth phases and stress conditions. Primer extension analyses identified four transcripts, T1, T2, T3, and T4. Transcriptional fusion studies using gfp showed that the respective putative promoter regions, P1, P2, P3, and P4, possessed promoter activity. T1, T2, and T3 were found to originate from the intergenic region between ftsZ and the upstream gene, ftsQ. T4 was initiated from the 3' portion of the open reading frame of ftsQ. RT-PCR analyses indicated co-transcription of ftsQ and ftsZ. The four transcripts were present in the cells at all growth phases and at different levels in the cells exposed to a variety of stress conditions in vitro. T2 and T3 were absent under hypoxia and nutrient-depleted stationary phase conditions, while the levels of T1 and T4 remained unaffected. These studies showed that ftsZ gene expression through multiple transcripts and differential expression of the transcripts at different growth phases and under stress conditions are conserved in M. smegmatis, like in other Actinomycetes.