Phenotypic resistance in mycobacteria: is it because I am old or fat that I resist you?

OBJECTIVES: We aimed to explore the phenomenon of phenotypic resistance to antimycobacterial antibiotics and to determine whether this was associated with cell age or the presence of lipid bodies. METHODS: The accumulation of lipid-body-positive [lipid-rich (LR)] cells was followed using cell staining and flow cytometry. LR cells of Mycobacterium smegmatis, Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium bovis (BCG) were separated from non-lipid-body-containing [lipid-poor (LP)] cells and their MBCs determined. We also compared the MBCs for LR and LP cells from 'old' and 'young' cultures. RESULTS: The LR cells of all species were more resistant to antibiotics than LP cells. For BCG, the susceptibility ratios were as follows: rifampicin, 5×; isoniazid, 16.7×; ethambutol, 5×; and ciprofloxacin, 5×. Phenotypic resistance was found in LR cells irrespective of cell age. CONCLUSIONS: We have shown that phenotypic antibiotic resistance is associated with the presence of lipid bodies irrespective of cell age. These data have important implications for our understanding of relapse in mycobacterial infections.

Real-time monitoring of live mycobacteria with a microfluidic acoustic-Raman platform.

Tuberculosis (TB) remains a leading cause of death worldwide. Lipid rich, phenotypically antibiotic tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse and the need for long-term TB treatment. We present a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis. We then perform optical analysis in the form of wavelength modulated Raman spectroscopy (WMRS) on the trapped M. smegmatis for up to eight hours, and also in the presence of isoniazid (INH). The Raman fingerprints of M. smegmatis exposed to INH change substantially in comparison to the unstressed condition. Our work provides a real-time assessment of the impact of INH on the increase of lipids in these mycobacteria, which could render the cells more tolerant to antibiotics. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to different conditions and stimuli.

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA).

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers' safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of <1 log10eCFU/mL (i.e., <10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

Enhanced Methodologies for Detecting Phenotypic Resistance in Mycobacteria.

Lipid droplets found in algae and other microscopic organisms have become of interest to many researchers partially because they carry the capacity to produce bio-oil for the mass market. They are of importance in biology and clinical practice because their presence can be a phenotypic marker of an altered metabolism, including reversible resistance to antibiotics, prompting intense research.A useful stain for detecting lipid bodies in the lab is Nile red. It is a dye that exhibits solvatochromism; its absorption band varies in spectral position, shape and intensity with the nature of its solvent environment, it will fluoresce intensely red in polar environment and blue shift with the changing polarity of its solvent. This makes it ideal for the detection of lipid bodies within Mycobacterium spp. This is because mycobacterial lipid bodies' primary constituents are nonpolar lipids such as triacylglycerols but bacterial cell membranes are primarily polar lipid species. In this chapter we describe an optimal method for using Nile red to distinguish lipid containing (Lipid rich or LR cells) from those without lipid bodies (Lipid Poor or LP). As part of the process we have optimized a method for separating LP and LR cells that does not require the use of an ultracentrifuge or complex separation media. We believe that these methods will facilitate further research in these enigmatic, transient and important cell states.

Detecting Phenotypically Resistant Mycobacterium tuberculosis Using Wavelength Modulated Raman Spectroscopy.

Raman spectroscopy is a non-destructive and label-free technique. Wavelength modulated Raman (WMR) spectroscopy was applied to investigate Mycobacterium tuberculosis cell state, lipid rich (LR) and lipid poor (LP). Compared to LP cells, LR cells can be up to 40 times more resistant to key antibiotic regimens. Using this methodology single lipid rich (LR) from lipid poor (LP) bacteria can be differentiated with both high sensitivity and specificity. It can also be used to investigate experimentally infected frozen tissue sections where both cell types can be differentiated. This methodology could be utilized to study the phenotype of mycobacterial cells in other tissues.

Centrifugation and decontamination procedures selectively impair recovery of important populations in Mycobacterium smegmatis.

Diagnosis and treatment monitoring of patients with tuberculosis (TB) requires detection of all viable mycobacteria in clinical samples. Quantitation of Mycobacterium tuberculosis (Mtb) in sputum is commonly performed by culture after sample decontamination to prevent overgrowth by contaminant organisms. Exponentially growing cultures have cells that predominately lack non-polar lipid bodies whereas stationary cultures have a predominance of cells with non-polar lipid bodies. This may reflect rapidly growing 'active' and non-replicating 'persister' sub-populations respectively in sputum from TB patients. We investigated the effect of decontamination on culture-based quantitation of exponential and stationary phase cultures of Mycobacterium smegmatis in an artificial sputum model. Exponentially growing populations were between 89 and 50 times more susceptible to decontamination than stationary phase cultures when quantified by most probable number and colony forming units. These findings suggest that decontamination selectively eliminates the 'active' population. This may impair diagnostic sensitivity, treatment monitoring, and compromise clinical trials designed to identify new antibiotic combinations with activity against all mycobacterial cell states.

Label-free optical vibrational spectroscopy to detect the metabolic state of M. tuberculosis cells at the site of disease.

Tuberculosis relapse is a barrier to shorter treatment. It is thought that lipid rich cells, phenotypically resistant to antibiotics, may play a major role. Most studies investigating relapse use sputum samples although tissue bacteria may play an important role. We developed a non-destructive, label-free technique combining wavelength modulated Raman (WMR) spectroscopy and fluorescence detection (Nile Red staining) to interrogate Mycobacterium tuberculosis cell state. This approach could differentiate single "dormant" (lipid rich, LR) and "non-dormant" (lipid poor, LP) cells with high sensitivity and specificity. We applied this to experimentally infected guinea pig lung sections and were able to distinguish both cell types showing that the LR phenotype dominates in infected tissue. Both in-vitro and ex-vivo spectra correlated well, showing for the first time that Mycobacterium tuberculosis, likely to be phenotypically resistant to antibiotics, are present in large numbers in tissue. This is an important step in understanding the pathology of relapse supporting the idea that they may be caused by M. tuberculosis cells with lipid inclusions.