On-Slide Heat Sterilization Enables Mass Spectrometry Imaging of Tissue Infected with High-Threat Pathogens Outside of Biocontainment: A Study Directed at Mycobacterium tuberculosis

Abstract

Mass spectrometry imaging investigations of tissues infected with agents that require high-security biocontainment, such as Mycobacterium tuberculosis, have been limited due to incompatible sterilization techniques. Here we describe an on-slide heat sterilization method that enables mass spectrometry imaging investigations of pharmaceuticals, lipids, and metabolites in infected tissue samples outside of biocontainment. An evaluation of different temperatures and incubation times determined that 100 °C for 1 h was essential to sterilize 5 times the bacterial burden observed in tuberculosis (TB) cavity sections. Laser-capture microdissection combined with liquid chromatography with tandem mass spectrometry quantitation, in addition to mass spectrometry imaging, showed that no degradation was observed following the on-slide heat sterilization protocol for a variety of drug classes covering a range of physicochemical properties. Utilizing the tissue mimetic model, we demonstrated that the detection of lipid and metabolite ions was not impacted by heat sterilization and that, for several metabolites, the on-slide heat sterilization method improved the sensitivity when compared to control samples. An application of the on-slide heat sterilization to M. tuberculosis infected tissue enabled the first detection and spatial distribution of lipids indicative of a lysosomal storage disease phenotype within TB granuloma macrophages, in addition to the differential distribution of metabolites central to the fatty acid oxidation pathway. These initial investigations detected a pronounced heterogeneity within the cellular regions and necrotic cores of individual TB granulomas and across different evolving granulomas. This study provides the framework for mass spectrometry imaging investigations of high-threat pathogens outside of biocontainment.

Keywords: MALDI; Mycobacterium tuberculosis; TB granuloma; drug distribution; heat sterilization; lipidomics; mass spectrometry imaging; metabolomics.

Figure 1

Schematic of the on-slide heat sterilization and validation for M. tuberculosis. Twenty microliters of the Mtb cell suspension from the control glass slides were serially diluted (10⁴–10⁷), and 25 μL of each dilution were plated in quadrants onto Middlebrook 7H11 agar; the entire cell suspension from the heat-treated slides were plated out (middle panel). Mtb growth was observed in two quadrants of the control plates following three weeks of incubation, whereas no growth was observed on the plates containing the heat-treated cell suspension (right panel).

Figure 2

(a) LCM-LC-MS/MS quantification of antimicrobial drug compounds in heat-treated vs control liver mimetic tissue sections dosed with 1000 ng/g of tissue. (b) LCM-LC-MS/MS quantification of antimicrobial drug compounds in heat-treated vs control TB lung lesions. RIF, rifampicin; RBT, rifabutin; RPT, rifapentine; PA-824, pretomanid.

Figure 3

(a) MALDI MS images of the antimicrobial rifamycin compounds in control and heat-treated liver mimetic sections. (b) The MSI images and average spectra of the BDQ ⁷⁹Br and ⁸¹Br isotopes. BDQ, bedaquiline; C, control; HT, heat-treated.

Figure 4

MS images of SMs of three heat-inactivated TB-infected rabbit lung samples (1–3). All species are in [M + H]⁺ ion form.

Figure 5

MS images of cholesterol, CARs, and TAGs of three heat-inactivated TB-infected rabbit lung samples (1–3). TAGs are in [M + Na]⁺ ion form; all other species are in [M + H]⁺ ion form.

Figure 6

Higher-magnification H&E images of caseous and foamy macrophage TB granuloma regions. MS images merged with histology display cholesterol, CARs, SMs, and TAGs. The scale bars for the higher-magnification regions of the TB-infected rabbit lung samples 1–3 are 100, 300, and 300 μm, respectively.