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A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.
Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.
The human microbiome is a highly dynamic ecosystem involving molecular interactions of bacteria, viruses, archaea, and other microbial eukaryotes. While microbial relationships have been intensely studied in recent years, much remains to be understood about microbial processes at the chemical level1,2. This is in part due to the unavailability of tools capable of accurately measuring complex microbial environments. Advances in the field of imaging mass spectrometry (IMS) over the past decade have enabled in situ and label-free spatial mapping of many metabolites, lipids, and proteins in biological substrates3,4. Matrix-assisted laser desorption/ionization (MALDI) has emerged as the most common ionization technique used in imaging mass spectrometry, involving the use of a UV laser to ablate material from the surface of a thin tissue section for measurement by mass spectrometry4. This process is facilitated by the application of a chemical matrix applied homogeneously to the surface of the sample, allowing for sequential measurements to be made in a raster pattern across the sample surface. Heat maps of analyte ion intensities are then generated following data acquisition. Recent advances in ionization sources and sampling techniques have enabled the analysis of non-traditional substrates such as bacterial5 and mammalian6,7,8 cellular specimens grown on nutrient agar. The molecular spatial information afforded by IMS can provide unique insight into the biochemical communication of microbe-microbe and host-microbe interactions during infection9,10,11,12,13,14.
Upon Clostridioides difficile infection (CDI), C. difficile is exposed to a rapidly changing microbial environment in the gastrointestinal tract, where polymicrobial interactions are likely to impact infection outcomes15,16. Surprisingly, little is known about the molecular mechanisms of interactions between C. difficile and resident microbiota during infection. For example, enterococci are a class of opportunistic commensal pathogens in the gut-microbiome and have been associated with increased susceptibility to and severity of CDI17,18,19,20. However, little is known about the molecular mechanisms of the interactions between these pathogens. To visualize small-molecule communication between these members of the gut microbiome, bacterial macrocolonies were grown herein on agar to simulate microbe-microbe interactions and bacterial biofilm formation in a controlled environment. However, obtaining representative metabolic distributions upon MALDI imaging mass spectrometry analysis of bacterial culture specimens is challenging due to the high water content and uneven surface topography of these samples. This is largely caused by the highly hydrophilic nature of agar and the non-uniform agar surface response during moisture removal.
The high water content of agar can also make it challenging to achieve homogeneous MALDI matrix coating and can interfere with subsequent MALDI analysis performed in vacuo21. For example, many MALDI sources operate at pressures of 0.1-10 Torr, which is a sufficient vacuum to remove moisture from the agar and can cause deformation of the sample. These morphological changes in the agar induced by the vacuum environment cause bubbling and cracking in the dried agar material. These artifacts reduce the adherence of the agar to the slide and can cause dismounting or flaking of the sample into the instrument vacuum system. The thickness of the agar samples can be up to 5 mm off the slide, which can create insufficient clearance from ion optics inside the instrument, causing contamination and/or damage to instrument ion optics. These cumulative effects can result in reductions of ion signal reflective of the surface topography, rather than the underlying microbial biochemical interactions. Agar samples must be homogeneously dried and strongly adhered to a microscope slide prior to analysis in vacuo.
This paper demonstrates a sample preparation workflow for the controlled drying of bacterial culture macrocolonies grown on agar media. This multi-step, slower drying process (relative to those previously reported) ensures that the agar will dehydrate uniformly while minimizing the effects of bubbling or cracking of agar samples mounted on microscope slides. By using this gradual drying method, samples are strongly adhered to the microscope slide and amenable for subsequent matrix application and MALDI analysis. This is exemplified using model bacterial colonies of C. difficile grown on agar and murine tissue models harboring CDI with and without the presence of commensal and opportunistic pathogen, Enterococcus faecalis. MALDI imaging mass spectrometry analyses of both bacterial and tissue models allow for the spatial mapping of amino acid metabolite profiles, providing novel insight into bioenergetic microbial metabolism and communication.
NOTE: Animal experiments were approved by the Animal Care and Use Committees of the Children's Hospital of Philadelphia and the University of Pennsylvania Perelman School of Medicine (protocols IAC 18-001316 and 806279).
CAUTION: Clostridium difficile (C. difficile) and Enterococcus faecalis (E. faecalis) are BSLII pathogens and should be handled with extreme caution. Utilize proper decontamination protocols when necessary.
1. Growth of bacterial culture macrocolonies and preparation for overnight shipment
2. Ambient drying of C. difficile + E. faecalis bacterial macrocolonies
3. Vacuum and heat-mediated drying of C. difficile + E. faecalis bacterial macrocolonies
NOTE: A custom-built vacuum drying apparatus (Figure 1) was built to facilitate the removal of excess moisture from the agar samples. This apparatus utilizes a rotary vane vacuum pump connected in line to a HEPA biofilter, a cold trap, and a stainless-steel chamber, where the bacterial samples are placed. A variable voltage transformer is connected to an insulated wire filament, which allows the user to heat the chamber to expedite the drying process.
4. MALDI matrix application via robotic spraying
NOTE: After the agarose samples have been thoroughly dried and the height of the culture sections have noticeably decreased, use a robotic matrix sprayer to homogeneously apply a thin coating of a chemical MALDI matrix compound. This procedure should be performed in a chemical fume hood and with proper personal protective equipment, including gloves, laboratory glasses, and a lab coat.
5. Preparing bacterial macrocolonies for MALDI imaging mass spectrometry data acquisition
NOTE: All imaging mass spectrometry analyses were performed using a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This instrument is equipped with a Nd:YAG MALDI laser system (2 kHz, 355 nm) .
6. Imaging mass spectrometry data analysis and compound identification
7. Preparation and shipment of noninfected control and C. difficile -infected mouse cecal tissues
8. Cryosectioning of noninfected control verus C. difficile -infected mouse cecal tissues
9. Preparation of noninfected control versus C. difficile -infected mouse cecal tissues for matrix application and MALDI imaging mass spectrometry
We have performed metabolite MALDI imaging mass spectrometry of model bacterial colonies and mice co-colonized with E. faecalis and C. difficile to study the role of amino acids in microbe-microbe interactions. Bacterial macrocolonies grown on agar serve as a well-defined model to analyze distinct biochemical changes in bacterial biofilm formation. It is important to ensure a controlled drying process for bacterial culture macrocolonies grown on agar media to minimize deformations and cracking in the ag...
During MALDI imaging mass spectrometry, it is important to have a flat sample surface to provide for a consistent focal diameter of the incident MALDI laser on the sample substrate. Deviations in sample height can cause the MALDI laser beam to shift out of focus, causing alterations in beam diameter and intensity, which can affect MALDI ionization efficiency. These alterations in ionization efficiency can result in differences in analyte intensity across the tissue surface that are not reflective of the underlying tissue...
The authors declare no competing financial interests.
This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) under award GM138660. J.T.S. was supported by the Charles and Monica Burkett Family Summer Fellowship from the University of Florida. J.P.Z. was supported by NIH grants K22AI7220 (NIAID) and R35GM138369 (NIGMS). A.B.S. was supported by the Cell and Molecular Biology Training Grant at the University of Pennsylvania (T32GM07229).
Name | Company | Catalog Number | Comments |
0.2 μm Titan3 nylon syringe filters | Thermo Scientific | 42225-NN | |
1,5-diaminonaphthalene MALDI matrix | Sigma Aldrich | 2243-62-1 | |
20 mL Henke Ject luer lock syringes | Henke Sass Wolf | 4200.000V0 | |
275i series convection vacuum gauge | Kurt J. Lesker company | KJL275807LL | |
7T solariX FTICR mass spectrometer equipped with a Smartbeam II Nd:YAG MALDI laser system (2 kHz, 355 nm) | Bruker Daltonics | ||
Acetic acid solution, suitable for HPLC | Sigma Aldrich | 64-19-7 | |
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9% | Sigma Aldrich | 75-05-8 | |
Ammonium hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metals basis | Sigma Aldrich | 1336-21-6 | |
Autoclavable biohazard bags: 55 gal | Grainger | 45TV10 | |
Biohazard specimen transport bags (8 x 8 in.) | Fisher Scientific | 01-800-07 | |
Brain heart infusion broth | BD Biosciences | 90003-040 | |
C57BL/6 male mice | Jackson Laboratories | ||
CanoScan 9000F Mark II photo and document scanner | Canon | ||
CM 3050S research cryomicrotome | Leica Biosystems | ||
Desiccator cabinet | Sigma Aldrich | Z268135 | |
Diamond tip scriber, Electron Microscopy Sciences | Fisher Scientific | 50-254-51 | |
Drierite desiccant pellets | Drierite | 21005 | |
Ethanol, 200 Proof | Decon Labs | 2701 | |
flexImaging software | Bruker Daltonics | ||
ftmsControl software | Bruker Daltonics | ||
Glass vacuum trap | Sigma Aldrich | Z549460 | |
HTX M5 TM robotic sprayer | HTX Technologies | ||
Indium Tin Oxide (ITO)-coated microscope slides | Delta Technologies | CG-81IN-S115 | |
In-line HEPA filter to vacuum pump | LABCONCO | 7386500 | |
Methanol, HPLC Grade | Fisher Chemical | 67-56-1 | |
MTP slide-adapter II | Bruker Daltonics | 235380 | |
Optimal cutting temperature (OCT) compound | Fischer Scientific | 23-730-571 | |
Peridox RTU Sporicide, Disinfectant and Cleaner | CONTEC | CR85335 | |
PTFE (Teflon) printed slides, Electron Microscopy Sciences | VWR | 100488-874 | |
Rotary vane vacuum pump RV8 | Edwards | A65401903 | |
Tissue-Tek Accu-Edge Disposable High Profile Microtome Blades | Electron Microscopy Sciences | 63068-HP | |
Transparent vacuum tubing | Cole Palmer | EW-06414-30 | |
Ultragrade 19 vacuum pump oil | Edwards | H11025011 | |
Variable voltage transformer | Powerstat | ||
Water, suitable for HPLC | Sigma Aldrich | 7732-18-5 | |
Wide-mouth dewar flask | Sigma Aldrich | Z120790 |
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