BACLIB measures extracted microbial lipids by MALDI mass spectrometry. Microbial membrane fingerprints were discovered and validated in the labs of Profs. Robert Ernst and David Goodlett. Initially a modification of the method of El Hamidi et al. was used. A rapid BACLIB extraction method was later developed by Pataigin in collaboration with the Goodlett and Ernst labs.

Each microbial species has a unique membrane lipid fingerprint that can be readily distinguished in MALDI mass spectra. Machine learning techniques identify the species in a specimen with high sensitivity and specificity.

At right is a MALDI target plate developed by Pataigin for BACLIB, with a 1 µL sample drop on a target.

Fast Lipid Analysis Technique

The modern BACLIB method performs lipid extraction directly on the surface of a stainless-steel MALDI plate, using an approach called FLAT (Fast Lipid Analysis Technique) as follows:
1. Dispense 1 µL from a liquid specimen onto a MALDI target.
2. Apply a buffer to the MALDI target.
3. Heat MALDI plate for 30 min.
4. Rinse with water.
5. Apply 1 µL matrix.

The MALDI plate is now ready to be inserted in the mass spectrometer.

Heating adheres bacteria and fungi to the MALDI target, and facilitates lipid extraction from membranes. In Gram-negative bacteria, buffer lowers pH, cleaving the KDO bond between lipid A and the LPS core.

Washing removes water-soluble cellular debris, enhancing lipid signals.

Sparse BACLIB Spectra facilitate ID

Shown at right are typical BACLIB spectra for the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida auris.

These spectra are notably sparse, which is an important advantage of the BACLIB method. Sparse, highly distinctive ion peaks can be detected even at low concentrations with a complex background –important for a direct-from-specimen method.

Baclib’s sparse spectra also facilitate analysis of polymicrobial specimens.

Further reading:

Rapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids
Sorensen M, Chandler CE, Gardner F, et al.
Sci Rep. 2020

Streamlined Analysis of Cardiolipins in Prokaryotic and Eukaryotic Samples Using a Norharmane Matrix by MALDI-MSI
Yang H, Jackson SN, Woods AS, et al.
J Am Soc Mass Spectrom. 2020

Pathogen Identification Direct From Polymicrobial Specimens Using Membrane Glycolipids
Fondrie WE, Liang T, Oyler BL, et al.
Sci Rep. 2018

Identification of the ESKAPE pathogens by mass spectrometric analysis of microbial membrane glycolipids
Leung LM, Fondrie WE, Doi Y, et al.
Sci Rep. 2017

Research applications:

Deep-sea microbes as tools to refine the rules of innate immune pattern recognition
Gauthier AE, Chandler CE, Poli V, Gardner FW, et al.
Sci Immunol. 2021

Colistin heteroresistance is largely undetected among carbapenem-resistant Enterobacterales in the United States
Band V, Satola S, Smith R, et al.
mBio 2021

Early evolutionary loss of the lipid A modifying enzyme PagP resulting in innate immune evasion in Yersinia pestis
Chandler CE, Harberts EM, Pelletier MR, et al.
Proc Natl Acad Sci U S A 2020

On-tissue derivatization of lipopolysacharide for detection of lipid A using MALDI-MSI
Yang H, Chandler CE, Jackson SN, et al.
Analytical Chemistry 2020

Bacterial medium-chain 3-hydroxy fatty acid metabolites trigger immunity in Arabidopsis plants
Kutschera A, Dawid C, Gisch N, et al.
Science 2019

Lipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship
Scott AJ, Oyler BL, Goodlett DR, Ernst RK.
Biochim Biophys Acta Mol Cell Biol Lipids 2017

Norharmane Matrix Enhances Detection of Endotoxin by MALDI-MS for Simultaneous Profiling of Pathogen, Host, and Vector Systems
Scott AJ, Flinders B, Cappell J, et al.
Pathog Dis. 2016

Unique structural modifications are present in the lipopolysaccharide from colistin-resistant strains of Acinetobacter baumannii
Pelletier MR, Casella LG, Jones JW, et al.
Antimicrob Agents Chemother. 2013

Influence of lipid A acylation pattern on membrane permeability and innate immune stimulation
Li Y, Wang Z, Chen J, Ernst RK, Wang X.
Mar Drugs. 2013