BACLIB Method

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.

A BACLIB MALDI target plate developed by Pataigin, with a 1 µL sample drop on one target spot
Fig. 1A MALDI target plate developed by Pataigin for BACLIB, with a 1 µL sample drop on a target.

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 five FLAT steps: apply sample, apply buffer, heat plate, rinse with water, MALDI matrix
Fig. 2Sorensen et al., Sci Rep 2020

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

BACLIB spectra of Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus and Candida auris, each with a few labelled peaks between m/z 1000 and 2400
Fig. 3Sorensen et al., Sci Rep 2020

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.

100015002000 m/z179618761377172819101047135413291357

E. coli · Gram-negative · lipid A

Fig. 4Schematic: the labelled peaks of the four spectra in Fig. 3 on a single axis. Each species shows only a few peaks, in different places. Peak positions and rough heights from Sorensen et al., Sci Rep 2020.