Rapid bacterial identification with medical and security applications

With the recent anthrax outbreak in the United States, rapid detection of dangerous bacteria has become a security aim of considerable importance. In general, vegetative cells of some Bacillus species form endospores when subject to environmental stressors, such as lack of nutrients. This survival mechanism renders them extremely resistant to heat, cold, sunlight, and certain chemicals. Easily airborne, they readily disperse and can cause illness when inhaled. In 2001 mail containing anthrax spores caused the Hart Senate Building in Washington DC and other locations to be shut down for up to several months.

The most common method of bacterial identification is through cultures, but this is a time-consuming process that takes hours or even days. It is difficult to quickly and reliably identify potentially harmful species or strains. Analytical techniques include Fourier transform infrared (FTIR), Raman spectroscopy, photoacoustic FTIR, and matrix-assisted laser desorption/ionization (MALDI). All have shown promise but each has shortcomings in terms of sensitivity, measurement time, or portability.

Infrared spectroscopy has been used successfully to distinguish between sporulated and vegetative states.^{1, 2} It can also differentiate species: crucial because some non-pathogenic Bacillus are indigenous to the environment.^{1, 3, 4} Spectra of the various sporulated species are all quite similar, though subtle yet reproducible spectroscopic differences do exist. A robust detection system requires reliable differentiation.

We at the Pacific Northwest National\ Laboratory (PNNL) and others have demonstrated that vegetative bacteria and endospores have unique IR signatures that can identify species and, in some cases, discriminate at even the strain level.^1–4 Using chemometrics, we have developed a classification scheme for several Bacillus samples sporulated in glucose broth.³ For example, Figure 1 shows the IR spectra of three different Bacillus species, all in the sporulated state. Although similar, some subtle differences—such as the broad sugar peak near 1100cm⁻¹—are quite reproducible. Position and amplitude of the small shoulder peak near 912cm⁻¹ also vary by species. Chemometric studies show that baseline correction and taking the second derivative significantly improve IR capacity to discriminate, and also to appropriately cluster the spectra for the identification of unknowns.^1–4 Further research has shown that even the culture medium can affect spectra.

Peaks responsible for distinguishing the sporulated from vegetative state, together with their potential assignments, have also been demonstrated. Figure 2 shows the IR spectra of both forms of the microbial insecticide Bacillus thuringiensis israelensis (Bti). Here we found that the quartet of peaks at 766, 725, 702, and 660cm⁻¹ (indicated by arrow and bracket) are consistently found only in the endospore spectra, but not in the IR spectra of the vegetative form. These four are likely associated with calcium dipicolinate.³ Conversely, spectra of the vegetative bacteria consistently indicate a peak (or strong shoulder) at 1738cm⁻¹ that is not present in the spores. This peak, in Figure 2 highlighted by the arrow at the left, is not associated with the Amide I or Amide II bands of proteins, but it shows up in the vegetative spectra of many Bacillus species.

PNNL has established a vigorous research program for identification and attribution of microbiological endospores using analytical techniques that make it possible to easily distinguish vegetative bacteria and endospores from interferents in a timely way. Such measures may eliminate the need to shut down buildings for long periods of time in cases of suspected attack. We plan to leverage our experience in developing vapor- and liquid-phase infrared spectral libraries to help construct a well-documented and vetted set of standard signatures. This will include spectra of species of interest, together with aerosols and other interferent materials, for use in food protection, medical applications, and homeland security.