Rapid bacterial identification with medical and security applications

Abstract

Recent studies at Pacific Northwest National Laboratory (PNNL) using infrared spectroscopy combined with statistical analysis have shown the ability to identify and discriminate vegetative bacteria, bacterial spores and background interferents from one another. Since the anthrax releases in 2001, rapid identification of unknown powders has become a necessity. Bacterial endospores are formed by some Bacillus species as a result of the vegetative bacteria undergoing environmental stress, e.g. a lack of nutrients. Endospores are formed as a survival mechanism and are extremely resistant to heat, cold, sunlight and some chemicals. They become airborne easily and are thus readily dispersed which was demonstrated in the Hart building. Fourier Transform Infrared (FTIR) spectroscopy is one of several rapid analytical methods used for bacterial endospore identification. The most common means of bacterial identification is culturing, but this is a time-consuming process, taking hours to days. It is difficult to rapidly identify potentially harmful bacterial agents in a highly reproducible way. Various analytical methods, including FTIR, Raman, photoacoustic FTIR and Matrix Assisted Laser Desorption/Ionization (MALDI) have been used to identify vegetative bacteria and bacterial endospores. Each has shown certain areas of promise, but each has shortcomings in terms of sensitivity, measurement time or portability. IRmore » spectroscopy has been successfully used to distinguish between the sporulated and vegetative state. [1,2] It has also shown its utility at distinguishing between the spores of different species. [2-4] There are several Bacillus species that occur commonly in nature, so it is important to be able to distinguish between the many different species versus those that present an imminent health threat. The spectra of the different sporulated species are all quite similar, though there are some subtle yet reproducible spectroscopic differences. Thus, a more robust and reliable method is needed for differentiation. Using chemometrics, a classification scheme was developed and performed on samples sporulated in glucose broth. PNNL has demonstrated that vegetative bacteria and endospores have unique infrared (IR) signatures that can be used to identify to the species-, and in some cases, even to the strain-level. We have shown that the IR spectra of spores of different species tend to be quite similar, yet the small but reproducible differences in the spectra allow for a certain degree of differentiation. Further studies have shown that the culture medium can also have an effect on the spectra. For the distinction between vegetative and endospores, we have consistently observed a series of four peaks at 766, 725, 702, and a fairly sharp peak (FWHM 7 cm-1) at 660 cm-1, present only in the endospore spectra.« less