Sunil Palchaudhuri, Ph.D., left, and Steven Rehse, Ph.D., prepare a bacterial culture for Laser Induced Breakdown Spectroscopy.
Sunil Palchaudhuri, D.Sc., Ph.D., a professor in the Department of Immunology and Microbiology in the School of Medicine, and Steven Rehse, Ph.D., an assistant professor of the Department of Physics and Astronomy, are using Laser Induced Breakdown Spectroscopy to identify the atomic “fingerprints” of bacteria. Their goal is to develop a computerized reference library of bacterial fingerprints to speed identification of infection and treatment, as well as assist in selection of specified antibiotics rather than an attack with broad-spectrum antibiotics.
Laser Induced Breakdown Spectroscopy, or LIBS, works like this: An intense pulse of laser light, Dr. Rehse explained, vaporizes a small amount of the targeted substance, in this case, bacteria. The vaporization causes a plasma plume in which the bacteria’s base elements are excited and ionized. As the plume cools, the atoms, ions and molecules lose energy by emitting optical wavelength photons. This resulting light, he said, is collected by a spectrometer, and the peaks are analyzed to identify all the elements that were initially present in the targeted substance. The process can be used to identify elements in the target or how much of a substance the target contained.
After a blood sample is taken and the bacteria are separated from the human cells, the bacteria sample is mounted on a glass slide or agar bed. The sample is then exposed to a laser beam produced by an infrared pulsed laser. In effect, the laser burns away all of the atoms in the bacteria, creating a glowing spark called a plasma. The light from the spark is collected by an optical fiber in an Echelle spectrometer and the information is fed into a computer for analysis. The computer then reads the resulting spectrum to produce an atomic fingerprint unique to each bacterial species. That fingerprint can be read against an ongoing library of such files until a match is found, identifying the strain of bacteria.
“The photons given off during spontaneous emission can be collected and spectrally analyzed, which provides a ‘spectral fingerprint’ of all the constituent elements of the target and the plasma,” Dr. Rehse said. “Since each element has a unique spectral fingerprint, relative and absolute elemental concentrations within the target material can be determined.”
Such technology, Dr. Palchaudhuri said, is “desperately needed to identify bacteria in clinical samples and patients at ‘time zero’ – the time when a clinical sample of blood, urine or sputum is obtained – with little or no sample preparation. This ability to identify the bacteria onsite will help doctors initiate treatment of the disease immediately, without waiting for offsite lab results to return.”
Ultimately, Dr. Palchaudhuri and Dr. Rehse said, physicians, nurses and technicians could take a sample from a patient and have an accurate diagnosis of bacterial infection within minutes or an hour instead of days or weeks.
Such technology could play a crucial role in outbreaks of illness such as tuberculosis or meningitis, not only in the treatment of patients, but in tracing the strain to help identify the origin and spread of the disease. The technology could also provide rapid identification of biochemical warfare agents used against the military or civilian targets.
“With LIBS, we hope to provide an immediate diagnosis of multiple pathogens in multiple sectors across our society -- medical, food and environment -- at a relatively low cost,” Dr. Palchaudhuri said.
As an example, he pointed to recent outbreaks of salmonella and E. coli strains in the nation’s food source. Some E. coli strains can be deadly if consumed via contaminated food. The various strains are closely linked, and physicians and lab technicians require days to develop cultures to identify the strain they are treating. A LIBS reference system, however, could quickly identify the trace atomic differences between the strains, helping identify not only the potential source of contamination, but also better inform physicians which antibiotic to prescribe.
Accurate identification could also minimize the use of antibiotics. Delay and uncertainty of identification, Dr. Palchaudhuri said, have led to “ever-increasing rates of food-borne and water-borne outbreaks globally. In consequence, overuse and abuse of broad spectrum antibiotics not only costs billions of dollars, but also helps evolve antibiotic resistant bacteria.”
Dr. Rehse points out that LIBS systems are already in use in a number of applications. The Army has a prototype system that can identify explosive powders on roadside objects from a distance of up to 100 meters. The system is mounted on a Humvee and could be used to detect the presence of roadside improvised explosive devices. Similar systems are in place in European factories, used to sort recyclable materials and for glass quality control analysis. A commercial instrument, the PharmaLIBS, incorporates the same technology to sort and classify pharmaceuticals in real time.
A LIBS system, Dr. Rehse added, is scheduled to go into space as part of the 2012 Mars Science Laboratory to chemically characterize rocks up to five meters from the surface rover, and search for the existence of exobiological signatures – signs of chemical evolution on the planet.
“The technology is mature, field-able, and ready for design and implementation now. This is not a ‘laboratory-only’ technique,” Dr. Rehse said. “But, up until now, Sunil and I are really the only people who are interested in its use to identify bacteria. We are applying for funding from multiple sources, but our proposal to the U.S. Defense Advanced Research Projects Agency would have us producing (in conjunction with private sector and Army collaborators) a working prototype in three to four years. This would be able to test a human specimen of blood, urine, or some other fluid and identify bacteria in it.”