Researchers Use New Method To Accurately Identify Bacteria

Charles Wilkins, Distinguished Professor of chemistry and biochemistry, Jack Lay, director of the Arkansas Statewide Mass Spectrometry Facility, and their colleagues reported their findings in a recent issue of Analytical Chemistry.

The most common way to identify bacteria involves isolating them, growing them and examining them under microscopes, but this method is time-consuming, sometimes taking weeks to produce an identification. After the anthrax outbreak in 2001 when people were exposed to the deadly bacteria through contamination of mail, more researchers seriously began to study rapid methods of bacteria identification. Many bacteria exist in both deadly and benign strains, so tests that identify bacteria must do so down to the strain level. Researchers hope to get at this level by looking at proteins within bacteria and finding proteins unique to each strain.

Researchers seeking to speed up and simplify the identification process started using a technique called matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to examine bacterial proteins in an attempt to identify bacteria from specific protein markers. This technique, called time-of-flight mass spectrometry (MALDI-TOFMS), relies on ionizing bacteria, shooting the particles down a tube and measuring the amount of time it takes to go down the tube, then calculating the masses using the time it takes particles of known mass to travel down the tube. Although the method works more rapidly than microscopy, it leaves a big margin for error, and often it cannot distinguish between specific proteins within the bacteria.

"There are other things besides mass that can affect how long it takes a particle to go down the tube," Wilkins said.

The researchers decided to compare the current method with a different type of MALDI called Fourier transform mass spectrometry (MALDI-FTMS)--a technique that Lay calls "the Cadillac of mass spectrometry." In this technique, a laser beam ionizes the bacteria, and the ions follow a circular path in a magnetic field, each one cycling at a specific frequency that is directly related to its mass and to the magnetic field strength. The researchers can measure frequency with precision, which allows them to make accurate calculations of protein masses.

The investigators used Escherichia coli, a well-characterized bacteria that lives in the gut of humans and other animals and occasionally causes illness in humans. The E. coli genome, which codes for 4,300 proteins, has been mapped, and researchers can access information about these proteins in a computer database. Thus, they can compare the masses obtained through mass spectrometry techniques with the known information in the database and see how closely they correspond.

They found that the FTMS method had an error of 26 parts per million as opposed to an error of 200 parts per million for the time-of-flight method.

Analyzing bacteria will continue to challenge researchers, because the organisms respond to their environment more rapidly than almost anything else in nature, Lay said. To offset this issue, researchers will need to reproduce the same conditions in studies each time.

Even if the mass spectrometry technique never becomes a standard diagnostic test, it could still prove useful to medical researchers. E. coli, like other bacteria, have both toxic and non-toxic strains. Wilkins and Lay hope to identify proteins that differentiate the benign and the disease-causing strains, and identify protein markers in antibiotic-resistant microorganisms.

"The data produced from our studies may simplify other tests down the road," Lay said.

Eventually, identifying protein markers in bacteria such as E. Coli may allow medical researchers to focus on certain proteins involved in disease, leading to a new generation of anti-microbial medicines.