Rapid and accurate identification of bacterial colonies is important in providing high-quality patient care. It is important for clinicians to know the name of bacteria or fungi that grow from their patients’ specimens to assist in making diagnoses and to decide if and how to treat their patients. Further, if not properly handled, bacterial colonies such as Neisseria meningitidis (often referred to as meningococcus) and the Brucella species pose an exposure risk to personnel working in clinical laboratories.
With the introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (a.k.a., MALDI-TOF MS), laboratories can rapidly identify a wide range of bacteria, including those that pose a risk if incorrectly handled. Once identified, bacteria can be inactivated using tube-based protein extraction preparatory or direct colony testing. While both methods aid in the deactivation of hazardous bacteria, Mayo Clinic researchers conducted a study to determine the most practical solution to work with potentially hazardous bacteria in the era of MALDI-TOF MS.
The study, published in the Journal of Clinical Microbiology, evaluated methods to safely work with organisms using MALDI-TOF MS. Researchers assessed bacterial inactivation times in 70 percent ethanol (EtOH) and bactericidal activities of 70 percent formic acid and MALDI-TOF MS matrix.
According to Robin Patel, M.D., first author on this paper, MALDI-TOF MS is optimal for bacterial identification. Dr. Patel is a Consultant in the Clinical Microbiology and Infectious Diseases Laboratories at Mayo Clinic and Chair of the Division of Clinical Microbiology.
“MALDI-TOF MS is a proteomic method that allows fast, accurate, and inexpensive identification of a wide range of bacteria and fungi. Over the past four years, this technology has become our go-to method for identifying most of these organisms in Mayo Clinic’s clinical microbiology laboratory,” says Dr. Patel.
While tube-based protein extraction performed in a biological safety cabinet (BSC) allows for bacterial identification, it requires clear, defined steps to ensure successful identification—often resulting in additional costs and decreased turnaround time. According to Dr. Patel, when this technology was originally developed, formal protein extraction or the so-called “tube-based protein extraction” method was standard. It involves a few steps, including centrifugation.
Alternatively, direct colony testing allows Mayo Clinic to test directly without much manipulation. By adding one step where technicians treat the colony on the plate with a small amount of formic acid, the laboratory can generally attain results equivalent to tube-based protein extraction.
“Direct colony testing has streamlined our workflow significantly,” addsDr. Patel. “With it, we are able to identify colonies growing on plates in minutes, which is much shorter than the turnaround time with alternative methods.”
The study results indicated that 70 percent formic acid and MALDI-TOF MS matrix each inactivated all bacteria tested. This suggests that laboratories may safely identify potentially hazardous bacteria by MALDI-TOF MS using either on-plate formic acid-based extraction or direct colony testing, provided that processing is performed in a BSC and that all bacterial material is treatedwith 70 percent formic acid and/or MALDI-TOF MS matrix before MALDI-TOF MS plates are removed from the BSC.
The results also suggest that when using tube-based extraction, non-spore-forming bacteria may be treated with 70 percent EtOH for 5 minutes in a BSC. The rest of the procedure can be performed outside of the BSC, but spore-forming bacteria tested using tube-based extraction should remain inside a BSC until treated with formic acid/acetonitrile.
In terms of safety, these results are positive because they suggest that special treatment of potentially hazardous bacterial cells, including tube-based extraction, is unnecessary. Use of common MALDI-TOF MS reagents with appropriate steps performed in a BSC provides an accurate and safe tool to rapidly identify potentially hazardous bacteria by MALDI-TOF MS using on-plate testing.
“While we have extensive safety practices in place in our laboratory, we don’t know for sure what the organism is until we’ve identified it. If there is a possibility that it could be hazardous, it requires special handling until such a time as we know it’s not a hazardous organism,” says Dr. Patel. “In this study, we showed a safe way to handle such organisms and get a rapid identification using MALDI-TOF MS.”