Rapid detection of bacterial pathogens without the need for culturing or time-consuming and costly genomic methods is evolving as an important goal in clinical diagnostics. Similarly, detection of novel or emergent pathogens is critically important to ensuring the safety of U.S. citizens and U.S. armed forces overseas who are susceptible to contracting diseases, such as pneumonia and meningitis (S. pneumoniae), persistent infections (P. aeruginosa), nosocomial infections (A. baumannii), cholera (V. cholerae), typhoid fever (S. typhi), and food poisoning (S. typhimurium). Current detection methods of bacterial culture or genomic sequencing are expensive, slow, and potentially inaccurate. Development of rapid and informative diagnostics will enable clinicians to quickly determine the most effective treatment for infected individuals. 

Identify pathogenic bacteria based on virulence-correlated enzyme activity with a rapid and easy fluorescence measurement.

To address some of these challenges, researchers have developed a fluorescent, enzyme-selective probe to screen directly for pathogenic phenotypes. Using a small molecule activity-based fluorescent probe, potential disease-causing bacteria can be quickly identified and isolated in tens of minutes compared to current detection methods that can take days and require culturing. Neuraminidase, a glycoside hydrolase capable of cleaving sialic acid residues, is a commonly conserved enzyme across many different species of pathogenic bacteria. Neuraminidases are common in bacterial biofilms and used by bacteria for host cell invasion.

A chemical probe capable of labeling pathogenic bacteria

Researchers at PNNL have synthesized a novel, patent-pending, activity-based probe for bacterial neuraminidase. This probe labels pathogenic bacteria for identification and subsequent isolation using fluorescent-activated cell sorting (FACS). This discovery would allow physicians, for the first time, to identify pathogenic bacteria based on virulence-correlated enzyme activity with a rapid and easy fluorescence measurement.

A fluorescent activity-based probe rapidly detects pathogenic bacterial presence, and FACS then can be used to isolate and culture the species to establish treatment.

Unlike a genomics approach, this approach is a robust and high-throughput method of detection, in which many classes and species of bacteria can be screened simultaneously. Neuraminidases are conserved among several gram positive and gram-negative bacteria, including S. pneumoniae, P. aeruginosa, A. baumannii, V. cholerae, S. typhi, and S. typhimurium. By targeting a commonly observed virulence factor, such as neuraminidase expression, researchers were able to develop an activity-based probe that could screen—for the first time—across a wide scope of pathogenic bacteria simultaneously. Such a capability would aid in rapid detection and isolation of pathogenic bacteria.

Activity-based probes for neuraminidase will

• rapidly identify pathogenic bacterial phenotypes
• expand on previously discovered knowledge regarding specific mechanisms by which these pathogens can cause disease
• isolate new or emergent microbes to study and, eventually, develop methods of treatment.

By coupling these together, scientists can now identify, investigate, and inform the way that this class of enzyme operates within the pathogenesis of these bacteria.