Trace detection of chemical markers for bacteria in clinical and environmental matrices

Muramic acid (Mur), a component of peptidoglycan in eubacteria, serves as a chemical marker for the presence of viable and non-viable bacteria as well as their degraded cell wall remnants in clinical and environmental samples. It has been hypothesized that bacteria and their cell wall remnants may play role in inflammatory diseases of unknown etiology. In order to evaluate this hypothesis, it must first be determined if Mur is present in normal mammalian tissues where previous reports have given conflicting results. Mur was not detected in the brains and spleens of normal rats using gas chromatographytandem mass spectrometry (GC-MS/MS). In contrast, Mur was readily detected in the cerebrospinal fluid of patients with pneumococcal meningitis. Human, culture negative spleens also did not generally contain detectable levels. However, Mur was detected at minute concentrations, 50 parts per billion [ppb], in one spleen by GC-MS/MS and the more advanced technique of GC-MS3. There can be an enormous difference in the levels of Mur found in different mammalian tissues and body fluids in health and disease. However, when it is present, this suggests that bacterial cell walls may be implicated in the underlying disease process. In an environmental application of GC-MS/MS techniques, lunar dust was analyzed for the presence of Mur and 3-hydroxy fatty acids, a chemical marker for bacterial lipopolysaccharide, as evidence of terrestrial biocontamination. The lunar sample collection has been in storage for the past 30 years. Both chemical markers were detected in one sample indicating the presence of bacterial contamination. The power of GC-MS/MS techniques in assessing the levels of terrestrial contamination of meteorites as well as in the future return of samples from Mars is demonstrated by this technique. Finally, in a continuing effort to decrease the limit of detection of Mur a stable, halogenated derivative was analyzed by negative ion chemical ionization GC-MS/MS. The utility of the technique was demonstrated in the trace detection of Mur in settled dust as well as airborne dust collected on filters. The limit of detection of Mur was comparable to previous methodologies and offers a complimentary technique in confirming its presence.