Identification of immobilized bacteria by aminopeptidase profiling

Abstract

Conventional aminopeptidase methods require cell concentrations of approximately 10s to 101°cells/mL and an Incubation period In the labeled substrates of 20 h. In order to obtain the large number of cells required to perform this assay, a 36-48-h growth period must precede the assay. An Improved procedure Is described that combines time-resolved fluorometry and a nondestructive whole cell Immobilization procedure. This method has reduced cell concentrations 80 000-fold from that required for the standard assay, has reduced the room-temperature incubation period from 20 h to 3 min, and has shortened the total turnaround time for the assay from 2.5 days to approximately 3-7 h.At present a major goal of "improved" microbial identification techniques is to reduce the time required for identification. The turnaround time can be broken into two specific elements. The first is the growth period required to isolate

Microorganism gram-type differentiation based on pyrolysis-mass spectrometry of bacterial Fatty Acid methyl ester extracts

“…Application of instrumentation-based techniques in microbiology to perform conventional procedures for the identification and differentiation of microorganisms has increased during the last decade. Gas chromatography (GC) (12,24), fast atom bombardment mass spectrometry (MS) (17), Curie-point pyrolysis (Py)-MS (8), Py-GC-MS (31), Fourier transform infrared spectroscopy (27), UV resonance Raman spectroscopy (7), flow cytometry (10), and laser-based enzyme profiling (3,20) have all been used to differentiate microorganisms. These techniques apply established analytical methods to measure a distinct biochemical property and/or chemical distribution of taxonomic significance.…”

Application of capillary electrophoresis coupling with electrochemiluminescence detection to estimate activity of leucine aminopeptidas

“…Assay of LAP activity in vitro was also carried out by fluorescence spectrometry using β ‐naphthylamlde derivative as substrate (Hughes et al ., ; Mantle et al ., ; Xiong et al ., ) This method was more sensitive for LAP activity assay in vitro , but the technical limitations caused by interfering effects of the matrix components in crude biological samples made the results inaccurate and irreproducible. Lytle's group further described a new technique referred to as electrophoretically mediated microanalysis, which allows enzyme assays to be performed in a single capillary electrophoresis column using time‐resolved laser‐induced fluorescence detection (Miller et al ., ) and on a microfabricated electrophoresis chip using two‐photon excited fluorescence detection (Zugel et al ., ), respectively.…”

Simultaneous Multiple Substrate Tag Detection with ESI-Ion Trap MS for In Vivo Bacterial Enzyme Activity Profiling

“…Aminopeptidases are a group of exopeptidase enzymes that hydrolyze single amino acid residues of a peptide at the amino terminus (N-terminus). Aminopeptidase enzyme profiles have been used to identify and differentiate different species of bacteria and genetically modified bacteria from the wild-type bacteria − and pathogenic fungi . In these studies, the microbial samples were incubated with a series of amino acid tagged derivatives for a specified amount of time, and the extent of each amino acid aminopeptidase activity related directly to the amount of free tag detected.…”