Rapid Detection of Bacteria using Mass Spectrometric Analysis

Abstract

Methods for the detection or diagnosis of a bacterial infection or colonisation utilising mass spectrometric analysis are provided. The methods involve short-term enrichment of samples followed by mass spectrometric analysis of biomarker profiles. Also provided are methods for preparing short-term enrichment cultures.

1 . A method for detection of the presence or absence of particular bacteria in a clinical sample, the method comprising:
 (i) enriching the clinical sample by culturing the bacteria present in the sample for a period of time less than 6 hours such that pre-modification biomarkers are produced in the sample, wherein enrichment is carried out by:
 inoculating the bacterial-containing clinical sample into a broth; 
 culturing the clinical sample-containing broth for a period of less than 6 hours; and
 separating an enriched clinical sample from the sample-containing broth by centrifugation, and 
 wherein said broth is prepared by a method comprising:
 reconstituting a powdered microbiological culture material in water; 
 autoclaving the reconstituted solution at a temperature between 80 and 150° C.; 
 leaving the solution to cool in the autoclave to a pre-determined temperature of between 60 and 90° C.; 
 removing the solution from the autoclave when the predetermined temperature is reached; and 
 allowing the solution to continue cooling at room temperature; 
 
 
   (ii) analysing the enriched clinical sample by mass spectrometry, to obtain biomarker profile data for the clinical sample, wherein the biomarker profile data comprises peaks of specific masses corresponding to pre-modification biomarkers produced by the bacteria in the sample;   (iii) correlating the clinical sample biomarker profile data with reference data to look for peaks having specific masses common to the sample biomarker profile data and to the reference data, to determine the presence of absence of said particular bacteria in the sample;   wherein a match between the masses of the peaks of the biomarker profile of the enriched clinical sample and the masses of the peaks of the biomarker profile of the reference clinical sample indicates the presence of the particular bacteria in the sample,   wherein the reference data relates to pre-modification biomarkers represented by peaks having specific masses produced by a reference sample of said particular bacteria which has been cultured for less than six hours, and   wherein the concentration of bacteria in the clinical sample before enrichment is about 10 7  cfu/ml or below.   
     
     
         2 . A method according to  claim 1 , wherein the pre-modification biomarkers are pre-modification proteins, peptidoglycans, glycoproteins, glycopeptides, carbohydrates, and/or lipopolysaccharides. 
     
     
         3 . A method according to  claim 2 , wherein the pre-modification proteins are pre-PTM (posttranslational modification) proteins. 
     
     
         4 . A method according to  claim 2 , wherein the pre-modification biomarkers comprise unglycated cell wall peptides. 
     
     
         5 . A method according to  claim 1 , wherein the pre-modification biomarkers comprise biomarkers with a molecular mass below 4000 Da, or from 1000 to 4000 Da. 
     
     
         6 . A method according to  claim 1 , wherein the culturing in step (i) is carried out for four hours or less. 
     
     
         7 . A method according to  claim 1 , wherein the culturing in step (i) is carried out for two hours or less. 
     
     
         8 . A method according to  claim 1 , wherein the said bacteria is a Gram positive bacteria. 
     
     
         9 . A method according to  claim 1 , wherein said bacteria is a  Staphylococcus.    
     
     
         10 . A method according to  claim 9 , wherein said bacteria is methicillin-resistant  Staphylococcus aureus.    
     
     
         11 . A method according to  claim 1 , wherein the sample is a clinical sample taken from a human patient. 
     
     
         12 . A method according to  claim 1 , wherein the sample is taken from a wound site, an ulcer, or a screening site. 
     
     
         13 . A method according to  claim 1 , wherein said biomarker profile data is proteomic profile data. 
     
     
         14 . A method according to  claim 1 , wherein the analysis by mass spectrometry is performed using MALDI-TOF mass spectrometry. 
     
     
         15 . A method according to  claim 1 , wherein the broth is a Brain Heart Infusion Broth, a Mueller-Hinton Broth, an anaerobic broth or a nutrient broth. 
     
     
         16 . A method according to  claim 1 , wherein culturing the sample comprises incubation in a water bath. 
     
     
         17 . A method according to  claim 1 , wherein the centrifugation step comprises:
 dividing the sample-containing broth into a plurality of aliquots;   centrifuging the aliquots;   discarding the resultant supernatants;   re-suspending the pellets in water;   pooling the resultant pellets; and   centrifuging a suspension of the pooled pellets, to separate the pellet containing the enriched sample.   
     
     
         18 . Apparatus for use in a method according to  claim 17 , comprising a mass spectrometer and a data processor, the processor being programmed with said reference data for identifying one or more particular bacteria, the reference data comprising peaks of the biomarker profiles of each particular bacteria, wherein the biomarker profiles of the particular bacteria have been obtained by enriching a sample of each particular bacteria by culturing for a period of time less than 6 hours. 
     
     
         19 . Apparatus according to  claim 18 , wherein the mass spectrometer is a MALDI-TOF mass spectrometer. 
     
     
         20 . Apparatus according to  claim 18 , comprising an automated system for culturing a sample for analysis in the mass spectrometer. 
     
     
         21 . Apparatus according to  claim 20 , wherein the automated system comprises:
 an incubator for heating the sample during culturing;   a temperature control device for controlling the temperature of the incubator;   a centrifuge for separation of the cultured sample from the culture medium.   
     
     
         22 . The method of  claim 1 , wherein the particular bacteria being detected is selected from the group consisting of  Staphylococci, Clostridium, Escherichia coli, Campylobacter, Salmonella, Pseudomonas, Shigella, Neisseria, Klebsiella, Vibrio, Legionella, H. Influenza, H. pylori, Bacillus, Listeria , and bacteria causing tuberculosis and leprosy.