Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
  • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes

Definitions

• the present invention concerns the identification of micro-organisms, and more particularly to the differentiation between strains of bacteria.
• Bio-typing has been used to try to provide the required differentiation. Bio-typing investigates the differences in, for example, toxin production and trytophan dependence between different strains. Even this method frequently fails to identify strains, even where the strains are derived from different patients and where a nosocomial incident (cross-infection) has occurred.
• MRSA Methycillin Resistant Staphylococcus Aureus
• the analysis of mycobacteria present particular problems.
• the pathogenic forms, such as Mycobacterium tuberculosis are typically slow to grow and accurate identification by traditional methods can take many weeks.
• early identification of tuberculosis is vital in the management of the infection in patients.
• Chromatographic analysis of derivatised mycolic acids and cell wall lipids is the current method for identification of mycobacteria.
• such a method requires relatively large amounts of culture for the extraction and derivatisation steps .
• MALDI-TOF-MS Matrix-Assisted Laser Desorption Ionisation Mass Spectrometry
• MALDI-TOF-MS spectral data for micro-organisms is spectral data which is dependent on the molecular structure of the microorganisms concerned. In effect, therefore, such data is a 'finger print' for the organism concerned. As a microorganism transmogrifies, its MALDI-TOF-MS spectral data will change to reflect the change in structure/composition of the organism.
• MALDI-TOF-MS matrix assisted laser desorption ionisation time of flight mass spectrometry
• apparatus for the screening of micro-organisms characterised in that the apparatus comprises :-
• the invention enables the identification of microorganisms quickly and without the labour intensive procedures hitherto employed.
• MALDI-TOF-MS is a high performance mass spectrometry tool, which may be operated by the non-specialist. Furthermore, the equipment required to perform the analysis is relatively inexpensive.
• the method and/or apparatus of the invention uses dried, solid samples which are easy to handle and store, and which are capable of analysis in accordance with the invention over a wide mass range.
• the micro-organism Prior to analysis, the micro-organism may be cultured by conventional means, for example, on a nutrient agar plate.
• the spectrometer samples may be prepared by taking microorganism cells e.g. from a culture medium and applying them directly • to a sample plate and a matrix added. Alternatively the cells may be admixed with the matrix prior to application so that cellular integrity may be retained .
• the dried matrix-sample mixture is bombarded with laser energy to create gas phase ionic species which are then pulsed into a flight tube. Both positive and negative ions may be generated and identified,
• the species may be identified by their mass to charge ratio (m/z), which can be identified with an accuracy of better than 0.1%.
• the m/z value of each spectral peak may be identified from the centr ⁇ id of the peak corresponding to the to the average molecular mass of the particular M+H ion (in the positive ion mode).
• the spectral data of the unidentified micro-organism is derived from a plurality of laser shots of the sample.
• the position of the laser energy impinging on the sample is varied, between shots, over the sample spot.
• This multiple shot technique may assist accurate, reproducible spectral data to be produced.
• spectra are taken from each and every tracking position over the sample area.
• a linear analyzer may be used to enhance the sensitivity of the data where limited amounts of sample are available .
• MALDI-TOF-MS produces unique spectra for each microorganism allowing identification and differentiation between different genera of micro-organisms and even within different strains.
• the present invention is also useful for the analysis of mycobacteria, since the MALDI-TOF-MS method requires very small amounts of material, can be completed within minutes, and requires no derivatisation step.
• each spectra compiled in the database is recorded as a graphical representation of a plot of intensity against mass to charge ratio.
• the spectral data may be recorded in graphic, numerical or electronic format, in digital or analogue form.
• each spectra is recorded on the storage medium of a computer or computer network.
• the storage medium may be magnetic, such as floppy disk, tape or hard disk.
• the storage medium may be optical, such as CD-ROM or laser-disc, in another embodiment the storage medium may be ROM microchips.
• each spectra is stored as a series of mass to charge ratios each corresponding to a centroid of the peaks of each spectrum.
• mass to charge ratios the minimum data required to characterize and/or identify the micro-oranisms is stored.
• the data may be arranged in groups of data corresponding to the genus of each micro-organism, with sub-divisions corresponding to the strain of micro-organism.
• the software may carry out the steps of acquiring and storing the unidentified micro-organism's spectral data by manual input or, preferably directly from the spectrometer, comparing the unidentified spectral data with the pre-characterised spectral data in the database and indicating where a match is found, or otherwise.
• the database may include spectral data grouped according to the genus of each micro-organism and the software may be carry out a search strategy which involves comparing spectral data first to identify the genus of the unidentified bacteria and then to identify the strain of bacteria. In this way the search process is optimized to improve efficiency where a large range of spectral data is stored in the database.
• the software may be capable of allowing for calibration errors in the unidentified spectral data, so that the trends (e.g. the difference in m/z value between adjacent peaks in the data) may be screened with the corresponding data in the database rather than the absolute values.
• the computer may be incorporated into the MALDI-TOF-MS Spectrometer so that data is transferred directly from the detector to the computer for analysis.
• the computer may be spaced apart from the spectrometer and linked by remote means.
• the present invention also includes a database of microorganisms comprising spectral data of each micro-organism obtained by MALDI-TOF-MS analysis for use in the method, or in conjunction with the apparatus, of the invention.
• Figure 1 is a graphical representation of partial spectra obtained from samples including SA, CF and ECC2.
• peaks A-K represent ⁇ number of possible isobaric species of mycoserates .
• the relative peak heights are related to the number of isobaric species present at each molecular weight.
• the peak letter, molecular weight, and one of the possible isobaric structures are listed as follows:
• the ten micro-organisms were each sub-cultured in duplicate onto separate nutrient agar plates. Each pair was incubated at 37 ⁇ C, for three and six days respectively .
• a single, visible colony of cells was regarded as sufficient material for analysis and was removed from the agar plate with a sterile loop. All samples were analyzed in a matrix of ⁇ -cyano-4-hydroxycinnamic acid ( ⁇ -CHCA). The organisms were emulsified in 50-100 ul of the matrix, dissolved in a mixture of water, ethanol and acetonitrile (in the ratio 1:1:1).
• a 1 ul aliquot was then applied to the sample target for the laser and allowed to dry before analysis.
• the samples were then analyzed by MALDI on a KRATOS" Kompact MALDI III time of flight mass spectrometer.
• a nitrogen laser giving a 337 nm output of 3 ns pulse width was use to ionize the species.
• the laser fluence was set just above the threshold for ion production.
• the mass spectrometer was used in the positive ion detection mode using an acceleration voltage of +20 KV. Samples were analyzed by using both the linear and reflectron analyzers.
• the detector used consisted of a discrete dynode combine with an electron multiplier.
• FIG. 1 shows the spectra of SA, CF and ECC2. There is a clear difference in the distribution of the peaks corresponding to ionic species from each microorganism.
• the spectra produced constitute a unique " ingerprint" which allows identification of icro- organisms ihter-species and inter-strain.
• the similarities in the features of each spectra allow the spectra to be grouped according to genus of micro- organis ,
• Figure 2 shows the positive ion spectrum of Mycobacterium smegmatis which shows an interesting pattern of peaks between 1150-1300 Da. Previous analyses of cell wall extracts from this organism suggest that these peaks are either mycolic acids or related lipids within the cell wall. The data probably relates to dimycocerosates of the phthiocerol family. These are non-covalently bound and are probably extracted into the matrix during the admixture process. The proposed structure of the mycoserates are shown in the legend of figure 2.
• Table 1 lists subsets of m/z values for all the micro- organisms examined.
• the m/z ratios of the spectral peaks of the four cultures of E.COLI bacteria are shown in the first column of table 1 for the m/z range 1058-1852 Da.
• the figure in bold type are peaks present in all the tested strains of the genus E.COLI (1102, 1231, 1316, 1360, 1489 and 1618).
• the strain may be identified by the absence or presence of further peaks.
• ECN has a peak at 1852 which differentiates from ECC1 and ECC2.
• ECK also has a peak at 1852, but may be differentiated by the additional peaks at 1058 and 1187.
• CF and KA Different species which are closely related to E.COLI are CF and KA. These have peaks 1102, 1231, 1360, 1489, 1618 in common with E.Coli, but may be differentiated therefrom by the absence of the peak at 1316 which is characteristic of E.Coli. CF and KA may be distinguished by the absence or presence of the peaks at 750, 751, 1314, 1445, 1480, 1575, 1582 and 1876.
• the spectra produced provide a "fingerprint" for each micro-organism.
• the fingerprint is sensitive to both inter-genus differences, and inter- strain differences.
• a database of these fingerprints provides a method of rapidly screening samples to identify the strain of micro-organism present. This has particular application in the field of medical microbiology, -but will find application in many other fields where the precise and rapid identification of micro-organisms is required.
• a database of MALDI-TOF-MS spectral data was prepared in respect of the following organisms :-
• Da t a aOOOl.l 17 Jul 97 17:18 Cal: test 25 Ap r 97 li - ⁇ Kr Ma.to» «s Kompa»c_t_.MA complicat_LDI 2: V v 5 o* 2 **•• 1 • + ri - u v .

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• 1996
  • 1996-08-27 GB GBGB9617852.0A patent/GB9617852D0/en active Pending
• 1997
  • 1997-08-26 CA CA002264535A patent/CA2264535A1/en not_active Abandoned
  • 1997-08-26 TR TR1999/00780T patent/TR199900780T2/xx unknown
  • 1997-08-26 EP EP97937712A patent/EP0922295A1/de not_active Withdrawn
  • 1997-08-26 AU AU40245/97A patent/AU4024597A/en not_active Abandoned
  • 1997-08-26 BR BR9711448-0A patent/BR9711448A/pt not_active Application Discontinuation
  • 1997-08-26 WO PCT/GB1997/002278 patent/WO1998009314A1/en not_active Ceased
  • 1997-08-26 CN CN97199172A patent/CN1234906A/zh active Pending
  • 1997-08-26 KR KR1019997001675A patent/KR20000035935A/ko not_active Withdrawn
  • 1997-08-26 JP JP10511367A patent/JP2001502164A/ja active Pending

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