Classifications

• • 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
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • 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
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • 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
  • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
  • C12Q2337/10—Anilides
  • C12Q2337/12—Para-Nitroanilides p-NA

Definitions

• This invention relates to a process for identification of microorganisms, especially bacteria, by way of their extracellular enzymes, especially proteases.
• the present process is suprisingly easy to carry out and as compared with previously known processes it is less time-consuming, substantially the same accuracy still being obtained.
• Processes for typing bacteria are based on culturing the bacteria in so many steps that a pure culture is obtained. A sample of the whole or part of such a purely growing colony is thereafter transferred to a special growth plate or a test container. The bacteria are either allowed to grow further on the plate or in the test container and react with reagent ( ⁇ ) added in advance or the bacteria are allowed to grow first and regent(s) added afterwards.
• Added reagents comprise material(s) reacting with substances secreted from the bacteria (extracellular.) or occurring on or in the bacterium cell in such a way that the absence or presence of the substance is apparent - mostly by way of a color change in the added reagent.
• substances secreted from the bacteria extracellular.
• bacterium cell in such a way that the absence or presence of the substance is apparent - mostly by way of a color change in the added reagent.
• a characteristic profile of the bacterium type is obtained which is the basis of the classification ("typing") thereof.
• enzymes examples of substances, the presence of which in bacteria cultures is interesting for typing bacteria, are enzymes.
• the enzymes may be such as react with carbohydrates, peptides or proteins.
• proteases i.e. enzymes, degrading proteins and peptides, has for instance been used in known processes for identification of bacteria.
• Such a previously described process based on protease determination is based on isolation of the bacteria after growth on a solid medium, e.g. an agar plate. Bacteria are thereafter allowed to grow on a special solid medium containing a protein that either can be precipitated or degraded. In both cases the medium must be transparent in order that the reaction might be read. Usually such a demand on the medium is difficult to combine with the demand that the medium should also contain nutrients giving optimal growth as in this case the plate will not be transparent because of such nutrients. Examples are blood and haemoglobin. Alternatively, the bacteria can be allowed to grow in a liquid medium where the growth conditions are often better than on solid media.
• protease determination is carried out in a special step where the liquid phase from the culture medium is isolated by filtering or centrifuging and samples of the liquid phase thus obtained are incubated with a substance, i.e. a substrate reacting with proteases.
• a substance i.e. a substrate reacting with proteases.
• Such substances can either be high-molecular, naturally occurring proteins or low-molecular synthetic so-called peptide substrates. Examples of natural proteins used are casein, gelatin and elastin. However, an occurring positive reaction is not particularly specific as a plurality of enzymes are capable of reacting with these proteins.
• a reagent can be obtained by accurate selection of the amino acids that are to constitute the synthetical peptide part, said reagent making protease reactions much more specific as compared with use of natural proteins according to the above description.
• the marker of the synthetical peptide substrate can be a conventional one such as an ester or amide-linked alcohol or amine that can be easily determined by means of chemical-physical determination methods, for example by means of polarography/electrode surface bound reactions or radioactivity determination or fluorometric, luminescence measuring or spectrophotometric methods.
• the marker is preferably easy to determine in a photometric method, either directly or after having been derivatized, e.g. by means of amines by way of e.g. diazotation or formation of so-called Schiff's bases.
• this object is achieved by means of a process having the characteristic features defined in claim 1.
• This invention is especially suitable for use when identifying bacteria, preferably on the basis of extracellularly secreted bacteria proteases. Accordingly the invention is described in greater detail with reference to such embodiments, however, without restricting the invention to that.
• the invention also relates to use of the present process for diagnosis of bacteria, e.g. Legionella, and especially Legionella pneumophila, in a biological sample.
• bacteria e.g. Legionella, and especially Legionella pneumophila
• the present process means that a colony is removed by a suitable tool from a culture plate on which a pure culture of the bacteria to be determined has been cultured, and an absorbent material is placed on the surface area from which the colony has been removed.
• This absorbent material which preferably consists of a cellulose containing material such as a paper disc but which also can be of cotton or of a synthetical material capable of absorbing liquid through capillary force may be designed in such a way that a definite volume is always obtained in the absorbtion process.
• a test container which for instance can be a cuvette, a test tube or a well in a so-called microtiter plate.
• Reagent is also added to the test container to prove the existence of protease, said reagent being comprised of a suitable synthetical peptide substrate and a buffer medium suitable for the reaction.
• the quantity of released marker is read on an instrument for this purpose, e.g. a fluorimeter for a fluorescent marker or photometer for a chromophore marker such as p-nitroaniline (pNA).
• pNA p-nitroaniline
• it may be suitable to carry out color amplifying reactions with the released marker e.g.
• a culture plate is per se - as distinguished from the conditions in a liquid medium - a mini-chemostat in that the nutrients of the plate medium diffuse continously towards the growing colony and the metabolites diffuse continuously away from it and therefore the enzyme generation always takes place under optimal local conditions.
• the advantages of the higher concentration of substances secreted from the bacteria into the plate under the colony mean that the culture medium in the plate - as distinguished from the conditions in a liquid medium - can be selected more freely and consequently more optimally since it is less important to avoid such media as contain components which could interfere with the measuring process, such as blood, serum and other colored components.
• a colony is removed from a bacteria culture on an agar plate by means of a tool.
• a circular piece of paper is thereafter placed directly on the agar plate for 3 min on the spot where the colony has been removed.
• the piece of paper is brought to a test tube to which 5 ⁇ l of Tris-buffer, 0.2 M, pH 7,8 and 10 ⁇ l of a solution of the substrate SucOMe-Arg-Pro-Tyr-pNA . HCl , 1 mM is added.
• 5 ⁇ l of Tris-buffer, 0.2 M, pH 7,8 and 10 ⁇ l of a solution of the substrate SucOMe-Arg-Pro-Tyr-pNA . HCl , 1 mM is added.
• After 30 min at 37°C 20 ⁇ l of concentrated acetic acid are added and the test tube is shaken. Thereafter 5 ⁇ l of a solution of 0.1 % sodium nitrite are added.
• test tube is shaken and stored for 3 min at room temperature, after which 5 ⁇ l 0.5 % ammonium sulfamate solution are added.
• test tube is shaken and stored for 3 min at room temperature after which 5 ⁇ l 0.1 % N-1-naphtylethylene diamine dihydrochloride are added.
• Red color is developed which confirms the presence in the agar plate of the protease similar to chymotrypsin and characteristic of Legionella pneumophila.

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• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Analytical Chemistry (AREA)
• General Engineering & Computer Science (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• Toxicology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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Citations (5)

• 1987
  • 1987-04-30 SE SE8701801A patent/SE8701801L/ not_active Application Discontinuation
• 1988
  • 1988-04-29 WO PCT/SE1988/000220 patent/WO1988008455A1/en unknown
  • 1988-04-29 AU AU17113/88A patent/AU1711388A/en not_active Abandoned

Patent Citations (5)

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