Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-12-19
2003-12-09
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S029000, C435S018000, C435S021000, C435S032000, C435S252310, C435S253300, C435S975000, C548S414000
Reexamination Certificate
active
06660494
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel compounds and substrates having utility for detection of microbial metabolites, i.e. substances secreted or otherwise produced by such microorganisms, by color formation upon contact with such metabolites, as well as to methods of producing such compounds and substrates and of using them for detection and identification of various microorganisms including bacteria.
PRIOR ART
Phospholipase C enzymes are found in a variety of microbes. These enzymes have been associated with the pathogenicity of the microbes to its host.
More specifically, it is known that an enzyme named “phosphatidylcholine-specific phospholipase C (also known as phosphatidylcholine cholinephosphohydrolase, or lecithinase C, termed PC-PLC herein for short; enzyme classification EC 3.1.4.3) can be found in a variety of microbes including
Clostridium perfringens, Clostridium novyi, Bacillus cereus, Bacillus thuringiensis, Pseudomonas aeruginosa
and
Staphylococcus aureus
(cf. J. G. Songer; Trends in Microbiology 5 (1997), 156) as well as
Bacillus anthracis
(cf D. M. Guttmann, D. J. Ellar, FEMS Microbiology Letters 188 (2000) 7),
Helicobacter pylori
(cf. J.-H. Weitkamp et al.; Zentralblatt für Bakteriologie 280 (1993), 11),
Legionella pneumophila
(cf. W. B. Baine; Journal of General Microbiology 134 (1988), 489), and
Listeria monocytogenes
(cf. A. Coffey et al.; Applied and Environmental Microbiology 62 (1996), 1252). Furthermore, PC-PLC has been found in yeasts, e.g.
Candida albicans,
and in molds, e.g.
Aspergillus fumigatus
(cf. M. Birch et al.; Infect. Immun. 64 (1996), 751).
Several procedures for assay of PC-PLC have been developed. Some of the more recent assays were reviewed by E. L. Krug and C. Kent (cf. Methods in Enzymology 72 (1981), 347). The most commonly used procedures detect choline phosphate produced by the phospholipase C reaction on the natural substrate phosphatidylcholine. For other methods special equipment is needed. All these methods allow only measurement of the total amount of enzyme present in a sample at a certain moment and therefore are discontinuous assay methods. Additionally, all these procedures are clearly not suitable for the direct detection of microbes secreting PC-PLCs.
In 1976, Kurioka et al. (cf. S. Kurioka, M. Matsuda; Analytical Biochemistry 75 (1976), 281) have reported a continuous spectrophotometric assay for PC-PLC using a substrate containing 4-Nitrophenyl choline phosphate (p-Nitrophenylphosphorylcholine). Kurioka first synthesized this compound in 1968 (cf. S. Kurioka; Journal of Biochemistry 63 (1968), 678).
However, this substrate has several disadvantages. The specific activity of the enzyme towards this substrate is extremely low. Only after the addition of sorbitol or glycerol in high concentrations (up to 60%) an assay of PC-PLC with reasonable, yet still low cleavage rates could be developed. Thus, as already stated by Krug and Kent, this procedure is only suitable for investigations with pure enzyme preparations. This means that this substrate is less suitable for direct detection of microbes or microbial secretes containing PC-PLCs.
Furthermore, this substrate cannot be used for plating media since the 4-nitrophenolate liberated upon enzymatic cleavage is water-soluble and thus would migrate into the medium. Additionally, the yellow color of 4-Nitrophenolate may interfere with the background in biological samples like body fluids or culture media.
To sum up, prior art assay methods are unspecific, not flexible and do not allow continuous measurement of actual PC-PLC concentrations in a sample.
Prior art methods for detection and identification of bacteria producing PC-PLC use freshly prepared egg yolk agar. Egg yolk contains a variety of phosphatides; the main constituents are phosphatidylcholine, phosphatidylethanolamine and inositol phosphatides. Phosphatidylcholine is cleaved by PC-PLC to form choline phosphate and water-insoluble diglycerides, the latter giving an opaque zone around the bacterial colonies producing PC-PLC on egg yolk agar.
These methods were improved by the use of Lecithin agar (cf. G. L. Chrisope et al.; Applied and Environmental Microbiology 31 (1976), 784) containing crude soybean lecithin. Bacteria secreting PC-PLC showed turbid halos around the colonies indicating the presence of the enzyme. The average time to produce a reaction of moderate degree was about two to three days. This detection method lacks specificity because other phospholipases, e.g. phospholipase A or phosphatidylinositol-specific phospholipase C (PI-PLC) may act on other components of lecithin producing opalescent zones around the colony, too. Furthermore, training is required to correctly screen the plates for such zones. Generally, these prior art methods are unspecific, laborious and time consuming and, hence, expensive.
Moreover, prior art detecting systems generally fail to distinguish between different bacteria. For example, there is no simple plating medium to discriminate between
Bacillus cereus
and
Bacillus thuringiensis,
in particular since data show that
Bacillus cereus
and
Bacillus thuringiensis
should be regarded as one species (cf. C. R. Carlson, Applied and Environmental Microbiology 60 (1994), 1719) where horizontal transfer of plasmid genome occurs.
OBJECTS AND SUMMARY OF THE INVENTION
Accordingly it is a primary object of the present invention to provide novel chromogenic compounds for easy and convenient detection of PC-PLC, which avoid the disadvantages of prior art.
It is another main object of the present invention to provide novel chromogenic substrates and methods for detection and/or identification of microorganisms producing PC-PLC by means of conventional spectrophotometric and/or histochemical assay methods including use in broth, and notably in plating media, and which substrates are substantially free from the disadvantages of prior art substrates or methods.
It is a further object of the present invention to provide means for detecting and/or identifying various pathological bacteria, such as
Clostridium perfringens, Pseudomonas aeruginosa, Helicobacter pylori, Legionella pneumophila, Bacillus cereus, Bacillus anthracis, Listeria monocytogenes
and others.
Yet another object of the present invention is a method to discriminate
Bacillus cereus
from
Bacillus thuringiensis.
The above and further objects and advantages apparent from the present disclosure will be achieved by means of certain novel chromogenic compounds of formula (I):
in which R is selected from the group consisting of hydrogen and C
1-4
alkyl, such as methyl, ethyl, and all possible isomers of propyl and butyl, while R
1
, R
2
, R
3
, and R
4
are radicals independently selected from the group consisting of hydrogen, halogen (e.g. fluorine, chlorine, bromine and iodine), cyano, nitro, carboxy, amino, amino substituted with one or two C
1-4
alkylgroups, aminomethyl, hydroxy, C
1-4
alkoxy, carboxyalkyl, and sulphonyl.
The term “chromogenic” as used herein with reference to compounds according to the invention is intended to indicate the capacity of such compounds to become colored, i.e. visibly or calorimetrically detectable upon interaction with bacteria and, specifically, upon interaction with PC-PLC.
The terms “detection” or “detecting” are intended herein to include detection methods and assay techniques, as well as substances or substrates for use in such methods or techniques.
As will be apparent to those experienced in the art, most preferred compounds within the scope of formula (I) above are those which will yield deeply colored indigo dyes when used as substrates for detecting PC-PLCs, and which, preferably, are easily available and generally suitable for histochemical uses. Typical examples of preferred formula (I) compounds will be given herein below.
While no limitation is intended by such explanation, effectiveness of compounds of formula (I) as substrates for detection of PC-PLC enzymes is believed to reside in the fact that cleavage of a compound according to t
Frampton Elon W.
Restaino Lawrence
Schabert Günter
Biosynth AG
Blank Rome LLP
Leary Louise N.
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