Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-07-17
2002-05-14
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S027000, C435S028000, C435S018000
Reexamination Certificate
active
06387648
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, in a general manner, to means for regulating the disinfection of liquids. It relates more particularly to means of regulation using the measurement of enzymatic activities.
BACKGROUND OF THE INVENTION
The assurance of the quality and safety of animals, such as water intended for consumption, dietary liquid, bathing water, water intended for pharmaceutical or biotechnological preparations, depends directly on the reliability and the sensitivity of the techniques used in measure the number of microorganisms surviving in the said liquid.
The methods currently used for regulating the disinfection of a liquid use the techniques for culturing on agar and/or microscopy techniques.
The techniques for culturing on agar consist in counting the number of bacterial colonies which develop on various standardized nutrient agar media [e.g. French Standard NF T 90-414, Essais des Eaux, Recherche et dénombrement des coliformes et des coliformes thermotolérants (Water analysis, Identification and Enumeration of coliforms and of thermotolerant coliforms)]. These techniques have several disadvantages.
In the first instance, there may be mentioned the fact that they give their results only after 24 hours on average, which delays by as much the possible adjustment of the disinfection method.
The techniques for culturing on agar necessitates, furthermore, the carrying out of series of microbiological cultures for the analysis of each liquid sample. Indeed, all the microbial, and in particular bacterial, families do not develop on the same nutrient medium; thus, it is possible to detect no coliform after culturing on the standardized nutrient medium for the detection of coliforms, but to find a good number of other bacteria after culturing on other nutrient media. The choice of the nutrient agar media therefore determines the quality of the analysis. This choice is all the more delicate since a larger number of colonies has sometimes been observed after culturing on a medium other than the standardized nutrient medium for the detection of these same colonies. It has, for example, been demonstrated that the counts of the viable aerobic bacterial flora were higher on R2A agar medium than on the corresponding standardized nutrient medium (see for example <<A new rapid medium for enumeration and subculture of bacteria from potable water>> by Reasoner D. J. and Godreich E. F., App. Environm. Microbiol. (1985) 49-p.1-7). To establish a reliable negative diagnosis, the techniques for culturing on agar therefore require a multiplication of the analyses. The techniques for culturing on agar furthermore do not easily allow the regulation of the method of disinfection to be automated.
Bacteria are furthermore capable, and in particular following an environmental stress such as the application of a disinfection method, of adopting a form of resistance in which they no longer multiply while providing a minimal metabolism; as soon as more favourable environmental conditions are restored, these bacteria could resume their multiplication. Such bacteria are said to be <<non-culturable but viable>>: they are not detectable by the conventional techniques for culturing on agar and can represent a biological risk for the consumer.
A method which is currently available for controlling with virtual certainty the efficiency of the disinfection of a liquid uses microscopy techniques combined with specific staining. This method allows discrimination between culturable bacteria, non-culturable but viable bacteria and dead bacteria. It requires nevertheless the use of several staining tests for each sample and is technically difficult to automate.
SUMMARY OF THE INVENTION
The present invention aims to overcome the disadvantages of the prior art techniques and proposes a method for regulating the disinfection of a liquid, characterized in that it comprises:
A. at one stage of the said disinfection, designated hereinafter stage 2, measuring the activity of at least one enzyme by bringing the microorganisms which may be present in the said liquid into contact with a substrate chosen as being capable of revealing the activity of this or these enzyme(s), in particular by the conversion of the said substrate to coloured fluorescent or luminescent compounds or by the disappearance of the said substrate, this enzymatic activity being called hereinafter specific activity,
B. at one stage, designated hereinafter stage 1, prior to the said stage 2, measuring the activity of the same enzyme(s) as in A, this activity being designated hereinafter initial activity,
C. translating, for each enzyme, the said specific activity and initial activity, into levels of surviving microorganisms in the said liquid at stage 2 of the said disinfection by means of a reference system preestablished with the aid of a sample of the said liquid collected at the said stage 1 and then exposed to increasing doses of disinfectant(s), as well as
D. adjusting, as a function of the said level of surviving microorganisms, of the nature and/or doses of physical or chemical agent(s) used for the said disinfection.
The expression surviving microorganisms is understood to mean in the present application culturable microorganisms and/or non-culturable but viable microorganisms.
The expression level of surviving microorganisms is understood to mean in the present application the ratio between the concentration of surviving microorganisms in the said liquid at the said stage 2 of a disinfection and the concentration of surviving microorganisms in the same liquid at the said stage 1. This level of surviving microorganisms is preferably expressed as reduction values in the form of negative powers of 10, or as log reduction which corresponds to −log
10
(reduction).
The said stage 1 may, for example, correspond to a stage <<before disinfection>> of the said liquid and the said stage 2 to any stage of the disinfection method (stages <<after disinfection>> of the said liquid included).
The method according to the invention relates to liquids in which the microorganisms which may be present are subjected to very specific conditions, namely disinfection conditions, which induce notable stress of the cells.
The method for regulating the disinfection of a liquid according to the invention relates particularly to liquids intended to be brought into contact with humans or animals, whether through simple contact, absorption, ingestion, instillation or injection. It applies particularly to liquids intended to be in contact with humans or animals, such as bathing water, water intended for consumption, water intended for pharmaceutical or biotechnological preparations, or a dietary liquid.
The said bringing of the microorganisms which may be present in the said liquid into contact with the said substrate may be carried out by bringing the said liquid or a sample of the said liquid directly into contact with the said substrate, or alternatively by bringing a concentrate of the said microorganisms which may be present, such as a filtrate or a centrifugation pellet of the said liquid or liquid sample, into contact with the said substrate.
Prior to the measurement of the activity of some enzymes such as glucose-6-phosphate dehydrogenase or glutathione reductase, the method according to the invention advantageously comprises the step of subjecting the said liquid, liquid sample or concentration to a lysis treatment, especially by sonication.
Prior to the measurement of the activity of other enzymes such as catalase or superoxide dismutase, the preliminary lysis stage may be avoided: the activity of these enzymes may be measured with the aid of a substrate which diffuses into the microorganisms, such as lucigenin or hydrogen peroxide.
According to an advantageous aspect of the invention, the enzyme(s) whose activity or activities is (are) measured exhibit(s) in the said liquid, liquid sample or concentrate a ratio between the specific activity and the initial activity in close r
Dukan Sam
Levi Yves
Touati Danièle
Connolly Bove & Lodge & Hutz LLP
Leary Louise N.
Suez Lyonnaise des Eaux
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