WO2002073154A2 - Method and the detection of bacteria and bacterial groups - Google Patents

Abstract

A method to detect bacteria or bacteria groups after a short pre-incubation followed with a short selective enrichment phase of that the presence of bacteria or bacteria groups are detected by the metabolisms, gases or gas like substances that they form or by enzymatic activity or by structures that bacteria cells have accordingly in an early growth phase synthesized for example immunologically. The detection is done during the actual enrichment, at its best already during the lag phase and by the latest when the population has reached its maximum growth speed.

Description

Method and the detection of bacteria and bacterial groups

Background of the invention

Microbiological detections are made from different sources among others within the healthcare sector, food and other industries, veterinary medicine, hygiene control and the follow-up of environmental conditions. For many purposes certain norms exist with which results are analysed and categorised but in addition the objective is often to get the information as soon as possible and in these cases new rapid methods are used in addition to the control methods approved by officials. These rapid methods are used to speed up not only the actual analysis meant to detect the microbe but also for example the enrichment with which the count and relative share of the desired microbe to be detected can be increased before the analysis. When studying for example the detection of Salmonella -bacteria with the method according to the international patent application PCT FI99/00192 the objective is especially the enrichment of specific, desired and from the viewpoint of detection reaction important antigens.

Hygiene control is still an important aspect of quality control in production, storage and delivery in many industries. Besides the medical industry requirements for a clean production environment are especially in cleanroom production where the objective is to produce and package devices, their components, various analysing equipment, reagents and the equivalent either in a sterile way or as microbe free as possible. Also in normal food production quality requirements have become more stringent in order to ensure clean food to consumers. Nowadays the control stretches from industrial plants all the way to production farms for instance broiler breeding establishments or milk farms and primary production is increasingly based on contracts. Thus in addition to fulfilling the normative control regulations set by the officials industrial production often tries to obtain more efficient control with the use of new rapid methods in order to minimise risks and for instance storage costs.

In Finland, in the microbiological control of milk products, norms set by the EU are followed as well as the Finnish National Veterinary and Food Research Institute recommendations on methods to be used. By acting in accordance with these instructions and recommendations only, the time needed for getting analysis results is nowadays often too long from the viewpoint of practical microbiological quality evaluation in dairy industry so that results could immediately be exploited in the dairy. Thus, in practical industrial production, the development of fast microbiological rapid methods has proved to be increasingly important. Especially in the dairy industry this is crucial because the preservability of the raw material and many products is relatively limited However also in many other industries the total bacterial content (TC) or coliform bacterial count or certain specific bacterial groups, genera or species or other microbes are aimed to be detected In addition the so called somatic cells count in milk is detected A hygiene norm of these contents exist The risen count of somatic cells may indicate among others cow's inflammation of the udder or other health changes in udders

In recent years the hygienic quality of raw milk from farms has improved continuously in Finland Nevertheless milk is a product that fairly easily gets spoilt and considerable health risks are related to its preservability, transport and storage These risks become emphasized in warm weather or warm climate conditions but contamination may also happen during different phases of the so called cold transport and storage chain or in shops' or consumers' refrigerators through the action of various bacteria that stand cold fairly well like many Liste a or Bacillus cereus strains This contamination risk applies to all fresh milk products (for example liquid milk products, unripened cheese, ice creams) and to lesser extent also to sour milk products (for example sour milk, soured whole milk, yoghurts and matured cheeses)

Slow microbiological control analysis methods for milk or milk products lead in practice to the fact that if batches with quality defects are found they have normally already left the dairy and the returning and removing of them cause great economical expenses Indirectly damage is caused by the reputation of the production plant and its products getting worse thus causing lower demand and the deterioration of company image Therefore it would be especially desirable that dairies would have methods to use for microbiological quality control which even indicatively could produce analyse results in a shorter time than presently With these methods it ought to be possible additionally to handle considerable numbers of samples economically and preferably in partly simple conditions in farms, in production lines or their vicinity because it is known that long sample transport and storage times weaken their quality and representativeness and increase the time needed for getting results

In the same way as in milk production and the dairy industry enhanced microbiological control is needed also in other food industries like in meat processing, milling industry, processed food industry, juice industry and other drink production, child food industry and other food industries and in primary production New rapid methods that reduce stock storage time and increase product safety are needed also in the fodder production industry Often, to detect microbe growth for example from different carbon and/or energy sources of various cultivation media, a relatively long enrichment time is needed to notice visible or optically measurable growth The measuring principle may also be the change of pH, redox- potential, electrical conductivity or other equivalent measurable parameter in the medium, in other words, cultivation medium Also in these cases, especially when conducted as cultivation in a test tube or in an equivalent way, relatively long cultivation times are needed On the other hand when using semi-automatic or automatic equipment suitable for analysing smaller liquid amounts, the price of the equipment together with its usage and maintenance expenses might be too big an economical investment for a small production unit like a dairy or a cheese dairy factory

When choosing the composition of a cultivation medium suitable for microbiological cultivation the aim is to make it meet the growth requirements of the detectable microbes as well as possible If the occurrence of for example bacteria or some other broad group is generally detected from a sample, the aim is to include to the cultivation medium as few selective factors as possible, for example only substances that generally prevent the growth of fungi and moulds When it is required to selectively grow a specific smaller group of microbes (for example coliform bacteria) or for example a certain species, like Salmonella, or a certain strain (for example E.coh 0157) also other selective factors that prevent the growth of non-desired species are added to the cultivation medium As a selective factor may act not only a liquid that is added directly to the cultivation medium or a solid material that is dissolved into the liquid but also a gas or gas mixture that is led into the cultivation medium Thus with the help of the enrichment method for example aerobic, anaerobic or microaerophilic bacteria can be selected by choosing a gas composition that suits other enrichment processes and desired enrichment objectives to be used in the enrichment

Although selective microbe cultivation tries to prevent, suppress or limit the multiplication or preservation of non-desired microbes from the viewpoint of detection or other purpose, the selective growth conditions or cultivation media components or their equivalent cause often strain also to those microbes whose appearance is supposed to be favoured compared to other microbe groups or strains Thus selective cultivation may have restrictive effects also to the multiplication or viability of bacteria to be detected Correspondingly strongly selective conditions may restrict the appearance of desired antigens or their molecules which are necessary for detection purposes With the help of temperature so called total coliforms (enrichment temperature 37°C) and fecal coliforms (enrichment temperature over 40°C ) can be for example sorted out from coliform bacteria Another example of using temperatures in enrichment as a selective factor is the cultivation of psychrophilic bacteria, bacteria that stand cold, for example in milk samples In addition to the examples described here temperature can be used as a selective factor also in many other microbe enrichments

The acidity of the cultivation medium is also a factor with which desired microbes can be selected in many different applications, the same with the osmolarity of the cultivation medium It has been generally stated that the existing optimum in relation to some environmental factor of a bacteria or the content of some component in the culture, is closer to the upper than lower limit of its tolerance Based on this for example fecal coliforms can be sorted out from total coliforms with the help of a fairly small disparity in enrichment temperatures

In many microbiological cultivation and enrichment methods it is favourable to use changeable cultivation conditions In this way it is possible for example to get microbe cells that have been subject to so called environmental stress to revive in difficult conditions On the other hand for example the germination of spores, in other words endospores, of sporogenous bacteria might be favourable to perform in different conditions than the actual growth

In earlier studies it has been found that many for example enteric bacteria strains form certain surface antigens in a very early phase of the cultivation, directly after the stationary growth period (international patent application PCT/FI99/00192). It is possible to exploit this property in the detection and verification of these and other similar microbes and in enhanced enrichment Thus the amount of cells in a sample does not yet have time to notably multiply but the number of certain detectable antigens is at its peak. It is possible to observe this growth phase for example with the help of a pH indicator that is added to the cultivation medium or another similar indicator and detection principle that reacts to changes caused by microbes' metabolism

Description of the invention

Because the synthesis of desired antigens, which is a basis for detection taking place with the use of antibodies, and the messenger RNA synthesis that prepares it begin already during the stationary phase, it is important from the viewpoint of the method's functioning, that as many as possible of the cells transfer immediately after sampling to the stationary phase and begin to prepare cell divisions. To enable this and to ensure the expression of early desired antigens pre- enrichment is used in the method according to this invention. Pre-incubation is carried out in the cultivation media and conditions that are as little selective as possible or otherwise disturb the growth or viability of the bacteria to be detected as little as possible or are as close as possible to the optimal growth conditions of the micro-organism under study. The pre-incubation is carried out in a way that it lasts a part or the whole of the lag phase's estimated duration and continues at least only a short time during the logarithmic growth phase. This pre-incubation phase may take only a few hours (for example 8 to 10 hours or less) or for example overnight ("overnight enrichment") or a similar time, ln some cases pre-incubation may take 4-5 hours or less. Only after that are selective factors added to the cultivation media and/or cultivation conditions changed to be more selective or microbes are further inoculated into another growth container with conditions that have changed this way. The actual detection reaction is carried out during the selective cultivation at the latest immediately after the population of the desired bacteria has reached its maximum growth speed in the used cultivation medium.

Based on the considerations described above, the pre-incubation of a bacteria sample or culture as though softens the strain to the bacteria cells to be detected caused by selective cultivation and at the same time ensures that in a detection reaction they form exploitable molecules and/or structures as much and as soon as possible and simultaneously. On the other hand pre-incubation might be meaningful to the recovery of bacteria cells to be detected in conditions that are often strenuous to them in clinical, industrial or environmental samples or their equivalent. In some cases when searching for a certain bacteria or bacterial group, a somewhat selective approach can be used in pre-incubation. In that case for example psychrophilic bacteria, bacteria that stand low temperatures can in pre-incubation be cultured in a relatively low temperature and after that transferred to be further cultivated in a higher temperature for example to speed up reactions. On the other hand in psychrophilic selection the temperature can also be lowered, if wanted, when transferring to further cultivation.

The advantage in the method according to this invention is that as large as possible a proportion of the microbe cells are made to get into metabolic state and to perform biosynthesis. At the same time the expression of certain desired antigens is made to happen in as many cells as possible and as simultaneously as possible which has a positive effect on the immunodetection of these antigens. Also correspondingly the biosynthesis phase that precedes the expression of certain messenger RNA (ribonucleic acid) molecule proteins can be made to happen as broadly as possible and synchronously in the cell population. This in respect is favourable for example to the use of detection methods that are based on the detection of the nucleic acid in question This detection might happen with the help of nucleic acid hybridisation and/or after PCR reaction or in another similar way What is essential is that a short pre-incubation enables that a short actual enrichment is adequate The time required by this enrichment may be, depending on application, 2 to 4 hours, less than 5 hours, less than 8 hours or less than 10 hours

Using the method according to this invention it is possible to detect simultaneously several different antigens or nucleic acid molecules providing that the timing of their molecular expression happens approximately in the same population growth phase Because with the help of pre-incubation, this detection reaction can be synchronised as efficiently as possible, the efficiency and sensitivity of the method increases On the other hand the synthesis of the molecules to be detected in the reaction can also grow faster due to pre-incubation and happen in broad scale in an earlier phase in relation to cell growth than without pre-incubation In regard to cross reactions caused by other molecule types, this decreases them and thus adds to the reliability of the method

Current enrichment methods usually try to obtain as efficient as possible growth of the microbes to be detected The main purpose in the method according to this invention is to produce in the earliest phase as possible as much as possible of the antigens to be detected onto the surface of the microbe cell or otherwise detectable molecule structures, for example specific RNA molecule, into the cell, onto the surface or other structures or free from the cell to the cultivation media The formation of these molecular structures mentioned before begins normally during a short, at the most a few hours, growth lag phase of the cell population In certain special cases, like when bacteria spores germinate or facultative bacteria change their metabolism from aerobic to anaerobic metabolic type or vice versa when the environmental conditions change, synthesis of new molecules happen also which can be exploited in microbe detection in the same way as components that have been formed already during the lag phase This transfer phase requires also often special conditions where the selection pressure directed on the population is consciously limited before the actual enrichment The synthesis of the molecules or molecular structures to be detected which has favourably begun already during the lag phase continues often strongly during the first part of the logarithmic growth phase when the detection reaction is performed at the latest The molecules to be detected can be gases or gas like substances that are released from the bacteria culture They can be detected for example chromatographically or with the help of an ion mobility spectrometer (IMS) The basic objective of immunological detection methods presently in use is usually to detect microbes as specifically as possible which in immunological methods leans on antibodies that are as specific as possible. In practise problems often occur due to cross reactions. The development work of many detection methods may completely come to an end when specific enough antibodies can not be produced. The advantage of the method according to this invention is that after a kind of short adaptation phase the microbes are transferred to grow in selective conditions that quickly eliminate and/or limit the appearance of non-desired microbes in respect to the detection reaction. In this case there are less expectations on the qualities of the antibodies which makes the development work of the method more economical and on the other hand also ensures the reliability of the detection reaction because the irrelevant microbes that cause cross reactions can efficiently be eliminated. The selection pressure can be directed after the short adaptation phase not only to cell growth but also to the expression of desired antigens. It is also possible to assume that the characters of these antigens might be different as soon as they have synthesised in young cultures compared with the antigens that longer cultivated enrichments contain. At best this fact may also add to the specification of the detection reaction and thus also decrease the requirements towards antibodies.

lt is possible to exploit this invention in the detection of certain bacteria groups more broadly than methods presently in use because, as distinct from these present methods, when using immunological detection antibodies in the method according to this invention, whose specificity is not as precisely limited as those in present immunological methods, can be exploited as antibodies. One of these bacteria groups is for example coliform bacteria whose significance as a group and as indicator organisms in different hygiene analysis is remarkable. The detection of total coliforms or fecal coliforms (which are able to grow for example in the maximum temperature of 40 - 45°C) comes into question in environmental analytics, the analytics of driving water, water for household use or drinking water, in waters meant for refreshment for example swimming pools or Jacuzzis, in the production of drink, food, spices or additives in the food industry as well as in the quality rating of raw materials, ingredients or semi-finished products, in the production of medicine or medicinal substances, in clean room production, in different storage and delivery chains in industry, in the hygiene control of buildings, their water distribution and ventilation channels, in primary production in farms, cattle breeding farms, fish farms or their equivalent, in biotechnical industry and in various sectors of health care ranging from hygiene control of hospitals and health centres to bed-side diagnostics and the analysis of patient samples. It is to be noted that in the study of all coliforms and especially fecal coliforms it is favourable to determine the optimal temperature for selective cultivation. Coliform bacteria are a group that apply as target organisms to the method according to this invention for many reasons, for example because the group comprises of several bacteria groups whose characters are difficult to distinguish or strains that have a lot of similar features. However only a few of these are often interesting in respect to a certain detection reaction based on their pathogenic ability or indicator value or other aspect. Often these interesting species are in the same samples and often as a minority in the middle of other microbes and particularly bacteria of the same group. When exploiting the method according to this invention it is possible to get to verify the bacteria of interest from relatively difficult conditions and from the middle of a mixed population better and faster than it is generally possible with presently known methods.

In addition to coliforms the method according to this invention enables the efficient enrichment and rapid detection of many other specific microbe groups even from difficult conditions better than it is usually possible with current technologies. These groups are for example sporogenous bacteria whose spore detection from a mixed population or other sample types becomes easier as well as some absolutely or facultatively anaerobic or microaerophilic bacteria in whose detection a short pre-incubation and a short enrichment phase that follows it can be exploited in the method according to this invention to improve the detection and verification of these bacteria. When it comes to the latter mentioned bacteria it is possible to use for example a device solution and its exploiting principles according to international patent application PCT FI98/00854. If the bacteria to be detected are for example facultatively anaerobic, a gas mixture containing oxygen can be used in pre-incubation and in the actual enrichment phase this can be replaced with a gas free from oxygen. Gas can be used as a selective factor also in the detection of anaerobic or microaerophilic bacteria (for example Camp lobacter).

Also many bacteria that are found for example in soil or waters and which can if necessary exploit slowly degradable organic compounds, can efficiently be detected with the method according to this invention. These bacteria are for example many of the Pseudomonas species bacteria which are important in clinical analytics (for example Pseudomonas aeruginosa) and important as indicators in hygiene and water analysis for example when evaluating the microbiological quality of industrial waters or industrial waste waters or chlorinated waters or other chemically treated waters meant for recreation use. In detecting these bacteria it is possible to carry out pre-incubation using some general microbiological cultivation media and after that carry out the selective enrichment on a more demanding cultivation media. In these cases the cultivation time might in combinations of many bacteria and nutrient alternatives increase but it is still when relatively observed shorter than the time required for the population to get to its maximum cell density.

The detection of a microbe at the right time, in respect to early detection reaction at an appropriate time, requires the identification of the growth phase of the bacteria growth during selective enrichment. For this purpose for example indicator colour pigment that is added to the cultivation medium can be exploited. This indicator colour pigment produces a colour change caused by the pH of the culture or change in redox-potential or substances produced into the cultivation by the microbe or by the effect of enzyme activities and which can be observed with visual or optical devices. The growth phase can also be detected by measuring the electrical conductivity or optical transparency of the culture or the formation of the culture's gases or gas like substances. This latter measurement may also produce information that is usable in respect to the identification of the bacteria because the formation of different gases from known cultivation media depends on the qualities of the bacteria to be studied.

Claims

Claims

1. A method to detect aerobic, unaerobic, sporogenous, coliform or similar bacteria or bacteria groups characterized in, that the bacteria are first incubated for a few hours or for example overnight in a cultivation medium or conditions that are as little selective as possible or otherwise as little disturbing to the microbe's growth or viability as possible, and are then transferred to selective enrichment conditions and/or a cultivation medium in which the desired bacteria group, species or strain is selected and it is detected with the help of antigens, that are verifiable in the first part of the cultivation after the inoculation, or other molecular structures or gas like metabolic products that have been formed before the bacteria cultivation has reached its maximum growth speed during the period of culturing the microbe after the transfer inoculation or immediately after reaching it.

2. A method according to claim 1 characterized in, that bacteria are cultivated first in conditions that are as close as possible to the optimal growth conditions of the desired bacteria strain or species or group.

3. A method according to claims 1 and/or 2 characterized in, that in the selection of bacteria to be detected gas is used to help as a selective factor.

4. A method according to one or more of the claims 1 to 3 characterized i n, that indicator colour, that reacts to the change of the pH or redox-potential or to the change caused by the bacteria's metabolism produces or equivalent or an enzyme formed by the bacteria or a metabolism produce in the cultivation medium, is added to the cultivation medium.

5. A method according to one or more of the claims 1 to 3 characterized i n, that the growth phase of the bacteria is detected with the help of electrical conductivity or optical transparency or an equivalent.

6. A method according to one or more of the claims lto3 characterized i n, that the growth and/or growth phase of the bacteria is detected with the help of the gas like metabolism produces produced by the cultivation. A method according to one or more of the claims lto6 characterized i n, that the presence of bacteria is found out immunologically right in the first part of the cultivation after the transfer inoculation when the proportion of desired surface antigens has reached an adequate level

A method according to claim 7 characterized in, that the bacteria are detected with the help of antibodies that are specific to desired surface antigens

A method according to claims 7 and/or 8 characterized in, that the possible antibody cross reactions with non-desired bacteria or their structures are eliminated when these are transferred into selective conditions and the desired bacteria and their structures enrich in selective cultivation

A method according to one or more of the claims ltoό characterized i n, that the molecule structure to be detected is a specific messenger RNA

A method according to claim 6 characterized in, that the formation of gas or gas like substances from the culture is detected cromatographically

A method according to claim 6 characterized i n, that the formation of gas or gas like substances from the culture is detected with the help of ion mobility spectrometer (IMS)

A method according to one or more of the claims 1 to 12 characterized i n, that the bacteria to be detected are coliform bacteria that are detected as a group

A method according to claim 13 characterized in, that the bacteria to be detected are fecal coliforms that are cultivated in an enrichment temperature of40-45°C

A method according to one or more of the claims 1 to 12 characterized in, that the bacteria to be detected are bacteria that form spores and whose spores germinate during the pre-incubation phase A method according to one or more of the claims 1 to 12 characterized i n, that the bacteria to be detected are anaerobic, facultatively anaerobic or microaerophilic bacteria

A method according to one or more of the claims ltol2 characterized i n, that the bacteria to be detected are bacteria that exploit organic molecules that are slowly degradable

A method according to one or more of the claims 1 to 17 characterized i n, that temperature is used as a selective factor in enrichment

A method according to claim 18 characterized in, that the bacteria to be detected are psychrophilic bacteria which are transferred to a selective cultivation in low temperature after pre-incubation in higher temperature

A method according to claim 19 characterized in, that the bacteria to be detected are psychrophilic bacteria which are transferred to a selective cultivation in higher temperature after pre-incubation in lower temperature

A method according to one or more of the claims lto20 characterized i n, that the bacteria to be detected are a minority among other microbes in the sample to be examined