METHOD OF AND GROWTH MEDIUM FOR DETECTING MICRO-ORGANISMS IN A SAMPLE

Abstract: A method of detecting micro-organisms in a sample comprising culturing the sample in a growth medium, and detecting the presence in the cultured sample of an indicator enzyme associated with growth of the micro-organisms is characterised in that the growth medium contains both PTS and non-PTS carbon and energy source compounds, and in that the initial concentration of the compounds together in the medium is not substantially greater than 20 gdm<-3>.

Description: METHOD OF AND GROWTH MEDIUM FOR DETECTING MICRO-ORGANISMS IN A SAMPLE Field of the Invention This invention relates to a method of detecting micro-organisms in a sample, for example a sample of a foodstuff or a prepared food product, and to a growth medium for use in such methods. Background to the Invention The food industry in the UK exists mainly to supply home markets and was worth some £73 billion in 1 995. The production of safe and wholesome food is, therefore, of fundamental importance to its continued success. Recent innovations in the industry (e.g. cook-chill food, just in time production) makes it an essential requirement to have efficient and reliable methods of analysis to ensure microbiological safety. Escherichia coli (£. coli) is a bacterium which has generated considerable interest since its discovery in the 19th Century. It is a bacterium which, as a species, is considered as an indicator of faecal pollution, both in water and in foods. In addition, the species contains a number of pathogenic strains. The food industry currently devotes large amounts of resources to analysing products for this organism, as its presence can indicate serious health and quality problems. There are numerous methods available on the market to detect micro-organisms and the introduction of new and rapid methods is a developing area in microbiology. Traditional microbiological methods for the detection and identification of E. coli rely on a two stage process, the first involving growth in a selective medium (e.g. McConkey broth) followed by a second identification stage (e.g. the production of indole at 43°C followed by t he API 20E method). Each stage usually requires a 24 hour incubation period, making the assay a lengthy process. This is not an ideal situation for analysis of short shelf-life products. Improvements to the traditional methods for the identification of £. coli have concentrated on adding 'identifier' markers to a growth medium, for example the addition of 4-methylumbellιferyl-β-D-glucuronιde (MUG) into a culture medium (Feng & Hartman, 1982; Schets & Havelaar, 1991 ). This compound acts as a substrate for glucuronidase (an extracellular enzyme expressed by E. coli ), forming the product 4- methylumbel ferone. This product fluoresces under long wave UV light. This addition has proved to be a successful method for the identification and enumeration of E. coli, and Oxoid (Basingstoke, UK) market a MUG supplement to add to microbiological media. However, the process still takes between 12-24 hours to obtain a result. This time delay relates to the need to obtain sufficient numbers of E.coli cells to enable enzyme expression to reach a level sufficient to produce an amount of the fluorescent product to be detected. The aim of the research, upon which the patent application is based, was to investigate the growth of E. coli and maximise the expression of β-giucuronidase. In theory this would allow the fluorescent product to be detected at a lower number of E. coli cells present in the medium, thus resulting in a reduction in time from inoculation of the medium to the reading of the results in comparison with methods involving the addition of the MUG supplement. The implementation of the method involves the design of the medium to achieve this high level of β-glucuronidase expression in a culture containing a relatively low number of E. coh cells (i.e. during the early exponential phase of growth). This involves the medium containing levels of compounds that result in a change to the biochemical pathways expressed. In order to assist understanding of the invention, a summary of the relevant biochemical processes in E. co is required. The Hexuronide/Hexuronate pathway in Eseheriehia coli β-glucuronidase is an enzyme that is part of a biochemical pathway responsible for converting methyl-β-D-glucuromde to pyruvate (Nemoz et al, 1 976). Pyruvate can be utilised, by the oxidative phosphorylation pathways, to generate energy for the E. coh cell. Bacteria commonly utilise a wide range of substrates as carbon and energy sources, ensuring a versatility which maximises survival. It follows that all of the energy of the bacterium would be expended in protein synthesis if every possible enzyme combination was present in the cell in high concentrations. Therefore, a complex regulatory process has evolved to enable those compounds that produce the most energy to be degraded preferentially. Thus, glucose would be degraded before methyl-β- D-glucuronide, and so in the presence of glucose the expression of β-glucuronidase is low or absent. It is thought that the Hexuronide/Hexuronate pathway is regulated by the phenomenon known as catabolite repression, which will bring about this preferential degradation within the cell. An object of the invention is to overcome the repression of the biochemical pathways that can be utilised for organism identification. It is possible to overcome catabolite repression by setting the carbon and energy source at an appropriate level. When this is combined with a compound that promotes cell recovery, and preferably an enzyme inducer, which will boost the levels of the enzyme system utilised in the detection protocol, it is possible to detect E. coh in 3-8 hours using the MUG system. Summary of the Invention According to the invention, there is provided a method of detecting micro organisms in a sample, comprising culturing the sample in a growth medium, and detecting the presence in the cultured sample of an indicator enzyme associated with growth of the micro-organisms, characterised in that the growth medium contains both PTS and non-PTS carbon and energy source compounds, and in that the initial concentration of the compounds together in the medium is not substantially greater than 20 gdm 3 . The invention also provides a growth medium for use in detecting micro¬ organisms in a sample, the medium comprising both PTS* and non-PTS carbon and energy source compounds, and the initial concentration of the compounds together in the medium being not substantially greater than 20 gdm 3 . The initial concentration of the compounds together in the medium is preferably less than 15 gd 3 , and more preferably less than or equal to 10gdm 3 . * This system is a mechanism by which compounds are phosphorylated as they cross the cell membrane - in brief it is a way of moving compounds across the membrane. It follows therefore that a damaged cell may not have this system in operation. The PTS and non-PTS compounds are present in the mixture in a ratio that selects for the micro-organism of choice. For example, for E. co the ratio of PTS to non- PTS compounds in the growth medium is suitably from about 1 :3 to about 1 :1 , and preferably the growth medium contains substantially equal proportions of PTS and non- PTS compounds. Other rapid methods can be developed by changing the target enzyme and the substrate/product. Where the micro-organism is Eschenchia coh, the PTS compound is preferably one or more of glucose, fructose, mannose, glucosamine, N-acetylglucosamine, β- glucosides and hexitols, and the non-PTS compound is one or more of maltose, melibiose, lactose, mannose-6-phosphate, a succinate, πbose, glucose-6-phosphate, fructose-6-phosphate, L-arabinose, xylose, a gluconate, a lactate, a pyruvate, glycerol and galacturonic acid. The growth medium preferably comprises an enzyme inducer compound, for example p-nιtrophenol-β-D-glucuronιde.. Traditional and rapid microbiological media containing MUG have a carbon and energy source set at levels which will repress the expression of β-glucuronidase during the initial growth stages. They do not contain a 'recovery compound' that promotes cell repair This is essential to minimise false negatives due to the damage food processing will inflict on the bacterial cells. Current methods will require a pre-enπchment step to achieve this. The £. co///MUG system has been used as a model to illustrate the possibilities for a new rapid assay. However, the invention is not limited to this system. The following Examples are provided by way of illustration of the method of the invention; the invention is not limited to these Examples: EXAMPLE 1 Defined Medium: The method of formulation The defined medium used in these studies was based upon the method of Cruickshank et al (1975) with the following modifications: The following were added to 600ml of distilled water: Potassium dihydrogen ortho phosphate. 3.0g Disodium hydrogen citrate. 0.5g Magnesium sulphate. 0.1 g Ammonium sulphate. 1.0g After dissolving the constituents the 600ml volume was divided into four 1 50ml volumes, bottled and autoclaved. The trace element solution was formulated as follows: The following were added to 9995ml of distilled water Ferrous sulphate. 0.5g Manganese sulphate. 0.5g Zinc sulphate. 0.5g Sulphuric acid 0.25M 5.0ml The trace element solution was then buffered to a pH value of 7.0 by adding 50%(w/v) NaOH. A 1.0%(w/v) calcium chloride solution was also made. Both solutions were autoclaved. 7.0g of dipotassium hydrogen orthophosphate was added to 200ml of distilled water. This was then dissolved and then dispensed into 50ml volumes and bottled. After- bottling, the four solutions were autoclaved. After autoclaving, 1.25ml of sterile trace element solution and 0.1 ml of sterile 1.0%(w/v) calcium chloride solution were added to the 50ml bottled volumes. The required carbon source was dissolved into 200ml of distilled water and filter sterilised through a 0.45μm pore sized cellulose filter. 50 ml of this solution was added to the 150ml volume of the defined medium. This produced a volume of 200ml. The solution of dipotassium hydrogen phosphate supplemented with trace elements and calcium chloride solutions was added to the 200ml volume above producing a final defined medium with a total volume of 250ml volume with a final pH value between 7.0 - 7.1. The carbon source level varied and was dependent on the study conducted (See results section). Carbon Source Utilisation Studies The procedure was as follows: 250ml of defined medium was prepared. Various carbon sources were used at a concentration of 2g/l. A 9ml nutrient broth was inoculated with a colony of E.coh (NCIMB 1 3058) from a nutrient agar slope. This was then incubated in a rotary shaker for 2 hours at 250 rev mm- 1 and 37°C±1°C. 1 ml of the 9ml pre-grown culture above was inoculated into the defined medium and incubated in a rotary shaker for 16 hours at 25°C±1°C and 50 rev mm After 16 hours the incubation temperature was increased to 37°C±1°C, however, all other parameters remained constant. At periodic intervals an aliquot was taken from the medium and an estimate of growth was obtained by taking an absorbance reading at 600nm. The Assessment of Enzyme Expression The differential rate of enzyme synthesis is the division of the rate of enzyme expression to growth. The observation is made graphically in Figure 1 , the units of growth being assigned to the x axis whilst the y axis is for the units of rate for enzyme synthesis. Rates of β-glucuronidasc synthesis in this cases are defined as the number of units of p-nitrophenol per mιn"1 per litre * 100 per unit volume of enzyme solution. Growth is expressed as absorbance at 600nm. This method has been used widely, with the exception that the mass of protein or dry cell weight has been used instead of absorbance (Novel et al 1 74; Paigen and Williams 1 70 and Jacob and Monod 1961 ) to estimate the amount of growth in a culture. However, the method of using absorbance (as an estimate of growth) was used by Pastan and Adhya (1976). The method of incorporating the use of absorbance measurements was chosen here because of its simplicity and rapidity in comparison with the other techniques. Construction of a differential rate plot. Experiments to obtain data to construct a differential rate plot were carried out as follows:- 250ml of defined medium was formulated as described hereinbefore. Varying types of carbon sources (see Results) were used to a concentration of 2g/l. Each defined medium was inoculated with a colony of E.coli (NCIMB 13001 or NCIMB 13058) from a nutrient agar slope. The medium was then incubated in a rotary shaker at 50 rev m ' and 25°C±1°C for 15 hours before the temperature was increased to 37°C±1°C for a further 8-14 hours. At the point when the temperature was increased to 37°C±1°C, the following analyses were conducted hourly: Absorbance at 600nm; a 3.0ml aliquot of the defined medium was centπfuged at 3000 rev mm 1 for 10 minutes. The supernatant was then collected and stored overnight at 5.0°C±1°C. The following day the amount of b- glucuronidase activity was determined. The results obtained were used to construct a differential rate plot Mixture Selection : The superior ratio to produce the highest β-glucuronidase expression The results from the basal activity study were examined and the carbon sources which showed the highest basal level of enzyme expression were chosen and formulated into three mixtures of varying ratios. These ratios were assessed to find the formulation which provided the maximal rate of β-glucuronidase expression in the presence of an inducer of enzyme expression, namely p-nitrophenol-β-D-glucuronide. The assessment of growth : The ability of E.coli serotypes to utilise three mixture types. Three defined media were prepared as stated above. Each medium contained a different supplement of the selected carbon sources. The total concentrations of the components used in the supplement was 2g/l: The supplement mixtures were as follows Mixture 1: 0.5g mannose; 1.0g galacturonic acid; glycerol 0.5 g* (0.4ml). Mixture 2: 1.0g mannose; 0.5g galacturonic acid; glycerol 0.5g* (0.4ml). Mixture 3: 0.5g mannose; 0.5g galacturonic acid; glycerol 1.0g* (0.8ml). * The density of glycerol = 1.25g/ml (Sigma 1996). After preparation of the 3 defined media, each 250ml voiume was then aseptically dispensed in 10ml volumes into pre-sterilised MacCartney bottles. Each 250ml defined media provided 25 10ml volumes. The procedure was as follows: Nutrient broth cultures for all NCIMB and NCTC Escherichia serotypes were used to inoculate, in duplicate, the 10ml volume of selected defined medium. The inoculum volume was 0.1 ml. The 10ml volumes were then incubated at 50 rev mm ' at 37°C±1 °C for 20 hours, after which, each 10ml defined media had an absorbance measurement at 600nm taken. The results are set out in Table 1 : Table 1 : β-Glucuronidase Activity: Selected Escherichia Serotypes Culture Type Mixture 3 NCIMB 9464 Positive Positive NCIMB 12129 Negative Negative NCIMB 13002 (Pathogenic) Positive Positive NCIMB 1 3001 (Pathogenic) Positive Positive NCIMB 13058 Positive Positive NCIMB 13003 (Pathogenic) Positive Positive NCIMB 1 1380 Weak Positive Weak Positive NCIMB 8545 Positive Positive NCIMB 9466 Positive Positive NCIMB 9472 Positive Positive NCTC 12080 (Pathogenic) Negative Negative NCIMB 9465 Positive Positive NCTC 10964 Positive Positive NCTC 12079 (E. Hermann) Negative Negative NCIMB 13059 Positive Positive (all Escherichia coli serotypes, except NCTC 12079) Determination of β-glucuronidase activity for all NCIMB and NCTC Escherichia cultures within mixture 3. The protocol described hereinbefore was used with the following amendment. The amount of β-glucuronidase was determined by a modified version of the Fishman (1961) β-glucuronidase assay. Results The ability of Escherichia coh NCIMB 13058 to utilise a range of carbon sources is illustrated in Fig 1. The higher the absorbance figure the greater the cell mass in the broth. This illustrates the variability in growth that can be obtained by varying the carbon source. We utilised this study to investigate which carbon sources would give a detectable ceil density with a corresponding high level of enzyme expression. These parameters were set in order to achieve the goal of a rapid method that would have a clear time advantage over existing methods. It is clear that growth on glucose (a traditional carbon and energy source for micro-organisms) results in a very low level of β-glucuronidase expression until approximately 18 hours (Fig 2). This relates to the onset of stationary phase (see Graph of growth rate Fig 2) and hence a relatively low glucose concentration. Thus a series of experiments were carried out, utilising a range of carbon and energy sources, to determine a differential rate of enzyme synthesis relating it to the growth of the organism (See method section for an explanation). These results are illustrated in From this we determined a ranking of carbon sources in relation to both growth and enzyme expression (Table 2). 98/07882 12 - Table 2: Preference of Carbon Source To Total Biomass of Growth E.co// NCIMB 13058 E. co// NCIMB 13001 Highest Total Fructose Sorbitol Biomass of Growth Lactose Fructose Glucose Glucose Sorbitol Lactose Galacturonic Acid Mannose Mannitol Mannitol Gluconic Acid Gluconic Acid Mannose Galacturonic Acid Xylose Glycerol Glycerol Galactose Ribose Xylose Pyruvate Pyruvate Malic Acid Ribose Galactose Malic Acid Lowest Total Succinate Succinate Biomass of Growth (The lines in between the columns denote the groups in which the carbon sources have been selected by the cultures of concern. Allowance should be made for experimental deviations. The selection between the two cultures does show a strong degree of homology.) This enabled us to discard a number of compounds that would not support growth at a sufficient level to meet the parameters we had set for the test. A detailed analysis of the results on rates of growth and the associated level of β-glucuronidase expression without the presence of an inducing compound (basal expression) led us to devise the three supplement mixtures to be tested. The rate of biomass produced by E.coiι on inoculation of each of the mixtures containing the supplement mixtures was determined. This enabled us to confirm that each of the mixtures would fall within one of the target parameters we had set, namely the production of adequate biomass to enable maximal enzyme expression to occur. However, we decided that the basal level of expression could be enhanced by stimulating the expression of β-glucuronidase. We therefore decided to add an inducer/substrate. This is a compound that will stimulate enzyme expression before being degraded itself. In effect it just enhances with respect to time the natural process of enzyme expression with the added advantage that it produces p-nitrophenol which can be used in a detection system alongside fluorescence. The concentration of this compound was set at an optimal level which has been reported in previous studies. The differential rate of synthesis of a number of E coh strains was determined and these are illustrated in Fig 4a, 4b and 4c. These results clearly indicated that supplement mixture 2 was the one that met the parameters set of maximal enzyme expression at a relatively low biomass of E. coh. The versatility of this medium was investigated with 9 strains of E. co all of which gave similar results to NCIMB 13508 It is clear from the data (Fig 5) that as the carbon and energy source increases, the enzyme expression diminishes to a level that eventually makes it unsuitable for a rapid method. This is due to the mimicking of the situation found with glucose (Figs 1 & 2) which we believe to be caused by catabolite repression. It would appear that the composition of the mixture added to a medium could vary within this range depending upon the micro-organism being detected. EXAMPLE 2 Comparison of Fluorescence Development Utilising a Growth Medium of the Invention and Tryptone Soya Broth containing MUG The method was as in Example 1 , with the following amendments: E. coh (NCIMB 1 3058) was diluted (in sterile nutrient broth) before inoculation into the growth medium and into Tryptone Soya Broth (TSB) with MUG supplement to give final inoculation levels of 100 CFU/ml, 1000 CFU/ml and 10 000 CFU/ml. TSB (Oxoid, Basingstoke, UK) was chosen as a medium for comparison because it is representative of traditional microbiological media, in that it has relatively high levels of compounds which are used as a carbon and energy source. MUG supplement (Oxoid) was added to the medium according to the manufacturers' instructions. Optical density at 600nm and fluorescence of the cultures were determined over a 19 hour incubation period. The results shown In Figures 6, 7 and 8 relate to the inoculation of 100 CFU/ml of E coli (NCIMB 13058) into each growth medium. As expected, this level of inoculation led to the slowest development of fluorescence early in the exponential phase for the new medium which quickly reached saturation point (i.e. the fluoπmeter reached its upper limit for fluorescence detection). Figure 6 illustrates that the development of fluorescence develops in early log phase. The optical density measurements are indicative of cell mass. By comparison, Figure 7 shows the results of the measurements for the TSB-MUG growth medium. It can be seen that fluorescence does not develop until the E coh culture is in the stationary phase of its growth cycle. This indicates that the expression of β-glucuronidase has been suppressed. Figure 8 is a graph showing comparative plots for the growth medium of the present invention and TSB-MUG, confirming the much earlier development of detectable fluorescence for the medium of the present invention. eferences. Cruickshank, R, Duguid, J P, Marmion, B P and Swain, RHA, (1975) Minimal medium of Davis and Mmigioli and its variants. In Medical Microbiology. Vol 2: The Practice of Medical Microbiology. 2nd edition, published by Churchill Livingstone, p 109. Feng, P.C.S. & Hartman P.A. (1 982) Journal of Applied and Environmental Microbiology 43 1 320-1329 Fishman, W H (1 65) β-glucuronidase In: Methods of Enzymatic analysis. Vol 2, Edited by Bergmeyer, H U, Published by Verlag Chemie, p929-943. Jacob, H & Monod, J. (19611 Journal of Molecular Biology 3 318-356 Nemoz, G.; Robert-Baudouy, J & Stoeber, F. (1976) Journal of Bacteriology 127(1) 706-718 Novel, G.; Didier-Fichet, M.L. & Stoeber, F. (1974) Inducibility of β-glucuronidase in wild type and hexuronate-negative mutants of Escherichia coh K-12, Journal of Bacteriology 120 89-95 Paigen, K. & Williams, B. (1 70) Advances in Microbial Physiology Vol 4 eds. Rose. A.H. & Wilkinson J.F. Published by Academic Press 251-324 Pastan. I. & Adhya, S. (1970) Bacteriological reviews 40 527-551. Schets, F M and Havalaar, A H, (1991 ) Comparison of mdole production and β- glucuronidase activity for the detection of Escheichia coli in membrane filtration methods, Letters in Applied Microbiology. Vol 1 3, p272-274.

Claims: CLAIMS 1. A method of detecting micro-organisms in a sample, comprising culturing the sample in a growth medium, and detecting the presence in the cultured sample of an indicator enzyme associated with growth of the micro-organisms, characterised in that the growth medium contains both PTS and non-PTS carbon and energy source compounds, and in that the initial concentration of the compounds together in the medium is not substantially greater than 20 gdm- 3 . 2. A method according to Claim 1. wherein the initial concentration of the compounds together in the medium is less than 1 5 gdm 3 . 3. A method according to Claim 2, wherein the initial concentration of the compounds together in the medium is less than or equal to 10gdm 3 . 4. A method according to Claim 1 , 2 or 3, wherein the ratio of PTS to non- PTS compounds in the growth medium is from about 1 :3 to about 1 :1. 5. A method according to Claim 1 , 2 or 3, wherein the growth medium contains substantially equal proportions of PTS and non-PTS compounds. 6. A method according to any preceding claim, wherein the micro-organism is Escherichia coli, the PTS compound is one or more of glucose, fructose, mannose, glucosamine, N-acetylglucosamine, β-glucosides and hexitols, and the non-PTS compound is one or more of maltose, melibiose, lactose, mannose-6-phosphate, a succinate, ribose, glucose-6-phosphate, fructose-6-phosphate, L-arabinose, xylose, a gluconate, a lactate, a pyruvate. glycerol and galacturonic acid. 7. A method according to any preceding claim, wherein the growth medium comprises an enzyme inducer compound. 8. A method according to Claim 7, wherein the enzyme inducer is p- nitrophenol-β-D-glucuronide. 9. A method of detecting micro-organisms in a sample, substantially as described with reference to the Examples. 10. A growth medium for use in detecting micro-organisms in a sample, the medium comprising both PTS and non-PTS carbon and energy source compounds, and the initial concentration of the compounds together in the medium being not substantially greater than 20 gdm 3 . 1 1. A growth medium according to Claim 10, in which the initial concentration of the compounds together is less than 15 gdm 3 . 12. A growth medium according to Claim 1 1 , in which the initial concentration of the compounds together is less than or equal to 10gdm 3 . 13. A growth medium according to Claim 10, 1 1 or 12, in which the ratio of PTS to non-PTS compounds is from about 1 :3 to about 1 :1. 14. A growth medium according to Claim 10, 1 1 or 12, which contains substantially equal proportions of PTS and non-PTS compounds. 15. A growth medium according to any of Claims 10 to 14, in which the PTS compound is one or more of glucose, fructose, mannose, glucosamine, N- acetylglucosamine, β- glucosides and hexitols, and the non-PTS compound is one or more of maltose, melibiose, lactose, mannose-6-phosphate, a succinate, ribose, glucose-6- phosphate, fructose-6-phosphate, L-arabinose, xylose, a gluconate, a lactate, a pyruvate, glycerol and galacturonic acid. 16. A growth medium according to any of Claims 10 to 1 5, which comprises an enzyme inducer compound. 17. A growth medium according to Claim 16, wherein the enzyme inducer is p-nitrophenol-β-D-glucuronide. 18. A growth medium for use in detecting micro-organisms in a sample, substantially as described in the Examples.

Inventors: BEERS PAUL JOHN, GLASCOCK STEPHEN JAMIE, MELDRUM RICHARD JAMES, BEERS, PAUL, JOHN, GLASCOCK, STEPHEN, JAMIE, MELDRUM, RICHARD, JAMES
Applicants: BEERS PAUL JOHN, GLASCOCK STEPHEN JAMIE, MELDRUM RICHARD JAMES, UNIV LINCOLNSHIRE & HUMBERSIDE, BEERS, PAUL, JOHN, GLASCOCK, STEPHEN, JAMIE, MELDRUM, RICHARD, JAMES, THE UNIVERSITY OF LINCOLNSHIRE & HUMBERSIDE
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1