AU2006345267A1

AU2006345267A1 - Mastitis and bacterial detection media

Info

Publication number: AU2006345267A1
Application number: AU2006345267A
Authority: AU
Inventors: Raymond Thomas Cursons; Richard John Wilkins
Original assignee: WaikatoLink Ltd
Current assignee: WaikatoLink Ltd
Priority date: 2006-06-29
Filing date: 2006-07-21
Publication date: 2008-01-03
Also published as: NZ548223A; AR059281A1; WO2008002156A1

Description

WO 2008/002156 PCT/NZ2006/000181 MASTITIS AND BACTERIAL DETECTION MEDIA TECHNICAL FIELD This invention relates to mastitis and bacterial detection media. Preferably, although not exclusively this invention relates to a growth medium for the isolation of micro-organisms. 5 Reference throughout this specification shall now be made to use of the present invention for the isolation of Streptococcus using a novel agar medium. However, this should not be seen as a limitation on the present invention in any way. BACKGROUND ART A number of methods exist by which the presence of a micro-organism in a sample can 10 be determined. These range from DNA testing, such as polymerase chain reaction (PCR) to the culturing of micro-organisms in a growth medium. PCR has the power to produce a rapid, accurate result for the presence of a specific targeted nucleic acid sequence. This sequence may be unique to a particular organism, 15 or may be common to a number of related bacterial species. If this nucleic acid sequence is present in a sample, then a positive result will be produced. However, PCR techniques can be both labour and resource intensive and prone to both false positives and false negatives. Contamination by other nucleic acids or nucleic acid degrading enzymes may mean that reactions need to be repeated, using further time and 20 resources. PCR also requires the use of specialised enzymes and reagents which if a large number WO 2008/002156 PCT/NZ2006/000181 of samples are to be routinely tested, may prove prohibitively expensive. PCR also cannot provide indications of the number of bacteria present in a sample, or whether other non-target bacterial species are present. Another method of determining the presence of a micro-organism is to examine a sample 5 directly using microscopy to determine the number of cells present. In order to more clearly see micro-organisms using a microscope, dyes are often used to stain the cells and improve their contrast. Differential stains such as the Gram Stain are widely used to separate bacteria into different groupings. The morphology shown by the bacterial cells may also provide an indication of the likely species present. 10 However, while staining is a useful tool, it is limited in the amount of information it can provide about the identity of bacterial species. To determine the presence of a micro-organism, another widely used method is to grow a bacterial culture of the organism under laboratory conditions and investigate the characteristics the culture exhibits. The culture medium provides all the necessary 15 nutrients for the micro-organism to grow. Culture media can be prepared for use either in a liquid state, or in a gel or semi solid state. Often, a liquid medium is converted to a semi solid state by the addition of a gelling agent, most commonly agar. Culture media containing agar can then be dispensed into Petri plates, slides etc before 20 being inoculated with a sample and incubated to allow the bacteria to grow. The colonies that form are often visible to the naked eye, allowing identification or further testing. Some species of bacteria may require very specific culture conditions and/or culture 2 WO 2008/002156 PCT/NZ2006/000181 media, including the provision of trace elements either from chemical compounds or by the addition to the media of unrefined animal or plant extracts. Other media may be classed as being either selective, differential, or both. A selective medium may include compounds added to selectively inhibit the growth of certain micro 5 organisms but not others, whereas differential medium generally has some sort of indicator added to allow the user to differentiate between various chemical reactions being carried out by the micro-organisms. Depending on the bacterial species, further tests may still need to be carried out to identify the species of interest. 10 There are a number of clinically used agar mediums for the selective isolation of a range of gram negative bacteria. Based on the growth on primary isolation media, it may be possible to restrict the identity of an unknown bacterium to a relatively small number of species. A range of biochemical tests can then be performed to help identify the organism, based 15 on the presence or absence of enzymes involved in the catabolism of substrates added to the medium. The isolation of selective bacterial species from a sample is often an essential prerequisite in the identification of specific species of bacteria. The ability to detect the presence of a specific enzyme using suitable substrates, 20 particularly chromogenic or fluorogenic substrates has led to the development of media specific identification of a number of bacterial species. However, while there is a range of selective and differential media available, they are 3 WO 2008/002156 PCT/NZ2006/000181 often limited to gram-negative bacterial species. At present, there are no known media that select and provide presumptive evidence for the isolation of a large proportion of gram-positive bacteria, particularly bacterial species such as Streptococcus. Many Streptococcus species in addition to being gram-positive are pathogenic to humans 5 or animals. Other species are important in food spoilage. Because current methods for the isolation and identification of Streptococcus are typically both time consuming and expensive, any advances in these techniques would be beneficial, especially with environmental samples where background contamination is often complex. All references, including any patents or patent applications cited in this specification are 10 hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these 15 documents form part of the common general knowledge in the art, in New Zealand or in any other country. It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. 20 that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process. 4 WO 2008/002156 PCT/NZ2006/000181 It is an object of the present invention to provide a relatively accurate, low cost and low labor method of identifying the level of gram-positive Streptococcus bacteria in a sample or at least to provide the public with a useful choice. Further aspects and advantages of the present invention will become apparent from the 5 ensuing description which is given by way of example only. DISCLOSURE OF INVENTION According to one aspect of the present invention there is provided a method for providing evidence for the presence of Streptococcus in a sample, characterised by the steps of, 10 inoculating a culture medium with the sample, wherein the medium contains a substance with which Streptococcus can react to form a visual indicator detectable under short wavelength ultra-violet radiation. Reference throughout this specification shall now be made to the sample as being a milk sample from an animal. However, this should not be seen as limiting in any way as the 15 sample may be any sample taken from the environment, such as grass, soil, or water; or the sample may be a clinical specimen taken for the purposes of clinical and diagnostic microbiological testing of humans and/or animals. Reference shall also now be made to the microbial species as being Streptococcus uberis. Once again, this has been given by way of example only and should not be seen 20 as limiting in any way. The term "short wavelength ultra-violet radiation" may be defined as ultra-violet (UV) light with a wavelength between 200 nm to 320 nm. To provide short wavelength UV light, a 5 WO 2008/002156 PCT/NZ2006/000181 UV transilluminator operating at 312 nm will preferably be used. Once again, this is given by way of.example only and should not be viewed as a limitation on the present invention as it is expected that visual indicators will also be visible to differing extents at other wavelengths within the short wavelength UV region of the spectrum. 5 It should be appreciated that the growth medium may also be used to isolate and identify any Streptococcus species, such as Group A and Group B streptococci, among others. In preferred embodiments the species of Streptococcus may be S. uberis. For ease of reference only the present invention will now be described in relation to S. uberis. The term "substance" may be defined as any chemical that S. uberis can react with to 10 produce a visual indicator detectable under short wavelength UV radiation. Preferably, the substance will act as a substrate for enzyme(s) produced by S. uberis, yielding the visual indicator. However, this should not be seen as a limitation as the visual indicator may be produced by the result of another chemical reaction occurring due to the presence of S. uberis. 15 In one preferred embodiment of the present invention the substance is 4 methylumbelliferyl-beta-D-glucuronide, which when cleaved by an enzyme of S. uberis produces a fluorogenic product visible under short wavelength UV light. In another preferred embodiment of the present invention the substance is esculin. Esculin when cleaved by an enzyme of S. uberis produces a product which gives S. 20 uberis colonies a visible "halo" under short wavelength UV light. A number of other related chemical compounds may also be cleaved by various enzymes of S. uberis to produce fluorogenic products or other visual indicators and as such these 6 WO 2008/002156 PCT/NZ2006/000181 should not be seen as a limitation in any way. Fluorogenic substrates have been used as microbial indicators for a number of years. Such substrates are highly sensitive and very specific for a given enzyme. Because of their pH-dependence, strong diffusion in solid media and the need of UV light, the use of 5 these substrates is limited. Methylumbelliferyl-substrates have previously been used as an indicator of 3-D glucuronidase activity to detect the presence of coliforms, which produce a fluorescent product visible when irradiated with long wavelength UV light (366 nm). Long wavelength UV radiation is commonly used as it has a low energy and for its 10 recognised ability to cause fluorogenic materials to admit visible light. The inventors have surprisingly found that if S. uberis is present, 4-methylumbelliferyl beta-D-glucuronide is cleaved to yield a product which exhibits fluorescence under shorter wavelength UV light, such as 312 nm. This was unexpected given that no fluorescence is detectable at 366 nm, the wavelengths commonly used to test for P-D 15 glucuronidase activity. Because methylumbelliferyl-substrates are known to have high pH dependence, it is thought that S. uberis may lower the pH in the culture media to optimal enzyme conditions for the production of the fluorogenic compound. Shorter wavelength UV light has higher energy, often enough to damage biological 20 tissues. UV light at 312 nm is commonly used to visualise DNA samples stained with fluorescent dyes such as ethidium bromide or SYBR green. However, because of its mutagenic effects on biological samples, such wavelengths are not routinely used for other techniques. 7 WO 2008/002156 PCT/NZ2006/000181 S. uberis is a major bacterial pathogen in agriculture where it is often found to be a causative agent in mastitis, being the inflammation of the mammary glands in animals. Mastitis is a major concern in the dairy industry worldwide. In addition to immediate on farm animal health concerns, mastitis may cost dairy farmers significantly through lower 5 grade or lost production, in addition to the time and expense in treating infected animals. Before a treatment can be administered, it is preferable to first identify the bacterial species present in order to better target the treatment response. Thus having a technique which provides rapid evidence for the presence of S. uberis in a sample would allow affected animals to be rapidly treated, limiting lost production times 10 and reducing the costs of treatment. According to another aspect of the present invention there is provided a method for providing evidence of S. uberis in a sample substantially as described above, including an initial step of providing a visual indicator for the growth of S. uberis in a culture medium which is detectable under visible light. 15 Preferably, the culture medium includes a carbohydrate source with which Streptococcus can react to form a visual indicator. The visual indicator will preferably be produced as a result of carbohydrate fermentation, though this should not be seen as a limitation in any way. Carbohydrate fermentation will preferably result in the production of an acid, the presence 20 of which can be shown with a pH indicator. Gas such as hydrogen and/or carbon dioxide may also be formed during fermentative growth of carbohydrates which can be determined by observing gas production either in 8 WO 2008/002156 PCT/NZ2006/000181 gas collection vials or through the presence of gas bubbles present in the media. In preferred embodiments of the present invention the carbohydrate source is inulin. However, this is given by way of example only and should not be seen as a limitation on the present invention in any way. S. uberis ferments a number of carbohydrates such as 5 glucose and mannitol which could theoretically be used in the present invention as a carbohydrate source. The pH of the growth medium will preferably be 5.6 to 8.0 to provide optimal conditions for S. uberis growth and enzyme function. However, this should not be seen as limiting as S. uberis growth and enzyme action will occur outside this pH range to a lesser extent. 10 In order to show that the carbohydrate is being utilised, a pH indicator is preferably included in the media to detect changes in pH. Fermentation of the carbohydrate source by S. uberis will cause the pH of the media surrounding the bacterial colonies to be lowered as the breakdown products of fermentation are produced. As this occurs, the pH indicator in the media surrounding the 15 colonies will change colour, prompting a user to further investigate these colonies as potential S. uberis. In preferred embodiments of the present invention the pH indicator is Bromocresol purple, though it should be appreciated that the indicator could be any indicator which may work around the desired pH range of 5.2-6.8. 20 The inventors have found that using any of the visual indicators described herein in isolation enables approximately 90% of S. uberis strains to be identified. However, by combining inulin and one of either of 4-methylumbelliferyl-beta-D 9 WO 2008/002156 PCT/NZ2006/000181 glucuronide or esculin into a single media gives a far greater likelihood of determining the presence of S. uberis in a sample, providing a synergy not previously used for the presumptive identification of S. uberis in a sample. While a number of other chemical substrates, for example P-galactosidase, have been 5 trialed for use by the applicants, they have found that inulin, in combination with either 4 methylumbelliferyl-beta-D-glucuronide or esculin provides the best evidence of the presence of S. uberis in a sample. -galactosidase has previously been used in prior art attempts to create differential media and as such chemical substrates on which P-galactosidase act were a starting point for 10 the inventors. P-galactosidase was however found to be difficult to use when attempting to isolate and identify S. uberis. The bacterial colonies which grew on media containing P3 galactosidase substrates were often found to be very small in comparison to colonies grown on different media, requiring the addition of inducing compounds in the media to 15 promote growth. The inventors also found difficulties when using media containing -galactosidase substrates in isolating S. uberis direct from environmental samples, where background contamination is often high, even when selective media was used.. Because environmental samples often have background contamination, pre-isolation 20 steps are often necessary prior to identification. According to another aspect of the present invention there is provided a culture medium which includes a substance with which Streptococcus can react to form a visual indicator detectable under short wavelength UV radiation. 10 WO 2008/002156 PCT/NZ2006/000181 In preferred embodiments the medium contains inulin and one of either of 4 methylumbelliferyl-beta-D-glucuronide or esculin with which S. uberis can react to yield products which produce visual indicators. In preferred embodiments the medium contains at least one selective inhibitor. 5 The term "selective inhibitor" used within the present specification should be understood to mean anything that selectively inhibits the growth of some species of micro-organisms while not affecting the growth of other species. These compounds are preferably added to selectively inhibit the growth of contaminating micro-organisms, especially when the background level of contamination is expected to 10 be high, such as in environmental samples. In more preferred embodiments of the present invention the selective inhibitor may be one or both of the chemical compounds known as gentamicin and/or thallous acetate. It should be appreciated that these are given by way of example only and should not be seen as a limitation on the present invention, for a wide range of chemical compounds or 15 active agents may be added to the growth medium as selective inhibitors, depending on the species of micro-organism of interest. By using selective media as a pre-isolation step prior to the identification of S. uberis from contaminated samples, a wide range of environmental samples can easily be monitored for the presence and enumeration of S. uberis. 20 Such techniques could be used to monitor levels of S. uberis in grass, soil etc, allowing better farm management. When S. uberis levels increase past a given threshold, a farmer can move animals onto areas with low S. uberis levels as a preventative measure, 11 WO 2008/002156 PCT/NZ2006/000181 further reducing costs to a herd due to lost production and treatment of mastitis. The incubation of bacteria on agar plates is a standard microbiological procedure. This invention is directed to improvements on an existing procedure for the rapid isolation and presumptive identification of S. uberis from samples. 5 The applicants have found that the combination of inulin and 4-methylumbelliferyl-beta-D glucuronide or esculin in a growth medium unexpectedly provides a method for the rapid detection of S. uberis. While not all strains of S. uberis will utilise one of inulin, 4-methylumbelliferyl-beta-D glucuronide or esculin, it is statistically highly improbable that a strain of S. uberis would 10 not use any of these substrates. The present invention has been described with specific reference to Streptococcus uberis and a number of chemical compounds and culture conditions. It should be appreciated however that variations to the present invention are possible and will fall within the scope of the present invention as herein described. 15 BEST MODES FOR CARRYING OUT THE INVENTION As defined above, the present invention is directed to a growth medium for the isolation of Streptococcus using a novel agar medium. The invention is based upon the inventors' investigation into a wide range of carbohydrate sources, selective agents, chemical substrates and identification means that when 20 present in a growth medium provides presumptive evidence for the isolation of Streptococcus species, especially from complex samples. Non-limiting examples of the invention will now be provided. 12 WO 2008/002156 PCT/NZ2006/000181 Example 1 To isolate and identify the presence of S. uberis in a sample, a method has been devised involving inoculating a growth medium containing a carbohydrate source and a chemical substrate with the sample, culturing the medium for a given period of time then examining 5 the medium to detect the presence of S. uberis. The growth medium is preferably a Brain Heart infusion media consisting of 2.5 g peptone, 8.5 g yeast extract, 0.5 g meat extract, 15.0 g pancreatic digest of casein and 7.5 g of NaCI. 37.0 g of the Brain Heart media is dissolved in 1L of water and the pH adjusted to 6.8. 10 10.0 g of agar is then added per litre of Brain Heart infusion media. The carbohydrate source, preferably inulin, is added to the growth medium at a concentration of 10.0 g/L. Either 0.25 g/L of esculin or 0.1 g/L of 4-methylumbelliferyl-beta-D-glucuronide is also added to the media, as the chemical substrate. 15 Bromocresol purple is also preferably added to the growth medium as a pH indicator in order to show that inulin is being fermented by S. uberis. Gentamicin (2.5 U/ml) and/or thallous acetate (1.0 g/L) are preferably added to the growth media as selective inhibitors to reduce the background contamination of other microbial species. 20 Example 2 In order to isolate and positively identify S. uberis in a sample, a Petri plate containing the 13 WO 2008/002156 PCT/NZ2006/000181 medium of the present invention is preferably inoculated with a sample and incubated at 372C for 24 hours. If strains of S. uberis are present in the sample, fermentation of the carbohydrate source by S. uberis will cause the pH of the media surrounding the bacterial colonies to be 5 lowered. This causes the pH indicator in the media surrounding the colonies to change colour from blue to yellow, prompting a user to further investigate these colonies as potential S. uberis. If S. uberis is present, the P-glucuronidase enzyme produced by the bacteria cleaves 4 methylumbelliferyl-beta-D-glucuronide to produce 4-methylumbelliferone, which will 10 fluoresce blue under ultraviolet (UV) light. A UV transilluminator operating at 312 nm will preferably be used to detect for the fluorescent product. Esculin when cleaved by S. uberis causes a halo to be seen around the bacterial colonies under ultraviolet (UV) light. In complex samples where background contamination by other micro-organisms is high, 15 such as environmental or faecal samples, an initial isolation and identification step can be carried out on media containing inulin, esculin and selective agent(s). This type of media reduces most of the background contamination, whereupon esculin positive colonies can be inoculated onto media containing inulin and 4-methylumbelliferyl-beta-D-glucuronide to confirm the isolation of S. uberis in a sample. 20 Three indicators have been used by the inventors to determine the presence of S. uberis. These are the fermentation of inulin, the ability to hydrolyse esculin and the ability to break down 4-methylumbelliferyl-beta-D-glucuronide to produce a fluorescent blue product. 14 WO 2008/002156 PCT/NZ2006/000181 While not all strains of S. uberis will utilise one of inulin, 4-methylumbelliferyl-beta-D glucuronide or esculin, it is statistically highly improbable that a strain of S. uberis would not use any of these substrates. The applicants have found that the combination of inulin and esculin or 4 5 methylumbelliferyl-beta-D-glucuronide in a growth medium unexpectedly provides a method for the rapid detection of S. uberis. The incubation of bacteria on agar plates is a standard microbiological procedure. This invention is directed to improvements on an existing procedure for the rapid isolation and presumptive identification of S. uberis from samples 10 Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the appended claims. 15

Claims

1. A method for providing evidence for the presence of Streptococcus in a sample, characterised by the steps of, inoculating a culture medium with the sample, wherein the medium contains a substance 5 with which Streptococcus can react to form a visual indicator detectable under short wavelength ultra-violet radiation.

2. A method according to claim 1 wherein the sample is a milk sample from an animal, a sample taken from the environment, such as grass, soil, or water; a clinical specimen taken for the purposes of clinical and diagnostic microbiological testing of 10 humans and/or animals, or the like.

3. A method according to claim 1 wherein short wavelength ultra-violet radiation is ultra-violet (UV) light with a wavelength of between 200 nm to 320 nm.

4. A method according to claim 5 wherein short wavelength ultra-violet radiation is ultra-violet (UV) light with a wavelength of substantially 312 nm. 15

5. A method according to claim 1 wherein Streptococcus is any species of the Streptococcus genus.

6. A method according to claim 3 wherein the Streptococcus species is Streptococcus uberis.

7. A method according to claim 1 wherein the substance acts as a substrate for 20 enzyme(s) produced by Streptococcus which can react with to produce a visual indicator detectable under short wavelength UV radiation. 16 WO 2008/002156 PCT/NZ2006/000181

8. A method according to claim 1 wherein the substance is 4-methylumbelliferyl beta-D-glucuronide, esculin, or the like.

9. A method for providing evidence for the growth and presence of Streptococcus in a sample, 5 characterised by the steps of, a. providing a visual indicator for the growth of Streptococcus in a culture medium which is detectable under visible light, and, b. inoculating a culture medium with the sample, wherein the medium contains a substance with which Streptococcus can react to form a visual indicator detectable under 10 short wavelength ultra-violet radiation.

10. A method according to claim 9 wherein the culture medium includes a carbohydrate source with which Streptococcus can react to form a visual indicator.

11. A method according to claim 9 wherein the visual indicator is produced as a result of fermentation of a carbohydrate source. 15

12. A method according to claim 11 wherein carbohydrate fermentation results in the production of an acid, the presence of which can be shown with a pH indicator.

13. A method according to claim 12 wherein the pH indicator is included in the media to detect changes in pH.

14. A method according to claim 9 wherein gas such as hydrogen and/or carbon 20 dioxide may also be formed during fermentative growth of carbohydrates which can be determined by observing gas production either in gas collection vials or through the 17 WO 2008/002156 PCT/NZ2006/000181 presence of gas bubbles present in the culture medium.

15. A method according to claim 11 wherein the carbohydrate source is inulin, glucose, mannitol, or the like.

16. A method according to claim 9 wherein the pH of the growth medium is in the 5 range 5.2 to 8.0 to provide optimal conditions for Streptococcus growth and enzyme function.

17. A method according to claim 11 wherein the pH indicator is Bromocresol purple.

18. A method according to anyone of the preceding claims wherein the method includes an additional pre-isolation step prior to Streptococcus identification. 10

19. A culture medium which includes a substance with which Streptococcus can react to form a visual indicator detectable under short wavelength UV radiation.

20. A culture medium as claimed in claim 19 wherein the medium contains inulin and one of either of 4-methylumbelliferyl-beta-D-glucuronide or esculin.

21. A culture medium as claimed in claim 19 wherein the medium includes at least 15 one selective inhibitor.

22. A culture medium as claimed in claim 21 wherein the selective inhibitor is gentamicin and/or thallous acetate. 20 18