WO2008002156A1 - Mastitis and bacterial detection media - Google Patents
Mastitis and bacterial detection media Download PDFInfo
- Publication number
- WO2008002156A1 WO2008002156A1 PCT/NZ2006/000181 NZ2006000181W WO2008002156A1 WO 2008002156 A1 WO2008002156 A1 WO 2008002156A1 NZ 2006000181 W NZ2006000181 W NZ 2006000181W WO 2008002156 A1 WO2008002156 A1 WO 2008002156A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- streptococcus
- sample
- culture medium
- uberis
- visual indicator
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
Definitions
- This invention relates to mastitis and bacterial detection media.
- this invention relates to a growth medium for the isolation of micro-organisms.
- DNA testing such as polymerase chain reaction (PCR)
- PCR polymerase chain reaction
- 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, 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.
- 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 resources.
- PCR also requires the use of specialised enzymes and reagents which if a large number 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 directly using microscopy to determine the number of cells present.
- 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.
- staining is a useful tool, it is limited in the amount of information it can provide about the identity of bacterial species.
- the culture medium provides all the necessary 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 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 media, including the provision of trace elements either from chemical compounds or by the addition to the media of unrefined animal or plant extracts.
- a selective medium may include compounds added to selectively inhibit the growth of certain micro- 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.
- a range of biochemical tests can then be performed to help identify the organism, based on the presence or absence of enzymes involved in the catabolism of substrates added to the medium.
- Streptococcus species in addition to being gram-positive are pathogenic to humans 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.
- sample as being a milk sample from an animal.
- the 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.
- short wavelength ultra-violet radiation may be defined as ultra-violet (UV) light with a wavelength between 200 nm to 320 nm.
- UV ultra-violet
- a UV transilluminator operating at 312 nm will preferably be used.
- the growth medium may also be used to isolate and identify any Streptococcus species, such as Group A and Group B streptococci, among others.
- the species of Streptococcus may be S. uberis.
- S. uberis For ease of reference only the present invention will now be described in relation to S. uberis.
- substrate may be defined as any chemical that S. uberis can react with to produce a visual indicator detectable under short wavelength UV radiation.
- the substance will act as a substrate for enzyme(s) produced by S. uberis, yielding the visual indicator.
- the visual indicator may be produced by the result of another chemical reaction occurring due to the presence of S. uberis.
- 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.
- the substance is esculin.
- Esculin when cleaved by an enzyme of S. uberis produces a product which gives S. 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 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 these substrates is limited.
- Methylumbelliferyl-substrates have previously been used as an indicator of ⁇ -D- glucuronidase activity to detect the presence of coliforms, which produce a fluorescent product visible when irradiated with long wavelength UV light (366 nm).
- UV radiation is commonly used as it has a low energy and for its recognised ability to cause fluorogenic materials to admit visible light.
- the inventors have surprisingly found that if S. uteris 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 ⁇ -D- glucuronidase activity.
- UV light at 312 nm is commonly used to visualise DNA samples stained with fluorescent dyes such as ethidium bromide or SYBR green.
- fluorescent dyes such as ethidium bromide or SYBR green.
- 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 grade or lost production, in addition to the time and expense in treating infected animals.
- a treatment Before a treatment can be administered, it is preferable to first identify the bacterial species present in order to better target the treatment response.
- 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.
- 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 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 gas collection vials or through the presence of gas bubbles present in the media.
- the carbohydrate source is inulin.
- S. uberis ferments a number of carbohydrates such as 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.
- 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 colonies will change colour, prompting a user to further investigate these colonies as potential S. uberis.
- 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.
- 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.
- ⁇ -galactosidase has previously been used in prior art attempts to create differential media and as such chemical substrates on which ⁇ -galactosidase act were a starting point for the inventors.
- ⁇ -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 ⁇ - 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 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..
- a culture medium which includes a substance with which Streptococcus can react to form a visual indicator detectable under short wavelength UV radiation.
- the medium contains inulin and one of either of A- methylumbelliferyl-beta-D-glucuronide or esculin with which S. uberis can react to yield products which produce visual indicators.
- the medium contains at least one selective inhibitor.
- 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 be high, such as in environmental samples.
- the selective inhibitor may be one or both of the chemical compounds known as gentamicin and/or thallous acetate.
- Such techniques could be used to monitor levels of S. uberis in grass, soil etc, allowing better farm management.
- S. uberis levels increase past a given threshold, a farmer can move animals onto areas with low S. uberis levels as a preventative measure, 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.
- 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 present in a growth medium provides presumptive evidence for the isolation of Streptococcus species, especially from complex samples.
- 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.
- 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.
- 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
- thallous acetate 1.0 g/L
- Gentamicin 2.5 U/ml
- thallous acetate 1.0 g/L
- a Petri plate containing the medium of the present invention is preferably inoculated with a sample and incubated at 37 9 C for 24 hours.
- 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 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.
- the ⁇ -glucuronidase enzyme produced by the bacteria cleaves 4- methylumbelliferyl-beta-D-glucuronide to produce 4-methylumbelliferone, which will 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.
- UV ultraviolet
- 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.
- S. uberis 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. 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.
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- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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Abstract
Description
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Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2006345267A AU2006345267A1 (en) | 2006-06-29 | 2006-07-21 | Mastitis and bacterial detection media |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ548223 | 2006-06-29 | ||
NZ54822306A NZ548223A (en) | 2006-06-29 | 2006-06-29 | Methods for detecting the presence of Streptococcus in a sample |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2008002156A1 true WO2008002156A1 (en) | 2008-01-03 |
Family
ID=38845831
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NZ2006/000181 WO2008002156A1 (en) | 2006-06-29 | 2006-07-21 | Mastitis and bacterial detection media |
Country Status (4)
Country | Link |
---|---|
AR (1) | AR059281A1 (en) |
AU (1) | AU2006345267A1 (en) |
NZ (1) | NZ548223A (en) |
WO (1) | WO2008002156A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101985648A (en) * | 2010-11-30 | 2011-03-16 | 深圳市爱杰特医药科技有限公司 | Group B streptococcus selective culture medium, identification culture medium and using method thereof |
WO2016137341A1 (en) * | 2015-02-27 | 2016-09-01 | Mastaplex Limited | Bacteria identification and antimicrobial susceptibility test |
CN106480161A (en) * | 2016-11-16 | 2017-03-08 | 吉林大学 | A kind of mastitis for milk cows detection kit |
-
2006
- 2006-06-29 NZ NZ54822306A patent/NZ548223A/en not_active IP Right Cessation
- 2006-07-21 WO PCT/NZ2006/000181 patent/WO2008002156A1/en active Application Filing
- 2006-07-21 AU AU2006345267A patent/AU2006345267A1/en not_active Abandoned
-
2007
- 2007-02-01 AR ARP070100428A patent/AR059281A1/en unknown
Non-Patent Citations (4)
Title |
---|
BEIGHTON D. ET AL.: "A scheme for the identification of viridans streptococci", JOURNAL OF MEDICAL MICROBIOLOGY, vol. 35, no. 6, 1991, pages 367 - 372 * |
MATTHEWS K.R. ET AL.: "Evaluation of two fluorogenic assays for identification of Streptococcus species isolated from bovine mammary glands", JOURNAL OF DAIRY SCIENCE, vol. 74, no. 2, 1991, pages 421 - 425 * |
PEARCE C. ET AL.: "Identification of pioneer viridans streptococci in the oral cavity of human neonates", JOURNAL OF MEDICAL MICROBIOLOGY, vol. 42, no. 1, 1995, pages 67 - 72 * |
SCHAUFUSS P. ET AL.: "Rapid differentiation of streptococci isolated from cows with mastitis", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 24, no. 6, 1986, pages 1098 - 1099 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101985648A (en) * | 2010-11-30 | 2011-03-16 | 深圳市爱杰特医药科技有限公司 | Group B streptococcus selective culture medium, identification culture medium and using method thereof |
WO2016137341A1 (en) * | 2015-02-27 | 2016-09-01 | Mastaplex Limited | Bacteria identification and antimicrobial susceptibility test |
US10829796B2 (en) | 2015-02-27 | 2020-11-10 | Mastaplex Limited | Bacteria identification and antimicrobial susceptibility test |
CN106480161A (en) * | 2016-11-16 | 2017-03-08 | 吉林大学 | A kind of mastitis for milk cows detection kit |
CN106480161B (en) * | 2016-11-16 | 2018-04-03 | 吉林大学 | A kind of mastitis for milk cows detection kit |
Also Published As
Publication number | Publication date |
---|---|
AR059281A1 (en) | 2008-03-19 |
AU2006345267A1 (en) | 2008-01-03 |
NZ548223A (en) | 2008-09-26 |
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