Patent Application
20130059320
The present invention relates to the field of selective growth media and to culture media for the detection and identification of bacteria using such selective growth media, and more particularly to such selective growth media and culture media for the detection and identification of Campylobacter bacteria.
Campylobacter bacteria are the cause of campylobacteriosis in humans and animals. Most people who become ill with campylobacteriosis get diarrhea, cramping, abdominal pain, and fever within two to five days after exposure to the organism. Campylobacteriosis generally requires a week to 10 days for the patient to recover.
Patients with such symptoms are common in the United States and foreign countries and Campylobacter is amoung the most prevelent causes of these symptoms. It has been estimated that campylobacteriosis affects over 2.4 million persons every year in the United States, or 0.8 % of the population. While campylobacteriosis seldom results in death or long term illness, treating the symptoms of the illness results in significant health costs to both government and private sector. Accordingly the need to care for patients with symptoms of campylobacteriosis requires prompt diagnosis, and the large number of patients requires test procedures to be conducted economically.
Campylobacteriosis is generally diagnosed by finding Campylobacter bacteria in association with the patient. Unfortunately the nature of the Campylobacter bacteria has impeded both a prompt diagnosis and an inexpensive diagnosis. Most Campylobacter related human illness is caused by the species Campylobacter jejuni or Campylobacter coli, but human illness can also be caused by other species. Campylobacter jejuni and Campylobacter coli are temperature sensitive, cannot tolerate drying and grow only in places with less oxygen than the amount in the atmosphere. As a result, culture media of the prior art have not only required long incubation times and have lacked specificity, but the colonies of Campylobacter jejuni and Campylobacter coli have appeared small on the incubated media of the prior art and difficult to locate against a background of more rapidly growing organisms.
It is an object of the present invention to provide a culture medium that overcomes the prior art deficiencies for the detection and identification of the principle strains of Campylobacter that produce campylobacteriosis, namely Campylobacter jejuni and Campylobacter coli.
More specifically, it is an object of the present invention to provide a culture medium in which incubated plates exhibit fewer colonies of non-target or background organisms.
Further, it is an object of the present invention to provide a culture medium in which colonies of Campylobacter bacteria appear larger and brighter on incubated plates than on prior art incubated plates.
These objects are achieved by an agar plating media that contains (1 ) a substrate that reacts with the enzyme C-2 esterase, but substantially no other organism, to release precipitate of a distinctive color into the medium, and (2 ) a selective growth media for Campylobacter jejuni and Campylobacter coli bacteria containing nutrients and growth facilitators for Campylobacter bacteria to enlarge and accelerate growth of target colonies, and inhibitors for non-target organisms.
The selective growth medium of the present invention contains nutrients to promote growth of Campylobacter bacteria. The inventor has found that a selective growth medium achieving the objects of the present invention contains a mixture including one or more of the ingredients of the group heart infusion broth, vitamin mixture, lactalbumin hydrolysate, casamino acids, α-ketoglutaric acid and yeast extract. The preferred embodiment of the present invention described hereinafter utilizes a mixture of all of the ingredients of the foregoing group.
The inventor has found that one or more ingredients of the following group improve aero tolerance of the Campylobacter, when added to the mixture: hemin, sodium metabisulfite, sodium carbonate and sodium pyruvate. The preferred embodiment utilizes all of the ingredients of the foregoing group in its mixture.
Also selective growth media according to the present invention include one or more inhibitors of a group that the inventor has found are not capable of inhibiting the growth of Campylobacter bacteria. An inhibitor for gram positive bacteria is utilized, and the inventor has found bile salts #3 to be preferable. Other inhibitors of gram positive bacteria can also be used. The inventor has also found that Irgasan DP 300 is desirable in a selective growth medium and in a Campylobacter plating media according to the present invention materially reduces the background display on cultured plates, especially enteric bacteria such as Escherichia coli. Also, the inventor found it desirable for the selective growth media to contain sodium cefsulodin hydrate to inhibit Pseudomonas and Aeromonas type bacteria without affecting the growth of Campylobacter bacteria, and cycloheximide to prevent the growth of yeasts and molds. In addition, the inventor found that sodium cefoperazone is effective in selective growth media according to the present invention to inhibit the growth of other Campylobacter strains than Campylobacter jejuni or Campylobacter coli. The above described growth inhibitors form a group suitable for use in selective growth media for Campylobacter bacteria and one or more of the inhibitors of this group may be used in any particular medium, with or without any other inhibitor. Other growth inhibitors of non-target organisms that do not adversely effect the growth of Campylobacter bacteria may also be used.
The inventor has found that a selective growth medium suitable for use in Campylobacter plating media that achieves the objects of the present invention is achievable with at least all of the following growth inhibitors: cycloheximide, Irgasan DP 300, bile salts, sodium cefsuladin hydrate and cefoperazone sodium salt.
The inventor's preferred identification system for Campylobacter bacteria utilizes a solid plating medium containing a substrate that reacts with the enzyme C-2 esterase (acetate hydrolysis) which is released by Campylobacter bacteria but does not react with many other enzymes, thereby discriminating against bacteria that release such other enzymes. The preferred substrate is Aldol 515 acetate (A-4673_POO) which is produced by Biosynth A. G. of Switzerland under the trade name ALDOL. On contact with this substrate, Campylobacter jejuni and Campylobacter coli bacteria release enzyme that cleave the ALDOL substrate from the acetate group to produce a dark salmon precipitate into the medium, but other strains of Campylobacter that may grow on the medium do not.
The preferred plating medium comprises a mixture of the novel selective growth medium and the identification system, and this mixture is solidified by mixing Bacto-agar at 17 grams/liter to reduce the swarming of Campylobacter with the other ingredients of the medium. In addition, the inventor mixes titanium (IV) oxide into the combined mixture resulting in a white opaque medium which causes a white opaque background and results in a better contrast with the dark salmon Campylobacter colonies.
The preferred embodiment of the plating medium contains the ingredients in the proportions set forth in the following Table I.
Except for sodium cefsulodin hydrate, sodium cefoperazone Aldol 515 acetate, the ingredients are mixed in any order, the pH adjusted to 6.8 to 6.9, boiled to sterilize the mixture, and the mixture is permitted to cool to 48 -50 degrees Celsius. Thereafter, the supplements listed in Table 1, in sterile condition and at room temperature, are added aseptically to the other ingredients. The composition is then poured into plates and permitted to dry for 48 hours in the dark, and the plates are then ready to be used. Storage time of poured plates is as much as 45 days at 2 to 8 degrees Celsius. The final pH is between 6.8 and 7.2.
The process of the present invention requires a plate or mass of the plating medium to be inoculated with the test sample, and the inoculated mass is then incubated at 41°-42° Celsius for 48 hours under microaerophilic conditions to permit growth of the microorganisms in the test sample to observable dark salmon colonies 1-2 millimeters in diameter. The inventor has found that with the preferred plating medium described above, a period of 24 to 48 hours of incubation is sufficient time for Campylobacter colonies present in a test sample to grow into colonies that are readily observable with the naked eye. It is believed that the abundant growth of microorganisms in the preferred plating medium is due to the nutrients provided by the ingredients listed above and in Table 1. The surface of the plating medium mass is then assayed and the presence and number of dark salmon colonies, 1 to 2 millimeters in diameter with and without a clear ring, are recorded.
It is to be noted that no special equipment is required to observe the incubated mass of plating medium. The time required to note the number and presence of dark salmon colonies is far less than required when other colonies are present. Also, there are no ingredients in the plating medium that are especially costly. Hence, an assay of a test sample may be made at reduced cost from assays made with prior plating media.
Table 2 sets forth the results of tests made using the medium of Table 1 as described above and 188 microbial strains.
Campylobacter jejuni
Campylobacter coli
Campylobacter lari
Acinetobacter calcoaceticus
Acinetobacter baumannii
Pseudomonas sp.
Enterobactar aerogenes
Enterococcus faecalis
Candida albicans
Acinetobacter grimontii, Acinetobacter sp., Aeromonas
hydrophila, Alcaligenes sp., Candida paludigena,
Citrobacter freundii(2), Escherichia vulneris,
Edwardsiella tarda, Enterobacter sp.(4), Escherichia coli
Hafnia alvei (2), Klebsiella pneumonia(2), Kluyvera
ascorbata, Moraxella sp.(4), Morganella morganii(2),
Ochrobactrum anthropi, Pantoea agglomerans, Proteus
mirabilis, Providencia alcafaciens, Pseudomonas sp.(9),
Rhodococcus rhodochrous, Saccharomyces cerevisiae,
Salmonella sp.(7), Serratia sp.(4), Shewanella
putrefacieni, Shigella sp.(2), Stenotrophomona
maltophilia, Vibrio parahaemolyticus, Yersinia
enterocolitica
Carnobacterium mobile, Corynebacterium matruchotii,
Enterococcus faecalis, Lactobacillus sp.(2), Leueonostac
mesenteroides, Listeria sp.(3), Micrococcus luteus,
Paenibacillus macerans, Salinicoccus hispanicus,
Staphylococcus aureus(2) and Streptococcus sp.
The art will devise other uses for the selective growth media of the present invention including as a source of Campylobacter bacteria for laboratory study or experimentation, or use as a part of a plating media with a different detection system than that disclosed in this application. Likewise those skilled in the art may utilize the plating media of the present invention for uses not specifically described in the foregoing specification, but within the scope of the present invention. It is therefore intended that the scope of the present invention be not limited by the foregoing specification, but rather only by the appended claims.
| Number | Date | Country | |
|---|---|---|---|
| 61575795 | Aug 2011 | US |
