1. Field of the Invention
This invention relates to media containing an indicator, 2,3,5-triphenyltetrazolium chloride, and a selective agent composition; and methods for the recovery and enumeration of Campylobacter species.
2. Description of the Related Art
Campylobacter species have been recognized as important causative agents of foodborne illness. There is a strong association of foods of animal origin in the transmission of disease to humans. Poultry is one such food with high carriage rates of Campylobacter contamination. Campylobacter jejuni, C. coli and C. lari are known to cause an estimated 2.2 million cases of foodborne gastroenteritis per year in the United States alone (Tauxe et al., American J. Public Health, Volume 77, 1219-1221, 1987). The vast majority of these cases are associated with the consumption of improperly prepared or handled foods. Although the origin of this disease in humans is primarily linked to poultry, the food microbiology and poultry communities have been slow in directing substantive attention toward the organism. This has been due, in part, to the unique physiological requirements of these organisms, impairing their culture and identification from foods and clinical specimens.
A variety of enrichment and culture media have been proposed for the isolation of Campylobacter species (Park et al., Campylobacter, In: Compendium of Methods for the Microbiological Examination of Foods, second ed., M. L. Speck (ed.), Am. Pub. Hlth. Assoc., Wash., D.C., 386-404, 1984xe2x80x94the contents of which are herein incorporated by reference). Because Campylobacter can be overgrown by other organisms present in sources, the use of selective media, incorporating antibiotics and/or antimicrobial agents, is essential for their isolation. Ideally, any culture medium selected should also be differential, allowing the characterization of the Campylobacter by distinctive colonial appearances in culture.
Rapid and sensitive methods for recovering Campylobacter would be useful for both epidemiological work and routine examination of food sources. The main drawback associated with numerous available procedures is the length of time needed for enrichment. Enrichment culture incubation ranges from 16 to 48 hours before plating on selective media, which then requires an additional 24 to 48 hours for isolation. This 3 to 4 day procedure is difficult to reconcile with rapid marketing strategies while maintaining interest in the public health.
The unique physiological requirements of Campylobacter species provide difficulties in culturing the microorganism. C. jejuni require special microaerobic atmospheres for growth (Kiggin et al, J. Bacteriology, Volume 72, 397-400, 1956), and its translucent colonies are frequently difficult to identify on dark, opaque Campylobacter agars.
Several agar media have gained prominence for the isolation of Campylobacter. Campy-Brucella Agar Plate (Campy-BAP), has been widely used and cited in the Compendium of Methods for Microbiological Examination of Foods (2nd Ed., American Public Health Association, Wash., D.C., M. L. Speck, ed., 386-404, 1984). Campy-BAP agar contains Brucella agar, lysed horse blood, vancomycin, polymyxin B, trimethoprim lactate, amphotericin B and cephalothin. Food samples assayed with Campy-BAP medium often yield large numbers of breakthrough flora. Butler developed a selective medium for C. jejuni containing a nutrient agar base, blood, and five selective agents, cycloheximide, cefazolin, bacitracin, colistin sulfate and novobiocin as described by Smibert (Campylobacter, In: Bergey""s Manual of Systematic Bacteriology, Krieg and Holt (eds.), Williams and Wilkins, Baltimore, Md., Volume 1, 111-115, 1984; the contents of which are herein incorporated by reference). Another agar, Campy-Cefex, allows for selective and differential culture of C. jejuni (Stern et al., Journal of Food Protection, Volume 55 (7), 514-517, Jul. 1992; U.S. Pat. No. 5,891,709, Apr. 6, 1999). Campy-Cefex agar contains Brucella agar, 0.05% ferrous sulfate, 0.02% sodium bisulfite, 0.05% sodium pyruvate, 33 mg/L sodium cefoperazone, 200 mg/L sodium cycloheximide, and 5% lysed horse blood. CCDA agar contains Nutrient broth No. 2, Bacteriological charcoal, casein hydrosylate, sodium desoxycholate, ferrous sulfate, sodium pyruvate, agar, yeast extract, sodium cefoperazone, and sodium cycloheximide. CCDA medium, also widely used and cited in Compendium of Methods for Microbiological Examination of Foods, (supra), was developed to replace the blood component which is specified in many Campylobacter recovery media, with charcoal. It uses cefoperazone as the selective antimicrobial agent acting in concert with a 42xc2x0 C. incubation temperature and microaerobic atmosphere to limit the proliferation of non-Campylobacter organisms. The 42xc2x0 C. incubation temperature greatly reduces the need for anti-gram-positive antimicrobials. The ferrous sulfate component of the medium has been used to enhance the growth and aerotolerance of Campylobacter spp. The main disadvantage of CCDA medium is its dark opacity, making it difficult to differentiate between Campylobacter spp. and non-Campylobacter spp. flora. Cefoperazone does not inhibit growth of molds and yeast on CCDA medium which can be associated with poultry samples.
Rothenberg et al (Applied and Environmental Microbiology, Volume 48(1), 78-80, Jul. 1984) disclose an attempt to develop an enrichment broth requiring only 7 hours of incubation and the comparison of their broth with that described by Doyle and Roman (Applied Environmental Microbiology, Volume 43, 1343-1353, 1982) and Park and Stankiewicz (Abstr. Assoc. Of Anal. Chem., Annu. Meet., volume 19, page 3, 1982). The Rothenberg et al. medium was a modification of the Doyle Roman broth and additionally contains 0.2% ferrous sulfate, 0.025% sodium metabisulfite, 0.05% sodium pyruvate, 0.1% sodium lauryl sulfate, and 0.075% agar. The Doyle and Roman broth contains Brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 xcexcg/ml), trimethoprim (5 xcexcg/ml), polymyxin B (20 IU/ml), and cycloheximide (50 xcexcg/ml). After a 16 to 18 hour incubation, the medium containing inoculum is plated directly onto Campy-BAP agar plates. The Stankiewicz broth contains vancomycin (20 mg/l), trimethoprim (10 mg/l), polymyxin B (5,000 IU/l for monophasic broth and 7,500 IU/l for diphasic medium), and lysed horse blood (5%, optional) in brucella broth. For the monophasic medium, 50 ml of the enrichment broth is placed in a 250 ml-Erlenmeyer flask. For the diphasic medium, brucella agar base (30 ml) with an overlay of the enrichment broth (50 ml) was made in a 500 ml-Erlenmeyer flask. Two other media are disclosed by Rothenberg. One contains 25 ml Brucella broth supplemented with 0.2% ferrous sulfate, 0.025% sodium metabisulfite, and 0.05% sodium pyruvate. The other, a selective medium, contains blood agar base no. 2 (Oxoid Ltd., London, England), 5% lysed horse blood, 10 xcexcg/ml vancomycin, 2.5 IU/ml polymyxin, 5 xcexcg/ml trimethoprim, and 15 xcexcg/ml cephalothin.
Castillo-Ayala (Journal of Food Protection, Volume 55(5), 333-336, May 1992) disclose enrichment broths for isolation of Campylobacter jejuni/coli from freshly deboned market chicken. The first broth, VTP broth, contained 25 ml of double strength Brucella broth, 0.025% of sodium metabisulfate, 0.025% sodium pyruvate, 20 xcexcg/ml vancomycin, 10 xcexcg/ml trimethoprim, 5 IU polymyxin B. The other, BCN, contained 25 ml of double strength Brucella broth, 0.025% sodium metabisulfate, 0.025% sodium pyruvate, 50 IU/ml bacitracin, 20 IU/ml novobiocin, and 10 xcexcg/ml cycloheximide. They concluded that the vancomycin-trimethoprim-polymyxin B mixture was not a suitable agent for use in an enrichment-plating procedure to recover Campylobacter from poultry.
Christopher et al (Journal of Food Protection, Volume 45(3), 260-262, Feb., 1982) disclose a method and media for isolation and enumeration of Campylobacter fetus subsp. jejuni that included a subculturing step that used Brucella broth containing 0.15% agar, 0.05% sodium pyruvate, 10 mg/liter vancomycin, 5 mg/liter trimethoprim, 2,500 IU/liter polymyxin B sulfate, 2 mg/l amphotericin B, and 15 mg/l cephalothin followed by subsequent streaking on plates of Brucella agar supplemented with 10% defibrinated horse blood and 10 mg/liter vancomycin, 5 mg/liter trimethoprim, 2,500 IU/liter polymyxin B sulfate, 2 mg/liter amphotericin B, and 15 mg/liter cephalothin.
U.S. Pat. No. 5,302,388 (Doyle et al; Apr. 12, 1994) discloses an enrichment broth for Campylobacter jejuni containing Brucella broth, 7% lysed horse blood, 0.3% sodium succinate or 0.01% cysteine hydrochloride, 15 xcexcg vancomycin/ml, 5 xcexcg trimethoprim/ml, 20 IU polymyxin B/ml, and 50 xcexcg cycloheximide/ml.
Luechtefeld and Wang (J. Clin. Microbiol., Volume 15(1), 137-140, January 1982) disclose the use of 1 mg or 400 xcexcg of 2,3,5-triphenyltetrazolium chloride (TTC) per ml of a medium containing brucella agar and sheep blood for differentiating C. fetus subsp. jejuni from C. fetus subsp. intestinalis. They report that all strains of C. fetus subsp. intestinalis were sensitive to 400 xcexcg/ml of TTC and all strains of C. fetus subsp. jejuni were insensitive to 400 xcexcg/ml TTC. The reference also discloses that Veron and Chatelain (Intl. J. Syst. Bacteriol., Volume 23, 122-134, 1973) found that none of the 18 strains of C. fetus subsp. fetus (C. fetus subsp intestinalis of Smibert (supra)) grew on blood agar containing 1 mg/ml of TTC in contrast to 9 strains of Campylobacter coli and 1 strain of C. jejuni. 
Hanninen (Acta vet. scand., Volume 23, 88-98, 1982) disclose testing the tolerance of the C. jejuni/coli group to 1 mg/liter of TTC. The reference states that most strains in their study tolerated TTC. It further states that according to Skirrow and Benjamin, C. jejuni strains are sensitive to TTC and C. coli strains tolerated it. The reference further states that there are differences in TTC tolerance between the Campylobacter strains investigated by different authors and that most of the TTC tests made earlier and in their studies have been performed on a blood agar substrate containing TTC. Hanninen concludes that the blood in the substrate may be one reason why so many of the present strains tolerated TTC and the reference also observed that Skirrow and Benjamin did not use a blood-containing medium in their tests. The reference further discloses that the optimum TTC concentration for TTC reduction of C. fetus and related vibrios is shown to be 400 xcexcg/ml and notes that TTC has been used in a concentration range of 400 xcexcg/ml to 1 mg/ml.
Existing media for the recovery and enumeration of colonies of Campylobacter species often contain many colonies of contaminating microorganisms and are difficult to enumerate because of the translucent nature of Campylobacter colonies. Therefore, there is a need in the art for media which recover high populations of Campylobacter with fewer contaminating colonies and which are easily enumerated. The present invention, described below, is improved media which provide selectivity for Campylobacter species while increasing the contrast of the colonies with the media to simplify counting procedures and is different from related art media.
It is therefore an object of the present invention to provide improved media for recovery and enumeration of Campylobacter species.
Another object of the present invention is to provide improved media for the recovery and enumeration of Campylobacter species, the improvement includes an indicator for facilitating accurate enumeration of Campylobacter colonies.
A further object of the present invention is to provide improved media for the recovery and enumeration of Campylobacter species wherein the indicator is 2,3,5 Triphenyltetrazolium chloride (TTC).
A still further object of the present invention is to provide improved media for the recovery and enumeration of Campylobacter species wherein the 2,3,5 Triphenyltetrazolium chloride concentration is about 200 xcexcg/ml or less.
A further object of the present invention is to provide improved media for the recovery and enumeration of Campylobacter species which is a blood-containing or a blood-free media.
A still further object of the present invention is to provide a media which contains a selective agent composition to provide improved selectivity for Campylobacter species.
Further objects and advantages of the present invention will become apparent from the following description.
The media of this invention may be used for the isolation and enumeration of Campylobacter species such as, for example, C. jejuni, C. coli, C. lari, etc., from a variety of sources. Although the media are particularly valuable for the growth and recovery of this microorganism from samples taken from poultry carcasses, especially chicken, it is understood that the media may also be used for the isolation of Campylobacter from any samples suspected of containing this pathogen. Without being limited thereto, other sources include animal carcasses such as cattle and sheep, food, milk, water, or environmental samples, or clinical sources such as blood or feces. The culture media contemplated for use in this invention may be prepared using techniques conventional in the art. The basal medium components including agar and/or nutrient media with an energy source are mixed, heated to boiling and sterilized by autoclaving. After cooling the sterilized medium to about 50xc2x0 C.-55xc2x0 C., blood and filter-sterilized supplements are added with mixing, pH asceptically adjusted to about pH 7.4 with, for example, 10N NaOH, and the medium finally poured into a culture container, such as a petri dish, for example, and cooled to allow the agar to solidify.
In one embodiment of the present invention, the basal medium components selected for use are not critical and may be readily determined by the practitioner skilled in the art, one of the improvements includes the addition of an indicator, 2,3,5-triphenyltetrazolium chloride in amounts which do not significantly inhibit the growth of Campylobacter species. Any nutrient medium and energy source effective to support growth of Campylobacter species may be used. Suitable nutrient media include, for example, but are not limited to Brucella agar (e.g. BBL, Cockeysville, Md.; Difco Laboratories, Detroit, Mich.;) CM 691 (Oxoid, Columbia, Md.), Campylobacter agar base (Difco), Blood agar base No. 2 (Oxoid), Brain-heart infusion agar (BBL;Difco), or Columbia Blood Agar Base. A variety of energy sources may also be employed, and may be incorporated into commercially available nutrient media or added separately. In the preferred embodiments of the present invention, Brucella agar (Acumedia) is the preferred nutrient medium. Suitable energy sources for use in the media are described by Sibert (INL Bergey""s Manual; supra, the contents of which are herein incorporated by reference) and include pyruvate, citrate, succinate, cis-aconitate, xcex1-ketoglutarate, fumarate, malate, and oxaloacetate.
For all embodiments of the present invention, the source of blood added to the medium also is not critical. While horse blood is preferred, it is understood that other blood sources may be used, such as for example, sheep blood. For blood-free media, hemin, yeast extract, sodium carbonate and xcex1-ketoglutaric acid are used to replace the blood component.
Selective agents, for use in all embodiments of the present invention, are used to prevent the growth of contaminating microorganisms present in samples to be tested, but which do not inhibit growth of Campylobacter species. The selective agents include any selective agent or combination thereof known to one of ordinary skill in the art. Improved preferred selective agents include vancomycin, trimethoprim, polymyxin B, cycloheximide, rifampicin, nystatin, amphotericin, and cefoperazone, their salts, and mixtures thereof. The combination of these preferred agents provides excellent selectivity for Campylobacter species.
An indicator, for use in all embodiments of the present invention, is included in Campylobacter media to facilitate manual or automated enumeration of Campylobacter colonies. The indicator 2,3,5-triphenyltetrazolium chloride (TTC; Sigma) is used in a concentration range of about 50 xcexcg/ml to about 250 xcexcg/ml. The preferred concentration is about 200 xcexcg/ml.
Other adjuvants, useful in all embodiments of the present invention, may also be incorporated into the media for enhancing growth and/or aerotolerance of Campylobacter. Preferred adjuvants enhancing aerotolerance are described by Smibert (supra) and include but are not limited to sodium pyruvate or pyruvic acid, ferrous sulfate, bovine superoxide dismutase, catalase and reducing agents such as sodium bisulfite or sodium metabisulfite. Particularly preferred for addition to medium are ferrous sulfate, pyruvic acid, and sodium bisulfite. It is understood that the use of blood in the media also enhances aerotolerance because it contains heme, catalase and superoxide dismutase.
The concentration and amount of each of the components of the Campy-TTC blood-containing medium, a particularly preferred embodiment of the present invention, are variable and may be readily determined by the practitioner skilled in the art. The amount of each component of the basal or nutrient media should be effective to promote growth of Campylobacter species, while the amount of the selective agents should be effective to inhibit growth of contaminating (non-Campylobacter) microorganisms without substantially inhibiting growth of Campylobacter species relative to culture medium lacking these selective agents. Without being limited thereto, preferred ranges of the selective agents include about 20-50 mg/liter cefoperazone, about 100-400 mg/liter cycloheximide, about 5-20 milligrams/liter vancomycin, about 2.5-10 milligrams/liter trimethoprim, about 5-20 milligrams/liter polymyxin, about 5-20 milligrams/liter rifampicin, about 10-40 milligrams/liter amphotericin, about 20-80 milligrams/liter nystatin, and about 100-250 mg/liter 2,3,5-Triphenyltetrazolium chloride.
In accordance with a particularly preferred formulation of blood-containing Campy-TTC, ranges of the amount of each component per liter include but are not limited to:
The final pH of the medium should generally be between about 6.5 to about 7.8, pH 7.4 is preferred.
In accordance with another particularly preferred formulation, blood-free Campy-TTC, ranges of the amount of each component per liter include but are not limited to:
The final pH of the medium should generally be between about 6.5 to about 7.8, pH 7.4 is preferred.
In accordance with another particularly preferred formulation, blood-free Campy-TTC, ranges of the amount of each component per liter include but are not limited to:
final pH of the medium should generally be between about 6.5 to 7.8, pH 7.4 is preferred.
In accordance with another particularly preferred formulation, blood-free Campy-TTC, ranges of the amount of each component per liter include but are not limited to:
The final pH of the medium should generally be between about 6.5 to 7.8, pH 7.4 is preferred. Effective amounts of each ingredient are those amounts which promote growth of Campylobacter species.
In accordance with another particularly preferred formulation, blood-free Campy-TTC, ranges of the amount of each component per liter include but are not limited to:
The pH of the medium should generally be between about 6.5 to 7.8, pH 7.4 is preferred. Effective amounts of each ingredient are those amounts which promote growth of Campylobacter species.
In accordance with another particularly preferred formulation, blood-free campy-TTC, ranges of the amount of each component per liter include but are not limited to:
The final pH of the medium should generally be between about 6.5 to 7.8, pH 7.4 is preferred. Effective amounts of each ingredient are those amounts which promote growth of campylobacter species.
In use, the sample to be analyzed is inoculated onto the culture medium using techniques conventional in the art and is incubated for a sufficient time and under conditions effective to promote growth of Campylobacter species. Suitable conditions may be readily determined by the practitioner skilled in the art and are described by Smibert (supra). Without being limited thereto, preferred conditions include a temperature between about 35xc2x0 C. to about 44xc2x0 C., especially about 42xc2x0 C. to about 43xc2x0 C., and a low oxygen tension (i.e., microaerobic), especially an oxygen concentration of between about 3-6%. Techniques for generating this reduced atmosphere are well known in the art and are described in, for example, Inoue (U.S. Pat. No. 4,904,597), or Hutchinson and Bolton (J. Clin. Pathol., Volume 36, 1350-1352, 1983), or Park et al. (supra), the contents of each of which are herein incorporated by reference.
Following incubation, generally after about 24-48 hours, the culture may be examined for the presence of colonies indicative of Campylobacter. Colonies of Campylobacter on any of the preferred Campy-TCC media of this invention are deep red to magenta in color. These colonies can be readily discriminated from non-Campylobacter breakthrough flora. Further confirmatory testing of the colonies and speciation can be conducted as described by Park et al (supra) or Smibert (supra). The use of these agars containing 2,3,5-triphenyltetrazolium chloride would be a benefit to technicians in enumerating and confirming Campylobacter colonies. The use of Campy-TTC agars, either blood-containing or blood-free with a preferred selective agent composition, also is a benefit because of the increased selectivity of the agars. Automated counting methods may also be made possible using triphenyltetrazolium chloride containing agars since the darker colonies contrast sufficiently with the agar base to allow detection by electronic means.