Selective media for recovery and enumeration of campylobacters

Details Selective media for recovery and enumeration of campylobacters Selective media for recovery and enumeration of campylobacters

2000-06-01

2002-04-09

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252100, C435S034000, C435S244000

Reexamination Certificate

active

06368847

ABSTRACT:

BACKGROUND OF THE INVENTION
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, 1984—the 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 42° C. incubation temperature and microaerobic atmosphere to limit the proliferation of non-Campylobacter organisms. The 42° 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 &mgr;g/ml), trimethoprim (5 &mgr;g/ml), polymyxin B (20 IU/ml), and cycloheximide (50 &mgr;g/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 &mgr;g/ml vancomycin, 2.5 IU/ml polymyxin, 5 &mgr;g/ml trimethoprim, and 15 &mgr;g/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 &mgr;g/ml vancomycin, 10 &mgr;g/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 &mgr;g/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
t

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