US-12618095-B2 - Medium for Bacillus cereus group detection

US12618095B2US 12618095 B2US12618095 B2US 12618095B2US-12618095-B2

Abstract

A medium for Bacillus cereus group detection, which is favorable in growth of Bacillus cereus regardless of the temperature condition, and further is excellent in selectivity; and a method for detecting a Bacillus cereus group using the medium. The medium for Bacillus cereus group detection includes a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical; and trimethoprim. The medium further includes a β-lactam antibiotic. The medium further includes an antifungal agent. The method for detecting further includes inoculating a sample into the medium to culture the sample; and determining a detectable colony on the medium.

Inventors

Yusuke KOYANAGI

Assignees

SHIMADZU DIAGNOSTICS CORPORATION

Dates

Publication Date: 20260505
Application Date: 20210330
Priority Date: 20200331

Claims (3)

1 . A medium for Bacillus cereus group detection, comprising, as concentrations at the time of detection: from 0.001 to 10 g/l of a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical; from 0.01 to 500 mg/l of trimethoprim; from 0.001 to 100 mg/l of a cephem antibiotic or a monobactam antibiotic; and from 0.001 to 100 mg/l of a polyene antifungal agent, wherein the cephem antibiotic comprises ceftazidime, the monobactam antibiotic comprises aztreonam, and the polyene antifungal agent comprises amphotericin B, wherein the Bacillus cereus group is at least one bacterium selected from the group consisting of Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus cytotoxicus , and Bacillus toyonensis.
2 . The medium for Bacillus cereus group detection according to claim 1 , wherein the phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical is at least one selected from the group consisting of 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate, 5-bromo-6-chloro-3-indoxyl myo-inositol-1-phosphate, 6-chloro-3-indoxyl myo-inositol-1-phosphate, 4-methylumbelliferone myo-inositol 1-phosphate, 4-nitrophenyl-myo-inositol-1-phosphate, luciferin-myo-inositol-1-phosphate, and a salt thereof.
3 . A method for detecting a Bacillus cereus group, comprising: inoculating a sample into the medium of claim 1 to culture the sample; and determining a detectable colony on the medium.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a National Stage entry under 35 U.S.C. § 371 of PCT/JP2021/013502, filed on Mar. 30, 2021, and claims priority to Japanese Patent Application No. 2020-064835, filed on Mar. 31, 2020, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION The present invention relates to a medium for Bacillus cereus group detection. BACKGROUND OF THE INVENTION Bacillus cereus is a gram-positive spore-forming bacillus, and in general, is widely distributed in the natural world such as soil and rivers. The range of contamination by the present bacterium is a broad range starting from, for example, grains and spices, which are foods having a close relationship with soil, to, for example, food products that are cross-contaminated by using such foods, for example, noodles such as chow mein, and spaghetti, cooked rice such as rice balls, and fried rice, gratin, pizza, fish and shellfish, processed products of fish and shellfish, meat, processed products of meat, a food raw material, confectionery, environment, and a clinical material. Contamination with the present bacterium sometimes causes rotting and deterioration. Further, the present bacterium is known to produce a vomiting toxin and a diarrhea toxin, and may cause food poisoning. Therefore, the control of the present bacterium is important also from the viewpoint of food hygiene and safety (Non Patent Literature 1). In general, as the medium used for detecting Bacillus cereus, for example, a NGKG (Nacl-Glycine-Kim-Goepfect) agar medium, and a MYP (Mannitol Yolk-Polymixin) agar medium (for example, Non Patent Literatures 1 to 3) are known. These media contain egg yolk in order to utilize egg yolk reaction that is one of the characteristics of Bacillus cereus, as one of the detection principles. The method for preparing the egg yolk-containing medium includes a step of sterilizing and dissolving a medium raw material such as agar other than egg yolk in advance, a step of cooling and keeping the dissolved material at around 50° C., a step of further aseptically adding and mixing collected egg yolk to the material, and a step of aliquoting the medium raw material mixed in this way into a Petri dish and solidifying the medium raw material. As described above, at least three steps are required when adding egg yolk, and the procedure is complicated. This is to prevent thermal denaturation of the egg yolk component, and in particular, in the cooling and keeping step, if the medium temperature in the addition of the collected egg yolk is extremely high, the egg yolk component is denatured, while if the medium temperature is extremely low, for example, solidification of the agar is caused, and thus, it is important to control the temperature, and a skilled experience is required. In addition, since the state of the collected egg yolk used in the detection principle largely and easily varies depending on, for example, the species, the individual difference, and the breeding environment, of a hen for egg collection, the medium performance is affected by the state of the collected egg yolk, and thus, it is required to have an empirical rule for discrimination of a colony having the egg yolk reaction after culture. In view of this, the present applicant has reported a medium containing four components of polymyxin B, trimethoprim, a lincomycin antibiotic, and 5-bromo-4-chloro-3-indoxyl-α-D-glucopyranoside to be a substrate of α-glucosidase, as the medium for Bacillus cereus group detection, which does not use egg yolk reaction (Patent Literature 1). Further, a medium for selective detection of Bacillus cereus and Bacillus thuringiensis, obtained by mixing, for example, lithium chloride, ceftazidime, polymixin B sulfate, in addition to a phosphatidylinositol-specific phospholipase C substrate having a detectable chromogenic or fluorescent free radical, and nutrient components has been reported (Patent Literature 2). CITATION LIST Patent Literature Patent Literature 1: JP 2011-004712 APatent Literature 2: U.S. Pat. No. 6,284,517 Non Patent Literature Non Patent Literature 1: Standard Methods of Analysis in Food Safety Regulation, Microorganisms 2004, under the supervision of Ministry of Health, Labour and Welfare, incorporated association JAPAN FOOD HYGIENE ASSOCIATION, pages 266 to 282Non Patent Literature 2: ISO11133 Microbiology of food, animal feed and water-Preparation, production, storage and performance testing of culture media (2014)Non Patent Literature 3: ISO7932 Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of presumptive Bacillus cereus—Colony-count technique at 30° C. (2004) SUMMARY OF THE INVENTION Technical Problem However, it has been found that as to the medium disclosed in Patent Literature 1, there may be a case where a clear detection of colony cannot be done because the growth of Bacillus cereus is insufficient in 24 hours under the condi