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＝Techniques＝

A Novel, Selective, Ready-to-Use Culture Medium for the Enumeration of Bacillus cereus

Yusuke KOYANAGI†, Katsuya TOYOTA, Shingo MIZUOCHI and Yoshitaka TANAKA（Central Research Laboratory, Nissui Pharmaceutical Co., Ltd.: Hokunanmoro, Yuki, Ibaraki 307‒0036; †

Corresponding author: （ORCID: 0000‒0001‒9077‒1885）(Received: May 19, 2020)

(Accepted: December 22, 2020)

Bacillus cereus is a Gram-positive, sporulating microorganism that causes food poisoning and / or spoilage. Hence, it is important to detect and enumerate B. cereus; this typically involves culturing with mannitol egg yolk polymyxin agar and NaCl glycine Kim Goepfert agar, both of which require the addition of aseptic egg yolk. To eliminate this unstable and variable step, we developed and evaluated a novel B. cereus group-specific ready-to-use dry medium, CompactDry BC. Inclusivity study results revealed that CompactDry BC showed growth comparable to classi-cal media. Exclusivity study results revealed no typical blue colonies on CompactDry BC. Relative trueness study results revealed that CompactDry BC showed a linear association and almost no negative bias compared to the classical media. Furthermore, we also report the comparison stud-ies of CompactDry BC with CompactDry X-BC, which detects the B. cereus group. Throughout the experiments, CompactDry BC showed higher growth of the B. cereus group than CompactDry X-BC.

Key words: B. cereus, ready-to-use media, food safety, MYP, NGKG

Bacillus cereus is a ubiquitous microorganism that causes emesis and diarrhea, as well as food spoilage and is commonly found in agricultural products1, 13）． Food contamination by B. cereus leads to the decomposition of food, which causes intestinal disease, and in some cases, even death11）． Hence, the detection and biocontrol of B. cereus are important for food processing companies and distributors.

There are at least eight closely related spe-cies comprising the B. cereus group. B. anthracis, B. cereus, and B. thuringiensis are the most well-studied species4）． B. anthracis, characterized by Koch and Pasteur, is the causative agent of an-thrax and is harmful to humans, whereas B. thur-ingiensis, characterized by Ishiwata, is used as a biopesticide.

Mannitol egg yolk polymyxin agar (MYP) and NaCl glycine Kim Goepfert agar (NGKG) are widely used as plating media for the detection of the B. cereus group. Egg yolk emulsion needs to be added to both media at the time of use7, 10）， which can cause bacterial contamination.

The CompactDry system9） is a unique, dehy-drated, pre-sterilized medium, comprising nutri-tional components and a gelling agent that can be incubated after applying 1 mL of the sample on the plate. CompactDry X-BC16） is a B. cereus group-specific dehydrated medium that applies the CompactDry system. However, this medium occasionally leads to a lower growth of B. cereus than that in classical culture media under some conditions because of the excess antibiotics in-tended to inhibit most microorganisms other than B. cereus. These antibiotics also constrain the growth of the B. cereus group strains. Therefore, we developed a new media for the B. cereus group.

Phosphatidylinositol (PI) comprises a family of lipids that are commonly found in eukaryotic plasma membranes. Phosphatidylinositol-specific phospholipase C （PI-PLC） can hydrolyze PI and has been reported in several microorganisms including Staphylococcus aureus3）， Clostridium novyi15）， Listeria monocytogenes8）， B. thuringien-sis6）， and B. cereus12）． 5-Bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate ammonium salt （X-IP） (Carbosynth), is a chromogenic substrate that is †E-mail: y-koyanagi@nissui-pharm.jp

日本食品微生物学会雑誌　Jpn. J. Food Microbiol., 38(1), 18‒24, 2021

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cleaved by PI-PLC and is an indicator for patho-genic strains such as those in the B. cereus group.

Here, we aimed to develop a novel B. cereus group-specific, comparable to classical media, ready-to-use medium using X-IP and compare its performance with MYP, NGKG, and CompactDry X-BC.

Materials and Methods

MediaTryptic soy agar (TSA), NGKG, and Com-

pactDry X-BC were obtained from Nissui Pharma-ceutical Co., Ltd. (Tokyo, Japan). MYP and its supplements, egg yolk emulsion and antibiotics, were purchased from Merck KGaA. (Darmstadt, Germany). Each medium was prepared following the manufacturer’s instructions. CompactDry BC of following composition was ultimately chosen: Peptone, 12％; meat extract, 6％; mannitol, 12％; NaCl, 6％; pyruvate, 1％; phosphate, 6％; antibiotics, 0.01％; gelling reagents, 55％, chromogenic sub-strate, 1％. CompactDry X-BC has following com-position: Peptone, 28％; meat extract, 7％; mannitol, 14％; NaCl, 7％; pyruvate, 1％; glycine, 14％; antibi-otics, 0.1％; gelling reagents, 28％, chromogenic substrate, 0.2％.

FoodsFive categories of food were tested as part of

a food matrix: Cat. 1, rice or barley (white rice, wheat); Cat. 2, nuts (mixed nuts, mixed beans); Cat. 3, bean curd (silken tofu, tofu skin); Cat. 4, meat (raw poultry, raw pork); and Cat. 5, vegeta-bles or fruits (vegetable salad, frozen vegetables, dried vegetables, pineapple). All foods were pur-chased from grocery stores.

Dilution and cultureSeeded food samples or cultured strains were

serially diluted in sterile 0.86％ saline, 1 mL of which was used to inoculate Petri dishes, Com-pactDry BC, or CompactDry X-BC plates, and then TSA was poured into Petri dishes. Mean-while, 0.1 mL of the dilutions were inoculated Petri dishes containing MYP or NGKG using the spread-plating method. CompactDry BC and each agar medium, except for CompactDry X-BC, was incubated for 24 ± 2 hr at 30℃. CompactDry X-BC was incubated for 24 ± 2 hr at 35℃.

Inclusivity and exclusivity studiesA total of 16 B. cereus group strains were

tested for inclusivity, including 15 B. cereus strains and one B. thuringiensis strain. Each strain was diluted to an appropriate concentration and inoculated onto plates as described in the dilu-tion and culture section.

A total of 50 strains were tested for exclusiv-ity, including 13 gram-negative strains, 35 gram-positive strains (including eight Bacillus species, other than the B. cereus group), and two fungi. The dilution and inoculation were performed as described in the dilution and culture section.

Relative trueness studyEach food sample was divided into 10 g aliquots

and placed in bags. Bacterial fluids of B. cereus ATCC 14579, 19637, or 11778 were seeded in the food sample at a level of 2‒7 Log colony forming unit (CFU)/g． Samples were stored in suitable conditions: Refrigerated storage, at least 48 hr at 2‒8℃; room temperature storage, at least 2 weeks at 20‒25℃; frozen food storage, at least 2 weeks at －20℃. The foods (Cat. 3, Cat. 4, vegetable salad, and pineapple) were stored under refrigerated storage. Frozen vegetables were stored under frozen food storage. Others were stored under

Fig. 1.　Colonies of Bacillus cereus ATCC 14579 on Com-pactDry BCInoculated 1 Log CFU/mL and incubated for 22 hr; B. cereus formed conspicuous, blue colonies.

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room temperature storage. After the storage pe-riod, 90 mL of saline was added to each bag and homogenized. Then each sample was diluted and inoculated as described above.

Statistical analysisOne-way analysis of variance and Tukey’s

test were performed using statistical software R (version 3.6.1.). p values <0.05 were considered statistically significant. Bland‒Altman plots2） were drawn to show comparable growth and signifi-cance versus corresponding media.

Results

Inclusivity and exclusivity studiesTo investigate the relative growth potential,

16 strains belonging to the B. cereus group, 15 B. cereus, and one B. thuringiensis, were cultured on CompactDry BC, CompactDry X-BC, and agar media. All strains appeared as 2‒10 mm blue colo-nies on CompactDry BC (Figure 1). They also ap-peared as typical colonies with halos on MYP and NGKG. Table 1 shows the results for logarithmic-transformed CFU/mL. The average values of Log CFU/g were 7.33, 7.50, 7.53, 7.37, and 7.12 for TSA, MYP, NGKG, CompactDry BC, and CompactDry X-BC, respectively. There was a significant differ-ence among the different media analyzed using one-way ANOVA (p = 0.035). Tukey’s pairwise comparison showed that CompactDry X-BC and

NGKG had a significant difference (p = 0.036) (Ta-ble 2).

To investigate the selective potential, eight Bacillus species, except the B. cereus group, were cultured on CompactDry BC, CompactDry X-BC, and agar media (Table 3). None of the strains formed blue colonies on CompactDry BC and X-BC, but several strains formed atypical colonies without halos on MYP and NGKG.

Table 4 shows the results for strains other than the Bacillus species, including 13 Gram-negative strains, 27 Gram-positive strains, and two fungi. No strains formed blue colonies on Com-pactDry BC; however, the inoculation of a high

Table 1.　Inclusivity study for B. cereus group using various media

Organism CodeLog CFU/mL

TSA MYP NGKG Compact- Dry BC

Compact- Dry X-BC

Bacillus cereus ATCC 14579 6.92 7.14 7.30 7.06 6.96Bacillus cereus ATCC 19637 6.75 6.98 7.17 6.93 6.28Bacillus cereus ATCC 11778 7.60 7.76 7.74 7.60 7.54Bacillus cereus NS 10609 7.50 7.51 7.67 7.36 7.24Bacillus cereus NS 10610 7.30 7.55 7.45 7.51 6.74Bacillus cereus NS 9116 7.91 8.10 8.20 7.93 7.97Bacillus cereus NS 5109 7.53 7.63 7.64 7.62 7.44Bacillus cereus NS 5201 7.02 6.99 7.03 6.81 6.49Bacillus cereus NS 5202 6.71 7.07 7.17 6.98 6.87Bacillus cereus NS 5203 6.85 6.95 6.81 6.67 6.39Bacillus cereus NS 5204 7.46 7.82 7.70 7.63 7.57Bacillus cereus NS 5205 7.51 7.82 7.71 7.51 7.36Bacillus cereus NS 5206 7.72 7.94 7.75 7.77 7.55Bacillus cereus NS 5207 7.51 7.62 7.61 7.60 7.39Bacillus cereus NS 5208 7.50 7.56 7.56 7.45 6.95Bacillus thuringiensis NBRC 101235 7.51 7.59 7.91 7.45 7.26

Average 7.33 7.50 7.53 7.37 7.12

Strains were derived from ATCC, American Type Culture Collection; NS, Nissui clinical strains; NBRC, NITE Biological Resource Center, Japan; JCM, Japan Collection of Microorganisms; CFU, colony forming unit; TSA, tryptic soy agar; MYP, mannitol egg yolk polymyxin agar; NGKG, NaCl glycine Kim Goepfert agar.

Table 2.　 Tukey multiple comparisons of means and 95％ family-wise confidence level

Difference95％

Lower limit

95％ Upper limit

p-Value

MYP-BC 0.134 － 0.251 0.519 0.866NGKG-BC 0.158 － 0.226 0.543 0.778TSA-BC － 0.035 － 0.420 0.349 0.999XBC-BC － 0.243 － 0.628 0.141 0.399NGKG-MYP 0.025 － 0.360 0.409 1.000TSA-MYP － 0.169 － 0.554 0.216 0.735XBC-MYP － 0.377 － 0.762 0.007 0.057TSA-NGKG － 0.194 － 0.578 0.191 0.625XBC-NGKG － 0.402 － 0.786 － 0.017 0.036XBC-TSA － 0.208 － 0.593 0.176 0.557

BC, CompactDry BC; XBC, CompactDry X-BC; TSA, tryptic soy agar; MYP, mannitol egg yolk polymyxin agar; NGKG, NaCl glycine Kim Goepfert agar.

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concentration of L. monocytogenes imparted a faint blue color to the medium. For CompactDry X-BC, the inoculation of a high concentration of Corynebacterium xerosis imparted blue colonies on the medium.

Relative trueness studyThree representative B. cereus strains ATCC

11778, 14579, and 19637, were seeded in food sam-ples of all five categories. The food samples were stored under the condition described in the mate-rials and methods. After 48 hr to 2 weeks, the samples were diluted in saline and inoculated onto each medium. Figure 2 shows the scatter plot for the number of logarithmic-transformed colonies on each medium. Growth on CompactDry showed a linear association with that on each classical medium. The correlation functions of CompactDry BC are 0.994 and 0.996 for MYP and NGKG, re-spectively, whereas for CompactDry X-BC they were 0.978 and 0.977 for MYP and NGKG, respec-tively.

Figure 3 shows Bland-Altman plots2） compar-ing CompactDry BC with MYP, NGKG, or Com-pactDry X-BC. CompactDry BC showed compara-ble growth and no significant negative bias versus classical media; (－0.036) for CompactDry BC vs. MYP and (－0.009) for CompactDry BC vs. NGKG. The differences at each storage temperature for CompactDry BC vs MYP was －0.010 for refriger-ated storage, －0.067 for frozen storage, and +0.170 for room temperature storage. Most points were within the 95％ confidence limits; still few points were outside, but within the expected range. In addition, a significant positive bias was detected between CompactDry BC and CompactDry X-BC +0.489.

Discussion

This study compared the performance and selectivity of CompactDry BC with MYP, NGKG, and CompactDry X-BC for the detection and enu-meration of B. cereus group bacteria.

For the inclusivity study, all B. cereus group strains grew on CompactDry BC, showing no sig-nificant difference of growth potential compared to classical media (p < 0.05). CompactDry BC could not distinguish between B. cereus and B. thuring-iensis, but could distinguish these from Bacillus species, except for the B. cereus group strains. CompactDry X-BC showed significantly less growth potential than NGKG (p = 0.036). In the exclusivity study, CompactDry BC and Compact-Dry X-BC showed precise selectivity indicating that both dry media suppress a wide range of bacteria and fungi, other than the B. cereus group.

For the relative trueness study, we seeded B. cereus to a wide range of food materials. Compact-Dry BC showed comparable growth potential to classical media, and higher growth potential than CompactDry X-BC, even when inoculated and cultured with debris derived from various food matrices. CompactDry X-BC contains several anti-biotics intended to inhibit microorganisms other than those from the B. cereus group; however, it results in the inhibition of B. cereus growth. It has been reported that the addition of 3‒5％ glycine to media also inhibits the growth of B. cereus strains14）．Glycine is added for the purpose of dif-ferentiating cereus in NGKG medium, but is not required in the new substrate system. Compact-Dry BC is not an improvement on CompactDry X-BC, but a new medium created from scratch based on the use of a highly specific chromogenic

Table 3.　Study for genus Bacillus other than the B. cereus group using various media

Organism CodeLog CFU/mL

TSA MYP NGKG Compact- Dry BC

Compact- Dry X-BC

Bacillus circulans ATCC 4513 6.98 6.70 N.D. N.D. N.D.Bacillus licheniformis ATCC 14580 8.29 8.26 7.76 N.D. N.D.Bacillus pumilus ATCC 7061 7.70 6.78 N.D. N.D. N.D.Bacillus sphaericus ATCC 14577 6.51 6.07 6.71 N.D. N.D.Bacillus subtilis ATCC 6633 5.90 N.D. N.D. N.D. N.D.Bacillus subtilis ATCC 9372 7.92 6.90 N.D. N.D. N.D.Bacillus brevis ATCC 8246 7.43 7.58 3.00 N.D. N.D.Bacillus polymyxa NS 5068 7.18 7.18 5.69 N.D. N.D.

N.D.: Not DetectedATCC, American Type Culture Collection; NS, Nissui clinical strains; CFU, colony forming unit; TSA, tryptic soy agar; MYP; mannitol egg yolk polymyxin agar; NGKG, NaCl glycine Kim Goepfert agar.

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substrate, which results in fewer antibiotics, com-bining high growth potential with high selectivity.

MYP has been used for the detection and enumeration of B. cereus in food samples10）． It contains nutrient components and three key fac-tors; polymyxin, egg yolk emulsion, and mannitol. Polymyxin inhibits most Gram-negative bacteria, and the egg yolk emulsion is a rich source of vita-

mins that aids B. cereus growth. In addition to the nutrient advantage, B. cereus colonies on MYP have precipitation zones or halos as they produce lecithinase. As mannitol is not used by B. cereus, it forms red colonies, whereas bacteria that fer-ment mannitol form yellow colonies because of acidic products.

Because of its glycine content, that inhibits

Table 4.　Exclusivity study for strains other than genus Bacillus

Organism CompactDry BC CompactDry X-BC

Gram negative Code 7 Log CFU/mL

3 Log CFU/mL

7 Log CFU/mL

3 Log CFU/mL

Citrobacter amalonaticus ATCC 25405 N.D. N.D. N.D. N.D.Enterobacter aerogenes ATCC 13048 N.D. N.D. N.D. N.D.Enterobacter sakazakii ATCC 29544 N.D. N.D. N.D. N.D.Escherichia coli ATCC 8739 N.D. N.D. N.D. N.D.Escherichia hermannii ATCC 33650 N.D. N.D. N.D. N.D.Klebsiella ozaenae ATCC 11296 N.D. N.D. N.D. N.D.Klebsiella pneumoniae ATCC 13883 N.D. N.D. N.D. N.D.Serratia rubidaea ATCC 27593 N.D. N.D. N.D. N.D.Morganella morganii ATCC 25830 N.D. N.D. N.D. N.D.Proteus vulgaris ATCC 13315 N.D. N.D. N.D. N.D.Pseudomonas aeruginosa ATCC 10145 N.D. N.D. N.D. N.D.Pseudomonas putida ATCC 12633 N.D. N.D. N.D. N.D.Acinetobacter calcoaceticus ATCC 19606 N.D. N.D. N.D. N.D.

Gram positive

Corynebacterium minutissimum ATCC 23348 N.D. N.D. N.D. N.D.Corynebacterium renale ATCC 19412 N.D. N.D. N.D. N.D.Corynebacterium xerosis ATCC 373 N.D. N.D. Blue colonies N.D.Enterococcus avium ATCC 14025 N.D. N.D. N.D. N.D.Enterococcus durans ATCC 19432 N.D. N.D. N.D. N.D.Enterococcus faecalis ATCC 19433 N.D. N.D. N.D. N.D.Enterococcus faecium ATCC 19434 N.D. N.D. N.D. N.D.Micrococcus luteus ATCC 9341 N.D. N.D. N.D. N.D.Staphylococcus aureus ATCC 12600 N.D. N.D. N.D. N.D.Staphylococcus auricularis ATCC 33753 N.D. N.D. N.D. N.D.Staphylococcus capitis ATCC 27840 N.D. N.D. N.D. N.D.Staphylococcus epidermidis ATCC 12228 N.D. N.D. N.D. N.D.Staphylococcus haemolyticus ATCC 29970 N.D. N.D. N.D. N.D.Staphylococcus hominis ATCC 27844 N.D. N.D. N.D. N.D.Staphylococcus lentus ATCC 29070 N.D. N.D. N.D. N.D.Staphylococcus saprophyticus ATCC 15305 N.D. N.D. N.D. N.D.Staphylococcus sciuri ATCC 29062 N.D. N.D. N.D. N.D.Staphylococcus simulans ATCC 27848 N.D. N.D. N.D. N.D.Staphylococcus warneri ATCC 27836 N.D. N.D. N.D. N.D.Staphylococcus xylosus ATCC 29971 N.D. N.D. N.D. N.D.Leuconostoc mesenteroides ATCC 27258 N.D. N.D. N.D. N.D.Listeria monocytogenes ATCC 15313 Blue faint N.D. N.D. N.D.Paenibacillus macerans JCM 2500 N.D. N.D. N.D. N.D.Paenibacillus polymyxa ATCC 842 N.D. N.D. N.D. N.D.Clostridium perfringens JCN 3816 N.D. N.D. N.D. N.D.Listeria innocua ATCC 33090 N.D. N.D. N.D. N.D.Listeria ivanovii JCM 7681 N.D. N.D. N.D. N.D.Fungi

Candida albicans ATCC 14053 N.D. N.D. N.D. N.D.Saccharomyces cerevisiae NBRC 10217 N.D. N.D. N.D. N.D.

N.D.: Not DetectedATCC, American Type Culture Collection; NS, Nissui clinical strains; NBRC, NITE Biological Resource Center, Japan; JCM, Japan Collection of Microorganisms; CFU, colony forming unit.

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several microorganisms,5） NGKG shows stronger selectivity of the B. cereus group than MYP. In the present study, we observed some atypical growth of strains other than those in the B. cereus group on NGKG and / or MYP. Furthermore, some B. cereus strains showed small and ambiguous halos on these agar media. Hence, distinguishing the B. cereus group bacteria from other non-cereus species based on their growth is difficult for inex-perienced users. Although there are other bacte-rial species with X-IP cleaving enzymes besides B. cereus, the composition of CompactDry BC was optimized for B. cereus. Strains other than those in the B. cereus group could not grow on Com-pactDry BC; even if they did, they formed white colonies. Hence, only the B. cereus group bacteria formed blue colonies, which can be detected easily.

MYP or NGKG require the addition of egg

yolk emulsion at the time of their use, and asepti-cally separating the egg yolk from the egg white is technically difficult. CompactDry BC does not need any supplement and can be used instantly. This saves the operation time and eliminates any egg-to-egg variation. Thus, compared to classical media such as MYP or NGKG, CompactDry BC has tremendous advantages to the detection and enumeration of B. cereus group bacteria.

Summary

We developed and evaluated a B. cereus group-specific ready-to-use dry medium, Compact-Dry BC. The B. cereus group formed conspicuous, blue colonies on CompactDry BC. No strains other than the B. cereus group formed blue colonies on CompactDry BC. Relative trueness study results revealed that CompactDry BC showed a linear

Fig. 2.　Scatter plots of the number of logarithmic-transformed colonies on CompactDry BC vs those on MYP (a), those on NGKG (b), or those on X-BC(c)All plots showed linear association between the growth on CompactDry BC and that on each classical medium or X-BC. MYP, mannitol egg yolk polymyxin agar; NGKG, NaCl glycine Kim Goepfert agar.

Fig. 3.　Bland-Altman plots comparing CompactDry BC with (a) MYP, (b) NGKG, (c) CompactDry X-BCCat. 1; rice or barley, Cat. 2; nuts, Cat. 3: bean curd, Cat. 4: meat, Cat. 5: vegetables or fruits. Solid line: ideal (no bias), dashed line; bias, dotted: 95％ upper or lower limit. There was no significant bias, namely, － 0.036 for CompactDry BC vs MYP and － 0.009 for CompactDry BC vs. NGKG. In contrast, there was significant positive bias, 0.489 for X-BC. MYP; mannitol egg yolk polymyxin agar; NGKG, NaCl glycine Kim Goepfert.

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association and almost no negative bias compared to the classical media. Furthermore, CompactDry BC showed a higher growth potential than Com-pactDry X-BC.

Acknowledgments

The authors thank their colleagues, Dr. Nao Kondo, for technical assistance.

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