O FFICE OF P2-33 Efficient Isolation and Identification of Enterotoxigenic cereus Group R EGULATORY Kristin M. Kotewicz, Sandra M. Tallent, Reginald Bennett S CIENCE U.S. Food and Drug Administration, College Park, MD

Abstract Methods Results Bacillus cereus is a group of ubiquitous facultative aerobic sporeforming Gram positive rods commonly A pilot study was initiated that compared colony morphology, time to detection, and ability to grow and Isolates of B. cereus and one B. anthracis Sterne strain grew as expected on all media, but two of the found in soil. The spores frequently contaminate a variety of foods including produce, meat, eggs and identify B. cereus from pure cultures and in the presence of selected competitive organisms. A variety of chromogenic agars, Brilliance and Bacara, were preferred based upon uniform colony morphology (Fig Figure 3. dairy products. Foodborne illnesses associated with toxins produced by B. cereus resulting in selective and/or differential media were evaluated using bacterial isolates obtained from American Type 1, Fig 2). Bacara was ultimately the media of choice due to time to detection (<24 hours) and the ability Shown is a Bacara plate with B. cereus self-limiting diarrhea and/or vomiting. The current methods recommended in the FDA Bacteriological Culture Collection (ATCC), Bacillus Genetic Stock Center (BGSC), and US FDA collections (Tables 1 to enumerate colonies even from lower dilutions when compared to MYP plates of the same dilutions. (large, pink colonies with halo) and Analytical Manual (BAM) to detect the presence of B. cereus from potentially contaminated food and 2). Isolates were retrieved from frozen glycerol stock cultures, streaked to Tryptic Soy Agar (TSA), The exclusivity study comparison of MYP, Brilliance and Bacara consistently revealed that Gram staphylococci (tiny, white colonies). products include cultivation using differential plating media mannitol egg-yolk (MYP) agar and and incubated overnight. A single colony of each strain was grown overnight in BHI with 0.1% glucose negative were inhibited on all media, however; Gram positive organisms such as B. subtilis, B. B. cereus is easily distinguishable a qualitative toxin assay that identifies the diarrheal toxin. and 10µL aliquots were streaked for isolation using the selected media. Dehydrated media purchased licheniformis, S. aureus, and Listeria monocytogenes were not inhibited on MYP agar (Table 2). The from the staphylococci. from Oxoid and prepared per manufacturer’s instructions included MYP, Polymyxin Egg Yolk Mannitol staphylococci grew on Bacara as pinpoint, lecithinase negative white or pink colonies that were easily The aim of this study was to improve the method for the isolation of enterotoxigenic B. cereus from food Bromothymol Blue agar (PEMBA) and Brilliance; likewise, Bacillus Chromogenic Media (BCM) was distinguishable from B. cereus (Fig 3). products. Confirmation of the bacteria from incriminated foods is complicated by the presence of purchased from R&F Laboratories (Downers Grove, Illinois) and prepared per manufacturer’s Aerobic spread plates of the cocktail mixtures and adulterated food samples plated on MYP were background flora since B. cereus is not competitive with other organisms. Therefore, the challenge of instructions. Bacara was purchased from AES Chemunex as prepared media. problematic. First the interpretation was difficult because B. cereus typically did not form discrete this study was to recover viable bacteria in less time. colonies, but coalesced into one large massive colony (Fig 2). Second, following overnight incubation Pure cultures were used in inclusivity and exclusivity studies in order to evaluate the growth of spread plates inoculated from cocktail mixtures with staphylococci and all colonies appeared to A pilot study evaluated and compared the growth of 50 Bacillus cereus isolates grown on MYP to growth characteristics of each organism. Cocktails of overnight broth cultures were prepared from be mannitol positive. Since the colonies of B. subtilis were shown to be mannitol positive with a halo, Figure 4. of the same isolates on four different media formulations including PEMBA, BCM, Brilliance, and 9 resuspended cell pellets of S. aureus, E. coli, and B. subtilis approximately (1x10 CFU/ml). Low, the culture would be interpreted as negative for the presence B. cereus (Fig 4). Shown are serially diluted adulterated food samples plated to MYP and Bacara plates. Competitive Bacara. All media were purchased and prepared from Oxoid’s dehydrated media except Bacara agar 1 4 6 medium and high dilutions (approximately 1x10 CFU/ml, 1x10 CFU/ml and 1x10 CFU/ml, respectively) organisms also grow on MYP and if mannitol positive the observation of mannitol negative B which was purchased as prepared plates from AES Chemunex. Two of the agar formulations, Brilliance . cereus of B. cereus were mixed with undiluted resuspended pellets of S. aureus, E. coli, and B. subtilis then colonies will be obscured. The characteristic growth of is evident on Bacara as orange and Bacara, satisfactorily inhibited competitive organisms. Further assessments comparing Brilliance B. cereus spread on culture plates. These cocktails were plated to each test medium and also used to spike a colonies with a white halo, but the contaminants evident on MYP are inhibited. and Bacara to MYP included an inclusivity and exclusivity study as well organism enumeration from variety of food matrices. The same media was used to screen food products and supplements received artificially adulterated food matrices. The typical colony grown on MYP is pink with a halo; grown on Figure 1. for confirmatory testing for the enumeration of B. cereus. MYP Brilliance is turquoise green; grown on Bacara is pink-orange with a halo. Typical growth characteristics of B. cereus streaked on MYP (pink colonies with pink halo); PEMBA (blue colonies with yellow halo); BCM (turquoise green colonies); Brilliance (green colonies); and Adulterated food samples included rice, macaroni and cheese dinner, chicken broth and milk. Dilutions Bacara (orange with white halo). The colonies indicated with a circle on the BCM agar were not uniform Brilliance and Bacara were both selective and differential for B. cereus; however, Bacara performed best of and cocktail mixtures of and were added to 300g food samples B. cereus B. subtilis, S. aureus, E. coli in size or color. overall. Cultivation and detection of B. cereus using Bacara was achieved 24 hours earlier than with and allowed to equilibrate at 4oC for 48 hours. An equal portion of each food sample was homogenized Brilliance due to a delayed color development. Additionally, Brilliance was inhibitory even against B. in a blender, serially diluted in Butterfield’s Phosphate Buffered water (BPB), spread to TSA, MYP and cereus. Bacara then incubated overnight at 30oC. The BAM method is laborious and time to results can be as long as two weeks. Use of chromogenic Table 1. Table 2. agar will simplify detection, quantification and identification of B. cereus. Ultimately, consumers will be Summary of Bacillus cereus isolates used A detailed summary of bacterial strains and the growth protected since contaminated food products will be identified quickly and can be removed from in this study. Growth characteristics characteristics noted following incubation at 30˚C. BACARA consumer channels. following incubation are noted.

BACILLUS BRILLIANCE: BACARA: BRILLIANCE: BACARA:24 CEREUS MYP: 24 hoursa STRAIN NO. BACTERIAL ISOLATE MYP: 24 hoursa 48 hours 24 hoursb 48 hours hours b STRAIN NO. MYP PEMBA BCM BRILLIANCE BACARA A11778 P/HALO GREEN O/HALO F872 Acinetobacter sp. NG NG NG A14579 P/HALO GREEN O/HALO F873 Acinetobacter sp. NG NG NG B6A1 P/HALO GREEN O/HALO F825 Aeromonas hydrophila NG NG NG Introduction B6A2 P/HALO GREEN O/HALO F176 Bacillus amyloliquifaciens Y/HALO NG NG B6A3 P/HALO GREEN O/HALO F177 Bacillus amyloliquifaciens Y/HALO NG NG Bacillus cereus has been detected and implicated in several contaminated food products and B6A4 P/HALO GREEN O/HALO F178 Bacillus amyloliquifaciens NG NG NG B6A8 P/HALO GREEN O/HALO F179 Bacillus amyloliquifaciens NG NG NG supplements since 1948 when the organism was isolated from both vanilla sauce and people became ill B6A10 P/HALO GREEN O/HALO F180 Bacillus amyloliquifaciens NG NG NG Figure 2. after consuming the product. Methods to detect and identify B. cereus in food matrices have been slow B6A15 P/HALO GREEN O/HALO F181 Bacillus amyloliquifaciens NG NG NG Cooked rice was adulterated with B. cereus, held for 48 hours at 4oC, and homogenized. Serial dilutions B6A17 P/HALO GREEN O/HALO F182 Bacillus amyloliquifaciens NG NG NG to change. The current method recommended in the U.S. Food and Drug Administration’s B6A18 P/HALO GREEN O/HALO F183 Bacillus amyloliquifaciens NG NG NG were prepared in BPB and 100µL was spread to TSA, MYP, Bacara, and Brilliance plates. All plates Bacteriological Analytical Manual (BAM) for the cultivation and identification of B. cereus includes B6A25 P/HALO GREEN O/HALO A61 Bacillus circulans Y/NO HALO NG NG were incubated at 30oC and inspected at 24 (Panel A) and 48 hours (Panel B). The colony morphology B3E1 P/HALO GREEN O/HALO FMYC Bacillus mycoides Y/HALO NG NG Figure 5. growth on mannitol egg yolk (MYP) media and biochemical characterization of typical isolates. The B6G1 P/HALO GREEN O/HALO F1267 NG NG NG observed following overnight incubation was uniform and typical if cultures grown on MYP and Bacara B6G2 P/HALO GREEN O/HALO F1268 Bacillus subtilis Y/HALO NG NG A comparison of MYP and Bacara inoculated by media used to grow is problematic primarily because other organisms, when present in the F1269 Bacillus subtilis Y/HALO NG NG B. cereus B6E1 P/HALO GREEN O/HALO plates, however, an additional 24 hours was required for the expected color change on Brilliance plates. spreading with approximately 1x108 competitive B6G1 P/HALO GREEN O/HALO F1270 Bacillus subtilis Y/HALO NG NG food matrix, are not inhibited and the presence of competitive organisms such as staphylococci or other Colony enumeration was easily accomplished from the Bacara or Brilliance plates even using the lower 6 B6G2 P/HALO GREEN O/HALO F1258 Bacillus thuringiensis P/HALO GREEN O/HALO strains S. aureus, E. coli and B. subtilis and 1x10 bacilli can alter the expected growth characteristics of typical B. cereus. Chromogenic media B6G3 P/HALO GREEN O/HALO F1260 Bacillus thuringiensis P/HALO GREEN O/HALO dilutions as shown, but not from the MYP plate. B6A9 P/HALO GREEN O/HALO F1262 Bacillus thuringiensis P/HALO GREEN O/HALO B. cereus. Estimated colony counts were not formulations that are specific and selective for the six different species of B. cereus group are reported F1264 Bacillus thuringiensis P/HALO GREEN O/HALO F3995A P/HALO GREEN O/HALO possible upon inspection of the conventional to be inhibitory to competitive organisms and require only visual colony inspection to determine F3998A P/HALO GREEN O/HALO F1266 Bacillus thuringiensis P/HALO GREEN O/HALO A. TSA MYP B. TSA MYP F4014A P/HALO GREEN O/HALO F1397 Bacillus thuringiensis P/HALO GREEN O/HALO MYP agar. More accurate estimates were easily presence or absence without further confirmatory testing decreasing the time to results. F4034A P/HALO GREEN O/HALO F1398 Bacillus thuringiensis P/HALO GREEN O/HALO obtained from inspection of Bacara plates. F4047A P/HALO GREEN O/HALO B6A21 Bacillus weihenstephanensis P/HALO GREEN O/HALO F4225A P/HALO GREEN O/HALO B6A22 Bacillus weihenstephanensis P/HALO GREEN O/HALO F4226A P/HALO GREEN O/HALO B6A23 Bacillus weihenstephanensis P/HALO GREEN O/HALO F4228A P/HALO GREEN O/HALO A64 Brevibacillus laterosporus P/NO HALO NG NG F4229A P/HALO GREEN O/HALO A25408 Citrobacter freundii NG NG NG F4230A P/HALO GREEN O/HALO A43864 Citrobacter koseri NG NG NG F13061 P/HALO GREEN O/HALO A13048 Enterobacter aerogenes NG NG NG MYP BACARA FM77 P/HALO GREEN O/HALO A33731 Enterobacter amnigenus NG NG NG FTJL14 P/HALO GREEN O/HALO A35956 Enterobacter asburiae NG NG NG F196668 P/HALO GREEN O/HALO A35030 Enterobacter cloaceae NG NG NG F180WPB P/HALO GREEN O/HALO F611 Enterobacter sakazakii NG NG NG F4227A P/HALO GREEN O/HALO F385 Escherichia coli NG NG NG F66 P/HALO GREEN O/HALO F386 Escherichia coli NG NG NG F60006 P/HALO GREEN O/HALO F387 Escherichia coli NG NG NG F1259 P/HALO GREEN O/HALO F392 Escherichia coli NG NG NG F1261 P/HALO GREEN O/HALO F1403 Listeria monocytogenes Y/HALO NG NG F1263 P/HALO GREEN O/HALO F1404 Listeria monocytogenes Y/HALO NG NG F1265 P/HALO GREEN O/HALO F1405 Listeria monocytogenes Y/HALO NG NG Conclusion F1396 P/HALO GREEN O/HALO F1406 Listeria monocytogenes Y/HALO NG NG F3A P/HALO GREEN O/HALO A29948 gordonae NG NG NG Chromogenic media prepared by AES Chemunex for B. cereus was compared to traditional F26 P/HALO GREEN O/HALO A8509 Paenibacillus macerans Y/NO HALO NG NG F96 P/HALO GREEN O/HALO A7070 Paenibacillus polymyxa Y/NO HALO NG NG media MYP and PEMBA as well as other chromogenic media. Bacara was selected because F4A P/HALO GREEN O/HALO F1 Salmonella enterica NG NG NG F6A P/HALO GREEN O/HALO F249 Salmonella enterica NG NG NG the colony morphology of B. cereus was uniform; the color developed after overnight F59 P/HALO GREEN O/HALO F336 Salmonella enterica NG NG NG FTOL-12 P/HALO GREEN O/HALO F522 Salmonella enterica NG NG NG incubation, and each colony was discrete and easy to identify. In contrast, MYP the traditional A: American Type Culture Collection (ATCC) F1161 Shigella sonnei NG NG NG B: Bacillus Genetic Stock Center (BGSC) A1766 Staph aureus Y/HALO NG W/NO HALO media recommended for food testing is less effective since competitive organisms are not F: U.S. Food and Drug Administration (FDA) collection A1767 Staph aureus Y/HALO NG W/NO HALO apink colony surrounded by halo BACARA BRILLIANCE BACARA BRILLIANCE borange colony surrounded by hal A12228 Staph epidermidis Y/HALO NG NG inhibited. The conventional MYP is less efficient due to the propensity of B. cereus colonies A49051 Staph. intermedius P/HALO NG P/NO HALO A14576 Virgibacillus pantothenicus NG NG NG to coalesce preventing accurate aerobic plate counts. Future studies will include other food A: American Type Culture Collection (ATCC) B: Bacillus Genetic Stock Center (BGSC) matrices and collaborative studies. F: U.S. Food and Drug Administration (FDA) collection a,bNG no growth; P pink colony; Y yellow colony; halo lecithinase positive; Photo courtesy of: CDC/ Dr. William A. Clark no halo lecithinase negative; O orange colony; W white colony.