efficient isolation and identification of enterotoxigenic ... · p2-33 bacillus cereus is a group...

P2-33

Bacillus cereus is a group of ubiquitous facultative aerobic sporeforming Gram positive rods commonly found in soil. The spores frequently contaminate a variety of foods including produce, meat, eggs and dairy products. Foodborne illnesses associated with toxins produced by B. cereus resulting in self-limiting diarrhea and/or vomiting. The current methods recommended in the FDA Bacteriological Analytical Manual (BAM) to detect the presence of B. cereus from potentially contaminated food products include cultivation using differential plating media mannitol egg-yolk polymyxin (MYP) agar and a qualitative toxin assay that identifies the diarrheal toxin.

The aim of this study was to improve the method for the isolation of enterotoxigenic B. cereus from food products. Confirmation of the bacteria from incriminated foods is complicated by the presence of background flora since B. cereus is not competitive with other organisms. Therefore, the challenge of this study was to recover viable bacteria in less time.

A pilot study evaluated and compared the growth of 50 Bacillus cereus isolates grown on MYP to growth of the same isolates on four different media formulations including PEMBA, BCM, Brilliance, and Bacara. All media were purchased and prepared from Oxoid’s dehydrated media except Bacara agar which was purchased as prepared plates from AES Chemunex. Two of the agar formulations, Brilliance and Bacara, satisfactorily inhibited competitive organisms. Further assessments comparing Brilliance and Bacara to MYP included an inclusivity and exclusivity study as well organism enumeration from artificially adulterated food matrices. The typical colony grown on MYP is pink with a halo; grown on Brilliance is turquoise green; grown on Bacara is pink-orange with a halo.

Brilliance and Bacara were both selective and differential for B. cereus; however, Bacara performed best overall. Cultivation and detection of B. cereus using Bacara was achieved 24 hours earlier than with Brilliance due to a delayed color development. Additionally, Brilliance was inhibitory even against B. cereus.

The BAM method is laborious and time to results can be as long as two weeks. Use of chromogenic agar will simplify detection, quantification and identification of B. cereus. Ultimately, consumers will be protected since contaminated food products will be identified quickly and can be removed from consumer channels.

AbstractAbstract

Bacillus cereus has been detected and implicated in several contaminated food products and supplements since 1948 when the organism was isolated from both vanilla sauce and people became ill after consuming the product. Methods to detect and identify B. cereus in food matrices have been slow to change. The current method recommended in the U.S. Food and Drug Administration’s Bacteriological Analytical Manual (BAM) for the cultivation and identification of B. cereus includes growth on mannitol egg yolk (MYP) media and biochemical characterization of typical isolates. The media used to grow B. cereus is problematic primarily because other organisms, when present in the food matrix, are not inhibited and the presence of competitive organisms such as staphylococci or other bacilli can alter the expected growth characteristics of typical B. cereus. Chromogenic media formulations that are specific and selective for the six different species of B. cereus group are reported to be inhibitory to competitive organisms and require only visual colony inspection to determine presence or absence without further confirmatory testing decreasing the time to results.

IntroductionIntroduction

A pilot study was initiated that compared colony morphology, time to detection, and ability to grow and identify B. cereus from pure cultures and in the presence of selected competitive organisms. A variety of selective and/or differential media were evaluated using bacterial isolates obtained from American Type Culture Collection (ATCC), Bacillus Genetic Stock Center (BGSC), and US FDA collections (Tables 1 and 2). Isolates were retrieved from frozen glycerol stock cultures, streaked to Tryptic Soy Agar (TSA), and incubated overnight. A single colony of each strain was grown overnight in BHI with 0.1% glucose and 10µL aliquots were streaked for isolation using the selected media. Dehydrated media purchased from Oxoid and prepared per manufacturer’s instructions included MYP, Polymyxin Egg Yolk Mannitol Bromothymol Blue agar (PEMBA) and Brilliance; likewise, Bacillus Chromogenic Media (BCM) was purchased from R&F Laboratories (Downers Grove, Illinois) and prepared per manufacturer’s instructions. Bacara was purchased from AES Chemunex as prepared media.

Pure cultures were used in inclusivity and exclusivity studies in order to evaluate the growth characteristics of each organism. Cocktails of overnight broth cultures were prepared from resuspended cell pellets of S. aureus, E. coli, and B. subtilis approximately (1x109 CFU/ml). Low, medium and high dilutions (approximately 1x101 CFU/ml, 1x104 CFU/ml and 1x106 CFU/ml, respectively) of B. cereus were mixed with undiluted resuspended pellets of S. aureus, E. coli, and B. subtilis then spread on culture plates. These cocktails were plated to each test medium and also used to spike a variety of food matrices. The same media was used to screen food products and supplements received for confirmatory testing for the enumeration of B. cereus. Adulterated food samples included rice, macaroni and cheese dinner, chicken broth and milk. Dilutions of B. cereus and cocktail mixtures of B. subtilis, S. aureus, and E. coli were added to 300g food samples and allowed to equilibrate at 4oC for 48 hours. An equal portion of each food sample was homogenized in a blender, serially diluted in Butterfield’s Phosphate Buffered water (BPB), spread to TSA, MYP and Bacara then incubated overnight at 30oC.

MethodsMethods

Chromogenic media prepared by AES Chemunex for B. cereus was compared to traditional media MYP and PEMBA as well as other chromogenic media. Bacara was selected because the colony morphology of B. cereus was uniform; the color developed after overnight incubation, and each colony was discrete and easy to identify. In contrast, MYP the traditional media recommended for food testing is less effective since competitive organisms are not inhibited. The conventional MYP is less efficient due to the propensity of B. cereus colonies to coalesce preventing accurate aerobic plate counts. Future studies will include other food matrices and collaborative studies.

ConclusionConclusion

Isolates of B. cereus and one B. anthracis Sterne strain grew as expected on all media, but two of the chromogenic agars, Brilliance and Bacara, were preferred based upon uniform colony morphology (Fig 1, Fig 2). Bacara was ultimately the media of choice due to time to detection (<24 hours) and the ability to enumerate colonies even from lower dilutions when compared to MYP plates of the same dilutions.The exclusivity study comparison of MYP, Brilliance and Bacara consistently revealed that Gram negative bacteria were inhibited on all media, however; Gram positive organisms such as B. subtilis, B. licheniformis, S. aureus, and Listeria monocytogenes were not inhibited on MYP agar (Table 2). The staphylococci grew on Bacara as pinpoint, lecithinase negative white or pink colonies that were easily distinguishable from B. cereus (Fig 3).Aerobic spread plates of the cocktail mixtures and adulterated food samples plated on MYP were problematic. First the interpretation was difficult because B. cereus typically did not form discrete colonies, but coalesced into one large massive colony (Fig 2). Second, following overnight incubation of spread plates inoculated from cocktail mixtures with staphylococci and bacilli all colonies appeared to be mannitol positive. Since the colonies of B. subtilis were shown to be mannitol positive with a halo, the culture would be interpreted as negative for the presence B. cereus (Fig 4).

Figure 1.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 Bacara (orange with white halo). The colonies indicated with a circle on the BCM agar were not uniform in size or color.

Figure 2.Cooked rice was adulterated with B. cereus, held for 48 hours at 4oC, and homogenized. Serial dilutions were prepared in BPB and 100µL was spread to TSA, MYP, Bacara, and Brilliance plates. All plates were incubated at 30oC and inspected at 24 (Panel A) and 48 hours (Panel B). The colony morphology observed following overnight incubation was uniform and typical if cultures grown on MYP and Bacara plates, however, an additional 24 hours was required for the expected color change on Brilliance plates. Colony enumeration was easily accomplished from the Bacara or Brilliance plates even using the lower dilutions as shown, but not from the MYP plate.

Figure 4.Shown are serially diluted adulterated food samples plated to MYP and Bacara plates. Competitive organisms also grow on MYP and if mannitol positive the observation of mannitol negative B. cereus colonies will be obscured. The characteristic growth of B. cereus is evident on Bacara as orange colonies with a white halo, but the contaminants evident on MYP are inhibited.

ResultsResults

Kristin M. Kotewicz, Sandra M. Tallent, Reginald BennettU.S. Food and Drug Administration, College Park, MD

Efficient Isolation and Identification of Enterotoxigenic Bacillus cereus Group

PEMBA BCM BRILLIANCE BACARAMYP

Photo courtesy of: CDC/ Dr. William A. Clark

STRAIN NO. BACTERIAL ISOLATE MYP: 24 hoursa BRILLIANCE: 48 hours

BACARA:24 hours b

F872 Acinetobacter sp. NG NG NG

F873 Acinetobacter sp. NG NG NG

F825 Aeromonas hydrophila NG NG NG

F176 Bacillus amyloliquifaciens Y/HALO NG NG

F177 Bacillus amyloliquifaciens Y/HALO NG NG

F178 Bacillus amyloliquifaciens NG NG NG






A61 Bacillus circulans Y/NO HALO NG NG

FMYC Bacillus mycoides Y/HALO NG NG

F1267 Bacillus subtilis NG NG NG

F1268 Bacillus subtilis Y/HALO NG NG



F1258 Bacillus thuringiensis P/HALO GREEN O/HALO







B6A21 Bacillus weihenstephanensis P/HALO GREEN O/HALO



A64 Brevibacillus laterosporus P/NO HALO NG NG

A25408 Citrobacter freundii NG NG NG

A43864 Citrobacter koseri NG NG NG

A13048 Enterobacter aerogenes NG NG NG

A33731 Enterobacter amnigenus NG NG NG

A35956 Enterobacter asburiae NG NG NG

A35030 Enterobacter cloaceae NG NG NG

F611 Enterobacter sakazakii NG NG NG

F385 Escherichia coli NG NG NG




F1403 Listeria monocytogenes Y/HALO NG NG




A29948 Paenibacillus gordonae NG NG NG

A8509 Paenibacillus macerans Y/NO HALO NG NG

A7070 Paenibacillus polymyxa Y/NO HALO NG NG

F1 Salmonella enterica NG NG NG




F1161 Shigella sonnei NG NG NG

A1766 Staph aureus Y/HALO NG W/NO HALO

A1767 Staph aureus Y/HALO NG W/NO HALO

A12228 Staph epidermidis Y/HALO NG NG

A49051 Staph. intermedius P/HALO NG P/NO HALO

A14576 Virgibacillus pantothenicus NG NG NG

A: American Type Culture Collection (ATCC)B: Bacillus Genetic Stock Center (BGSC)F: U.S. Food and Drug Administration (FDA) collectiona,bNG no growth; P pink colony; Y yellow colony; halo lecithinase positive; no halo lecithinase negative; O orange colony; W white colony.

BACILLUS CEREUS

STRAIN NO.MYP: 24 hoursa BRILLIANCE:

48 hoursBACARA: 24 hoursb

A11778 P/HALO GREEN O/HALO

A14579 P/HALO GREEN O/HALO

B6A1 P/HALO GREEN O/HALO










B3E1 P/HALO GREEN O/HALO

B6G1 P/HALO GREEN O/HALO


B6E1 P/HALO GREEN O/HALO





F3995A P/HALO GREEN O/HALO










F13061 P/HALO GREEN O/HALO

FM77 P/HALO GREEN O/HALO

FTJL14 P/HALO GREEN O/HALO


F180WPB P/HALO GREEN O/HALO















FTOL-12 P/HALO GREEN O/HALO

A: American Type Culture Collection (ATCC)B: Bacillus Genetic Stock Center (BGSC)F: U.S. Food and Drug Administration (FDA) collectionapink colony surrounded by haloborange colony surrounded by hal

Table 1.Summary of Bacillus cereus isolates used in this study. Growth characteristics following incubation are noted.

Table 2.A detailed summary of bacterial strains and the growth characteristics noted following incubation at 30˚C.

TSAA. MYP

BRILLIANCEBACARA

TSAB. MYP

BRILLIANCEBACARA

MYP

BACARA

Figure 5.A comparison of MYP and Bacara inoculated by spreading with approximately 1x108 competitive strains S. aureus, E. coli and B. subtilis and 1x106 B. cereus. Estimated colony counts were not possible upon inspection of the conventional MYP agar. More accurate estimates were easily obtained from inspection of Bacara plates.

MYP BACARA

Figure 3.Shown is a Bacara plate with B. cereus (large, pink colonies with halo) and staphylococci (tiny, white colonies). B. cereus is easily distinguishablefrom the staphylococci.

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efficient isolation and identification of enterotoxigenic ... · p2-33 bacillus cereus is a group...

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