Denitration of glycerol trinitrate by resting cells and cell extracts of Bacillus thuringiensis/cereus and Enterobacter agglomerans
Min MengWendell Q. SunLinda A. GeelhaarGuneet KumarAnami PatelGregory F. PayneMarilyn K. SpeedieJ. R. Stacy
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A number of microorganisms were selected from soil and sediment samples which were known to have been previously exposed to nitrate ester contaminants. The two most effective bacteria for transforming glycerol trinitrate (GTN) were identified as Bacillus thuringiensis/cereus and Enterobacter agglomerans. For both isolates, denitration activities were expressed constitutively and GTN was not required for induction. Dialysis of cell extracts from both isolates did not affect denitration, which indicates that dissociable and depletable cofactors are not required for denitration. With thin-layer chromatography and high-performance liquid chromatography, the denitration pathway for both isolates was shown to be a sequential denitration of GTN to glycerol dinitrate isomers, glycerol mononitrate isomers, and ultimately to glycerol. GTN was observed to be completely converted to glycerol during a long-term incubation of cell extracts.Keywords:
Cereus
1. REVIEW OF THE LITERATURE 1 1.1. Taxonomic view on B. cereus 1 1.1.1. B. cereus group 1 1.1.2. The species B. cereus (sensu stricto) 1 1.2. B. cereus spore 2 1.2.1. Ultrastructure of the Bacillus spore 2 1.2.2. Properties of the Bacillus spores and those of B. cereus spores 3 1.2.3. Sporulation and germination of Bacillus spores 5 1.3. Diversity of B. cereus 6 1.4. Interactions of B. cereus with living and nonliving environment 7 1.4.1. Adhesion of B. cereus to nonliving and living surfaces 7 1.4.2. Antagonistic property of B. cereus towards microorganisms 8 1.4.3. B. cereus as a probiotic 8 1.5. Human virulence 10 1.5.1. B. cereus as a human pathogen 10 1.5.2. Cereulide, the emetic toxin of B. cereus 11 1.5.2.1. Structure and properties of cereulide 11 1.5.2.2. Toxic mechanisms and detection of cereulide 13 1.5.2.3. Toxicity of cereulide in whole animals 14 2. AIMS OF THE STUDY 20 3. MATERIALS AND METHODS 21 4. RESULTS AND DISCUSSION 22 4.1. Strategies for isolating and identifying cereulide producers among strains and isolates of B. cereus 22 4.2. Cereulide productivity of B. cereus isolates of geographically and temporally diverse origins 23 4.3. Occurrence of cereulide producing B. cereus in some environmental niches ..24 4.4. Potential for cereulide production in selected infant food formulas 25 4.5. Strategies of B. cereus spores for persistence in the dairy process environment 33 4.6. Are the cereulide producing B. cereus successful in competition with the non producing B. cereus in food? 34 5. SUMMARY AND CONCLUSIONS 36 6. ACKNOWLEDGEMENTS 38 7. REFERENCES 39
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Bacillus(B) cereus is regarded as a major foodborne pathogen which is widely distributed in the nature. In addition, it plays an important role in the contamination of ready-to-eat and dairy products. B. cereus causes the two different types of food poisoning in human: the diarrheal and the emetic type. The aim of this study is detection of hbl complex genes in B. cereus isolated from cow raw milk in Northwest of Iran. In the present study, the number of the samples collected from cow raw milk were 120. All the isolates already had been identified phenotypically, and they were assessed for molecular confirmation by using the PCR method. B. cereus isolates were determined by detecting the hbl genes complex in the isolates. The result of this study showed that B. cereus were found in the raw milk samples 117 (97.5%) from the 120 samples. The frequency of the hblA, hblC, and hblD genes found in B. cereus isolates were 105 (89.7%), 102 (87.1%), and 102 (87.1%) , respectively. 99 isolates (84.6%) harboured 3 tested genes simultaneously. 12 B. cereus isolates (10.3%) lacked these genes. The results of current study showed that B. cereus isolated from raw milk have high potential in causing food poisoning and therefore the use of the procedures to reduce the bacterial contamination during the processing of dairy product is required.
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본 연구는 우리나라 장수지역에서 제조된 전통된장을 수거하여 식중독세균 오염도와 미생물 군집 현황을 분석하고, 분리된 미생물 군집의 Bacillus cereus 길항 효과를 평가함으로써 전통된장의 미생물학적 안전성을 평가하고자 하였다. 전통된장의 미생물학적 안전성을 평가하기 위해 일반세균, 대장균군, 식중독세균을 실험한 결과 일반세균수는 10SUP6/SUP ~10SUP9/SUP CFU/g의 분포를 나타내었고 대장균군은 불검출 되었다. 식중독세균 중 B. cereus는 전통된장 10개 중 7개에서 검출되었으나 기타 식중독균은 불검출 되었다. 실험 결과 장수지역 전통된장에 가장 문제가 되는 미생물은 B. cereus로 나타나 전통된장의 안전성 확보를 위해 B. cereus 제어가 중요한 것으로 판단되었다. 전통된장의 미생물 군집 분석결과 Bacillus subtilis가 155균주(47.4%)로 가장 높은 분포를 나타내었으며, B. licheniformis 68균주(20.8%), B. amyloliquefaciens 46균주(14.1%)로 높은 분포를 나타내는 등 총 327균주가 동정되었다. B. cereus에 대한 길항 효과를 평가한 결과 분리균주 327균주 중 20균주(6.1%)에서 9~12 mm 생육저지환이 관찰되었으며, B. subtilis가 12균주로 가장 많았고, B. amyloliquefaciens 5균주, B. licheniformis 3균주 순으로 분포하였다. 전통된장의 경우 B. cereus 오염도가 식품공장 제조 된장에 비해 높지만 B. cereus 길항효과를 나타내는 균주가 많은 것으로 판단되었다. 이러한 결과를 종합해 볼 때 전통된장에 오염된 B. cereus 위험성은 기존의 생각보다 낮을 것으로 판단되며, 향후 다수의 전통된장을 대상으로 한 B. cereus 길항 효과 실험이 필요한 것으로 판단되었다.
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Bacillus cereus (B. cereus) is a spore forming bacteria with a highly adhesive ability on the food surface and which expresses concern about commercial sterilization due to heat resistance. In this study, the heat challenge was performed at 60℃ for 60 min to derive the importance of the inactivation of bacteria by time during heating 14 isolates of B. cereus from the tomato farm environment. As a result, the final cell counts of B. cereus reduced after heating were –3.6 Log CFU/mL on average (minimum: –2.8 Log CFU/mL, maximum: –4.7 Log CFU/mL). Reduced cell counts of B. cereus after heating for 60 min ranged from 0.5 Log CFU/mL to 2.4 Log CFU/mL. Remained B. cereus after heating can recover in an appropriate environment, and cause foodborne illness. B. cereus existed in the farm environment during the cultivation stage of agricultural products can be transferred to agricultural products through various routes such as soil, toilet handles and worker gloves, thus, the management of B. cereus in a farm environment is essential. In addition, improper sterilization, manufacturing, processing and packaging in the food processing stage cannot be expected to inhibit the complete growth of foodborne pathogenic bacteria, and the risk of foodborne illness can be increased. Therefore, thorough management of these processes is considered important.
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본 연구에서는 인터넷에서 판매되는 배달이유식의 위생안전성을 평가하고, B. cereus의 독소유전자 분포를 파악하여 즉석조리식품으로 구분되는 이유식의 B. cereus 기준설정을 위한 기초자료를 제공하고자 하였다. 기타 영 유아식 27건, 즉석조리식품 79건 등 총 106건에 대한 실험결과는 다음과 같다. 1. 이유식 106건의 세균수 검사결과 기타 영 유아식에서는 12건(44%)에서 최대 $2.5{\times}10^3CFU/g$ , 즉석조리식품에서는 46건(58%)에서 최대 $1.1{\times}10^6CFU/g$ 으로 대체적으로 즉석조리식품에서 세균수가 많이 검출되었다. 2. 106건의 검체 중 총 9건에서 B. cereus가 $10{\sim}3.6{\times}10^3CFU/g$ 범위로 검출되었으며 모두 즉석조리식품이었다. 3. B. cereus의 독소유전자 중 cytK는 6주(66.6%), nheA 및 entFM은 8주(88.9%), bceT는 7주(77.8%), hblC는 3주(33.3%)에서 검출되었으며 구토독소인 cer은 2주(22.2%)에서만 증폭밴드가 나타났다. 4. 분리된 균주는 B. cereus와 B. thuringiensis를 구분하기 위한 시험법에서 모두 B. cereus로 나타났다. Bacillus cereus is food poisoning bacteria frequently occured in starch food. Most of the delivery foods for infant is classified as ready-to-cook food. But unlike food for infant and young children, there are no standards and specifications of Bacillus cereus in ready-to-cook food. The purpose of this study is to examine the presence of Bacillus cereus, aerobic bacteria and coliforms in the food for infant and young children sold through internet. B. cereus was detected in 9 samples (8.3%), total aerobic bacteria was detected over $10^6CFU/g$ in 4 samples and coliforms were not detected in any samples. This will provide basic data for standards and specifications of Bacillus cereus in ready-to-cook food.
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当院では2005年度に血液培養でBacillus cereusの増加傾向がみられた.血液培養でB. cereusが検出された患者の背景を調べた結果,大半の患者で末梢カテーテルを留置していた.さらに血液培養でB. cereusが検出された数名の患者の末梢カテーテル先端からもB. cereusが検出され,B. cereusによる血流感染ありと判断した.また文献的に検索するとB. cereus菌血症についてはリネン等の汚染や留置カテーテルの取り扱いが指摘されているが,カテーテルの汚染とリネンの汚染との結びつきについては不明な点が多い.そこで今回我々はカテーテルにおける菌の定着と増殖の原因について,輸液製剤の種類による菌の発育速度と温度の影響,環境因子の影響について調査を行ったので報告する.輸液製剤の種類や保管時間の影響に関しては,B. cereusがStaphylococcus aureusやStaphylococcus epidermidisと比較して増加しやすいという傾向が見られた.環境検査ではタオルの培養で≦102~1×106 cfu/mLのB. cereusが検出された.落下菌の測定では,一般環境より病室で多く検出される傾向が見られた.さらにリネンの出し入れ後の落下菌の検査を行うと短時間で多数のB. cereusが検出された.以上より輸液製剤の保管の影響よりも,薬剤の調合やカテーテルの刺入,ルート交換を行う環境や取り扱いに問題があると考えた.
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布製の再生利用可能な清拭タオルのBacillus cereus汚染が洗濯方法に影響を受けるのか検討するために，複数施設間における清拭タオルのB. cereus汚染状況と洗濯方法を調査した．さらに，清拭タオルのB. cereus汚染状況と血液培養結果との関連性についても検討した．対象は新潟県内の病院6施設として，乾燥した清拭タオルを毎月3枚ずつ収集し，B. cereusの菌濃度を測定した．また，清拭タオルの洗濯方法と保管方法，血液培養からのB. cereus陽性件数を調査した．各施設の菌濃度は0から1142 CFU/mLであり，平均菌濃度と血液培養陽性率に正の相関傾向（R=0.72，P=0.11）を認めた．洗濯方式では連続式と比較しバッチ式で菌濃度が低い傾向を認めた．さらに，洗濯工程に次亜塩素酸ナトリウム浸漬がある場合にB. cereus菌濃度が有意に低かった（P＜0.01）．従って，清拭タオルのB. cereus汚染は洗濯方法の違いに影響を受け，清拭タオルのB. cereus汚染が血液培養陽性率に影響する可能性がある．血液培養のB. cereus陽性件数が多い施設では，洗濯方法を確認し変更することで，血液培養陽性件数を減少できることが示唆された．これにより，B. cereusによる血流感染を減らせる可能性がある．
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The growth profile of Bacillus cereus in ready-to-eat (RTE) food products of animal origin was examined under different temperature and incubation conditions. In sandwiches and Kimbab, B. cereus did not grow or exhibited only minimal growth at 4 and $10^{\circ}C$ , but it grew rapidly at ambient temperature. In sandwiches, B. cereus did not grow efficiently at $25^{\circ}C$ , however, in ham, the main ingredient of sandwiches, B. cereus growth was observed at the same temperature, with bacterial levels reaching 7.94 Log CFU/g after incubation for 24 h at $25^{\circ}C$ . Toxigenicity of B. cereus was observed only at temperatures above $25^{\circ}C$ . In Kimbab, B. cereus produced toxin after 9 h at $30^{\circ}C$ and after 12 h at $25^{\circ}C$ . Ingredients of sandwiches and Kimbab were collected from 3 different Korean food-processing companies to investigate the source of contamination by B. cereus. Among the 13 tested food items, 6 items including ham were found to be contaminated with B. cereus. Of these ingredients, B. cereus isolates from 3 items produced enterotoxins. None of these isolates harbored the emetic toxin-producing gene. The findings of the present study can be used for risk assessments of food products, including ham and cheese, contaminated with B. cereus.
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Food microbiology
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즉석섭취 편의식품은 일반적으로 소비자가 별도의 조리과정 없이 직접 신선한 상태로 섭취하기 때문에 병원성 미생물에 오염되어 있을 경우 식중독을 일으킬 수 있다. 특히 B. cereus는 자연계에 널리 분포하여 대부분의 식품에 쉽게 오염되어 식중독을 유발 시킬 수 있는 독소형 식중독균 중의 하나이다. 이에 본 연구는 유통 판매중인 즉석섭취 편의식품류 및 식중독이 발생하여 의뢰된 보존식에서 B. cereus의 분리율 및 독소 종류의 분포형태를 파악하였다. 식품 총 619건 중 263건(42.5%)의 B. cereus를 분리하였지만, 식품의약품안전처의 식품공전에서 즉석섭취 편의식품류에 대한 B. cereus에 대한 규격기준이 1,000 CFU/g 이하로, 본 실험에서는 규정 범위를 초과한 식품은 없었다. B. cereus 총 263개 분리균주에 대한 9종류의 장내독소 및 1종류의 구토독소를 분석한 결과, NHE complex (nheA, nheB, nheC)중 3개의 nhe 유전자를 모두 보유한 B. cereus는 236개 균주(89.7%), 1개만의 독소를 가지고 있는 균주는 2개(0.8%)뿐이었다. 또한, HBL complex (hblA, hblB, hblD) 중 3개의 hbl 유전자를 보유한 균주는 175개 균주(66.5%), 그리고 한개 혹은 두 개의 hbl 유전자를 가진 균주는 59개 균주(21.7%), 3개 유전자를 모두 가지고 있지 않은 균주는 29개 분리균주(11.0%)로 나타났다. NHE complex 유전자의 빈도가 HBL complex 유전자 보다 더 높게 나타나는 특징을 확인할 수 있었다. 장내독소 entFM, $cytK_2$ , bceT 유전자의 보유율은 각각 100, 100, 43.0% 순으로 확인되었다. B. cereus의 구토 독소인 CER 유전자는 평균 50.2%를 보유하고 있었으며, 식품유형에 따라 식중독 보존식, 즉석섭취 식품, 편의식품에서 각각 100, 59.4, 35.6% 순으로 나타났다. 식품 중 분리한 B. cereus가 산생하는 구토독소를 가지고 있는 B. cereus 균주 중 29.4%에서 8개의 장내독소를 보유하고 있었으며, 나머지 균주들도 대부분 5-8개의 장내독소를 가지고 있는 것으로 확인되었다. 따라서 본 연구 결과를 종합해보면, 식품에서 분리한 B. cereus는 대부분의 균주가 설사형 및 구토형 독소를 보유하고 있어, 즉석 섭취 편의식품의 미생물 오염 요인들은 계절에 관계없이 지속적으로 위생적인 관리가 필요할 것으로 사료된다. 또한, 식품의 미생물적 안전성 확보를 위해서는 업체의 생산단계부터 저장 및 운반을 포함한 유통단계 등에서 식중독을 유발 할 수 있는 오염원들이 내재되어 있기 때문에 철저한 개인 및 환경 위생관리가 필요할 것으로 사료된다. Toxin-producing Bacillus cereus is the causative agent of two different types of food poisoning: the emetic and the diarrheal types. This study was conducted to investigate the presence of enterotoxin and emetic toxin genes in 263 B. cereus isolated from 619 different ready-to-eat food items. Hemolytic enterotoxins hblA, hblC, and hblD were detected in 85.6, 41.1, and 76.8%, respectively, of the B. cereus isolates. About 67.0% (175/263) of the isolates presented all of three genes. Non-hemolytic enterotoxins nheA, nheB, and nheC were detected in 100, 97.0, and 68.4% of the isolates, respectively. Approximately 90.0% (236/263) of the isolates presented all of these three non-hemolytic enterotoxin genes. Emetic toxin gene, CER, was detected in 132 of 263 (50.2%) isolates. Computer-assisted cluster analysis of Rep-PCR profiles showed a high genetic diversity among the isolates. All B. cereus isolates from food samples tested in this study carried at least 6 of 10 toxin genes.
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