Characterization of Bacillus cereus Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to Bacillus anthracis and Harbor B. anthracis Virulence Genes
Alex R. HoffmasterKaren K. HillJay E. GeeChung K. MarstonBarun K. DeTanja PopovićDavid SuePatricia P. WilkinsSwati AvashiaRahsaan DrumgooleCharles H. HelmaLawrence O. TicknorRichard T. OkinakaPaul J. Jackson
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ABSTRACT Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A , B , and C genes required for capsule biosynthesis in B. anthracis . Both clinical isolates expressed a capsule; however, neither was composed of poly- d -glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.Keywords:
Cereus
Bacillus anthracis
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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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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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본 연구에서는 지금까지 알려진 바와 달리 항균활성의 적용범위가 모호한 actinonin을 대표적 식중독균인 B. cereus에 적용함으로써 식중독균에 대한 항균 효과에 대해 연구하였다. 계대배양된 대수기의 B. cereus에 actinonin을 처리하여 생장 저해 여부를 확인하였고, 96-well plate를 이용한 broth micro-dilution 방법과 agar disk diffusion 방법을 통해 actinonin의 B. cereus에 대한 항균활성을 측정해본 결과, actinonin의 농도에 비례하여 B. cereus의 생장이 저해되는 것으로 나타났다. 또한, actinonin의 처리에 따른 B. cereus의 세포 독성 측정 결과 actinonin이 B. cereus의 세포 독성 또한 억제하는 것을 확인하였다. 따라서, actinonin은 천연항균물질로서 B. cereus에 의한 식품 오염을 억제하고 식중독 예방 및 잠재적 치료제로서 적용 될 가능성이 있는 것으로 관찰되었으며, 나아가 다른 식중독균들에 대한 적용 가능성 탐색과 관련한 연구가 필요할 것으로 생각된다. The objective of this study was to investigate the anti-Bacillus cereus activity of actinonin. Actinonin inhibited the growth of B. cereus in a dose dependent manner. The growth-inhibitory activity of actinonin was evaluated using a broth micro-dilution method, and minimum inhibitory concentration (MIC) and agar disk diffusion tests. B. cereus showed high susceptibility to actinonin in a concentration-dependent manner and MIC was determined to be $0.192{\mu}g/mL$ . Additionally, 1 and 2 mM actinonin induced formation of B. cereus inhibition zones. In addition, as compared to B. cereus alone, B. cereus added with $10{\mu}M$ actinonin showed a lower level of cytotoxicity in HeLa cells in vitro. Thus, this study revealed that actinonin could be a potential source of a natural antimicrobial agent or a pharmaceutical component against B. cereus.
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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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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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The behaviour of the sporulating soil-dwelling Bacillus cereus sensu lato ( B. cereus sl ) which includes foodborne pathogenic strains has been extensively studied in relation to its various animal hosts. The aim of this environmental study was to investigate the water compartments (rain and soil water, as well as groundwater) closely linked to the primary B. cereus sl reservoir, for which available data are limited. B. cereus sl was present, primarily as spores, in all of the tested compartments of an agricultural site, including water from rain to groundwater through soil. During rain events, leachates collected after transfer through the soil eventually reached the groundwater and were loaded with B. cereus sl . In groundwater samples, newly introduced spores of a B. cereus model strain were able to germinate, and vegetative cells arising from this event were detected for up to 50 days. This first B. cereus sl investigation in the various types of interrelated environments suggests that the consideration of the aquatic compartment linked to soil and to climatic events should provide a better understanding of B. cereus sl ecology and thus be relevant for a more accurate risk assessment of food poisoning caused by B. cereus sl pathogenic strains.
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