Bacterial Intoxification

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1 Bacterial Intoxification
黃顯宗 東吳大學

2 Microbial Intoxication
細菌、真菌、藻類等都可能引起中毒 部份致病菌很難明確劃分中毒型或感染型，如Bacillus cereus、Clostridium perfringens

3 Bacillus cereus 譯名錯誤，非“仙人掌桿菌“，cereus=wax 從土壤到食物，分布極廣的腐生菌
常在澱粉質食物中發生，如飯、麵等因此外人常將它與中餐館相聯 G+ acerbic rods，強lecithinase有endospore，D100c=8min因此經加熱後的食物較易發生

4 INTRODUCTION The Genus Bacillus was established in 1872 with B. subtilis as type species. B. cereus was added fifteen years later Several accounts of food poisoning attributed to members of the genus Bacillus appeared in the European literature before 1950

5 INTRODUCTION An accumulating number of reports implicate both B. subtilis and B. licheniformis as potential food poisoning agents.

6 OCCURRENCE IN FOOD AND ENVIRONMENT
B. cereus has a wide distribution in nature, frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals It has been isolated from foods that were not involved in foodborne illness outbreaks. It is also present in the stools of 14 to 15% of healthy humans It is frequently isolated from milk and dairy products. In milk, B. cereus causes a defect known as 'bitty' cream or sweet curdling. It is found in rice, rice products, oriental dishes and ingredients

7 OCCURRENCE IN FOOD AND ENVIRONMENT
A variety of foods have been implicated in food-poisoning Emetic syndrome caused by B. cereus is highly associated with rice and rice products

8 OCCURRENCE IN FOOD AND ENVIRONMENT
B. cereus was isolated from 9, 35, 14 and 48% of raw milk, pasteurized milk, Cheddar cheese and ice cream samples, respectively In a local study, B. cereus occurred in 17% of fermented milks, 52% of ice creams, 35% of soft ice creams, 2% of pasteurized milks and pasteurized fruit- or nut-flavored reconstituted milks, and 29% of milk powders, mostly in fruit- or nut-flavored milk mixes (Wong et al., 1988a). B. cereus was found in 71.4% and 33.3% in spring and in autumn samples of full-fat milk in mainland China, respectively, and the average count among the positive samples was 11.7 MPN/ml (Zhou et al., 2008). Dried milk products and infant food are known to be frequently contaminated with B. cereus, 261 samples of infant food distributed in 17 countries were collected and 54% were contaminated with B. cereus reaching levels from 0.3 to 600/g (Becker et al., 1994).

9 OCCURRENCE IN FOOD AND ENVIRONMENT
Chinese 'take-out' foods appear to be particularly vulnerable to B. cereus infection and it has been shown that suspensions (2%) of seed flours and meals from diverse botanical origins were found to be excellent sources of nutrients for growth (Beuchat and Ma-Lin, 1980)

10 OCCURRENCE IN FOOD AND ENVIRONMENT
Of 433 honey samples collected in Argentina, 27% yielded B. cereus isolates and 14% yielded other species of Bacillus. Results showed a high degree of diversity, both phenotypic and genotypic among the isolates of B. cereus (Lopez and Alippi, 2007).

11 OCCURRENCE IN FOOD AND ENVIRONMENT
The B. cereus isolates from food are highly toxigenic. All the isolates from local dairy products lysed rabbit erythrocytes; 98% showed verotoxicity, 68% showed cytotonic toxicity for CHO cells (Wong et al., 1988a). In another study of 136 strains of B. cereus isolated from milk and cream, 43% and 22% showed toxicity to human embryonic lung cell when the isolates were cultured in brain heart infusion and milk, respectively (Christiansson et al., 1989). In milks, B. cereus growed rapidly and produced cytotonic and cytotoxic toxins (Wong et al., 1988b). Toxin production of B. cereus in milk at low temperature was also evaluated (Christiansson et al., 1989).

12 OCCURRENCE IN FOOD AND ENVIRONMENT
For the B. cereus isolated from seafood, 48% isolates produced both the hemolysin BL (HBL) and nonhemolytic (NHE) enterotoxins, and 94% and 50% produced NHE or HBL toxins, respectively. Only one B. cereus isolate possessed the cereulide synthetase gene, ces (Rahmati and Labbe, 2008).

13 OCCURRENCE IN FOOD AND ENVIRONMENT
The enterotoxin genes hblA, hblC, hblD(前面幾種兼溶血), nheA, nheB and nheC (前面幾種不溶血)occurred in B. cereus isolates from full-fat milk products with frequencies of 37.0%, 66.3%, 71.7%, 71.7%, 62.0% and 71.7% respectively Nine B. thuringiensis isolates were also identified from six pasteurized milk samples, and most of them harbored six enterotoxic genes and the insecticidal toxin cry1A gene. The single B. mycoides isolate harbored nheA and nheC genes (Zhou et al., 2008).

14 CHARACTERISTICS AND TAXONOMY
B. cereus is a Gram-positive, motile, facultative, aerobic sporeformer. Dimensions of vegetative cells are typically μm by μm. The ellipsoidal spores are formed in a central or paracentral position without swelling the sporangium. The organism does not ferment mannitol and has a very active phospholipase (lecithinase) system. B. cereus is keyed as citrate(+), arabinose (-), Gram (+), aerobic sporeformer.

15 CHARACTERISTICS AND TAXONOMY

16 CHARACTERISTICS AND TAXONOMY

17 CHARACTERISTICS AND TAXONOMY
Plasmids have been identified in B. cereus. Plasmids of molecular weight ranged from 1.6 to 105 MDa. Bacteriocin production could be attributed to a 45 MDa plasmid (pBC7), and tetracycline resistance to a 2.8 MDa plasmid (pBC16)

18 SPORE AND GERMINATION B. cereus produces elliptical shaped endospore with dominant central position, no distended sporangium. The spore when liberated from the sporangium is encased in a loose fitting exosporium. On germination the spore coat undergoes rapid lysis while the vegetative cell is emerging. Since spores of B. cereus may survive heat processing, spore germination is important in B. cereus study.

19 GERMINATION STEPS Once the initial 'trigger reaction' has been activated, germination continues in the absence of the inducer. After the 'trigger' steps, the various spore properties are changed sequentially in the following order: loss of heat resistance, release of dipicolinic acid (DPA) and Ca2+ into the medium, increase in spore stainability, beginning of phase darkening and decrease of the optical density of spore suspension as cortex peptidoglycan is hydrolyzed and the products released to the medium Finally, the onset of metabolic activity as measured by oxygen uptake.

20 Role of trypsin-like enzyme
The germination of B. cereus spore is partially prevented by several inhibitors of trypsin-like enzymes (leupeptin, antipain, and tosyl-lysine-chloromethyl ketone) A synthetic substrate of trypsin also inhibited germination (Boschwitz et al., 1983).

21 SPORE AND GERMINATION Inactivation of B. cereus spores during cooling from 90C occurs in two phases, one phase occurs during cooling from 90 to 80C; the second occurs during cooling from 46 to 38C. No inactivation occurs when spores are cooled from a maximum temperature of 80C.

22 SPORE AND GERMINATION Germination of B. cereus spores is more extensive in rice than in trypticase soy broth at <15C and is generally more extensive for diarrheal strains in either medium than emetic strains Germination of B. cereus spores was also inhibited by the growth of lactic acid bacteria or the organic acids produced (Wong and Chen, 1988).

23 SPORE AND GERMINATION B. cereus spores germinate in inosine or in l-alanine as sole germinants

24 Confirmation of outbreak
B. cereus strains of the same serotype should be present in the epidemiologically food, feces and/or vomitus of the affected persons. Or Significant numbers (>105 CFU/g) of B. cereus of an established food poisoning serotype should be isolated from the incriminated food, or feces, or vomitus of the affected persons. or Significant numbers (>105 CFU/g) of B. cereus should be isolated from the incriminated food, together with detection of the organism in the feces and/or vomitus of the affected persons.

25 Bacillus cereus 中毒分類 下痢型，無嘔吐 Diarrheal type 嘔吐型，無下痢 Emetic type
屬於感染型 嘔吐型，無下痢 Emetic type 屬於中毒型，短時間發 病 混合型，嘔吐下痢 Intermediate type 潛伏期1~18小時，中毒往往攝取到百萬個菌量以上。 Emetic toxin MW=2000 嘔吐型再食品中產生

26 Bacillus cereus 血清型 依照鞭毛抗原分為多種H型 H1 型主要與嘔吐型有關 其他H型與嘔吐以外的致病有關

27 Bacillus cereus 毒素 有多種溶血素 cereolysin
emetic toxin “cereulide”：小peptide，耐熱 enterotoxin：分離不易，不耐熱與蛋白 酵素 Emetic toxin MW=2000 嘔吐型再食品中產生

28 Enterotoxins hemolysin BL (HBL) enterotoxins nonhemolytic (NHE) enterotoxins

29 Bacillus cereus 培養利用強的lecithinase，培養平板培養基加入 蛋黃 生化鑑定 毒素和毒素基因鑑定
其他成分，營養與呈色 生化鑑定 毒素和毒素基因鑑定 reversed passive latex agglutination (BCET-RPLA) kit (Oxoid), and the Duopath Cereus Enterotoxins (Merck) gene

30 Detecting Cereulide cereulide synthetase gene (ces)
liquid chromatography-tandem mass spectrometry analysis HEp-2 cell vacuolation test mitochondrial respiratory uncoupling activity sperm micro assay(boar spermatozoa )

31 如何分離內生孢子 不好的環境促進孢子的形成 加熱，如80C，殺死營養細胞，留下耐熱的孢子 熱可以刺激萌發 平板培養基培養

32 Staphylococcus aureus 金黃色葡萄球菌
小球菌，腸發現在人類皮膚，鼻腔膿傷等處，動物中也常有 耐乾，Aw = 0.86以上可生長，亦可耐10% NaCl，可在攝氏45度生長 除腸胃炎外，亦引起腦膜炎、骨髓炎、肺炎、乳房炎、toxic shock syndrome

33 Staphylococcus aureus
污染食品多是肉類，沙拉，蛋，糕餅等營養豐富的食物，特別是經熱處理後，少了其他競爭性細菌 潛伏期1~8小時，症狀： 腸炎、下痢、嘔吐、噁心、腹痛、虛脫，很少維持12~15小時 鑑定方式：菌與毒素

34 Staphylococcus aureus毒素
Coagulase，coagulate citrate plasma Alpha-exotoxin，細胞毒素 Beta-toxin，a hot-cold 溶血素 FS toxin，可破壞白血球的毒素 Hyaluronidase，spreading factor Toxic shock syndrome toxins toxin, TSST Enterotoxins Coagulase 使血漿凝集

35 Enterotoxins 腸毒素 分成A、B、C1、C2、C3、D、E、F、G等型
新型腸毒素SEG→SElX陸續被發現，部分新型腸毒素被證明具有嘔吐活性(如SER, SES, SET)，尚未證實具有嘔吐性者，稱為staphylococcus enterotoxin-like toxin (SEL) 有些如SElX可感染人、牛、羊並造成兔子的壞死性肺炎，因此新型腸毒素在食物中毒中相當重要

36 Enterotoxins 腸毒素 中毒以A，B兩型為主 耐熱 A 60度 20min B 100度 5min可減低一半毒性
對蛋白酵素穩定pepsin，trypsin，chymotrypsin，rennin，papain等

37 Staphylococcus aureus Enterotoxin
                                                             SEB

38 金黃色葡萄球菌偵測 推定試驗是先在Baird-Parker Medium及TSA 上培養檢體，依照菌落的型態觀察判斷疑似金黃色葡萄球菌；其次，進行生化試驗，檢測微生物產生凝固的特性；第三為輔助試驗，分別進行觸試驗、溶菌試驗、厭氧下葡萄糖之利用、厭氧下甘露醇之利用、熱安定型核酸分解試驗

39 金黃色葡萄球菌偵測 培養特性 耐鹽性( %) 耐亞碲酸鹽(Potassium tellurits，K2TeO3)耐受性( %) 耐 %之氯化鋰(Lithin chloride，LiCl) 耐高濃度( %)之甘胺酸Glycine 耐polymycin(40ug/ml) 典型菌落：(1)利用該菌種對egg yolk及glycine之分解能力使菌落周圍成透明。(2)利用該菌還原K2TeO3的作用使菌落呈現黑色

40 金黃色葡萄球菌中毒 腸毒素耐熱，可能毒素存在，菌體死滅 活菌和毒素的測定 基因 毒素蛋白質 PCR等 PCR-ELISA
生物晶片（microarray） 毒素蛋白質

41 Clostridium botulinum肉毒桿菌
肉毒桿菌按照毒素而分成幾型 Type A,B,E，人類疾病 A型較毒 E型與水海產相關為主 Type C,D，家禽中毒者 Type F，丹麥發現 Type G，阿根廷發現

42 Clostridium botulinum
G+ rods，anaerobic，耐熱 有胞子 pH 4.87以上生長 pH 5.01以上胞子發芽 pH 4.5以下所有生長產毒停止 15~48℃生長

43 Clostridium botulinum
發生率不高，主要是罐頭製造時加熱不良，特別是家庭製罐或一些醃肉食品也有可能發生，死亡率高，由於送醫早，治療得法，死亡率較低 食物中毒多樣，蔬果肉類最多。

44 Clostridium botulinum 中毒

45 Botulism肉毒桿菌中毒毒素 毒素產生時是大分子蛋白質，無毒，保護毒物經過消化道，經蛋白質酵素作用較小（100萬多），變成有毒性，靠本身的酵素或外在的蛋白質酵素，如trypsin

46 Botulinum Toxins

47 Botulinum Toxin A

48 Action of Botulinum Toxin
毒素是神經毒素，作用再neuromuscular junctions peripheral automatic synapses阻礙了acetylcholoine的釋放 中毒在18~36小時呈現症狀：暈眩、噁心、嘔吐、神經失調四肢行動不協調，視覺模糊，吞嚥困難，痲痹而死 個人致死量0.1~1.0 μg

49 Action of Botulinum Toxin
Ayres et al., 1980

50 Prevention of Outhbreaks
使用適當熱處理之罐頭 拒用氣脹的罐頭gassy (swollen) 不吃懷疑食物 避免用已熱，放置過久，再熱不良 可腐敗者宜冷藏冷凍 再熱不良 加熱不夠

51 Infant Botulism

52 Infant Botulism 1970年前發現 毒素在體內產生（in vivo） 乳鼠餵食中毒菌後，半數老鼠腸內會產毒
嬰兒（6個月以下）食用毒菌後，腸道的正常flora未全下，會萌發產毒 症狀：weakness、lack of suckling、 lose of head control，反射作用弱 可疑食物，如蜂蜜

53 近期報導

54 肉毒桿菌檢測 厭氧培養 小白鼠毒性偵測 Trypsin treatment. Toxins of nonproteolytic types, if present, may need trypsin activation to be detected Conduct parallel tests with trypsin-treated materials and untreated duplicates Inject each of separate pairs of mice intraperitoneally (i.p.) with 0.5 ml untreated undiluted fluid and 0.5 ml of each dilution of untreated test sample

55 肉毒桿菌檢測 厭氧培養 小白鼠毒性偵測 Typing of toxin. USE monovalent antitoxins
BAM