Assigned text book: Medical Microbiology, 8 th ed., by Murray et al.

Transcription

1 Assigned text book: Medical Microbiology, 8 th ed., by Murray et al. 1

2 陳怡原 X3352 Microbiology March 6/14, 2018 Ch. 14 Mechanisms of Bacterial Pathogenesis 2

3 Terminology Pathogenicity (Virulence): the ability of a pathogen to cause disease Colonization: bacterium occupies and multiplies in a particular area of the body Infection: colonization of the human body by a bacterium capable of causing disease Disease: infection that produces symptoms Carrier: an infected person who does not have symptoms Opportunists: bacteria that normally do not cause disease in healthy people but can cause disease in people with impaired defenses Pathogenicity island: complex structures or virulence factors encoded by a gene cluster 3

4 Establishing an Infection 1. enter the human body 2. adhere, colonize, invasion 3. tissue damage 4

5 Entry into the human body Bacteria can enter human body through mouth, nose, respiratory tract, ears, eyes, urogenital tract and anus Human defence: 1. Natural defence mechanisms include skin, mucus, ciliated epithelium, lysozyme and other antibacterial secretions in tears and mucus, sweat, acid and bile in the GI tract, secretory IgA, and innate immunity 2. Specific defence: T and B cells infection shedding 5

6 Entry into the human body Route Ingestion ( 食入 ) Inhalation ( 吸入 ) Trauma ( 傷口 ) Needlestick ( 針扎 ) Arthropod bite ( 節肢動物叮咬 ) Sexual transmission ( 性交 ) Examples Salmonella spp., Shigella spp., Yersinia enterocolitica, enterotoxigenic Escherichia coli, Vibrio spp., Campylobacter spp., Clostridium botulinum, Bacillus cereus, Listeria spp., Brucella spp. Mycobacterium spp., Nocardia spp., Mycoplasma pneumoniae, Legionella spp., Bordetella, Chlamydophila psittaci, Chlamydophila pneumoniae, Streptococcus spp. Clostridium tetani, Staphylococcus aureus Staphylococcus aureus, Pseudomonas spp. Rickettsia, Ehrlichia, Coxiella, Francisella, Borrelia spp., Yersinia pestis Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum 6

7 群聚 (colonization) 黏附 (adherence) 及侵入 (invasion) 多數病毒感染及部分細菌感染是利用微生物之特殊沾合器與宿主細胞受器結合, 如金黃葡萄球菌之 clumping factor A A 族鏈球菌之 M 蛋白質 E. coli 的 type 1 fimbriae 淋病雙球菌的纖毛 (fimbriae) 部分腸內細菌可利用侵染素蛋白質 (invasin) 與宿主細胞結合, 進而刺激宿主細胞進行吞嗜動作 7

8 Colonization, Adherence, Invasion Microbe Adhesin Receptor Staphylococcus aureus Clumping factor A Fibrinogen Staphylococcus spp. MSCRAMM Extracellular matrix components (fibronectin, laminin, collagen, etc.) Group A Streptococcus Extracellular matrix LTA M protein complex components (fibronectin, F protein, MSCRAMM laminin, collagen, etc.) Streptococcus pneumoniae Adhesins and other proteins N-Acetylhexosamine-galactose Escherichia coli Type 1 fimbriae D-Mannose Colonization factor antigen fimbriae GM ganglioside 1 P fimbriae P blood group glycolipid Neisseria gonorrhoeae Fimbriae GD 1 ganglioside Treponema pallidum P 1, P 2, P 3 Fibronectin Chlamydia trachomatis Cell surface lectin N-Acetylglucosamine Mycoplasma pneumoniae Protein P1 Sialic acid Vibrio cholerae Type 4 pili Fucose and mannose LTA, Lipoteichoic acid MSCRAMM, microbial surface components recognizing adhesive matrix molecules 8

9 Bacterial Pathogenic Action 致病機轉 (pathogenesis): 細菌的代謝產物 外毒素 ( 包括超級抗原 ) 分解酵素 毒性蛋白質 內毒素及免疫病理 ( 過度發炎反應 ) 逃避寄主防禦機制 抗藥性 9

10 Pathogenic Action: Tissue Damage 細菌的代謝產物如發酵所產生的酸 氣體及其他副產品 cytolytic enzymes ( 溶細胞酵素 ) 10

11 Pathogenic Action: Exotoxin A-B toxins: 包含 A 及 B 二部份, 其中 B 負責與細胞表面的受器結合而 A 部份具抑制細胞功能的活性 Super antigens ( 超級抗原 ): 可不具專一性地活化 T 細胞, 造成大量細胞激素分泌, 引發類似自體免疫反應 也可刺激 T 細胞, 使具 T 細胞專一性受器之 T 細胞凋亡, 進而失去免疫反應 11

12 A-B toxin: Diphtheria toxin 12

13 A-B toxin: Cholera toxin ( 過度活化 ) 霍亂毒素 13

14 Pathogenic Action: LPS and cell wall components The molecular patterns in the structures of bacterial cell wall bind to Toll-like receptors (and other molecules) and stimulate cytokine production. Gram negative bacteria: LPS (endotoxin) binds to CD14 and TLR4 to activate acutephase and inflammatory reactions (cytokine production) LPS also stimulates the growth of B cells Gram positive bacteria: Peptidoglycan, teichoic acid and lipoteichoic acids could stimulate endotoxin-like responses 14

15 Immunopathogenesis 因感染後的發炎反應及過量的免疫反應所造成之症狀 由內毒素引發之敗血 活化之補體可造成 anaphylatoxins 的產生, 進而改變血管通透性, 引起 capillary breakage 由 superantigens 或 LPS 引發之大量 cytokine production 結核桿菌感染所引發之肉芽腫 A 族鏈球菌 M 蛋白質所引發之風濕性熱 15

16 The activities of LPS Disseminated intravascular coagulation (DIC): Is characterized by the intravascular activation of coagulation with loss of localization arising from different causes can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction 16

17 Mechanism for Escaping Host Defense 1. Encapsulation: antiphagocytic activity, enhancing biofilm formation and etc. 2. Antigenic mimicry 3. Antigenic shift 4. Production of anti-immunoglobulin protease: IgA protease 5. Destruction of phagocytes 6. Inhibition of chemotaxis 7. Inhibition of phagocytosis 8. Inhibition of phagolysosome fusion 9. Resistant to lysosomal enzymes 10. Intracellular replication 17

18 Mechanisms for escaping phagocytic 1: Fail to phagocytosed effectively killing 2: Fail to destroy the internalized bacteria 18

19 吞噬細胞的清除作用及細菌的逃脫 莢膜可抗吞噬 利用分解酶降低調理作用 ( 以降低吞噬 ) Opsonization ( 調理作用 ) 分泌酵素破壞趨化作用 分泌酵素毒殺吞噬細胞 分泌酵素降低溶酶體的殺菌作用 逃脫溶酶體並在細胞質內繁衍 抑制溶酶體的融合 19

20 Ch. 16 Laboratory Diagnosis of Bacterial Disease 20

21 Bacterial Detection and Identification I. General procedures: Microcopy Detection of antigens Detection of specific bacterial nucleic acids Culture Detection of an antibody response to the bacteria II. Antimicrobial Susceptibility Tests Broth dilution tests Agar diffusion tests 21

22 Ch. 17 Antibacterial Agents 22

23 常用名辭定義 Antibacterial spectrum ( 抗菌範圍 ) 廣效性 (Broad spectrum) 的抗生素可抑制多種革蘭氏陰性及陽性細菌 ; 窄效性 (Narrow spectrum) 的抗生素則只針對幾種細菌 Bacteriostatic antibiotic 抑制但不會殺死細菌之抗生素 Bactericidal antibiotic 可殺死細菌之抗生素 Minimum bactericidal concentrations(mbc, 最小殺菌濃度 ) 殺死 99.9% 標準濃度細菌所需最低抗生素濃度 Minimum inhibitory concentrations (MIC, 最小抑菌濃度 ) 最低抑制細菌生長所需之抗生素濃度 Synergism ( 抗生素相乘作用 ) 兩種藥物合併使用時有增強效應, 較兩種藥物分開更具殺菌活性 Antagonism ( 抗生素拮抗作用 ) 兩種藥物合併使用時互相干擾, 總體活性較任何單一藥物殺菌活性低 23

24 Agar diffusion tests ( 瓊脂擴散試驗 ) 此菌對測試之四種藥物具不同的敏感度, 其中藥物 1 及 3 抑菌效果好於 2 及 4 利用具梯度之抗生素紙錠同時測定最小抑制濃度 MIC: minimal inhibitory concentration 24

25 抗生素相乘作用 (Synergism) PABA + Pteridine Dihydropteroic acid Dihydropteroate synthetase inhibited by sulfisoxazole Dihydrofolic acid Dihydrofolate reductase inhibited by trimethoprim Tetrahydrofolic acid 25

26 Basic sites of antibiotic activity Inhibiting fatty acids biosynthesis 6. Acting on cell membrane

27 抑制葉酸新陳代謝 磺胺藥與 PABA 結構相似, 故可用來干擾細菌 PABA 之攝取進而抑制核苷酸及胺基酸的合成 27

28 Resistance to Antibiotics Penicillinase e.g. Vancomycin resistance Methicillin resistance e.g. Tetracycline resistance Adapted from Microbiology, Tortora et al, 11 th ed. 28

29 微生物的抗藥性產生機制 1. 因抗生素僅對生長活躍的細菌具殺菌或抑菌效果, 故因環境影響而導致代謝緩慢的細菌不會對藥物敏感, 此 抗藥性 不是來自遺傳物質的改變, 屬暫時性抗藥, 也不會遺傳到子代 2. 抗藥性是由遺傳物質的改變或增加而出現, 屬永久性抗藥, 也會遺傳到子代 i. 突變及篩選 ii. 藉由轉形 (transformation), 轉導 (transduction) 或接合 (conjugation) 造成抗藥基因的移轉 3. 交叉性抗藥 : 對結構相似的藥物具抗性 29

30 Now 我們要進入細菌個論了... 30

31 Ch. 18 and 20 Aerobic Gram (+) cocci ( 球菌 ) 31

32 課本 p.108 革蘭氏陰性細胞壁 革蘭氏陽性細胞壁 Teichoic acid 32

33 Ch. 18 Staphylococcus spp. & Related Organisms 葡萄球菌及其他類屬細菌 33

34 Aerobic, catalase positive cocci: Staphylococcus and Micrococcus 微球菌屬 (Micrococcus spp.): Coagulase negative Opportunistic infections 葡萄球菌屬 (Staphylococcus spp.): Coagulase positive/negative Important pathogens 34

35 Outline Morphology and physiology Pathogenesis (virulence factors) Epidemiology Clinical diseases Laboratory diagnosis Treatment, prevention and control 35

36 葡萄球菌屬之形態與生長特性 1. Gram (+) cocci in clusters; non-motile 2. Some produce capsule ( 莢膜 )/slime layer 3. Peptidoglycan contains endotoxin like activity 4. Aerobic or microaerophilic 5. Tolerate high NaCl concentrations (10%) 6. Grow at o C 7. Catalase ( 觸酶 ) positive 8. Major pathogens include: S. aureus: coagulase ( 凝固酶 ) 陽性 produce yellow-pigment possesses protein A ferment mannitol ( 木蜜醇 ) S. epidermidis S. haemolyticus S. lugdunensis S. saprophyticus 機會性感染, 均為 coagulase 陰性 36

37 葡萄球菌屬的特性 凝固酶 (coagulase) MSA plate M: 木蜜醇 (mannitol), S: 7.5% NaCl 觸酶 (catalase) 金黃葡萄球菌 (S. aureus) 觸酶 H 2 O 2 H 2 O + 1/2O 2 葡萄球菌 (Staphylococcus spp.) 表皮葡萄球菌 (S. epidermidis) 37

38 金黃葡萄球菌的傳染病學 Normal flora on skin and mucosal surfaces Person-to-person spread results from direct contact Risk factors are the presence of a foreign body, previous surgical procedures, and the use of antibiotics (which may suppress the normal flora) Patients at risk include infants, young children with poor personal hygiene, and patients with comprised pulmonary function 38

39 金黃葡萄球菌的致病能力 1. Able to evade immune clearance 2. Able to adhere to host tissues 3. Production of toxins and hydrolytic enzyme to cause tissue destruction 39

40 金黃葡萄球菌毒性因子之調控 Expression of virulence factors in staphylococci is under the control of the Agr (accessory gene regulator) system (a quorum-sensing [bacterial density] control system) 課本 p.126 S. aureus expresses adhesion proteins to promote tissue colonization when the population density is low. Proteins for tissue invasion and damage (hydrolytic enzymes and toxins) are produced when the population density is high. The innate immune regulation of bacterial virulence is mediated by apolipoprotein B, the major structural protein of very low- (VLDL) and low-density lipoproteins (LDL), which binds to AIPs and suppresses Agr signaling. 40

41 Oligopeptide mediated quorum sensing in Gram-positive bacteria Sensor kinase (HK) Response regulator (RR) 41

42 The Agr quorum-sensing accessory gene regulator system Trends in Microbiology , DOI: ( /j.tim )

43 I. Defenses against innate immunity: Capsule ( 莢膜 ): inhibiting phagocytosis by PMN Slim layer/biofilm: binding to tissues and foreign bodies Peptidoglycan ( 胜肽聚醣 ): stimulating pyrogen production; activation of complement; stimulating IL-1 production from monocytes; leukocyte chemoattractant (abscess formation) Teichoic acid/lipotechoic acid: binding to fibronectin MSCRAMM (microbial surface components recognizing adhesive matrix molecules): surface adhesion proteins for adherence to host matrix proteins 金黃葡萄球菌的毒素分子 -1 Protein A: inhibiting antibody-mediated clearance by binding to the Fc receptor of IgG1, 2, and 4 Fibronectin binding protein A Clumping factor proteins A and B 43

44 金黃葡萄球菌的毒素分子 -2 II. Toxins: Cytotoxins ( 細胞毒素 ): a (cause b-hemolysis), β, δ, γ, P-V leukocidin Exfoliative toxins ( 脫皮毒素 ): ETA & ETB Serine protease (split desmoglein 1); responsible for SSSS Enterotoxin ( 腸毒素 ): multiple types Superantigens (p.142) Heat stable and resistant to hydrolysis by gastric and jejunal enzymes Toxic Shock Syndrome (TSS)Toxin-1 ( 毒性休克症候群毒素 -1) Superantigens Heat- and proteolysis-resistant; responsible for TSS 44

45 Superantigen Superantigen can nonspecifically activate T cell, and lead to massive release of cytokines by both macrophages (IL-1b and TNF-a) and T cells (IL-2, IFN-g, and TNF-b). Release of TNF-a and TNF-b is associated with hypotension and shock, and fever is associated with IL-1b release. 45

46 金黃葡萄球菌的毒素分子 -3 III. Enzymes: Coagulase ( 凝固酶 ): convert fibrinogen to fibrin Catalase ( 觸酶 ): convert H 2 O 2 to H 2 O and O 2 Hyaluronidase ( 玻璃醣炭基酸酶 ) Fibrinolysin (Staphylokinase, 葡萄球菌激酶 ): dissolve fibrin Lipase ( 脂肪酶 ) Proteinase ( 蛋白酶 ) Penicillinase ( 青黴素酶 ) 46

47 金黃葡萄球菌的臨床致病 Toxin production Proliferation of the bacteria, leading to abscess and tissue destruction Presence of foreign body Congenital diseases associated with impaired chemotactic or phagocytic response 47

48 金黃葡萄球菌的臨床致病 -1 Benign skin infections are common, serious disease is not; however, infections are more serious in compromised individuals, e.g. surgical, burn wards and nurseries. 1. Cutaneous infections: One of the most common bacterial infection in humans. The most superficial situation is folliculitis. Others include boils, carbuncles, and impetigo. Staph scaldedskin syndrome usually occurs in infants. 48

49 金黃葡萄球菌所引起之皮膚感染 Folliculitis ( 毛囊感染 ) Deep folliculitis Furuncle Carbuncle 嚴重程度 Impetigo 膿泡症 Scalded-skin syndrome 脫皮症候群 49

50 Pustular Impetigo Impedigo is a superficial infection that mostly affects young children, occurs primarily on the face and limbs. 50

51 Bullous Impetigo: a localized from of SSSS Caused by phage type 71 51

52 Staphylococcal Scalded Skin Syndrome (SSSS) or Ritter s disease 脫皮症候群 脫皮症候群 Exfoliative toxins A and B ( 脫皮毒素 ) are responsible for the pathology. 52

53 金黃葡萄球菌的臨床致病 Food Poisoning: One of the more common forms of bacterial food poisoning. Caused by ingesting food that contains preformed toxin. Food can be contaminated during preparation and processing. Food is normal in odor, appearance, and taste. Heating food will NOT inactivate enterotoxin. Symptoms occur shortly after ingestion (30 min to 8 h): sever cramping, abdominal pain, nausea, vomiting, followed by diarrhea. Spontaneous remission occurs after 24 hours. 53

54 金黃葡萄球菌的臨床致病 Toxic Shock Syndrome (TSS, 毒性休克症候群 ): A fatal infection with cutaneous and soft tissue involvement. An acute febrile illness usually in young adults and children. Patients become hypotensive following vascular coagulation, renal failure, acidosis, and rash. It can be fatal. 54

55 金黃葡萄球菌的臨床致病 Bacteremia ( 菌血症 ) & Endocarditis ( 心內膜炎 ) 5. Pneumonia and Empyema: Staphylococcal pneumonia is most often seen as a complication of other bacterial or viral infections. 6. Osteomyelitis ( 骨髓炎 ) & Septic Arthritis ( 化膿性關節炎 ): i. Resulted from hematogenous spread or as a secondary infection. ii. S. aureus is the primary cause of septic arthritis in young children and adults with mechanically abnormal joints or receiving intra-articular injections. 55

56 表皮葡萄球菌及其他凝固酶陰性 葡萄球菌的臨床致病 Endocarditis Staphylococci are the major cause of endocarditis of artificial valves. S. lugdunensis is the staphylococcal species that is most commonly associated with native valve endocarditis, although this disease is more commonly caused by streptococci. Catheter and shunt infections Biofilm formation enhances the infection. Prosthetic joint infections Infections of artificial joints, particularly the hip Urinary tract infections S. saprophyticus is the common cause for UTI in young, sexually active women and is rarely responsible for infections in other patients. 56

57 葡萄球菌的抗藥性 MRSA (methicillin-resistant S. aureus) and MRSE (methicillin-resistant S. epidermidis) Resistance occurs as the result of acquisition of meca, a gene encoding PBP2A (penicillin binding protein 2A). PBP2A is not recognized by methicillin and thus the resistance. Vancomycin resistance Low level resistance: is observed in S. aureus strains with a thicker, more disorganized cell wall. High level resistance: is mediated by the vana gene operon that was acquired from vancomycin-resistant enterococci. 57

58 meca (gene) encoding PBP2A (protein) 具 meca 之菌種在有 methicillin 下,PBP2A 仍可正常運作, 故具抗性 (meca product) PNAS 2001;98:

59 葡萄球菌感染之實驗診斷 Microscopy: Gram positive cocci in clusters Nucleic acid-based test Culture: colonial characteristics on MSA and chromogenic agar Identification: MALDI-TOF 59

60 葡萄球菌感染之預防及治療 Localized skin and soft-tissue infections: incision and drainage of the abscesses Systemic infections: Oral antibiotic and intravenous vancomycin therapy The spread of staphylococci from person to person is more difficult to prevent. An example of this is surgical wound infections, which can be caused by relatively few organisms, because foreign bodies and devitalized tissue may be present. 60

61 葡萄球菌屬 Staphylococcus spp. 型態 : 革蘭氏陽性球菌成堆排列培養 : 好氧分類 : 金黃葡萄球菌 表皮葡萄球菌及其他抗原 : 細胞壁多醣類 胞壁酸主要病菌 : 金黃葡萄球菌金黃葡萄球菌之毒性因子 : 觸酶 凝固酶 葡萄球菌激酶 玻璃醣炭基酸酶 脂肪酶 蛋白酶 青黴素酶 細胞毒素 ( 溶血素 殺白血球素 ) 腸毒素 脫皮毒素 毒性休克症候群毒素臨床致病 : 化膿性皮膚炎 食物中毒 毒性休克症候群 心內膜炎 菌血症 肺炎 骨髓炎 化膿性關節炎實驗診斷 : 鏡檢 PCR 培養 fluorescent in situ hybridization (FISH) 治療預防 : 抗生素 (vancomycin) 抗藥性 : 青黴素酶 meca 61

62 位性皮膚炎患者其皮膚經常會受到微生物感染, 下列那一種病原菌最常見?(103-1) A. 金黃色葡萄球菌 (Staphylococcus aureus) B. A 群鏈球菌 (group A streptococcus) C. 黴菌類 (fungi) D. 大腸桿菌 (Escherichia coli) 關於 Staphylococcus aureus 的特徵, 下列何者錯誤?(104-1) A. Gram-positive cocci B. β-hemolysis C. Catalase-positive D. Coagulase-negative 戴隱形眼鏡導致之角膜潰瘍, 下列何種致病菌最常見? A. 淋球菌 (gonococci) B. 綠膿桿菌 (Pseudomonas aeruginosa) C. 金黃色葡萄球菌 (Staphylococcus aureus) D. 念珠菌 (Candida) 62