Medical Microbiology

Li Xiaoxia Department of Microbiology November, 2006

1 Chapter 10 Anaerobic bacteria

2 Classification

• spore­forming anaerobes Clostridium G + • non­spore­forming anaerobes G + , G ­ cocci, bacilli

3 Content

• Clostridium – C. tetani – C. botulinum – C. perfringens – C. difficile • Non­spore­forming anaerobes

4 I. Clostridium • General characteristics – gram­positive, spore­forming bacilli – obligate anaerobes – motile ­­ peritrichous flagella (exception: C. perfringens—nonmotile) – the sporangia– swollen – typical clinical symptoms

5 I. Clostridium • C. tetani • C. botulinum • C. perfringens • C. difficile

6 C. tetani

7 C. Tetani Characteristics • anaerobic gram­positive rod that forms terminal spores • motile with peritrichous flagella

• tetanospasmin

8 C. Tetani Pathogenicity • portal of entry: wound • conditions of infection regional anaerobic environment – deep and narrow wound, contamination of soil or foreign bodies – necrotic tissues – contamination of aerobes or facultative anaerobes

9 C. Tetani Pathogenicity

• Virulence factors – Tetanospasmin • Protein (neurotoxin) • Heat­labile (65℃, 30min) • Mechanisms

10 Mechanisms of tetanospasmin

toxin → inhibitory interneuron → blocks the release of neurotransmitters from the presynaptic membrane of inhibitory interneurons, e.g., glycine and γ–aminobutyric acid ( inhibit the motor neuron) → spastic paralysis (rigid paralysis) excitatory transmitter: acetylcholine inhibitory transmitter: glycine and γ–aminobutyric acid

11 Mechanisms of tetanospasmin

spastic paralysis (rigid paralysis)

12

C. Tetani­­Pathogenicity • Disease­tetanus(neonatal tetanus) latent period: 4­5d ～ several weeks typical symptoms: rigid paralysis

Opisthotonos

Lockjaw, sardonic smile Drooling, sweating, irritability 17 C. tetani­­ Pathogenicity

• Disease­neonatal tetanus – a frequent cause of death in developing countries – most common causes: cutting the umbilical cord with unsterilized instruments or infection of the umbilical stump – the fatality rate: around 50% – the common death cause: respiratory failure

18 C. tetani­­ Immunity

• Antitoxin immunity • Weak potent exotoxin rapid combination with target cells • Toxoid vaccine

19 C. tetani­­ Control • Proper care of wounds: surgical debridement • Active immunization: tetanus toxoid for children: basic immunization: DPT(diphtheria toxoid, pertussis vaccine, tetanus toxoid) for a high­risk group : toxoid booster • Passive immunization: tetanus antitoxin urgent prevention (along with toxoid) As soon as possible • Special treatment – administration of antibiotics – supportive measures 20 C. perfringens

21 C. perfringens Characteristics

• Shape and structure – Subterminal endospore – Capsule – Nonmotile

22 C. perfringens­­Characteristics

• Classification – five toxigenic types (A through E) – αtoxin: the most potent toxin→exhibits lecithinase activity→destroys erythrocytes, leukocytes, and platelets→ hemolysis, tissue necrosis

Type α, Alpha β, Beta ε, Epsilon ι, Iota

A +

B + + +

C + +

D + +

E + + 23 C. perfringens --Characteristics

• Cultivation anaerobic double zones of hemolysis carbohydrate fermentation (lactose)

Inner zone: θ toxin complete Outer zone: α toxin Incomplete

24 C. perfringens ­­ Pathogenicity

• Virulence factors – αtoxin • produced by all strains • acts as a lecithinase • diagnosis: Nagler reaction­­egg yolk agar

25 C. perfringens--Pathogenicity • Virulence factors – Enterotoxin • produced by types A(most), C, and D • heat­labile – Others • collagenase, hemolysin, proteinase, DNase (deoxyribonuclease)

26 C. perfringens Pathogenicity • Disease – Gas gangrene • Occurrence • Transmission: trauma • Pathogens: 60～80％ cases by type A • Conditions: localized anaerobic environment • Manifestation: sudden outset, emphysema, edema, necrotic tissues, foul­smelling, toxemia, shock

27 C. perfringens -- Pathogenicity

• Disease – Food poisoning • transmission: gastrointestinal tract • pathogens: type A • manifestation: short incubation period (10hrs) diarrhea self­limiting

28 C. botulinum

29 Biological weapon

• After Gulf War (1991), United Nations inspecting team reported Iraq admitted to having 19,000 L of concentrated botulinum toxin

30 C. botulinum

• What is it? • What it can do?

31 C. Botulinum Characteristics

• Gram positive rod • Subterminal endospore • Noncapsule • Obligate anaerobe

32 C. botulinum­­­ Pathogenicity

• Virulence factor—botulinum toxin – neurotoxin – relatively heat­labile: 100℃, 10min; 80℃, 20min – types: A, B, C, D, E, F, G – the most potent toxic material known potassium cyanide(KCN) 10,000 times

qmechanism of action: blocking the presynaptic release of the neurotransmitter acetylcholine → flaccid paralysis

33 Mechanisms of botulinum toxin

flaccid paralysis

34 C. Botulinum ­­­ Pathogenicity

• Disease—Botulism – from Latin botulus, "sausage" Endospore → germinate → toxin → flaccid paralysis Sausages, seafood products, milk, and p Food poisoning canned vegetables p Infant botulism Honey p Wound botulism

35 C. botulinum­­­ Pathogenicity

• Disease – Food poisoning • transmission: ingestion of toxin­contaminated food • manifestation: flaccid paralysis: double vision, dysphagia, difficulty in breathing and speaking rare gastrointestinal symptoms cause of death: respiratory failure

36 C. botulinum­­­ Pathogenicity

• Disease – infant botulism • transmission: ingestion of organism­contaminated food • manifestation: constipation, poor feeding, difficulty in sucking and swallowing, weak cry, loss of head control. Floppy baby • prevention: free of honey

37 C. botulinum­­­ Pathogenicity

• Disease – wound botulism • Rare • Transmission: trauma

38 Medicine

Blepharospasm

39 C. difficile

40 C. difficile ­­­ Pathogenicity • Virulence factor exotoxin A: enterotoxin exotoxin B: cytotoxin

• Disease pseudomembranous colitis: dysbacteriosis

antibiotic­associated diarrhea

41 C. difficile ­­­ Control

• Treatment discontinuation of causative antibiotics administration of sensitive antibiotics

• Prevention no vaccine use antibiotics only in necessary

42 non­spore­forming anaerobes

43 Characteristics

• include both G+ and G­ bacilli and cocci. • members of the normal flora • cause: endogenous infection

44 Conditions causing disease

• Change of habitat • Decrease of host defense • Dysbacteriosis • Local anaerobic environment formation

45 Characteristics of infections

• endogenous infection throughout body, most chronic • nonspecific manifestations, most pyogenic • foul­smelling discharge, sometimes gas formation • direct smear positive, aerobic culture negative • have no response to some antibiotics such as aminoglycisides

46 Diseases • septicemia • infections in central nervous system • dental sepsis • pulmonary infections • intraabdominal infections • infections of the female genital tract

47 Question 1. Make a list of the major pathogenic Clostridial species, indicating the virulence factors and diseases they cause. 2. What are the principle of treatment and prevention for the infection by C.tetani? 3. Make a list of conditions in which can cause disease infected by non­spore­forming anaerobes. 4. What effect do tetanospasmin and botulinum toxin have on muscles? 5. Choose the best answer 1) the primary toxin associated with invasive C.perfringens infection is a. neuraminidase b. alpha toxin c. hyaluronidase d. coagulase 2) a neurotoxin that causes flaccid paralysis is a. tetanospasmin b. botulinum toxin c. αtoxin d. enterotoxin occurrence development of anaerobic environments (e.g., deep wound) spores → vegetative cells ↓ tissue destruction and necrosis;

carbohydrate fermentation and gas (H2; , CO 2 ) formation and accumulation in the tissue ↓ restrict the blood supply (flow) → increases the tissue necrosis

49 endospore germinate under the favorable environment

sporulate under the unfavorable environment