1. Batch 17 2 March 2012 Dr S Gokul Shankar
Anaerobic
Infections
Batch 17
2 March 2012
Dr S Gokul Shankar

2. Lecture 26: Anaerobic Infections
OBJECTIVES:
The objectives of this lecture are to
describe the general properties of anaerobic bacteria
describe the virulence factors and characteristic clinical features of infections caused by anaerobic bacteria
discuss the classification of clostridium.
discuss the clinical categorization of clostridium infections.
discuss antibiotic induced diarrhoea and Clostridium difficile
describe the laboratory identification of anaerobic bacteria, using clostridium infections as example.

3. Learning outcomes
At the end of the lecture, students should be able to:
describe the general properties of anaerobic bacteria
describe the virulence factors and characteristic clinical features of infections caused by anaerobic bacteria
describe the morphology of common Clostridium pathogens
categorize clostridium infections clinically into the gas gangrene, tetanus, and food poisoning groups.
describe the laboratory identification of anaerobic bacteria, using clostridium infections as example.

4. WHAT ARE ANAEROBIC BACTERIA?
Anaerobic bacteria are those that grow in the absence of oxygen, because they cannot tolerate the presence of oxygen

5. Anaerobic infections Common May be serious and life-threatening
Anaerobes are the predominant components of bacterial flora of normal human skin and mucous membranes
Difficult to isolate from infectious sites and are often overlooked. Isolation requires appropriate methods of collection, transportation and cultivation of specimens
Treatment is complicated by slow growth of these organisms, by the commonly polymicrobial nature of the infection and by the growing resistance of anaerobic bacteria to antimicrobial agents

6. Classification of anaerobes
OBLIGATE ANAEROBE
- is any organism that does not require oxygen for growth and die in its presence
AERO-TOLERANT ANAEROBES
Can survive in presence of oxygen
which cannot use oxygen for growth, but tolerate the presence of it
FACULTATIVE ANAEROBES
- which can grow without oxygen but can utilize oxygen if it is present.

7. Protections of bacteria against oxygen
Bacteria possess protective enzymes, catalase and superoxide dismutase.
Catalase breaks down hydrogen peroxide into water and oxygen gas.
Superoxide dismutase breaks superoxide down into peroxide and oxygen gas.
Anaerobes missing one or both; slow or no growth in the presence of oxygen.

8. WHY ANAEROBIC BACTERIA ARE NOT ABLE TO TOLERATE OXYGEN?

9. Properties of Anaerobic bacteria
die in presence of oxygen due to the absence of the enzymes superoxide dismutase and catalase
obligate anaerobes use alternate electron acceptors for cellular respiration such as sulfate, nitrate, iron, carbon monoxide, etc
The energy yield of these respiratory processes is less than oxygen respiration.
Can be spore forming/non spore forming .

12. Organism
Site of infection
Gram +ve cocci
Peptostreptococcus spp.
Respiratory tract, intra-abdominal & soft tissue infections
Microaerophilic streptococci (not true anaerobes)
Sinusitis, brain abscess
Gram +ve non spore-forming bacilli
Actinomyces spp.
Intracranial abscess, chronic mastoiditis, aspiration pneumonia, head & neck infections
Propionibacterium spp.
Shunt infections, infections associated with foreign body
Bifidobacterium spp.
Chronic otitis media, cervical lymphadenitis, intra- abdominal infection
Arachnia propionica
Eubacterium lentum
Gram +ve spore-forming organisms
Clostridium perfringens
Soft tissue infection, food poisoning
Clostridium septicum
Neutropaenic enterocolitis
Clostridium ramosum
Soft tissue infection
Clostridium botulinum
Botulism
Clostridium tetani
Tetanus
Clostridium difficile
Colitis, antibiotic-associated diarrhoea
Gram –ve bacilli
Bacteroides spp.
Intra-abdominal infection, gynaecological infection, neonatal infection
Prevotella spp
Orofacial infection, aspiration pneumonia, periodontitis, intra-abdominal infection, gynaecological infection
Fusobacterium spp.
Aspiration pneumonia
Porphyromonas spp.

13. CLOSTRIDIA Gram positive spore forming bacilli ubiquitous
intestines of man and animals
animal and human faeces, contaminated soil and water
Several species associated with human disease

14. Pathogenesis of anaerobic infections
Contamination of site with spores
Factors which promote anaerobiasis
‘crush’ injuries with interruption of blood supply, contaminaton with foreign bodies (dirt), tissue damage
Germination of spores
Toxin release
Binding of toxin to receptor
Resulting effect produces symptom(s) of disease

15. Clostridium perfringens
Morphology
Large rectangular rod shaped Gram positive bacillus
Spores seldom seen in vivo or in vitro
non motile
Pathogenesis
Produces several toxins
alpha (lecithinase), beta, epsilon
enterotoxin
Causes a spectrum of human diseases
Bacteraemia
Myonecrosis
food poisoning
enteritis necrotica (pig bel)

16. Myonecrosis (Gas gangrene)
Clostridium perfringens is the most common bacterial agent, while C. novyi, C. septicum, and C. histolyticum cause most of the remaining cases
Gangrene is necrosis and putrefaction of tissues. Gas production forms bubbles of gas in muscle (crepitus) and smell in decomposing tissue.
After rapid and destructive local spread (which can take hours), systemic spread of bacteria and bacterial toxins may result in death. This is a problem in major trauma and in military contexts.

17. The incubation period - short: almost always <3 days and frequently <24 h.  
Typically, begins with the sudden onset of pain in the region of the wound
wound must be deep, necrotic, and without communication to the surface. 
Soon after pain develops, local swelling and oedema—accompanied by a thin, often hemorrhagic exudate—appear.  
Patients frequently develop marked tachycardia, but elevation in temperature may be only minimal.
Gas is usually not obvious/absent at this early stage 
Frothiness of the wound exudate may be noted.
Profuse serous discharge

19. Diagnosis Myonecrosis (Gas gangrene) Food poisoning 3rd common
Gram stain of exudate - typical organisms no pus cells
Culture -growth of C perfringens (and/or other clostridia associated with this clinical condition)
Food poisoning
3rd common
abdominal pain, diarrhoea and vomiting 8-18 hours after a suspect meal. Self limiting

20. Enteritis necroticans (Clostridial necrotizing enteritis) -Pigbel
Rare, can be fatal
severe abdominal pain, bloody diarrhoea (C perfringens type C)
Caused by Beta toxin
life-threatening sequence of severe abdominal pain, vomiting, bloody stool, ulceration of the small intestine with leakage (perforation) into the peritoneal cavity and possible death

21. Treatment and prevention
Myonecrosis
Proper wound debridement and ensure adequate blood supply
Penicillin
antitoxin and hyperbaric oxygen - no proven value
Food poisoning
Proper preparation and storage of food
self limiting disease -antibiotics not indicated
Pigbel
Proper cooking of food
immunization of susceptible population

22. Clostridium tetani
Small motile spore forming Gram positive bacillus with round terminal spores (tennis racket appearance)
Causes tetanus - a disease characterized by painful muscular spasms that can lead to respiratory failure and in up to 40% of cases, death.
C. tetani produces two exotoxins
tetanolysin
tetanospasmin

23. leads to muscle contraction and spasm )
Pathogenesis:
produces tetanospasmin during stationary phase which is released when cell lysis occurs
binds to ganglioside on neuronal membranes
toxin internalized and moves from peripheral to central nervous system
crosses synapse and localized within vesicles
acts by blocking release of neurotransmittors (eg GABA, Glycine
leads to muscle contraction and spasm
Characteristic features are a rigid smile, trismus (lock-jaw"), and opisthotonus (rigid, arched back)

25. Prevention and treatment
DPT Vaccine (diphtheria-pertussis-tetanus)
Active/passive immunization of ‘risk’ injuries
management of wound
tetanus toxoid
Anti-tetanus serum (ARS -horse serum) or Human Tetanus ImmunoGlobulin (HTIG)
Penicillin or Metronidazole
Management of patient with tetanus
respiratory and CVS support

26. Clostridium difficile
Clostridia are motile bacteria that are ubiquitous in nature and are especially prevalent in soil.
appear as long, irregularly (often "drumstick" or "spindle") shaped cells with a bulge at their terminal ends.
Gram positive, spore forming

27. Clostridium difficile
Associated with human disease in mid-1970’s
Found in human GIT in small numbers
With antibiotic use, increase in number in GIT
Clindamycin, ampicillin, cephalosporins
proton pump inhibitors are a risk factor for C. difficile infection 
Produces 2 entero toxins
Toxin A -enterotoxin & Toxin B –cytotoxin
responsible for the diarrhoea and inflammation
Diagnosis
detection of toxins in stools /toxin genes by PCR
culture of organism
presence of C. Diff in the stool by C. Diff GDH antigen)

28. Antibiotic induced diarrhoea
Fluoroquinolones are more strongly associated with C difficile infections
Other antibiotics including clindamycin, 3rd generation cephalosporins and beta-lactamase inhibitors
Medication to suppress gastric acid production: H2 receptor antagonists and proton pump inhibitors - increased the risk
Resistance development to ciprofloxacin and levofloxacin

29. Treatment - omit antibiotic if possible -
Clinical - Pseudomembranous colitis - the generalized inflammation of the colon and the development of pseudomembrane, a viscous collection of inflammatory cells, fibrin and necrotic cells.
Treatment - omit antibiotic if possible -
metronidazole or oral vancomycin

30. Clostridium botulinum
Fastidious spore forming anaerobic Gram positive bacillus
Produces 8 antigenically distinct toxins
Human disease described with types A, B, F & E
Clinical
Food borne botulism (weakness, dizziness, ocular palsy and progressive flaccid paralysis)
infant botulism (floppy baby)
wound botulism
75 ng of toxin can kill a healthy human being

31. Other anaerobic G-ve bacilli
Bacteroides, Prevotolla, Porphyromonas and Fusobacterium
Present in GI tract -form large component of normal flora
>80% of human infections associated with B fragilis
virulence factors - capsule, LPS, agglutinins and enzymes
Clinical - Endogenous infections
Intra-abdominal pyogenic infections
pleuro-pulmonary infections
genital infection

32. Lab identification of C. botulinum
In laboratory isolated in tryptose sulfite cycloserine (TSC) growth media
Always in an anaerobic environment with less than 2% of oxygen. Commercial kits that use a chemical reaction to replace O2 with CO2 (E.J. GasPak System).
C. botulinum is lipase negative
It grows pH values of 4.8 and 7
Cannot use lacose as a primary carbon.

33. Clostridium perfringens
Roberson’s cooked meet medium
Clostridium perfringens from food/ wound

34. Colitis and C.difficile
Patients with C. difficile colitis often have elevated white blood cell counts
Detect toxins produced by C. difficile in a sample of stool
Flexible sigmoidoscopy and colonoscopy – look for pseudomembranes in the rectum and the sigmoid colon