1. Anaerobic bacteria
Norazli Ghadin

2. Learning objectives
Able to describe basic characteristic of anaerobic bacteria
Able to discuss oxygen toxicity, ROS formation during respiration and enzyme involve in eliminating ROS
Able to discuss the virulence factor of anaerobic bacteria
Able to describe the clinical manifestation of anaerobic bacteria infection.
Able to correlate important anaerobic bacteria with human health
Clostridium sp.
others

3. What Are Anaerobic Microorganisms
Anaerobic microorganisms are widespread and very important
Do not require oxygen for growth - often extremely toxic

4. Defining Anaerobes
Facultative anaerobes - can grow in the presence or absence of oxygen
Obtain energy by both respiration and fermentation
Oxygen not toxic, some use nitrate (NO3-) or sulphate (SO42-) as a terminal electron acceptor under anaerobic conditions

5. Strict Anaerobic Bacteria
Obligate (strict) anaerobes - oxygen is toxic to these organisms, do not use oxygen as terminal electron acceptor.
Archaea such as methanogens and Bacteria, e.g Clostridia, Bacteriodes etc. etc.

6. The Requirements for Growth: Related to Oxygen
Oxygen (O2)
Table 6.1

7. Anaerobes and Oxygen Anaerobes generate energy by fermentation
Lack the capacity to utilize O2 as a terminal hydrogen acceptor
Some are sensitive to O2 concentration as low as 0.5% O2
Most can survive in 3%-5% O2
A few can grow poorly in the presence of air  aero tolerant anaerobes
Many are members of the normal flora
 created by presence of facultative
anaerobes

8. Oxygen Toxicity
Oxygen is used by aerobic and facultatively anaerobic organisms as its strong oxidising ability makes it an excellent electron acceptor
During the stepwise reduction of oxygen, which takes place in respiration toxic and highly reactive intermediates are produced reactive oxygen species (ROS).

9. ROS production during respiration
O2 + e- => O superoxide anion
O2- + e- + 2H+ => H2O hydrogen peroxide
H2O2 + e- + H+ => H2O + OH Hydroxyl radical
OH. + e- + H+ => H2O water

10. Chemical Dynamics in Anaerobic Bacteria
Organisms that use O2 have developed defence mechanisms to protect themselves from these toxic forms of oxygen - enzymes
Catalase: H2O2 + H2O2 => 2H2O + O2
Peroxidase: H2O2 + NADH + H+ => 2H2O NAD+
Superoxide dismutase: O2- + O2- + 2H+ => H2O O2

11. Anaerobic environments exist in Nature too
Anaerobic environments (low reduction potential) include:
Sediments of lakes, rivers and oceans; bogs, marshes, flooded soils, intestinal tract of animals; oral cavity of animals, deep underground areas, e.g. oil packets and some aquifers
Anaerobes also important in some infections, e.g. C. tetanii and C. perfringens important in deep puncture wound infections
Dr.T.V.Rao MD

12. Anaerobic Environments

13. FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN
1. Toxic compounds are produced e.g. H2O2 , Superoxide's 2. Absence of catalase & Superoxide dismutase 3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them

14. ANAEROBES OF CLINICAL IMPORTANCE
CLOSTRIDIA
C tetani; C perfringens; C difficile; C botulinum
BACTEROIDES
B fragilis;
Prevotella
Porphyromonas
ACTINOMYCES
FUSOBACTERIUM
ANAEROBIC STREPTOCOCCI

15. Sites and Infection produced by Anaerobes

17. Anaerobic Bacteria of Medical Interest
MORPHOLOGY
GRAM STAIN
GENUS
Spore forming
(+)
Clostridium
Non-spore forming bacilli
(-)
Actinomycetes,
Bifidobacterium,Eubacte-rium,Propionibacerium,
Mobilncus,Lactobacillus
Bacteroides,Fusobacterium
Prevotella,Porphyromonas
Non-sporefoming cocci
Peptococcus,
Pepto-streptococcus
Streptococcus
Veilonella

18. Gram-positive anaerobes
Actinomyces (head, neck, pelvic infections; aspiration pneumonia)
Bifid bacterium (ear infections, abdominal infections)
Clostridium (gas, gangrene, food poisoning, tetanus, pseudomembranous colitis)
Peptostreptococcus (oral, respiratory, and intra- abdominal infections)
Propionibacterium (shunt infections)

19. Gram-negative anaerobes
Bactericides (the most commonly found anaerobes in cultures; intra-abdominal infections, rectal abscesses, soft tissue infections, liver infection)
Fusobacterium (abscesses, wound infections, pulmonary and intracranial infections)
Porphyromonas (aspiration pneumonia, periodontitis)
Prevotella (intra-abdominal infections, soft tissue infections)

20. ANAEROBIC GRAM NEGATIVE BACILLI
Bactericides, 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

21. FACTORS RESPONSIBLE FOR THEIR VIRULENCE
1. Lipopolysaccharide - promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinase * develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. succinic acid – reduces phagocytic killing
Dr.T.V.Rao MD

22. CLINICAL MANIFESTATION
Clinical hints
1. odor
2. tissue
3. location
4. necrotic tissue
5. endocarditis with (-) blood culture
6. infection associated with malignancy
7. black discoloration
8. blood containing exudates
9. associated with sulfur granules
10. Bacteremic feature with jaundice
11. human bites

23. Common Human Anaerobic Infections

24. 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

25. Clostridium perfringens
Large rectangular Gram positive bacillus
Spores seldom seen in vivo or in vitro
non motile
Produces several toxins
alpha (lecithinase), beta, epsilon
enterotoxin
Causes a spectrum of human diseases
Bacteraemia
Myonecrosis
food poisoning
enteritis necrotica (pig bel)
Dr.T.V.Rao MD

26. Clostridium tetani
Small motile spore forming gram positive bacillus with round terminal spores
Causes tetanus
Pathogenesis:
produces tetanospasmin during stationary phase which is released when cell lysis occurs
heavy chain binds to ganglioside on neuronal membranes
toxin internalized and moves from peripheral to central nervous system by retrograde axonal transport
crosses synapse and localized within vesicles
acts by blocking release of inhibitory neurotransmitters (eg GABA)

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
Produces 2 entero toxins
Toxin A -enterotoxin & Toxin B -cytotoxin
Diagnosis
Detection of toxins in stools, culture of organism
Clinical - AAC Pseudomembranous colitis
Treatment
omit antibiotic if possible
oral vancomycin (125mg qds or metronidazole

28. Clostridium botulinum
Fastidious spore forming anaerobic gram positive bacillus
Produces 8 antigenically distinct toxins
Human disease described with types A, B & E
Heavy chain binds to ganglioside receptor
Toxin internalized and prevents release of acetyl choline from vesicles
Clinical
Food borne botulism (weakness, dizziness, ocular palsy and progressive flaccid paralysis)
infant botulism (floppy baby)
wound botulism

29. ANAEROBIC GRAM NEGATIVE BACILLI
Bactericides, 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

30. ACTINOMYCES
Strict anaerobic Gram positive bacilli typically arranged in hyphae which fragment into short bacilli
Normal flora of upper respiratory tract, GI tract and female genital tract.
Low virulence
produce disease when mucosal barrier is breached (eg: following dental trauma or surgery) ENDOGENOUS
Establishes chronic infection that spreads through normal anatomical barriers
Clinical -cervicofacial, abdominal and thoracic
Diagnosis:
Gram stain of ‘sulpher’ granules
culture