anaerobic infection ppt.

laboratory approach for anaerobic infection

1. Anaerobic Infection

◦By-
Dr. Ravi Bhushan

2. ◦Introduction
◦Definition and Classification of anaerobes
◦Anaerobic bacteria as commensal flora
◦Diseases caused by anaerobic bacteria and site of
infection
◦Virulence factor
◦Pathogenesis
◦Incidence of infection
◦Laboratory diagnosis
◦Antibiotic susceptibility test
◦Antimicrobial activity

3. Introduction
◦Anaerobes are indigenous flora of skin and mucous
membranes.
◦Cause infections involving every organ & anatomic region of
the body.
◦Deep seated abscesses and necrotizing lesions, are
polymicrobial, and may include obligate anaerobes,
facultative anaerobes, or microaerophiles.
◦Within past few decades endogenous anaerobic infections
have become far more common, as Compromised host
immune response due to immunosuppressive drugs.
◦Antimicrobial agents are used empirically due to inadequate
anaerobic culture techniques, poor quality control in vitro
susceptibility results, and difficulty in obtaining test results within
a useful time frame.

4. Why it is essential to isolate and identify anaerobic bacteria?
1)Associated with high morbidity & mortality.
2)Treatment varies with bacterial species involved.
◦Currently > 3/4th of anaerobes isolated from different clinical
specimens are Bacteroides fragilis group, Prevotella,
Porphyromonas, Fusobacterium, anaerobic cocci, and the
anaerobic gram-positive, non-spore forming rods.
◦Most of them are resistant to penicillin and its analogues; they
are resistant to many cephalosporins including third gen.,
tetracyclines, aminoglycosides, also emergence of resistance
to newer quinolones and clindamycins.

5. Definitions
◦Anaerobes
◦Bacteria that require anaerobic conditions to initiate and
sustain growth
◦Strict (obligate) anaerobes
◦Unable to grow if > than 0.5% oxygen
◦Moderate anaerobes
◦Capable of growing between 2-8% oxygen
◦Microaerophillic bacteria
◦Grows poorly in air, but better in anaerobic conditions
◦Facultative bacteria (facultative anaerobes)
◦Grows both in presence and absence of air

7. Classification of anaerobes
BACILLI COCCI
GRAM
POSITIVE
SPORING
Clostridium spp.
NON SPORING
Actinomyces
Bifidobacterium
Eubacterium
Lactobacillus
Mobiluncus
Propionibacterium
Peptococcus
Peptostreptococcus
Finegoldia
GRAM
NEGATIVE
Bacteroides
Prevotella
Porphyromonas
Fusobacterium
Leptotrichia
Veillonella
Megaspharea

8. Anaerobic bacteria as commensal flora
Oral cavity
and upper
respiratory
passages
Pigmented Prevotella sp; Porphyromonas sp.
Nonpigmented Prevotella sp. (P. oralis)
Bacteroides sp. (B. ureolyticus), Fusobacterium sp. (F.
nucleatum)
Anaerobic cocci,Veillonella sp., Actinomyces and
Propionibacterium sp.
Stomach and
small intestine
Lactobacilli
Large intestine
and terminal
ileum
Bacteroides fragilis group
Porphyromonas sp., Fusobacterium sp.
Clostridium sp. , Eubacterium sp. ,Bifidobacterium sp.,
Propionibacterium sp.
GUT, vagina
and cervix
Pigmented Prevotella sp; Porphyromonas sp.
Nonpigmented Prevotella sp.
Bacteroides sp., Eubacterium sp., Propionibacterium sp.
Urethra
(male and
female)
Propionobacterium sp., Anaerobic cocci, Bacteroides sp.,
Fusobacterium sp.
Skin Propionibacterium sp., Anaerobic cocci

9. Diseases caused by anaerobic bacteria
Mouth, head and neck Sinusitis, otitis media,
periodontitis, root canal infection,
periodontal abscess, ocular
infections
Thoracic cavity Empyema, lung abscess,
aspiration pneumonitis,
necrotizing pneumonia
Abdominal cavity Peritonitis, intra-abdominal
abscess, appendicitis, liver
abscess, wound infection, biliary
tract infections
Pelvic cavity/ vagina PID, pelvic abscess, bacterial
vaginosis
Skin and soft tissue
infections
Diabetic foot ulcers, cutaneous
abscess, gas gangrene, bite
wound infections
Central nervous system Brain abscess, subdural
empyema, epidural abscess

10. Sites of anaerobic infections

11. PATHOGENESIS OF
ANAEROBIC INFECTIONS
◦Infections caused by anaerobes are generally a result of
the breakdown of a mucosal barrier and the subsequent
leakage of indigenous polymicrobial flora into previously
sterile closed spaces or tissue.
◦Three major factors are involved:
-virulence factors of the organisms,
-bacterial synergy, and
-mechanisms of abscess formation.
◦The predominant gram-negative anaerobes in these
infections include B. fragilis, Prevotella, Fusobacterium, and
Porphyromonas spp.

13. Incidence of anaerobes in various
infections
S. N.Type of infection Incidence (%)
1. Lung abscess, necrotizing pneumonia 62-93
2. Bacteremia 6-10
3. Brain abscess 60-89
4. Chronic sinusitis 52
5. Thoracic empyema 76
6. Intra abdominal/pelvic abscess 60-100
7. Perirectal abscess 75
8. Gas gangrene 85-95
9. Post appendectomy 40

14. continue..

17. FEATURES OF ANAEROBIC INFECTIONS
◦Characterized by Putrid odors & foul-smelling exudates
◦Gas in tissues and exudates
◦Infections are always near to the site of the body which are
habitat.
◦Necrotic tissue (gangrene), abscesses
◦Black discoloration or black pigment containing exudates
◦Polymicrobial.
◦Failure to grow organism from pus if not culture anaerobically.
◦Failure to respond to usual antibiotics like aminoglycosides,
fluoroquinolones.
◦Infection secondary to human or animal bite.
◦Detection of "Sulfur granules“ in discharge (due to
actinomycosis).

18. LABORATORY LABORATORY
DIAGNOSISDIAGNOSIS

19. Specimen collection and Transport
Specimens suitable for anaerobic culture:
◦Material aspirated from abscesses (the best specimens are from
loculated or walled off lesions)
◦Decubitus ulcer (if obtained from base of lesion after thorough
debridement of surface debris)
◦Sulfur granules from discharging fistula
◦Tissue obtained at biopsy or autopsy
◦Fluid from normally sterile site (e.g. joints fluid)
◦Pulmonary specimens (Bronchial washings obtained with double-
lumen plugged catheter, Percutaneous lung aspirate or biopsy,
Thoracocentesis fluid, Transtracheal aspirate)

20. ◦Gastrointestinal specimens (Bile, Peritoneal fluid)
◦Genitourinary specimens (Suprapubic bladder aspirate, Uterine
contents, Biopsy of endometrial tissue obtained with an endometrial
suction curette, Culdocentesis aspirate)
◦Blood, Bone marrow, CSF

21. Specimens unsuitable for anaerobic culture:
◦Bronchial washing or brush
◦Coughed (expectorated) sputum
◦Feces (except for Clostridium difficile)
◦Gastric or small bowel contents (except in blind loop syndrome)
◦Ileostomy or colostomy drainage
◦Nasopharyngeal swab, throat swab
◦Rectal swab, urethral swab, vaginal or cervical swab
◦Secretions obtained by nasotracheal or orotracheal suction
◦Voided or catheterized urine

22. Aspiration is ideal;
Avoid Swabs
II. Collection by needle
aspiration is preferable
than swab culture
because of;
a. better survival of
pathogen
b. greater quantity of
specimen
c. less contamination
with extraneous
organism are often
achieved

23. Transport system:
◦Stuarts transport media (Na thioglycollate, Na glycerophosphate,
CaCl
2
, Agar, Methylene blue, Distilled water)
◦PRAS ( Pre-reduced anaerobically sterilized ) plated media (Na
thioglycollate, NaH
2
PO
4
, NaCl
2
)
◦Syringe and needle sealed with a sterile rubber stopper
◦CO
2
vials
◦Bactec anaerobic bottle for blood culture
◦Vacutainer anaerobic transport
◦Hungate tube

24. Stuarts transport media

25. Common Anaerobic Media
Non-selective
1.Anaerobic blood Agar: non-selective medium for
isolation of anaerobes and facultative anaerobes;
contain 5% sheep blood, hemin, L- cysine, and vit. K1
1.Supplemented Brain Heart Infusion Agar:
Enriched with addition of yeast extract, Vit. K1, Hemin
solution, L-Cystine.

26. Liquid Media
1.Robertson cooked meat broth: Ground beef,
yeast extract, Trypticase, KH
2
PO
4
; L-cystine, resazurin
solution, distilled water
2.Peptone yeast extract broth: nonselective for
cultivation of anaerobic bacteria for gas-liquid
chromatography
3.Thioglycollate broth: for cultivation of anaerobes,
as well as facultative anaerobes and aerobes.

27. •Selective
1. Bacteroides bile esculin agar (BBE)
Selective and differential for Bacteroides fragilis
group; gentamicin inhibits aerobic organisms, 20% bile
inhibits most anaerobes, esculin hydrolysis turns medium
brown.
2. Laked Kanamycin-Vancomycin blood agar
(LKV)
Selective for isolation of Prevotella and Bacteroides grp;
kanamycin inhibits facultative gram-negatives,
vancomycin inhibits gram-positives and Porphyromonas,
laked blood allows early detection (with 48 hr) of
pigmented Prevotella.

28. 3. Cycloserine cefoxitin fructose agar (CCFA)
Selective for Clostridium difficile; cycloserine and
cefoxitin acts as inhibitory of normal intestinal flora, and
differential by fructose and neutral red as pH indicator.
4. Anaerobic phenylethyl alcohol agar (PEA)
Selective for inhibition of enteric gram-negative rods
and swarming by some clostridia.

29. METHODS OF
ANAEROBIOSIS

30. Anaerobic systems for cultivation:
◦Conventional anaerobic jar with evacuation and replacement of
gases with hydrogen, nitrogen and carbon dioxide.
◦GASPAK anaerobic system (sealed foil satchet with 2 tablets of: citric
acid and sodium bicarbonate & sodium borohydride and cobalt
chloride)
◦Anoxomat (uses single jar with a mixture of gases)
◦PRAS (Pre-reduced anaerobically sterilized media)
◦Anaerobic cabinet and Glove Box.

31. Anaerobic incubation
◦85% Nitrogen
◦10% Hydrogen
◦5% Carbon dioxide

32. Anaerobic Jar Techniques- Jars are used primarily with primary plated
media or subculture plates. Oxoid jar has a metal lid, valves & a pressure
gauge.
It can be used either as an evacuation-replacement jar or, it can be
used with a disposable gas generator (Gaspak).

33. Gaspak
◦Method of choice for preparing anaerobic jars. It is available as
disposable envelope, containing chemicals which generate H2 & CO2
on addition of water.
◦After the inoculated plates are kept in the jar, Gaspak envelope, with
water added, is placed inside & the lid screwed tight.
◦Presence of a cold catalyst in the envelope permits combination of H2
& O2 to produce an anaerobic environment.
◦Gaspak is simple, effective, & eliminates the need for drawing a
vacuum & adding H2.
◦Reduced Methylene blue is used as indicator.

34. Gaspak

35. Anoxomat

36. Anaerobic glove box

37. PRAS
Roll tube method

38. Anaerobic Disposable Plastic
Bags

39. Roll Streak System
FIG. A) Side view of roll tube, butyl rubber closure and
screw-on cap. B) Top view of closed roll tube.

40. Newer anaerobic systems
◦Recently, anaerobic gas-generating systems have been introduced
that don’t require either catalyst or the addition of water to activate
these systems.
◦AnaeroPack, absorbs O2 and generates CO2, but doesn’t generate
H2.
◦Appear to be an excellent alternative to the GasPak and other
established anaerobic incubation systems.
◦Another type of commercially available catalyst free-system i.e.,
Anaerocult (Merck, Germany), makes use of iron filings in a sachet to
which water is added, producing an O2 free, CO2-rich atmosphere.

41. Methods for diagnosis of anaerobic
infections
A) Direct examination of specimens and staining
B) Culture
C) Metabolic product detection by gas-liquid chromatography
D) Molecular methods like PCR
E) Rapid systems.

42. A) Direct examination of
clinical materials
◦A foul odor, purulent appearance of fluid
specimens, & the presence of necrotic tissue &
gas or sulfur granules are valuable for suspicion of
anaerobes.
◦Background & cellular characteristics of smear.
◦Acridine orange stains are useful for detecting
bacteria in blood cultures, CSF, pleural fluid, joint
fluid, and exudates.

43. Direct Microscopic Examination
(Gram stain)
◦Polymicrobial infection characteristic of anaerobic bacteria, and
multiple distinct morphotypes of gram-negative and gram-
positive bacteria suggestive of anaerobic infection.
◦Bacteria seen in smears, but no growth when cultured aerobically

44. Bacteroides, Porphyromonas, Prevotella: Faintly staining
gram-negative cocco-bacilli (enhanced staining with
carbol fuchsin as counterstain)

45. Fusobacterium nucleatum: Thin gram-negative bacteria with
tapered (pointed) ends.
Fusobacterium necrophorum: Pleomorphic, long gram-
negative rod with round ends and bizarre shapes (filaments,
coccoid forms or round bodies)

46. Clostridium perfringes: Large boxcar-shaped gram-
positive bacilli with blunt ends

47. Veillonella: Tiny gram-negative cocci with gram
variability

48. Actinomyces: Branching filamentous bacilli with
beaded gram-positivity

49. Bacterial Vaginosis
Gram variable cocco-bacilli

50. B) Culture
◦Colonial appearance of common anaerobes on culture media;

51. Colony of Bacteroides fragilis on
anaerobic blood agar
◦Non-hemolytic
◦Semi opaque
◦Grey colony with concentric whorls inside

52. Bacteroides fragilis on
Bacteroides bile esculin agar
(BBE)

53. Porphyromonas spp. on anaerobic
blood agar
◦Tan to buff colonies : brown-black pigment
◦Brick-red fluorescence (UV)

54. Prevotella
◦Brown to black colonies on BA
◦Brick –red fluoescence (Long wave UV)
◦Produce indole
◦Ferments glucose

55. Actinomyces
◦Colonies of Actinomyces israelii are 0.5-2mm in
diameter, white or grey-white, smooth, entire or
lobulated resembling molar tooth

56. Growth of yellow colonies of Clostridium difficile on
cycloserine-cefoxitin-fructose agar (CCFA) with horse-
stable odor

57. Clostridium perfringens
Double zone of hemolysis on blood agar (smaller
zone of complete hemolysis due to theta-toxin, outer
zone of partial hemolysis due to alpha toxin), with
opacification of egg yolk agar (due to phospholipase
C).

58. Clostridium septicum
Gray to translucent, markedly irregular
swarming (rhizoid margins with Medusa head
pattern) over the surface of blood agar with
underlying β-hemolysis.

59. C) Gas- Liquid Chromatography
◦Used to detect anaerobes in exudates & body fluids.
◦A major amount of butyric acid in a specimen that contains only
thin, pointed, gram-negative rods would suggest Fusobacterium
spp.
◦A major peak of succinate & the presence of only gram-negative
rods would suggest Bacteroides spp., Prevotella spp.
◦A major propionate peak in a positive blood culture containing
pleomorphic, non spore forming gram-positive rods would be most
consistent with Propionibacterium spp.
◦Direct GLC provides only presumptive clues, & should be interpreted
cautiously in polymicrobial infections.

60. D) PCR
◦PCR amplification procedure appear promising, but are not well
commercialized.
◦Anaerobes identified by colony PCR and sequencing of the 16S
rRNA gene using universal primers (LiPuma et al. 1999).

61. Molecular diagnosis for Clostridium
Cl. perfringens
◦Detection of phospholipase C (lecithinase
activity) gene PCR
◦Detection of alpha, beta, epsilon and iota
toxin gene by Multiplex PCR

62. E) Rapid methods for diagnosis of
anaerobes
◦Two rapid systems are available for quick diagnosis of
anaerobes.
1)RapID ANA by Innovative diagnostic systems
2)AnIDENT by Analytal Products, Inc.
◦These both systems rely on preformed enzymes and only four
hours of anaerobic incubation is required.
◦Disadvantage is costly, and variable response.

63. Presumptive Identification of
Anaerobic Gram-Negative Bacilli
1
Van Kan Col BIL IND
B. fragilis group R R R + v
2
Pig Prevotella R R
s
v – v
Non-Pig Prevotella R R v – v
Pig Porphyromonas S R R – v
Fusobacterium R S S v
3
+
1
BIL=growth on bile esculin agar, IND=spot indole,
R=resistant, R
s
=resistant rarely susceptible,

S=susceptible, v=variable
2
B. fragilis indole –, B. thetaiotamicron indole +
3
F. nucleatum –, F. necrophorum v

64. Anaerobic Gram-Positive Cocci:
Presumptive Identification
◦Gram-positive, gram-variable, or gram-negative
cocci or cocci bacilli (confirm as gram-positive
cocci by susceptibility to 5-µg vancomycin disk
with inhibition zone >10 mm)
◦Peptostreptococcus anaerobius: Growth inhibition
by sodium polyanethol sulfonate (SPS) (zone of
inhibition >12 mm around a SPS disk)

65. Definitive Species Identification of
Anaerobic Bacteria
◦Biochemical reactions in prereduced
anaerobically sterilized (PRAS) liquid media
◦Fermentation end-product analysis and/or
cell wall fatty acid profiling by gas liquid
chromatography (GLC)
◦16S rRNA gene sequencing

66. Antimicrobial susceptibility testing
TEST CONDITIONS AGAR DILUTION BROTH
MICRODILUTION AND
MACRODILUTION
Medium Brucella agar
supplemented with
hemin ( 5mg/mL),
vitamin K (1mg/mL) and
5% laked sheep blood
Brucella agar
supplemented with
hemin (5mg/mL),
vitamin K (1mg/mL)
and lysed horse
blood
Inoculum size 1X10
5
CFU/spot 1X10
6
CFU/spot
Incubation
conditions
Anaerobic, 35
0
-37
0
C Anaerobic, 35
0
-37
0
C
Incubation
duration
48 hrs 48 hrs

69. Thank you