Recent advances in cultivation & identification of anaerobic

1. RECENT ADVANCES IN CULTIVATION
& IDENTIFICATION OF ANAEROBIC
BACTERIA OF CLINICAL
SIGNIFICANCE
Speakar: Dr.Priyanka Tiwari
Moderator: Dr. Vikas Manchanda

2. Objectives
• Challenges encountered in detecting anaerobic infection
• Classification of anaerobic bacteria
• Infections caused by anaerobic bacteria
• Major clues for anaerobic infections
• Isolation of anaerobic bacteria
• Methods for diagnosis of anaerobic infections
• Anaerobic culture techniques
• C. difficile-Associated Intestinal diseases: Laboratory Identification
• Commercial microsystems for the identification of anaerobes

3. INTRODUCTION
 Major component of Human microflora
 Association with life-threatening conditions (Botulism,
tetanus)
 Difficulties in diagnosis
 Antibiotic resistant microbes

4. Challenges encountered in detecting anaerobic
infection
• Rarely sought for by the clinicians
• Longer turnaround time - use of metronidazole as empirical
therapy
• Limited anaerobic workup facilities
• Decrease in susceptibility to metronidazole - clinical failure &
antibiotic resistance

5. • Based on ability to grow on solid medium, usually
blood agar in four different environments set up in
laboratory
– Room Air 21%O2 0.03%CO2
– CO2 incubator 15%O2 5-10%CO2
– Microaerophilic 5%O2 10% CO2 + 85%N2
– Anaerobic system 0%O2 + Other Gases
Relationships of Bacteria to O2 and CO2

6. Category Growth condition O2 Conc. Examples
Obligate
Anaerobe
X Room air
X CO2
incubator.
Strict : killed
if >0.5%
C. tetani, C. haemolyticum, C. novyi type
B, certain Treponemes
Moderate
grow at 2-8%
(avg 3%).
Several hrs
B. fragilis, Prevotella, Porphyromonas
spp., Fusobacterium spp., C. perfringens,
Eubacterium, Peptostreptococcus spp. ,
most strains of Veillonella parvula.
Aero-
tolerant
Anaerobe
↓Room air/
CO2 incubator,
 anO2/micro
0-5% C. histolyticum, C. tertium, C. carnis.
Most strains of Propionibacterium.
Facultative
Anaerobe
 Presence/
absence of O2
0-21% Enterobacteriaceae, most Staphylococci,
Streptococci
Microaero-
phile
X Room air
↓anaerobic
 Low O2
5% O2 Actinomyces spp., Bifidobacterium spp.,
Campylobacter jejuni, Neisseria, H.
pylori.
Obligate
Aerobe
 Room air/
CO2 incubator
15-21% Pseudomonas, Vibrio cholerae,
Micrococcus, Mycobacteria, Fungi
Capnophilie 5-10% CO2 15% Brucella abortis, Neisseria, Haemophilus.

7. CLASSIFICATION OF ANAEROBIC BACTERIA
Anaerobic
Bacteria
Gram
positive
Gram
positive
cocci
Gram
positive
rods
Sporing
Non
sporing
Branching
Non
branching
Gram
negative
Gram
negative
rods
Gram
negative
cocci

8. INFECTIONS CAUSED BY ANAEROBIC BACTERIA
Gram-positive spore-forming rods
Species
• Clostridium botulinum
• Clostridium difficile
• Clostridium perfringens
• Clostridium septicum
• Clostridium sordellii
• Clostridium tetani
• Clostridium histolyticum
Clinical syndromes/Infections
• Botulism: Food-borne, wound,
infant
• C. difficile colitis
• Gas gangrene, necrotizing
fascitis, food poisoning, skin &
soft tissue infections
• Bacteremia, necrotizing fascitis
• Bacteremia, postabortion
infections, necrotizing fascitis
• Tetanus
• Gas gangrene

9. Gram-positive non–spore forming rods
Branching
• Actinomyces israelii,
Actinomyces naeslundii,
Actinomyces odontolyticus
• Bifidobacterium dentium
• Mobiluncus spp.
• Thoracic, cervicofacial,
abdominal and pelvic
actinomycosis
• Oral/dental infections
• Bacterial vaginosis,
bacteremia

10. Non branching
• Atopobium vaginae
(Lactobacillus vaginae)
• Atopobium
rimiae(Lactobacillus rimae)
• Eggerthella lenta
(Eubacterium lentum)
• Propionibacterium acnes
• Propionibacterium
propionicum
• Bacterial vaginosis
• Oral infection, bacteremia
• Bacteremia
• Shunt-associated infections,
endophthalmitis following
cataract surgery, prosthetic
joint infections, endocarditis
• Actinomycosis-like infection,
lacrimal canaliculitis

11. Gram-positive cocci
• Anaerococcus prevotii
(Peptostreptococcus
prevotii)
• Finegoldia magna
(Peptostreptococcus
magnus)
• Peptostreptococcus
anaerobius
• Septic arthritis
• Bacteremia,
endocarditis, soft tissue
infections, prosthetic
joint infections
• Bacteremia,
endocarditis

12. Gram-negative rods
• Bacteroides fragilis group
• Bacteroides ureolyticus
• Bilophila wadsworthia
• Intra-abdominal abscess,
sepsis, pericarditis,
bacteremia associated
with malignanacies or in
post surgical infections
• Periodontal disease,
sinusitis
• Perforated and
gangrenous appendicitis,
surgical abdominal
infections, bacteremia

13. • Fusobacterium nucleatum
• Porphyromonas spp.,
Prevotella spp.
• Fusobacterium
necrophorum
• Leptotrichia spp.
• Sutterella wadsworthensis
• Pleuropulmonary infections,
bacteremia, abscess,
infections of GI tract, GU
tract, joints, brain
• Pleuropulmonary infections
• Otitis media & mastoiditis in
children, peritonsillar
abscess in adolescents,
sinusitis in adults
• Bacteremia, peritonitis,
endocarditis
• Peritonitis, intra-abdominal
abscess, bacteremia

14. Gram-negative cocci
• Veillonella parvula • Meningitis, osteomyelitis,
prosthetic joint infections,
endocarditis, bacteremia

15. Major clues for anaerobic infections
CLINICAL CLUES
 Putrid odors & foul-smelling exudates
 Necrotic tissue (gangrene), abscesses
 Gas in tissues and exudates
 Black discoloration or black pigment containing exudates
 Presence of sulfur granules in discharge
 Fight/bite wounds or trauma at sites where normal flora exists
(skin/mucosa)
 Infection secondary to human or animal bite
BACTERIOLOGICAL CLUES
 Unique morphology on gram stain
 Non-response to aminoglycosides, fluoroquinolones, other
antibiotics which lack efficacy for anaerobes

16. Isolation of anaerobic bacteria
• Tissue biopsy or by aspiration using a needle and syringe.
• Swab specimen should be discouraged.

17. Specimens suitable for anaerobic culture
Material aspirated from abscesses (best if lesions loculated )
2) Decubitus ulcer(base of ulcer)
3) Sulphur granules from discharging fistula
4) Tissue obtained at biopsy or autopsy
5) Fluid from normally sterile site (e.g. joints)
6) Pulmonary specimens (Percutaneous lung aspirate or biopsy,
Thoracocentesis fluid, Transtracheal aspirate)
7) Gastrointestinal specimens (Bile, Peritoneal fluid)
8) Genitourinary specimens (Suprapubic bladder aspirate, Uterine
contents, Biopsy of endometrial tissue, Culdocentesis aspirate)
9) Blood, Bone marrow, CSF

18. Specimens unsuitable for anaerobic cultures
1)Bronchial washing or brush(except if collected with a double lumen plugged
catheter)
2)Coughed sputum
3)Feces(except for Clostridium difficile)
4)Gastric or small bowel contents(except in blind loop syndrome)
5)Ileostomy colostomy drainage
6)Nasopharyngeal swab, Rectal swab, Swab of superficial skin lesions, Throat
swab, Urethral swab , Vaginal or cervical swab
7)Nasopharyngeal and oropharyngeal secretions
8)Voided or catheterised urine

19. TRANSPORT OF SPECIMEN
 Rapid transport
 As always, transport specimens to lab. in a biohazard bag.
 Purulent samples
 Pieces of tissues
Various transport methods include
1. For liquid specimens (fluid / exudates)
 Anaerobic transport vials
– PRAS media
– Indicator (usually resazurin)
OXYPLATES
• Self contained anaerobic chambers
• Incubated in standard aerobic incubator
• Media is PRAS
• Anaerobic environment- Oxyrase Enzyme System

20. Oxyplate
The Oxyrase Enzyme System was discovered by late Dr. Howard
Adler who found that the cell membrane fraction derived from
E.coli and other related microorganism were specific for oxygen
reducing it directly to water and capable of producing anaerobic
environment when added to a liquid or solid media.

21. Anaerobic transport vials

22.  Sterile tubes - if the above not available
– Purulent samples >1ml
– Clear fluids
– Tissue pieces and curettings
• If aspirate is to be transported
– Specimen collected in air tight syringe and needle is
plunged into a sterile rubber cork to seal it and sent
immediately to lab.
2. For tissue biopsy samples
 Sealable plastic bags
 Sterile urine cups- For very large pieces of tissue
3. For transport of swab specimens
 Soft agar deeps. (PRAS medium)
Organism can survive in swab for upto 48 hrs.
 Stuart medium or Amies medium
Viability of anaerobes for approximately 24 hrs
 Vacutainer anaerobic specimen collector

23. Methods for diagnosis of anaerobic infections
 Gram stain of specimen
 Culture characteristics, haemolysis , pigment
 Biochemicals
 Vitek anaerobic identification
 GLC
 HPLC
 MALDI TOF MS
 Proton-MR Spectroscopy
 16s ribosomal RNA sequencing

24. ANAEROBIC CULTURE TECHNIQUES
LIQUID MEDIA
Cooked meat broth (Robertson’s cooked meat broth)
Liquid media used for blood culture
Various liquid media include:-
A) Tryptic soy broth
B) Thiol broth
C) Columbia broth,
D) Thioglycollate medium
E) PRAS with brain-heart infusion yeast extract broth

25. • Automated anaerobic blood culture bottles
A) Bactec Plus Anaerobic/F
B) Bactec Lytic/10 Anaerobic F,
C) Bactec Anaerobic F
D) BacT/Alert and BacT/Alert FN,
E) ESP 80N Anaerobic Broth and ESP 40N Anaerobic Broth.

26. Solid culture media for primary isolation of
anaerobes from clinical specimens
Medium Major ingredients Purpose
CDC Anaerobe blood agar Trypticase soy agar, 5%
sheep blood; yeast extract,
hemin, vitamin K1, L-
cystine for anaerobes
requiring additional growth
factors.
Non selective medium for
the primary isolation of all
types of anaerobes
Anaerobe phenylethyl
alcohol blood agar (PEA)
Above contents + 2.5 gm/L
of Phenylethyl alcohol
Selective isolation of most
gram positive and gram
negative obligate
anaerobes. Facultatively
anaerobic gram positive
bacteria also grow well
Anaerobe laked
kanamycin-vancomycin
blood agar (KV)
Above + 100 mg/L of
kanamycin & 7.5 mg/L of
vancomycin.
Selective isolation of most
Bacteroides spp.,
Prevotella spp.,
Fusobacterium spp.,
Veillonella spp.

27. Medium Major ingredients Purpose
Anaerobe paromomycin-
vancomycin blood agar
(PV)
Above + 100 mg/L of
paromomycin & 7.5 mg/L
of vancomycin.
Selective primary isolation
of Bacteroides fragilis
group, pigmented and non
pigmented Prevotella spp.,
F. nucleatum, F.
necrophorum, F.
mortiferum, Veillonella
Cycloserine-cefoxitin
fructose agar (CCFA)
Tryptic soy or proteose
peptone base containing
fructose and neutral red
indicator+ cycloserine
(500mg/L) +cefoxitin
(16mg/L)
Selective isolation of C.
difficile from stool
specimens or other
intestinal materials
Bacteroides Bile Esculin
Agar (BBE)
Digest of casein and
soybeans. Hemin, vitamin
K1, bile, gentamicin
Selective & presumptive
identification of
Bacteroides fragilis group
Enriched thioglycolate
medium (THIO)
BBL-0135C formula
thioglycolate
medium(without indicator)
with hemin and vitamin K1
Non inhibitory, primary
isolation of actinomycetes,
isolation of slow growing
or fastidious organisms

28. Medium Major Ingredients Purpose
Egg Yolk agar Lecithin, triglycerides C. Perfringens and other
clostridia
Anaerobic Culture Techniques- Traditional
1. Vaccum desiccator
2. Displacement of O2 with other gases
3. Absorption of O2 by chemical or biological means

29. Anaerobic Culture Devices
Devices used Principle/Method Indicator used Example
Anaerobic jars or
boxes
Replacement of O2
with a gas mixture
having H2 that
combines with O2 in
presence of catalyst
to produce water.
Methylene blue
strips, Resazurin
strips, Biological
indicators: Strict
anaerobes (C. tetani
or B. fragilis) and
strict aerobe (Ps.
aeruginosa)
McIntosh Fildes
anaerobic jar,
Gaspak jar, Oxoid
anaerobic jar,
Anaeropack(catalys
t free),
Anaerocult(catalyst
free)
Plastic anaerobic
bags/pouches
H2-CO2 gas
generator system
generates an
atmosphere on
adding water + cold
palladium catalyst
Resazurin strips Anaerobic Bag,
Anaerocult pouch,
Anaerogen, Anabag

30. McIntosh and Fildes
Anaerobic jar

31. Gaspack

32. Devices used Principle/Method Indicator used Examples/Types
Roll-tube technique PRAS media
prepared in tubes
with rubber
stoppers
Agar or broth
indicator system
Hungate tube
Anaerobic chamber
or glove box
Final air fill: 10% H2
to get rid of any O2
and serves as
electron donor + 5-
10% CO2 + 85-90%
N2
Methylene
blue/resazurin strip.
Biologic indicator-
Simmon’s citrate
slant inoculated
with Pseudomonas
aeruginosa
Flexible vinyl plastic
anaerobic chamber
Anaerobic Holding
Jars
Air removed by a
vaccum of 25mm
Hg 3 times and jar
filled with N2 after
first two
evacuations and
final replacement
by 80-90% N2, 5-
10% H2 and 5-10%
CO2
O2 sensitive
Fusobacterium
nucleatum
Anoxomat

33. ANOXOMAT

35. PRAS
Roll tube method

36. Approach to presumptive identification of organisms
Organism Specimen
collection
Media used Colony
characteristics
Identification
C. difficile Feces
(unformed/
liquid)
Cycloserine-
cefoxitin
fructose agar
(CCFA)
Larger
yellowish
rhizoid colonies
having
birefringent
crystalline
internal
structure
Gram stain-
Gram positive
bacilli with
subterminal
spores, Gelatin
hydrolysis +,
Glucose
fermentation +
C. perfringens Food & fecal
material(transp
orted at 4⁰C)
Egg Yolk agar opaque zone
(white)
in medium
around
bacterial
growth.
Lecithinase +,
lipase -, indole
-, Gelatin +
Bacteroides
fragilis
Pus, wound
swab
Bacteroides
Bile Esculin
Agar (BBE)
Black zone
around positive
colonies
Vancomycin &
Kanamycin
resistant

37. Organism Specimen
collection
Media used Colony
characteristics
Identification
Peptostreptoco
ccus prevotti
Pus, wound
swab, blood
CDC Anaerobe
blood agar
Small colonies Gram stain-
cells in clumps
or tetrads, SPS
disk resistant,
Indole -, urease
+, alpha
galactosidase +
Propionibacteri
um acnes
Pus, wound
swab, aspirate,
blood
CDC Anaerobe
blood agar
White to pink,
shiny, opaque,
entire edge,
aerotolerant
Gram stain-
pleomorphic,
club shaped,
pointed ends,
nitrate
reduction +/-,
catalase +/-,
indole
production +/-

38. C. difficile-Associated Intestinal diseases:
Laboratory Identification
• Incidence of CDI varies
• Common in the post-bone marrow transplantation period in
India
• Increased prevalence of CDI after antibiotic usage in the
ulcerative colitis group
• Increased C. difficile carriage in psoriatic patients given either
methotrexate or mesalamine
• Important pathogen in younger children with AAD
– A decrease in the number of C. difficile positive cases due to
stringent surveillance and improved antibiotic policy adopted by
the hospital during a five-year study period has been reported from
north India.

39. Specimen
collection
• Watery or semisolid, unformed fecal specimens (about 5 gm or 1020 ml of
liquid stool)
Specimen
transport
• in leak-proof plastic containers at room temperature (ideally < 2 hours).
• delayed >2 hrs - specimen is refrigerated in an anaerobic
environment(transport vial or bag)
• If sent to reference laboratory for a toxin assay, - shipped on dry ice and stored
in lab at -70ºC before shipment.
Laboratory
diagnosis
• Culture methods and cytotoxin identification
• Enzyme immunoasays
• Nucleic acid amplification test
• C. difficile GDH antigen testing

40. • C. difficile- Gram + to Gram variable with subterminal spore,
motile.
• On GLC metabolic product analysis- acetic, isobutyric,
propionic, butyric, valeric, isovaleric, isocaproic acids.
• Biochemicals-
 esculin and gelatin are hydrolyzed.
 Indole, nitrate and urease -
Two new culture media to select for strains of toxigenic C.
difficile-
• Cdifftox plate assay- Toxigenic strains; blue colour and non
toxigenic; white colony. Results available in 24 to 48
hours(99.8% accuracy).
• Commercially produced chromogenic media chromID C.
difficile.

41. Enzyme Immunoassays for Toxin Detection
• Detect toxin A and toxin B
• Sensitivity as low as 52%
• Specificity is good, in the range of 91 to 94 % or greater
Nucleic acid amplification test
• Amplification of tcdB or tcdA genes
• Good sensitivity and specificity (Can significantly increase
sensitivity over EIA by as much >50% with good specificity as
compared to culture)
• Turn around time ranges from 45 to 180 minutes
C. difficile GDH antigen testing
• Highly sensitive(>99%) but not specific
• C. DIFF CHEK-60 ELISA can be performed in 60 minutes
• C. DIFF QUIK CHEK Complete(Rapid membrane EIA)
• Latex agglutination assay: sensitivity 97% & specificity 87%

42. • Antigen assay: negative screen, and confirmation of the
samples that are positive
• A three-step approach is followed: positive antigen results are
followed up with toxin A/B EIA; if the two are positive, results
are signed out as positive for toxigenic C. difficile, but if
negative, further confirmation testing with PCR or culture.

43. COMMERCIAL MICROSYSTEMS FOR THE
IDENTIFICATION OF ANAEROBES
Vitek Anaerobic Identification
Uses 20 chromogenic substrate tests and 8 modified
conventional tests
Gas Liquid Chromatography
• “liquid stationary phase” & “moving gas phase”
• Definitive identification of many species of Bacteroides and
Fusobacterium, Actinomyces, Bifidobacterium
• Major peaks in different anaerobic bacteria
 Fusobacterium mortiferum- butyric acid and acetic acid
 Clostridium difficile- acetic, propionic and isocaproic acid
 Peptostreptococcus anaerobius- acetic and isocaproic acid

44. The tank of carrier gas
Injection port of sample
The column
Detector with appropriate read out

45. Typical volatile acid standard
chromatogram
The time elapsed between the injection of an ether extract of the
standard solution and the peak for each acid (retention time) is
used to identify the acids. Eg retention time for acetic acid is 1.8
min

46. High performance liquid chromatography
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution.
• The potential advantages of HPLC over GLC are-
a) a single sample preparation step not requiring methylation
b) the simultaneous determination of both VFA and NVFA in a
single chromatographic run
c) increased sensitivity for detection of NVFA

47. HPLC instrumentation and processing

48. The TOF mass analyzer measures the time it takes for the ions to
fly form one end of the analyzer to the other and strike the
detector. The flying speeds of ions are proportional to their mass-
to-charge ratio.
Matrix assisted laser desorption ionization
time of flight mass spectrometry

49. Proton Magnetic resonance spectroscopy
 Magnetic resonance spectroscopy (MRS) is a specialised technique
associated to magnetic resonance imaging (MRI).
 Also known as nuclear magnetic resonance (NMR) spectroscopy
 Has been used to study metabolic changes in brain tumors, strokes,
pyogenic brain abscesses, seizure disorders, Alzheimer's disease,
depression and other diseases affecting the brain.
MRS equipment can be tuned (just like a radio receiver) to pick up
signals from different chemical nuclei within the body. The most
common nuclei to be studied are protons (hydrogen).

52. 16s ribosomal RNA sequencing
• Pyrosequencing of the 16S ribosomal RNA gene revealed the
taxonomic identity of bacteria to the species level.
• Predict the functional capability of a microbiome.

53. THANK YOU