The document discusses various rapid methods for identifying anaerobic bacteria, which are important pathogens but difficult to culture. It describes several biochemical, chromatographic, and spectrometric techniques, including gas liquid chromatography, MALDI-TOF mass spectrometry, electrospray ionization, VITEK 2 identification cards, and commercial kits like RapID ANA II and BBL Crystal that can identify anaerobes based on enzymatic substrate reactions within 4 hours through color or fluorescence changes. Rapid identification is crucial for appropriate treatment of anaerobic infections.

1. METHODS FOR RAPID
IDENTIFICATION OF ANAEROBES
- Presented by Dr. Devyashree Medhi, Second Year PGT, Dept. of Microbiology,
GMCH

2. CONTENTS:
 Definition of anaerobes
 Anaerobic Bacteria of Clinical Importance
 Anaerobic Bacteria and Associated
Representative Infections
 Methods of rapid identification of anaerobes

3. DEFINATION OF ANAEROBES:
Strict anaerobes:
 Organisms that do not use
oxygen for growth and
metabolism but obtain their
energy from fermentation
reactions.
Facultative anaerobes:
 Organisms that can grow
either oxidatively, using
oxygen as terminal electron
acceptor, or anaerobically,
using fermentation
reactions to obtain energy.

4. Anaerobic Bacteria of Clinical Importance:
Bacilli
 Gram negative
• Bacteroides fragilis group
• Prevotella melaninogenica
• Fusobacterium
 Gram positive
• Actinomyces
• Propionibacterium
• Clostridium
Cocci
 Gram positive
• Peptoniphilus
• Peptostreptococcus
• Peptococcus

5. Anaerobic Bacteria and Associated Representative
Infections:
Brain abscesses Peptostreptococci, Fusobacterium nucleatum etc.
Oropharyngeal
infections
Actinomyces, Prevotella melaninogenica, Fusobacterium species
Pleuropulmonary
infections
Peptostreptococci, Fusobacterium species, Prevotella melaninogenica,
Bacteroides fragilis
Intra-abdominal
infections
Liver abscess: Mixed anaerobes in 40-90%
Abdominal abscess: Bacteroides fragilis; other GI flora
Female genital tract
infections
Vulvar abscess: Peptostreptococci etc.
Tubo -ovarian and pelvic abscesses: Prevotella sp., Peptostreptococci etc.
Skin, soft-tissue, and
bone infections
Mixed anaerobic infection, Propionibacterium acnes
Bacteremia Bacteroides fragilis; Peptostreptococci; Propionibacteria, Fusobacteria;
Clostridium etc.
Endocarditis Bacteroides fragilis, Actinomyces

6. Methods for rapid identification of
anaerobes:
 GAS LIQUID CHROMATOGRAPHY
 MALDI-TOF
 ELECTROSPRAY IONISATION
 VITEK 2 ANC CARD
 RapID ANA II SYSTEM
 RAPID ID 32 A SYSTEM
 MICROSCAN RAPID ANAEROBE IDENTIFICATION PANEL
 BBL CRYSTAL ANAEROBE IDENTIFICATION

7. Gas Liquid Chromatography:
 The production of short-chain
fatty acids (SCFA) is a known
metabolic activity of anaerobic
bacteria.
 Such end products can be
detected by GLC in both clinical
samples and bacterial colonies
grown anaerobically.
 GLC gives a rapid and
presumptive identification of
anaerobes.

8. GLC
 Mobile Phase: Inert or
unreactive gas called the
carrier gas.
 Stationary Phase:
Microscopic layer of non-
volatile liquid or polymer on
an inert solid support, inside
glass or metal tubing.
 The mobile phase flows
through the stationary phase
and carries the components
of the sample with it.
 Different components of the
sample travel at different
rates.

9. MALDI-TOF MS:
 Matrix Assisted Laser Desorption/Ionization- Time of Flight Mass spectrometry is an
ionization technique that uses a laser energy absorbing matrix to create ions from
large molecules with minimal fragmentation.

10. MALDI-TOF MS:
 First, the sample is mixed with a
suitable matrix material and applied
to a metal plate.
 Then a pulsed laser irradiates the
sample,
triggering ablation and desorption of
the sample and matrix material.
 Subsequently, the analyte molecules
that are being are ionized by the
generated heat can be accelerated
into the TOF mass spectrometer used
to analyze them.
 The time that it takes for the ion to
reach the detector at a known
distance is measured.
 This time will depend on the velocity
of the ion, and therefore is a measure
of its mass-to-charge ratio as velocity
of an ion is proportional to its mass
to charge ratio.
 From this ratio and known
experimental parameters, one can
identify the ion.

11. ELECTROSPRAY IONISATION
 ESI and MALDI-TOF both transform
biomolecules into ions and separate
them based on their mass to charge
ratio.
 One of the main difference however
is the state in which the samples are
introduced.
 In ESI, samples are coated with liquid
solvent at the source region and high
pressure and heat is applied.
 The heated microdroplet evaporates
and the sample enters the analyzer in
the form of molecular ions.

12. VITEK®2 ANC ID card:
 The VITEK 2 is an automated
microbiology system utilizing growth-
based technology.
 VITEK®2 ID cards are convenient and
safe self-contained, disposable
cards designed for use with the VITEK®2
family of instruments.
 It can identify approximately 90
anaerobic and coryneform bacteria.
 VITEK 2 systems use Advanced
Colorimetry™, an identification
technology that enables identification of
routine clinical isolates

13. Working mechanism of VITEK 2 compact
system:
FILLING CHAMBER
 Once organism suspension is prepared
and relevant cards are put against the
suspension, the load is inserted into the
filling chamber.
 The negative pressure followed by
positive pressure applied in the filling
chamber pushes the suspension inside
the wells of the card containing
dehydrated reagents.
 After filling is completed , the cards are
transferred to the loading/incubation
chamber.
LOADING CHAMBER
 The scanner in the loading chamber
scans the code of the batch and each
card individually.
 The cutter cuts tubes of each card.
 The cards are then pushed into the
incubator rotator one by one.
 At every 15 minutes each card come to
the colorimetric scanner for scanning of
color development that occur due to
biochemical reaction.
 Once identification is finalized the
relevant card is put to waste tray.

14. RapID™ ANA II System
 RapID™ ANA II System is a qualitative
method employing conventional and
chromogenic substrates for
identification of medically important
anaerobic bacteria isolated from
human clinical specimens.
 It comprises of(1) RapID ANA II Panels
and (2) RapID ANA II Reagent.
 Each RapID ANA II Panel has several
reaction cavities molded into the
periphery of a plastic disposable tray,
each of which contain dehydrated
reactants.
 The tray allows the simultaneous
inoculation of each cavity with a
predetermined amount of inoculum.
 A suspension of the test organism in
RapID Inoculation Fluid is used as the
inoculum and incubated.
 After incubation, each test cavity is
examined for reactivity by noting the
development of a color. In some cases,
reagents must be added to the test
cavities to provide a color change. The
color change occur due to microbial
degradation of specific substrate.
 The resulting pattern of positive and
negative test scores is used as the basis
for identification of the test isolate by
comparison of test results to reactivity
patterns stored in an Electronic RapID
Compendium (ERIC™) database or by
use of the RapID ANA II Differential
Chart.

15. RapID™ ANA II System

16. Rapid ID 32A system
 Rapid ID 32 A and RapID ANA II
System are similar.
 They both identify anaerobes in 4
hours using preformed bacterial
enzymes for the hydrolysis of
chromogenic substrates.
 Rapid ID 32 A uses 29 miniaturized
enzymatic tests and a database.

17. Microscan Rapid Anaerobe
Identification System
 It is a 4 hour microdilution system.
 It is similar to the RapID-ANA system
except that the panels can be read both
visually and by the automated reader, the
AutoScan-4.

18. BBL™ Crystal™ Anaerobe identification system
 The BBL Crystal™ Anaerobe (ANR) Identification
(ID) System is a miniaturized identification
method employing modified conventional,
fluorogenic and chromogenic substrates.
 The BBL CRYSTAL ANR ID kit is comprised of :
I. BBL CRYSTAL ANR ID panel lids containing 29
dehydrated substrates and a fluorescence control
on tips of plastic prongs.
II. BBL CRYSTAL bases having 30 reaction wells.
III. BBL CRYSTAL™ ANR, ID Inoculum Fluid (IF) tubes

19. Working mechanism:
 Test inoculum is prepared with the inoculum
fluid and is used to fill all 30 wells in the
base.
 When the lid is aligned with the base and
snapped in place, the test inoculum
rehydrates the dried substrates and initiates
test reactions.
 Following an incubation period of 4hrs, the
wells are examined for color changes or
presence of fluorescence that result from
metabolic activities of the microorganisms.
 The resulting pattern of the 29 reactions is
converted into a ten-digit profile number
that is used as the basis for identification.
 The type of primary plate used to prepare
the inoculum will determine the appropriate
database.
 Calculation of the ten-digit profile
number, an example:
(4A is the fluorescent negative
control)
A B C D E F G H I J
4 * + - - + + + - + -
2 - + + + - + - + + -
1 + - + - + - - + + -
1 6 6 2 5 6 4 3 7 0

20. THANK YOU