Vitek, Bact/Alert, Bactec

1. AUTOMATION IN BACTERIOLOGY
PRESENTED BY:
Dr. Sumesh Kumar Dash
MODERATOR:
Prof (Dr.) Kundan Kumar Sahu

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The technique of making an apparatus, a
process, or a system operate automatically
AUTOMATION
Webster's Dictionary

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• Clinician need quicker result
• Faster identification & AST
• Saves time & Manpower
• Remove/Decrease manual error
• Helpful in processing whenever increased sample load
NEED OF AUTOMATION

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• Time consuming
• Tedious
• Run the risk of introducing contaminants during procecing
• Incomplete Identification
• A limited no. of AST can be detected on one plate.
• MIC may not be determined
PROBLEM WITH
CONVENTIONAL METHOD

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• Automated blood culture
• Vitek System
• Phoenix System
• MicroScan Walkaway system
• MALDI-TOF
VARIOUS AUTOMATED
SYSTEMS

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AUTOMATED BLOOD CULTURE

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AUTOMATED BLOOD CULTURE
• Automatically continuous reading of blood through computerized
system.
• Three such systems
• BacT/ALERT 3D (bioMérieux, Durham, NC)
• BD BACTEC (BD Diagnostic Systems)
• VersaTREK (ThermoScientific, Cleveland, OH)
• Each of these systems alerts if the culture is positive in the bottle.
• After that we have to do a subculture, Gram stain, Identification & AST

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• First non radiometric continuous
monitoring blood culture system.
• Contains incubator, shaker &
detection device, with a capacity
to hold either 120 or 240 bottles.
• Sample: Blood, Sterile fluid,
Respiratory & Non-respiratory
sample
• Detect: Aerobic & Anaerobic
BacT/ALERT 3D

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Principle
Detects of CO2
Lowering of the pH of the medium,
Colour change in a sensor
Detected by photosensitive detector
(Every 10mins)

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Media
AEROBIC CULTURE ANAEROBIC
MEDIA
MYCOBACTERIUM
MEDIA
BactAlert FA
Plus
BactAlert PA
Plus
BactAlert FN
Plus
BactAlert MB Process
SAMPLE
Blood & Sterile
Fluid
Blood Blood & Sterile
Fluid
All Specimens Except
Whole Blood
BOTTLE
CONTAINS
• 30 ml of Broth [BHI and TSB]
• Soduim poly-an-ethol-sulfonate
• Nutrients, Amino acids, Carbohydrate substrates
• APB (Adsorbent Polymeric Beads)
 In anaerobic media 40ml
• 10 ml Middle brook
7H9
• Pancreatic Digest of
casein.
• Bovin serum albumin.
• Catalase in purified
water
SAMPLE
VOLUME
5 to 10ml 1 to 4ml 1 to 10ml 0.5 ml after
decontamination

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Procedure

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Positive culture

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consists of a self-contained incubator, agitator and detection device.
• BACTEC 9050
• BACTEC 9120
• BACTEC 9240
• BACTEC FX
• BACTEC LX
BD BACTEC

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Principle

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Differs from the other
1. Monitored production of CO2
2. Both gas consumption and
production are monitored
3. Changes in the concentrations of
H, and O, also detected
Units with a capacity of 96 to 240 or
up to 528 bottles are currently
available.
VersaTREK

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Pressure of the head-gas is continuously monitored
(Every 12 minutes)
Consumption of H and O / Production of CO2
Change in pressure
Reading by electrostatic precipitator (ESP) system
Lights are illuminated for positive bottle.
Principle

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• REDOX 1 (aerobic)
• REDOX 2 (anaerobic)
• MYCO
Media

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• Detects different gases produce or consume
• Detects organism when they enter to log phase.
• Detection microorganisms, that may not produce sufficient CO2.
Advantages

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VITEK SYSTEM

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• VITEK 2 is the most widely used automated system in India
• Can perform both identification and antimicrobial susceptibility testing
(AST) of bacteria and yeast
VITEK 2

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Colorimetric reagent card
Each well contains an individual test substrate.
Substrates in the well measure various metabolic activity
Reaction pattern compared with the database
Identification within 4–6 hours.
Principle For Identification

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Microbroth dilution card
Optical reading of growth in presences of AMAs
(every 15min)
Algorithmic analysis of the reading performed by software
MIC
Principle For AST
AES analyzes MIC give rise to sensitivity pattern, recognized the resistance
pattern & also detects phenotypes of most organism in 4 -16hrs

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Card
AST N280 AST N281 AST N235 AST P628
Amikacin Amikacin Amikacin Ampicillin
Amox/clav Aztreonam Amoxicillin/Clav Ciprofloxacin
Ampicillin Cefepime Ampicillin Clindamycin
Cefepime Cefoperazone/sulbactam Cefalotin Daptomycin
Cefoperazone/sulbactam Ceftazidime Cefixime Erythromycin
Cefuroxime Ciprofloxacin Cefoxitin Gentamycin
Ceftriaxone Colistin Ceftazidime Gentamycin HL
Ciprofloxacin Gentamicin Ceftriaxone Levofloxacin
Colistin Doripenem Ciprofloxacin Linezolid
Ertapenem Imipenem Ertapenem Nitrofurantoin
Gentamicin Levofloxacin Fosfomycin Oxacillin
Imipenem Meropenem Gentamycin Rifampicin
Meropenem Minocycline Nalidixic Acid Teicoplanin
Nalidixic acid Piperacillin/tazobactam Nitrofurantoin Tetracycline
Nitrofurantoin Co-trimoxazole Ofloxacin Tigecycline
Piperacillin/tazobactam Ticar/Clav Norfloxacin Cotrimoxazole
Tigecycline Tigecycline Piperacillin/Tazobactam Vancomycin
Co-trimoxazole Ticarcillin
Trimethoprim/
Sulfamethoxazole

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Procedure
0.5-0.63 MCF
3 ml 0.45% saline
Results
AST
• GN - 145µL
• GP – 280µL
Gram Stain

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• Standardized procedure
• Fast turn around time
• Species level bacterial and fungal identification
• AST based on MIC
• Detection resistance pattern
• Validation of every result every time
Advantages

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Which drug is better ???

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PHOENIX SYSTEM

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• Largest capacity automated
system.
• Performed both identification &
AST with wide range.
• Manufactured by Becton
Dickenson Diagnostics,
Baltimore MD .
• Can determine AST upto16-22
antimicrobials
BD PHOENIX

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• Uses redox indicator system.
• Panels tray contains 126 wells (Identification:AST::51:85)
• Instrument reads panels every 20 mins
• Turbidity is measured as growth indicator,
• Turbidity causes the redox indicator to change from an oxidized (blue)
state to reduce (pink).
• BCXpert & BD EpiCenter software interpret MIC value according to
data base.
• Gives result in 6-16hrs
Principle

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Procedure
Label a Phoenix ID Broth
Mix pure colony
Vertex
0.20-0.30M BD PhoenixSpec
Nephelometer
Pour to ID Panel
Label the Phoenix AST Broth
Add 1dp Phoenix AST indicator
Mix properly
Add 25 µL standardized ID
broth
Pour to AST Panel
Place it to
instrument

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MICROSCAN WALKAWAY
SYSTEM

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• Developed in 1980s
• Uses broth microdilution method to determine the MIC.
• Initially there is 2 major type
1. MicroScan: Reads turbidimetrically, over night incubation
2. Walkaway: Reads fluorometrically, 3.5 – 15hrs
• Type of panel
o MIC panel
o MIC combination panels
o Breakpoint combination panels
 Synergies Plus (2005)
• All system uses LabPro software to generate result.
MicroScan Walkaway

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MASS SPECTROMETRY

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• Matrix-assisted laser desorption/
ionization time-of-flight
• Identify by a, mass spectrometric profile
of protein and largely rRNA proteins of
organism.
• Mass spectrometry is an analytical
technique in which chemical compounds
are ionized into charged molecules and
ratio of their mass-to-charge (m/z) is
measured.
• 2 systems are commercially available:
VITEK MS (bioMérieux)
Biotyper system (Bruker)
MALDI-TOF

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Working Principle

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• Organic compound: α-cyano-4-hydroxycinnamic acid (or)
2,5-dihydroxy benzoic acid (or)
3,5-dimethoxy-4-hydroxycinnamic acid
• Solvents: Ethanol/Methanol or Acetonitrile
• Water
• The solvents penetrate the cell wall of microorganisms and extract out
the intracellular proteins.
• When the solvent evaporates, ‘co-crystallization’ of protein molecules
and other cellular compounds entrapped within the matrix solution takes
place (Horneffer et al., 2001)
Matrix

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• Numerous gas and solid state lasers have been developed for use .
• Most MALDI devices use a pulsed UV laser
• N2 source at 337 nm
• Neodymium- yttrium aluminium garnet (Nd:YAG)
• Emits at 355 nm
• IR lasers are also used.
• Erbium doped- yttrium aluminium garnet (Er:YAG).
Laser

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• Sample preparation for identification depends upon the its cell wall.
• Some microbes might be identified directly by MS, called Direct Cell
Profiling.
E.g. Neisseria spp. (Ilina et al., 2009)
Yersinia spp. (Stephan et al., 2011)
Vibrio spp. (Eddabra et al., 2012)
• Preparatory extraction was necessary for identification of Gram-positive
bacteria by MALDI-TOF MS, but not for Gram Negative bacteria
(Alatoom et al., 2011; Saffert et al., 2011).
• A ‘preparatory extraction’ of microbes with Formic Acid increased the
ability of MALDI-TOF MS for identifying Gram-positive species.
Sample Preparation

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• Mycobacterial colonies collected in screw-cap tubes containing water
and 0.5% Tween 20, were inactivated by heating at 95◦C for 1 h.
• Inactivated samples were centrifuged and vortexed with glass beads to
disrupt the mycobacterial cell wall.
• The resultant pellet was re suspended in formic acid, acetonitrile, and
centrifuged again.
• Finally, the supernatant was deposited onto the MALDI target plate and
overlaid with matrix.
• It a procedure which combined inactivation and sample preparation.
Mycobacteria
EI Khéchine et al. (2011)

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Procedure
Andrew E. Clark et al. Clin. Microbiol. Rev. 2013;doi:10.1128/CMR.00072-12

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ADVANTAGE DISADVANTAGE
• Fast • High initial cost of the equipment
• Accurate • Identification from pure colony only
• Broad mass range • Not useful on specimen
• Soft ionisation • AST can’t determined
• Less expensive
• Trained laboratory personnel not
required
Adv & Disadv

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