This is a work in progress!

The future of Laboratory Microbiology

(and in particular bacteriology)

Overview• Background/history• Plate streakers• Automated Urine Analysers• Blood cultures• Automated Susceptibility Testing• Automated ID testing• Malditof• TLA• Molecular assays in bacteriology• Automation and volume• Automation and commerce

Bacteriology: The Origins

• 1881-Nutrient Gelatin first demonstrated by Robert Koch

• 1887 agar plates developed

• 1905 - MacConkey agar first used

• 1900s Dyes used to make media selective

• 1919 - Blood Agar first used to study haemolysis of streptococci

So what's changed??, not much really

• Agar plates are still the main media for the majority of microbial sub-culturing and the backbone of the bacteriology lab.

• Disc diffusion is still used in the vast majority of laboratories – if not all to some degree

• Automated ID/Sens machines (VITEK and PHOENIX) have improved – however many of the concepts are still the same, using broth dilution breakpoint methodology.

• Development in molecular diagnostics.

What about some other techniques?

• Manual streaking of plates has not really changed since solid agar plates were first used

• Microscopy is mostly unchanged –• Incubators while probably more reliable are

essentially the same• Plate reading has not really changed over years

– although you are not supposed to sniff plates anymore….but we know it happens

Pre-analytical – Plate Streakers

• Select appropriate media• Loads the samples• Spreading the inoculum to obtain isolated

single colonies following incubation• Suppliers: WASP (Copan) Previ-Isola

(BioMerieux) Innova (BD) and Inoqula (KIESTRA)

• Not all systems include Gram stain preparation

Automated Urine Analysers• Automated Dip-strip inoculation and

reading

• Cell counts performed automatically – either by flow cytometry or (more recently) high resolution optics taking pictures of cells

• Present now in many labs

Blood Cultures

• Standard in most diagnostic labs, varying sizes.

• Better detection times

• More advanced media

• Reduced total incubation before calling a bottle a final negative

• Suppliers: BacT/Alert (BioMerieux) BACTEC FX (BD)

The Automation of Susceptibility Testing• 1940s started playing with putting

antimicrobial agents into agar, both for selective culture and susceptibility testing.

• Late 1940s: diffusion techniques on filter paper, 6mm disc 1947

• 1966 Kirby/Bauer attempt to standardise with their disc diffusion technique

• 1975 This technique becomes the basis for the NCCLS (CLSI) standards.

The Automation of Susceptibility Testing

• 1974 – First automated AST marketed by Pfizer called the Autobac

• 1977 Abbott introduce the MS 2 System• 1977 McDonnell Douglas Corporation

launch the AMS System – this was the predecessor to the first Vitek Machine

• 1977 First standarised microtitre plates with Antibiotics introduced – leads the way for Microscan, Sensititre and BBL Spector

Automated ID and Sens

• Automated ID testing available since 1977• Biochemical substrates miniaturised and read by

colourmetric or fluormetric means• Available in many bigger labs• Multiple Antibiotics in different dilutions available

on Cards or panels to ascertain MIC – Expert Functions

• Suppliers: Microscan Walkaway (Dade Behring) Vitek2 (BioMerieux) BD Phoenix (BD)

• Cost neutral on ID part but not on susceptibility testing. What will happen to these systems with introduction of Maldi-tof?

MALDI-TOF

• Protein based spectral identification of bacteria• Identifications available in literally minutes – not

hours• Tiny amount of bacterial growth needed – not

affected by media or incubation conditions• Minimal cost per test, virtually no consumables• Suppliers : BD/Bruker, BioMerieux

What is coming?

• What is next in the world of Microbiology Automation??

• Some companies are in the process of producing TLA for Microbiology – similar to those seen in Biochem/Haem.

• Putting specimens on a track – with no human intervention until plate reading time – and even then its not like you know it…

Hands Off Microbiology!!

Spanner in the works….• All of this new automated technology

assumes that bacterial culture on agar plates will remain the cornerstone of microbiological diagnosis for the forseeable future.

• However molecular assays becoming more commonplace….even in bacteriology.

• Virology now becoming increasingly molecular. Viral culture will eventually disappear from the diagnostic scene.

Some of the current molecular assaysavailable commercially for bacteriology

• Chlamydia + gono PCR • Illumigene (LAMP) for C.difficile toxin, Gp B strep• PCR for Group B strep and MRSA and VRE &

ESBL enzymes• PCR Bordetella and C.difficile toxin• PCR and gene probe for mycobacteria• DNA probes for enteric specimens.• DNA probes for candida/BV/trichomonas• 16sRNA identification

What is in the pipeline for molecular assays in bacteriology?• Microarrays

Still in research phase.

Still need extraction and amplification step.

Why is bacteriology not completely molecular?

• Cost: Cost of Culture even including labour is inexpensive.

• Culture allows Quantification of bacteria (but does it matter that much really?)

• Looking for a range of different pathogens. Current methods aren’t that good at it. PCR gives up at about 5.

Will the agar plate eventually become defunct?

• Yes, but possibly not in my working lifetime.

• The companies producing culture automation are not daft. If they thought that agar plates would disappear in the next ten years, then they would not be R&Ding plate streakers, smart incubators etc.

• Leasing, not buying outright such equipment would be prudent.

Automation and Volume

• Automated methods most cost-effective when handling large volumes of samples.

• Favours larger laboratories.

• Interfacing is key

• Most of the cost savings are in labour.

Automation and Commerce

• Two main market players currently in this field, BD and Biomeurieux.

• As the number of pieces of automated equipment in a lab increases, there is more and more incentive for laboratories to “affiliate” with a company for all their automation requirements.

• Interesting times ahead….