Microbiological Identification with

MALDI-TOF MS | IVT

By

Tim Sandle, Ph.D.

Oct 20, 2015 7:30 am PDT

Peer Reviewed:

Microbiology

Introduction

Microbial identification plays an important role in pharmaceutical processing. Microbial

identification can be defined as "microbial characterization by a limited spectrum of tests pre-

chosen and appropriate to the problem being studied.” (1) For identification there is a range

of techniques available, dating back to the pioneering differential cell wall staining of

Christian Gram (2), to recent advanced molecular biology techniques (3). Current microbial

identification systems are divided between the phenotypic and genotypic.

Matrix-Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry (MALDI-

TOF MS) is one of the more recent microbial identification systems made available to

laboratories. While the system is „phenotypic‟ it, in some senses, bridges the gap between the

reliability of test results produced from a biochemical based phenotypic systems and a

genotypic identification system. The system is also very fast, making it a good example of a

'rapid microbiological method.' (4)

The basis of the MALDI-TOF technique is rooted in analytical chemistry, having first been

put forward in 1988 by Hillenkamp and Karas as a method for analyzing proteins (5). Mass

spectrometry is a chemical analysis technique that is used to measure the mass of unknown

molecules by ionizing, separating and detecting ions according to their mass-to-charge ratios

(dividing them into positive and negative ions.) The data are recorded as mass spectra.

The 'time-of-flight' element is based on the principle that time is related to mass and that this

can be measured under high vacuum conditions. Because the higher the mass, the lower its

velocity and the longer it takes before the ion strikes the detector, this leads to different

microorganisms forming different patterns or 'spectra.' The resultant protein spectra can be

compared to a database and a match made. The time taken to do this is often less than two

minutes.

This paper discusses the MALDI-TOF technology and outlines the advantages and

disadvantages with the system.

Microbiological identification methods

The objective of microbial identification is to differentiate one microbial isolate from another

and then to place that isolate into a family and a species (which is the best that can be

achieved at the phenotypic level of identification) or even as a particular strain (through

genotypic identification).

Microbial identification is the determination of whether an organism should be placed within

a group of organisms known to fit within some classification scheme. Identification methods

can be divided into two groups: phenotypic and genotypic. The genotype–phenotype

distinction is drawn in genetics. "Genotype" is an organism's full hereditary information, even

if not expressed. "Phenotype" is an organism's actual observed properties, such as

morphology, development, or behavior (6).

Phenotypic methods are the most widespread due to their relatively lower costs for many

laboratories. It should be recognized, however, that expressions of the microbial phenotype,

that is, cell size and shape; sporulation; cellular composition; antigenicity; biochemical

activity; sensitivity to antimicrobial agents and so on, frequently depend on the media and

growth conditions that have been used. Phenotypic reactions typically incorporate reactions

to different chemicals or different biochemical markers. These rely on the more subjective

determinations. The reliance upon biochemical reactions and carbon utilization patterns

introduces some disadvantages to the achievement of consistent (repeatable and reproducible)

identification. To improve on the classical methods of biochemical identification several

developments have been made and refined in recent years. Collectively these methods are

considered as modern biochemical identification techniques (7).

An example of biochemical profiling is the API identification system or the alternative BBL-

Crystal system (microtubes containing dehydrated substrates). Many laboratories now adopt

semi-automated phenotypic identification systems, such as VITEK or OmniLog (a

miniaturized system utilizing the microtiter plate format). Such phenotypic methods tend to

work on the process of elimination. If test A is positive and B is not then one group of

possible microorganisms is included and another is excluded. From this, tests C and D are

performed, and so on. The test results are compared against databases which work on the

basis of a dichotomous key (8).

Genotypic methods are not reliant upon the isolation medium or growth characteristics of the

microorganism. Genotypic methods have considerably enhanced databases of different types

of microorganisms. Before the advent of genotypic methods microbiologists speculated that a

number of taxa were present and unculturable (so-termed “viable but non-culturable” strains).

Genotypic methods have opened up a whole new set of species and subspecies, as well as

reclassifying species and related species (thus taxa are often similarly grouped by phenotypic

methods are actually polyphyletic groups, that is they contain organisms with different

evolutionary histories which are homogonously dissimilar organisms that have been grouped

together). Genotypic methods utilis one of two alternatives: hybridization or sequencing

(most commonly of the gene coding for 16S rRNA). With hybridization, DNA-DNA

homology (or how well two strands of DNA from different bacteria bind [hybridize]

together) (9).

An example of this technology is the Riobprinter (manufactured by Dupont Qualicon), an

automated Southern Blot device which uses a labeled ssDNA probe from the 16sRNA codon.

The Riboprinter uses a restriction enzyme and strains can be identified and/or characterized

by analyzing the ribosomal DNA banding pattern.

Another rapid method is a polymerase chain reaction (PCR) system which uses a form of

„bacterial barcodes‟ where the amplified genetic sequence is separated by gel electrophoresis

and visualized to give a „barcode‟ specific to that strain. PCR is a technique, which uses a

DNA polymerase enzyme to make a huge number of copies of virtually any given piece of

DNA or gene. It facilitates a short stretch of DNA (usually fewer than 3000 'base pairs') to be

amplified by about a million-fold. With this comparative test, differences in the DNA base

sequences between different organisms can be determined quantitatively, such that a

phylogenetic tree can be constructed to illustrate probable evolutionary relatedness between

the organisms. An example of such a system is the MicroSeq manufactured by Applied

Biosystems (10).

MALDI-TOF

The basis of MALDI-TOF is mass spectrometry. A mass spectrometer is composed of three

main components: an ion source to ionize and transfer sample molecules ions into a gas

phase, a mass analyzer device that separate molecules depending to their mass and a detector

to monitor all separated ions. Matrix - assisted laser desorption ionization – time of flight

mass spectrometry (MALDI-TOF MS) refers to the soft ionization technique used in mass

spectrometry. MALDI is deemed to be a „soft ionization technique‟ because it deploys a short

nitrogen laser pulse, instead of continuous laser, to ionize molecules. This "mild" ionization

means that the formed ions have low internal energy. This allows for the observation of

ionized molecules with little or no fragmentation.

As one system, the method allows for the analysis of biomolecules (such as DNA, proteins,

peptide and sugars) and large organic molecules (such as polymers, dendrimers and other

macromolecules). These molecules tend to be fragile and fragment when ionized by more

conventional ionization methods. The ionization is triggered by a laser beam. The system

scans for microbial proteins that primarily fall within the range of 4000 to 20,000 Daltons

(60% to 70% of the dry cell weight of bacteria) (11). The optimal reproducibility in microbial

identification by MALDI-TOF MS is based on the assessment of ribosomal proteins, which

are commonly abundant in the cell.

Based on these principles, the system is a rapid and highly reliable analytical tool for the

characterization of a diverse collection of microorganisms found in pharmaceutical and

healthcare facilities (12).

The method has been commercialized to analyze the protein composition of a microbial cell.

Comparative studies have shown MALDI-TOF MS to be a comparatively effective

identification technique; this claim being based on its reproducibility, speed and sensitivity of

analysis (13). One important advantage of MALDI-TOF MS, when compared with other

identification methods, is the time-to-result. With MALDI-TOF results are generally

available within minutes. In keeping with the relatively rapid testing; sample preparation is

also fairly quick and straightforward.

There are two main providers of the instrument:

bioMérieux - the Vitek MS;

Bruker Daltonics - the MALDI Biotype.

Method

With the method, there are two approaches in relation to the test microorganism. Typically

pure colonies are prepared on an appropriate agar plate. As with most identification method,

the colonies should be grown over night and not be more than 24-hours old when used.

However, unlike most other methods it is recommended that plates are not held at 2-8°C prior

to testing. Cold storage can affect the quality of spectra. As an alternative, in the clinical

setting, clinical specimens, such as blood culture material, can be used alongside a special

extraction kit.

The process of using the MALDI-TOF MS has been summarized by Patel, and it can be

broken down into five steps (14, 15):

1. The target plate is placed into the ionization chamber of the mass spectrometer. Spots

to be analyzed are shot by an ultraviolet nitrogen laser desorbing microbial and matrix

molecules from the target plate. The laser, operating at 337 nm, is generated by

nitrogen oxide (16). The majority of energy is absorbed by the matrix, converting it to

an ionised state.

2. Through random collision in the gas phase, charge is transferred from matrix to

microbial molecules

3. The cloud of ionized molecules is funneled through a positively charged electrostatic

field (20 kV) into the time off-light mass analyzer, a tube under vacuum.

4. The ions travel toward an ion detector with small analytes traveling fastest, followed

by progressively larger analytes. The pulsed laser takes individual 'shots' rather than

working in continuous operation.

5. As ions emerge from the mass analyzer, they collide with an ion detector generating a

mass spectrum representing the number of ions hitting the detector over time.

Although separation is by mass-to-charge ratio, because the charge is typically single

for the described application, separation is effectively by molecular weight. This

means that minor ions reach a TOF detector before larger ions.

Figure 1: Simplified diagram of the MALKDI-TOF MS method (source: Matt F. Traxler)

When running the method, positive and negative controls are recommended. Controls can be

used either for daily calibration or incorporated into each test run. Positive controls are

microorganisms of relevance to the laboratory. The negative control is a blank, used to show

that the target plate is clean.

Figure 2: Example of spectra relating to different types of bacteria (source: Anagnostec

GmbH)

Strengths and weaknesses of MALDI-TOF

As with any of the commercially available microbial identification systems, the MALDI-TOF

MS has its advantages and disadvantages. These are considered next.

Strengths

Various users of MALDI-TOF systems, primarily from the clinical setting, have presented

the strengths of the system as (17, 18, 19):

1. The system can identify a broad spectrum of bacteria, including Gram-positive cocci

and rods and fermentative and nonfermentative Gram-negative rods.

2. The system works well with yeasts. The system is more limited with fungi; however,

Aspergillus, Fusarium, and Penicillium can be identified accurately at the species

level.

3. The system works well with anaerobic bacteria.

4. The test only requires a single colony in most instances (greater amounts of microbial

culture are needed for yeasts or mucoid colonies).

5. Bacteria regarded as difficult to culture have a high success of being identified using

the system. An example is with Mycobacteria.

6. The system also has some success in the identification of viruses (20).

7. Exposure risk, in terms of laboratory safety, are low. This is because samples are

inactivated by extraction before use.

8. Only minimal consumables are required. There is a relatively high cost associated

with purchasing the instrument; however, running costs are low.

9. The system can be expanded as necessary.

10. The outcomes are generally reproducible.

Weakness

The main weaknesses of the system have been identified by the U.K. health agency Public

Health England and others (21). These are:

1. Spectral interference can occur as a result of the presence of endospores with bacteria

like Bacillus species. To overcome this, younger cultures can be used (22).

2. Sometimes mass spectrometry spectra cannot readily differentiate similar or closely

related organisms (such as Escherichia coli and Shigella species; of with different

yeasts).

3. For clinical laboratories, discrimination between different antibiotic resistant and

sensitive strains of the same species is not possible (23).

4. Some microorganisms cannot always be identified; the system appears weakest with

Mycobacteria, Burkholderia species, Acinetobacter species, Corynebacteria and β-

haemolytic streptococci. This is due genetic similarity (24).

5. As with any identification system, the reliability in identifying is based on how

comprehensive the database is (25). MALDI-TOF databases have a clinical bias and

need to be strengthened for use with industrial and pharmaceutical microbiology. This

is down to the quality of the reference spectra (26). However, by learning from the

data patterns, machine learning techniques should be able to exploit the information

embedded in the data so that previous 'unknowns' can be re-recognized.

6. Microorganisms that possess capsules are more resistant to cell lysis. This can lead to

a low extraction yield and hence lower quality spectra and thus mis-or null-

identification. Powell and colleagues reported on a weakness with MALDI-TOF MS

in differentiating between Streptococcus pneumoniae and Streptococcus mitisas and

Haemophilus influenzae and Klebsiella pneumoniae (27). Due to these potential

errors, some users recommended testing in duplicate and averaging the spectral result.

7. The type of media used can lead to interference. The media selected during method

verification should be used as standard throughout all subsequent identifications. This

media issue emphasizes why MALDI-TOF MS is prone to the weaknesses of other

phenotypic methods.

Method verification

It is important that when any microbiological identification method is introduced into the

laboratory that it is verified (qualified). To begin with the automated instrument is should be

taken through the following steps:

a) Installation qualification: This is the documented evidence that the equipment and

associated systems, such as software, hardware, and utilities are properly installed and

relevant documentation is checked. Documentation may include manuals, certificates,

procedures, and calibration records.

b) Operational Qualification: This verifies that the system or subsystem performs as intended

throughout all anticipated operating ranges and documents the information.

c) Performance Qualification: This proves the system performs consistently as intended

during normal operational use and remains in compliance with regulatory and user

expectations or requirements. Performance of automated microbial identification system is

very elaborate and time consuming due to multiple factors such as choice of isolates, operator

variability and the reproducibility of the system itself.

Following validation, or with non-automated systems, verification of the test is required in

order to show that it is suitable. Verification typically consists of (28):

a) Parallel testing with approximately fifty microbial isolates using an existing system.

b) The testing of twelve to fifteen representative stock cultures of commonly isolates species

(ensuring that these are of a broad enough range to cover the majority of the instruments test

array). Here type strains should ideally be used.

c) Confirming that twenty to fifty microbial identifications, including fifteen to twenty

different species, agree with the results of a reference laboratory testing split sample.

The key criteria to be assessed are (29):

a) Accuracy, which is expressed as a percentage of the number of correct results divided by

the number of obtained results, multiplied by 100.

b) Reproducibility, which is similarly expressed as a percentage. Here the number of correct

results in agreement is divided by the total number of results multiplied by 100.

Summary

This paper has outlined how mass spectrometry can be orientated towards the identification

and classification of microorganisms by using protein 'fingerprints' (characteristic protein

expression patterns which are stored and used as specific biomarker proteins for cross-

matching).

As the paper has shown, when identifying bacteria with a device like a Matrix Assisted Laser

Desorption Ionization Time-Of-Flight (MALDI-TOF) instrument, a single isolated colony or

simple cell extract is spotted onto a stainless steel target plate and overlaid with an ultra violet

absorbing molecule. The target plate is inserted into the MALDI-TOF. Nitrogen pulsed laser

ionization is then applied to the sample and the proteins are ionized. They are then separated

based on their mass/charge ratio. The resulting spectra, a protein finger print (which falls

within the 2,000 –20,000 Dalton range) are compared to a database of known spectra.

This method is, with the limitations presented notwithstanding, fast and efficient and it is

suitable for laboratories that need to process a high-volume of samples and are satisfied with

the determined result being based on phenotypic expression.

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