Bacterial Identification From the Agar Plate to the Mass. 2013

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Cite this: RSC Advances, 2013,3, 994

Bacterial identification: from the agar plate to the massspectrometer

Received 6th September 2012,

Accepted 24th October 2012

DOI: 10.1039/c2ra22063f

www.rsc.org/advances

Patricia Aparecida Campos Braga,a Alessandra Tata,a Vanessa Gonalves dos Santos,a

Juliana Regina Barreiro,b Nicolas Vilczaki Schwab,a Marcos Veiga dos Santos,b

Marcos Nogueira Eberlina and Christina Ramires Ferreira*a

For more than a century, bacteria and fungi have been identified by isolation in culture followed by

enzymatic reactions and morphological analyses. The identification of environmental microorganisms,

however, remains a challenge because biochemical and staining protocols for bacteria identification are

tedious, usually stepwise, can be long (days) and are prone to errors. Molecular techniques based on DNA

amplification and/or sequencing provide more secure molecular identification of specific bacteria, but

identification based on mass spectrometry (MS), mainly on MALDI-MS, has been shown to be an

alternative accurate and fast method able to identify unknown bacteria on the genus, species and even

subspecies level based profiles of proteins and peptides derived from whole bacterial cells. Breakthroughs

such as non-culture-based identification of bacteria from biological fluids and MS detection of antibiotic

resistance have recently been reported. This review provides an overview of the traditional bacterial and

fungal identification workflow and discusses the recent introduction of MS as a powerful tool for the

identification of microorganisms. Principles and applications of MS, followed by the use of high-quality

databases with dedicated algorithms, are discussed for routine microbial diagnostics, mainly in human

clinical settings and in veterinary medicine.

Introduction

If a microbiologist working in the first decades of the 20thcentury stepped into a current microbiology laboratory, it is

likely that he would not have a hard time feeling at home. In

fact, most methods for bacterial isolation and identification

have remained unchanged and are still based on the use of

specific culture media for isolation and on classical morpho-

logical, staining and biochemical enzymatic assays for micro-

organism identification.1

Improved assays, high-throughput microbiological analysis

with automated systems, molecular technologies based on

DNA amplification/sequencing for microorganism identifica-

tion and the detection of antimicrobial resistance are

available.2,3 However, challenges such as the urgent identifica-

tion of microorganisms and their antibiotic resistances in

septicemic patients and in infants, the occurrence of genetic

rearrangements in microorganisms that alter their behavior in

enzymatic assays, and the need to identify less frequent or rare

microorganisms are difficult to tackle with classical micro-

biological approaches and may lead to incorrect identifica-

tions.

In response to these challenges, a paradigm break formicrobiology has been the introduction of mass spectrometry

(MS)-based microorganism identification. This strategy

emerged after the development of electrospray ionization

(ESI)4 and matrix-assisted laser desorption/ionization

(MALDI)5 at the end of the 1980s. Since the early 1990s, more

than 13 000 Pubmed indexed manuscripts on microbiology

associated with MS have been published. However, micro-

organism identification by MS is not only performed for

research purposes. Dedicated instruments equipped with

automated database search functions for almost real-time

microorganism identification are being installed in hospitals,

clinical institutes and commercial settings, mainly in Europe.

In the United States, the Food and Drug Administration has

still not approved any MALDI-MS system for organism

identification, which limits the widespread implementation

of this approach in this country.6

This review provides a general overview of classical

microbiological approaches and the MS ionization strategies

that have been mostly applied in microbiology. The most

recent achievements in MS-based microorganism identifica-

tion, such as the identification of uncommon pathogens and

non-fermenting bacteria, non-culture identification of bacteria

in biological fluids, identification of antibiotic resistance and

aThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of

Campinas, Campinas, 13083-970, SP, Brazil. E-mail: [email protected];

Fax: +55 19-3521 3073; Tel: +55 19-35213049bUniversity of Sao Paulo, School of Veterinary Medicine and Animal Science,

Pirassununga, 13635-900, Sao Paulo, Brazil. E-mail: [email protected];

Fax: +55 19-5616215; Tel: +55 19-3565 4240

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mixed culture analysis, are also described and discussed.

Finally, real-world perspectives on moving bacterial identifica-

tion from the agar plate to the mass spectrometer are

presented.

Traditional bacterial and fungal identification based on

bacterial isolation by culture and enzymatic assays

There are several methods for routine microbial identification

in clinical and research microbiology laboratories, but micro-biologists have continuously pursued efficient systems for

microorganism identification. These methods are mainly

based on morphological and biochemical characteristics of

bacterial colonies. For example, bacterial colony morphology

can be evaluated under defined growth conditions to show

hemolytic capacity, by Gram staining (Fig. 1ab), and by

examining growth performance on selective media that allow

only specific bacteria to multiply, the potential to ferment

sugars, specific biochemical reactions (Fig. 1c), metabolic

characteristics, antigenic and pathogenic capacity, and anti-

biotic susceptibility.7

When determining the characteristics of a microorganism to

provide its identification, a pure culture population of

identical cells is needed; this means that these cells originate

from the same parental cell. However, microorganisms in

nature are typically found in mixed cultures with many

different species occupying the same environment.

Therefore, in a microbiological laboratory, the first step in

the microorganism identification workflow is the isolation of

the various species contained in a specimen. For this purpose,

there is an extensive list of available commercial tools, and the

chosen strategy depends on numerous factors. The main

considerations are the source of the sample, the species that

are expected to be present and the nutritional needs of these

microorganisms. For example, the isolation medium may

contain specific compounds that inhibit or prevent general

microbial growth yet simultaneously are appropriate for

growing the species that are determined to be present

(Fig. 1a).8 After isolation of the microorganism, phenotypic

characteristics such as enzymatic profiles, sensitivity to

antibiotics and chromatographic analysis of fatty acids can

be performed to characterize the strain.9

Microscopy is frequently used to characterize microorgan-

isms (Fig. 1b), and there are two main microscopic techniques:

light microscopy and electron microscopy. Light microscopy isroutine in the microbiology setting and can be performed with

bright field, dark field, fluorescence, and phase control.

Constant development and refinement of the techniques used

for optical microscopy allow one to perform additional

specialized functions, such as evaluating biochemical pro-

cesses occurring within living cells.10

For close to a century, bacterial identification relied on the

interpretation of a skilled microbiologist using a microscope,

specific media and antibiotics, but technological improve-

ments over the past 50 years have allowed for both automation

and simplification of analysis.2

Automated testing for bacterial identification in laboratories

has become necessary as microbiologists attempt to answerhigher demands and the need for fast diagnoses, such as in

cases of patients with septicemia or neonatal infections.

Automation also allows increasing numbers of specimens to

be studied.11 Usually, the microorganism cell count can be

determined in real time by incubators equipped with a system

to measure the light absorption of liquid cultures because

turbidity can be related to the number of cells. Another

strategy of an automated laboratory system is based on testing

the susceptibility of bacteria to a large number of antibiotics.10

Advances in the molecular biology field in the early 1980s

resulted in novel approaches for microbial identification and

characterization based on polymerase chain reaction (PCR) to

amplify specific gene sequences of bacteria. PCR allowsin vitroamplification of specific DNA or RNA sequences, the latter

being performed following the synthesis of complementary

deoxyribonucleic acid (cDNA). PCR is a technique with high

specificity and applicability, and hundreds of methods have

been described.12 The most important feature of PCR is its

ability to exponentially amplify copies of DNA from small

amounts of material. For PCR analysis, nucleic acids must be

extracted, and several protocols using specific reagents and

different strategies have been described for this purpose.10

Though time-consuming, costly and difficult in the case of

multiplex assays, PCR-based bacterial identification is now a

common and often indispensable technique used in medical

and biological research for a variety of other applications,including forensic DNA typing, clinical diagnosis, DNA

amplification for cloning or sequencing, paternity testing,

construction of DNA libraries and detection of mutations.1214

For example,Campylobacter, which is the most common cause

of acute bacterial gastroenteritis in the world, may be detected

in pork samples using this approach. Sixty Campylobacter

strains isolated from porcine rectal swabs and from different

areas in a pork processing plant were shown by PCR analysis to

be mostlyC. coli(86.9%) and C. jejuni.(13.1%).15

PCR is less prone to errors compared to traditional methods

of identifying microbes that rely exclusively on phenotypic

Fig. 1 (a) Staphylococcus aureus (left) and Staphylococcus spp. (right) on blood

agar. Note the hemolysis caused by S. aureus; (b) Bright light microscopy

analysis: Gram-positive stained cocci; (c) Biochemical tests used to identify

bacteria usually include color codes.

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features and some the morphological characteristics of the

organism to identify the strain, such as enzyme profiles,

antibiotic sensitivity profiles, and chromatographic analyses of

fatty acids.9 However, the expanding and impressive capabil-

ities of MS-based microorganism identification are causing a

revolution in the field of microbiology.

Even though bacterial identification represents the largest

interest in routine microbiology, invasive fungal disease plays

an important role in the morbidity and mortality of immuno-compromised patients.16 Approximately 60 years ago,

Wickerham described a broth method that is specific for

metabolic assimilation and fermentation testing of yeasts.17,18

Assimilation tests determine the ability to use various

substrates as the sole source of carbon (e.g., sucrose) or

nitrogen (e.g., KNO3). This method is still considered a

powerful tool and was used to characterize and determine

the taxonomy of yeasts. Most Wickerham media are not

commercially available and are produced in the laboratory.

This process is usually long and arduous and is employed only

by a few laboratories because basal media and various

substrates need to be prepared, sterilized, and dispensed

prior to inoculation. Since many types of yeast can carry-overnutrients from the isolation medium, one must run negative

controls for each test type and organism. Metabolic assimila-

tion tests are read for turbidity, fermentation and gas

production for up to four weeks. Due to their time-consuming

nature, these gold standard assays have been replaced by

more practical methods that are currently available.

The first technical progress to improve the speed and

sensitivity of yeast identification from fungal blood cultures

and histological practices was the development of highly

sensitive and specific molecular techniques, including PCR. At

the molecular level, genetic sequence variation offers an

alternative to culturing for the detection and identification of

fungi. For example, ribosomal genes have conserved sequence

regions that are ideal for primer targeting as well as regions of

variability that are useful for species identification. DNA

amplification techniques, with subsequent species-specific

probing of the amplicons or PCR-enzyme immunoassay, have

also been introduced to overcome the problems of sensitivity,

specificity, and delay that are encountered with conventional

methodology. These methods have already shown great

promise in the field of diagnostics. The use of species-specific

probes, however, is not always an efficient approach in

mycology, given the large number of potentially pathogenic

fungi.19,20

In parallel with the development of molecular methods,there has also been an emphasis on improving commercial

kits based on substrate utilization or hydrolysis. The results

are determined by increased turbidity, the generation of

colored products, or the detection of fluorescent products.

Some colored products require the addition of reagents to

reveal the color, while others are self-revealing. These kits

enable presumptive identification of the most important

etiologic agents of yeast infections. Another focus of these

rapid tests has been to screen for species that are commonly

associated with resistance to antifungal compounds.21 Some

kits are read manually, while others are read automatically.

Many systems employ the aid of computer algorithms for rapid

and reproducible data analysis.21

Merits of mass spectrometry in microorganism identification:

time-saving and reliablity

ELECTROSPRAY-BASED BACTERIAL IDENTIFICATION (PCR-ESI-QTOF).

Scientific advances in MS, such as the development of the

soft ionization techniques MALDI5 and ESI,4 have allowed

the ionization, detection and characterization of large intactbiomolecules. An improved understanding of the limitations

associated with MS analysis of nucleic acids led to the

ionization of intact PCR products by ESI.4,22 This capability

resulted in the development of MS analysis of nucleic acids for

microorganism identification, which was first described in

2005 by Hofstadler and co-workers.2326

This approach, previously termed TIGER (Triangulation

Identification for Genetic Evaluation of Risks) or PCR-ESI-

QTOF-MS, uses broad-range primers for PCR analysis to

amplify products from diverse organisms, such as viruses,

bacteria, fungi and protozoa within a taxonomic group, that

are present in samples combined with PCR product calcula-

tions using MS (Fig. 2).The Ibis T5000 Universal Biosensor is an automated platform

for pathogen identification that is based on TIGER technology.

In its commercial form, Ibis T5000 is capable of identifying and

strain typing a broad range of pathogens in a blinded panel

from human or animal samples.2830 Because the Ibis T5000

provides digital signatures of identified microorganisms, this

technology allows the collection and dissemination of epide-

miological information in real-time.3133 A major advantage of

this methodology is the ability to characterize an organism

without prior knowledge by the instrument operator as well as

rapid sample preparation. Since rapid pathogen identification

significantly reduces rates of patient mortality, technologies for

the correct and timely diagnosis of bloodstream infections areurgently needed. PCR-ESI-MS has been used as a new strategy

for detecting bloodstream infections and has provided high

concordance with results from standard methods, particularly

at the genus level. The results from this technique can also be

obtained in five to six hours, whereas culture and biochemical

characterization techniques typically require one to five days for

confirmation of microbial identification.34,35

Rapid detection and identification of Ehrlichia species, a

tick-borne pathogen responsible for causing Ehrlichiosis

disease, was performed by PCR-ESI-MS directly from crude

blood samples without microorganism culture. The results

from an enzyme immunoassay that was also performed

showed 100% agreement with the PCR-ESI-MS results.36

Thedetection of bloodstream infections can be biased, as blood

cultures are reported to be negative in more than 50% of the

cases where bacteria are believed to exist. This approach

allows the detection and identification of both culturable and

unculturable organisms by the same method, in addition to

the identification of mixed populations of bacteria. The PCR-

ESI-MS platform not only identifies organisms that are present

in a clinical sample but is also capable of providing

information about the strain type.37 This approach has been

applied for the genotypic characterization ofS. aureusisolates

and to detect the presence or absence of genetic elements that


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encode potential virulence factors and antibiotic resistance

elements. PCR-ESI-MS can also distinguish S. aureus from

other coagulase-negative staphylococci (CoNS) isolates.38,39

Members of the genus Acinetobacter, which are aerobic

Gram-negative organisms that are widely distributed in soil

and water in the natural environment and are important

nosocomial (hospital-acquired) pathogens, were isolated from

infected soldiers and civilians involved in an outbreak in the

military health care system associated with the conflict in Iraq.

Through the PCR-ESI-MS technique, it was possible to

distinguish at least 16 Acinetobacter species, and the

genotyping of A. baumannii showed a genetic relationshipbetween endemic European isolates and many of the isolates

found in patients and in military hospitals, indicating that this

approach provides a better understanding of the origins of

these infections and will improve infection control and

prevention measures.40

The application of this technique has also been described

for the genotyping of pathogens related to food-borne ill-

nesses, and it proved to be highly effective in differentiatingC.

jejuniisolates in a panel of 50 Campylobacterisolates as well as

determining the correct classification of C. coliinstead of C.

jejuni(Fig. 3).41

The Ibis 5000 platform has also been applied in aquatic

environmental analysis to identify different Vibrio species

directly from natural aquatic samples. From 278 total water

samples that were screened, nine differentVibriospecies were

detected, and 41% of samples were positive for V. cholerae, a

pathogen responsible for cholera disease. The results also

indicated that V. mimicus could be correctly identified and

distinguished from the close species V. Cholerae.42 PCR-ESI-

MS has been described as a high-throughput method to

simultaneously identify, based on genotype, a number of

bacterial species from complex mixtures in respiratory

samples taken from military recruits during respiratorydisease outbreaks and follow up surveillance at several

military training facilities.43 PCR coupled to ESI-MS has also

been described as a powerful tool for detecting other

microorganisms such as viruses.4450

Use of ambient desorption/ionization techniques for direct

bacterial identification

Ambient desorption/ionization describes a new set of MS

techniques that are performed in an open atmosphere directly

on samples in their natural environments or matrices or by

using auxiliary surfaces. Ambient MS has greatly simplified

Fig. 2 An overview schematic of the traditional bacterial identification workflow and the recent integration of mass spectrometry techniques. Figure adapted from

Drake et al., 201127 with permission from John Wiley and Sons.


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and increased the speed of MS analysis, and especially after

2004, this approach has experienced a large trend towards real-

world rapid chemical analysis of untreated samples in

ambient conditions.51,52 Recently, several new high-through-

put ambient desorption/ionization methods have been

reported. One of the most studied ambient ionization methods

is desorption electrospray ionization (DESI), which was

introduced by Cooks and co-workers in 2004.

53

DESI involvesspraying untreated samples with ionized solvent droplets from

a pneumatically-assisted electrospray. Desorption and ioniza-

tion of analytes occurs through interactions of the charged

droplets with the surface from which they pick up organic

molecules, and they are delivered as desolvated ions into the

mass spectrometer (Fig. 4a).

Another well-explored ambient ionization technique is the

direct analysis in real time (DART) method, first described by

Cody and co-workers in 200554 (Fig. 4b). Due to the increasing

importance of rapid identification of bacteria for food,

biosafety and medical analysis, ambient desorption/ionization

methods are of substantial interest.55 Both the DESI and DART

techniques allow direct and rapid analysis of condensed phasesamples without any sample preparation or the need to

introduce the samples into the vacuum system of the mass

spectrometer. DESI and DART have been widely utilized in

many different applications, including bacterial analysis and

identification.51 The most important characteristic of DESI

and DART-MS approaches is the absence of sample prepara-

tion, so the real-time identification of microorganisms by

these approaches appears to be feasible.56,57

Cooks and co-workers were the first to recognize the

potential of these ambient desorption/ionization methods for

microorganism identification when they performed DESI-MS

on freshly harvested cells from untreated E. coli and

Pseudomonas aeruginosa samples deposited on a polytetra-

fluoroethylene (PTFE) target. The characteristic DESI mass

spectra for each microorganism that was analyzed demon-

Fig. 3 Deconvoluted ESI-TOF mass spectra of PCR amplicons of thetkt

housekeeping genes from six differentC. jejunistrains. Both the forward (e) and

the reverse (#) strands of the PCR amplicons from each strain are clearly evident

in the spectra (e.g., for strain RM4197, the forward strand is A49, G22, C26 and

T45 and the reverse strand is A45, G26, C22 and T49). As can be observed in the

stacked spectra, differences due to variations in the sequence (and thus the base

composition) are readily discernible. Note that any mass differences resulting

from changes in the number of guanosines are enhanced by the use of 13C

guanosine (G*). The T5000 software automatically determines the base

composition of each strain and provides a strain association by using a set of

eight primer pairs. Reproduced from ref. 41 with permission from the American

Society for Microbiology.

Fig. 4 Schematic of ambient MS techniques used for direct microorganism

analysis. These approaches usually make use of untreated samples, which are

desorbed and ionized from surfaces or solutions under normal atmospheric

conditions. (a) For DESI, an ionized solvent is pneumatically sprayed onto the

sample, forming a thin film in which the sample molecules are dissolved.

Secondary droplets containing ionized analytes are then delivered in the

direction of the mass spectrometer inlet. (b) DART uses an electrical potential

applied to a gas with a high ionization potential (typically nitrogen or helium) to

form a plasma of excited-state atoms and ions, which desorbs low-molecular

weight molecules from the surface of a sample. (c) In LTP-MS, there is no need

for any solvent. The ion source consists of a glass tube with an internal

grounded electrode centered axially and an outer electrode of copper tape

surrounding the outside of the glass tube. An alternating voltage is applied to

the outer electrode with the center electrode grounded to generate the

dielectric barrier discharge. The discharge AC voltage is provided by a custom-built power supply with total power consumption below 3W. Helium is used as

the discharge gas, and it is fed through the glass tube to facilitate the discharge

to direct the plasma onto the sample surface and to transport analyte ions to

the mass spectrometer.


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strated the potential of the technique for microbiological

application.58

The similarity of the spectra for different samples of the

same culture or for E. colidifferent cultures was evaluated and

indicated that DESI-MS for microorganisms was reproducible.

The characteristic constituents of bacteria were observed

without chemical derivatization; in particular, acylium ions

of fatty acids were observed directly, not as the usual methyl

esters. Principal component analysis (PCA) was performed onthe DESI-MS data to differentiate the bacteria studied into two

well-separated groups that could be identified based on the

first principal component (PC1), which corresponds to the

differentiation betweenE. coliandS. typhimurium. Because the

mass spectra were recorded directly from freshly harvested

microorganisms, and no chemical reagent or other processing

step was used to disrupt the cells before MS analysis, any

variations in the final spectra associated with sample

pretreatment were eliminated in collaboration with the

separation. Although only two different species were evalu-

ated, this study demonstrated the possibility of performingin

situ identification using DESI-MS, including sub-species

differentiation of microbiological agents.56

Meetani et al. (2007) also applied DESI-MS to bacterial

identification for a larger number of different samples. In this

study, seven different bacterial species were evaluated on the

basis of their spectra profiles. Incubated bacterial cells were

transferred from agar plates, washed to remove media

components, applied on glass slides and directly subjected

to DESI-MS analysis. The mass range from 50500 was used,

and the observed ions in the mass spectra revealed the

presence of free fatty acids, such as palmitic acid (16:0) atm/z

257 for the protonated molecule and m/z279 for the sodium

adduct. Further comparisons of the mass spectra with low-

mass matrix-assisted laser/desorption ionization (MALDI)

mass spectra of bacteria did not show common ion signals,indicating the likely complementary nature of DESI-MS and

MALDI-MS for whole-bacteria identification. The mass spectra

of negative ions were also evaluated, showing a greater

number of detected ions than their counterpart positive ion

spectra throughout the measured m/zrange.57

In vivo recognition of bacteria was evaluated in another

study from the Cooks group that examined direct profiles of

intact biofilms ofBacillus subtilisby DESI-MS, noting that the

biofilms were still viable after the experiment. The authors

reported that the DESI plume primarily desorbs materials

from the bacterial cell envelopes outer layers together with

excreted metabolites. Bacteria with a rigid cell wall can

withstand the impinging sprayed droplets. This assumptionwas corroborated by experimental results from Gram-negative

and Gram-positive bacteria. The outermost layer of Gram-

negative bacteria is the cell outer membrane, while the

outermost layer of Gram-positive bacteria is a thicker cell

wall. For Gram-negative species, the outer membrane is

relatively easy to break, and its major phospholipids (PL) can

be readily ionized, which leads to PL dominance in the

resulting DESI mass spectra. However, the thicker cell walls of

Gram-positive species are more difficult to break, and their

major components, which correspond to 90% of glycans, are

much more easily ionized than PL. Consequently, the excreted

metabolites are observed as the dominant species in the

resulting DESI mass spectra, especially those lipopeptides that

are produced in large quantities, are surface-active and ionize

with high efficiency, such as the surfactins.59

Fernandez and co-workers have also applied DART-MS to

two different bacterial samples.60 They describe the detection

of fatty acid methyl ester (FAME) ions from whole bacterial cell

suspensions and their identification by accurate-mass ortho-

gonal TOF-MS. This study is interesting because the goldstandard methods routinely used in bacterial taxonomy and

classification are based on the determination of microbial

FAME composition after culturing, a process that forms the

basis of the commercial Sherlock microbial identification

system (MIDI Inc., Newark, Delaware, US). Routine FAME

analysis involves lengthy sample preparation, starting with the

hydrolysis of bacteria cells followed by fatty acid methylation.

Gas chromatography coupled to mass spectrometry (GC-MS) is

then used for separation and the detection of FAME composi-

tion. Each GC-MS run generally takes 20 to 30 min, whereas

the total DART-MS analysis takes less than 10 min.55,61 FAME

were generated from approximately 107 cellmL21 Streptococcus

pyogenesand E. coli. After incubation, cells were washed withTRIS-sucrose buffer, suspended in water, and diluted with a

solution of tetramethylammonium hydroxide (TMAH) to

produce thermal hydrolysis and methylation of bacterial

lipids. An aliquot of the whole bacterial cell suspension mixed

with TMAH was deposited in the bottom of the capillary tube.

The capillary was positioned so that the bottom of the tube

came in contact with the DART He stream directly in front of

the mass spectrometer inlet orifice after sliding the sample

holder arm. The protonated FAME C9:0, C10:0, C11:0, C12:0,

C14:0, C15:0, C17:1/cycloC17:0, and C19:1/cycloC19:0 were

found to be present only in E. coli, while C11:1 was uniquely

detected inS. pyogenes. C17:1/cycloC17:0 and C19:1/cycloC19:0

were found in E. coliat relatively high abundances but werenot detected in the S. pyogenes spectrum, which is in

accordance with the membrane characteristics of Gram-

negative bacteria. Some FAME ions were common to E. coli

and S. pyogenes; however, clear differences existed in the

relative abundances of these ions in the mass spectra.

Differences among samples were thus observed in the spectral

temporal and intensity domains.60

Recently, a new ambient ionization technique termed low

temperature plasma mass spectrometry (LTP-MS) was applied

for bacterial identification.60 This ambient ionization method

was introduced in 2008 by Cooks and co-workers, and the

absence of any solvent is the distinguishing feature of this

plasma-based method (Fig. 4c).55

LTP-MS was employed todetect fatty acid ethyl esters (FAEE) from bacterial samples in a

direct way. Positive ion mode FAEE mass spectrometric

profiles for 16 different bacterial samples were obtained

without extraction or other sample preparation. Data were

examined by PCA to determine the degree of possible

differentiation among the bacterial species. Growth media

effects were observed, but in this case, they did not interfere

with species recognition based on the PCA results.55

Ambient desorption/ionization techniques can therefore be

applied to bacterial identification, but in some cases, such as

for DESI that uses a high velocity nebulizing gas, it is necessary


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to secure or fix bacterial cells onto the DESI probe surface to

prevent sample dispersion and/or aerosolization during

analysis. This procedure is required for the analysis of

pathogenic bacteria, and heating (for example, at 220 uC for

30 s) or long-term drying (.1 h at room temperature) of

bacterial cells are effective means of fixing the sample onto a

glass slide surface.57 Different groups using the same ambient

ionization technique have presented different results due to

the dependence of mass spectral profiles of intact bacteria onthe experimental design and the way in which growth media

was used and/or prepared. Future investigations in this area

appear to require detailed evaluation of the experimental

design and its influence on the data output.57 However, the

successful demonstration of identification by ion composition

from whole bacterial cells via ambient MS analysis, mainly for

segregating bacterial strains according to Gram status,

constitutes the first step that could in the future lead to the

successful development of new approaches for high-through-

put microbial identification from a variety of biological, food,

and water samples in the open air with minimum sample

preparation.57

MALDI-MS based platforms: real-world breakthroughs in

microbiology

PROTEOMIC STUDIES FOR THE IDENTIFICATION OF BIOMARKERS.

Approaches that use proteomics as a tool for studying

expressed proteins are increasingly being utilized to address

diverse biomedical questions. Via the identification of

specific and conserved biomarkers, such as ribosomal

proteins, peptides and lipids, it is possible to provide cancer

diagnoses, study inflammatory and degenerative diseases and

to determinate pathogens responsible for a broad range of

diseases.6266

Protein profiles obtained from direct MALDI-MS analysis of

intact microorganisms or protein extracts have revealed robustbiomarkers that are mostly related to conserved and specific

ribosomal proteins. Currently, bacterial species identification

by MALDI-MS is the MS approach that has the highest impact

in the field of microbiology.67,68 This approach is based on the

acquisition of ribosomal protein fingerprints directly from

protein extracts from intact organisms. Interestingly, these

protein profiles, which primarily contain ribosomal proteins,

have been found to vary considerably and allow the proper

characterization of different microorganisms. These protein

markers are rapidly being incorporated into human clinical

microbiology routines due to the availability of bioinformatics

tools for databank searches, allowing secure identification and

high laboratory reproducibility.67,68

MALDI-MS has beenproved by many reports to be easier, faster and sometimes

more reliable than classical protocols even when compared to

more sophisticated DNA analysis-based technologies.69,70

MALDI-MS-based microorganism identification has rapidly

been introduced in laboratory and clinical settings and

delivers fast and reliable diagnostic results, not only for genus

level identification but also at the species level for bac-

teria,63,7081 fungi,16,8291 algae,92 viruses9396 and protozoa.97

An essential step in the identification of microorganisms at

the species level by MALDI-MS has been the use of dedicated

databases with rigorous data quality control and powerful

algorithms for comparison with mass fingerprinting. These

databases can be run in parallel with MS acquisition data,

giving almost real-time bacterial identification results.33,98,99

Because much effort has focused on MALDI-TOF-MS, this

approach is already in use in clinical diagnostic labora-

tories.68,100107 The observed biomarkers in the mass spectrum

enable not only the detection of pathogenic bacteria but also

the ability to distinguish them from corresponding non-

pathogenic species.108

ManyCampylobacter species and Helicobacter strains cause

gastrointestinal diseases and can be discriminatedvia protein

profiles observed by MALDI-MS.109113 Biomarker assignment

also makes it possible to distinguish subspecies of members of

the Enterobacteriaceaefamily so that their fingerprints can be

used as family-specific biomarkers for accelerated bacterial

identificationvia database searches.114

Biomarker monitoring by MALDI-MS can also be used to

identify environmental toxin producers. MS analyses of

peptides and polyketides from intact cyanobacteria were used

to identify toxic and nontoxic water blooms115117 and

pathogens isolated from seafood, which are associated with

food-borne diseases. This approach was also used to furtherstudy the different protein profiles of azaspiracid toxin

biomarkers in contaminated and non-contaminated blue

mussels (Mytilus edulis).118

Burkholderia cepacia, which are important agents of chronic

pulmonary disease in cystic fibrosis patients and are proble-

matic to accurately identify due to their complex taxonomy,

have been successfully discriminated by MALDI-MS.119122

Mycobacterial species, which are responsible for causing

significant morbidity in humans by diseases such as tubercu-

losis, and Haemophilusspp., which are well known etiological

agents of pneumonia, meningitis and conjunctivitis, have

been identified by MALDI-MS via their protein profile

spectra.123125

In the veterinary field, bacteria isolated from cows present-

ing subclinical mastitis, a common and easily disseminated

disease in dairy herds, were diagnosed in a few minutes

through the analysis of ribosomal protein biomarkers isolated

from microorganisms present in milk samples by MALDI-MS

with the use of the Biotyper database, a commercial software

for MALDI-MS-based microorganism identification that allows

earlier treatment with appropriate antibiotics.126

Immunoproteomic analyses ofMannheimia haemolytica, the

most important bacterial pathogen associated with bovine

pneumonia, have been performed by MALDI-MS to search for

and identify biomarkers from outer membrane proteins that

may hold potential as candidate vaccine antigens.127

Pigments and proteins from chlorosomes, the light-harvest-

ing organelles from the photosynthetic green sulfur bacterium

Chlorobium tepidum, were characterized directly from orga-

nelles and bacteriochlorophyll, and homologs were detected to

provide fingerprints for these biomarkers.128

MALDI-MS analysis was used to detect the increased

expression of cold shock proteins in bacteria collected from

the Siberian permafrost, and distinct proteins and peptide

profiles were observed as a function of temperature. The

recent capability of MALDI-MS imaging has been used to study

synergism and antagonism in microbial communities in the

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nests of leaf-cutting ants through the identification of

antimicrobial and antifungal compounds that are produced

to defend a symbiotic fungus, which is a major food source for

ant species.129

These numerous examples of MALDI-MS applications are

supported by the availability of dedicated instrumentation and

software containing fingerprinting databases for bacterial

identification. Such instruments are operator-friendly, and

technicians can operate them in microbiological settings andhospitals with diagnostic purposes with minimal specializa-

tion in MS or microbiology and with high-throughput, efficient

and trustworthy results. As a result, diverse human clinical

settings start by comparing the results of traditional methods

versus MALDI-MS-based bacterial identification, and, after

some time, they move to a solely MALDI-MS-based approach

because of the many advantages and the desire to avoid

numerous time-consuming and tedious assays.

Identification of uncommon bacterial pathogens, fungi and

yeast by MALDI-MS

Microorganisms still represent the largest reservoir of biodi-

versity yet to be studied. It has been estimated that onlybetween 1 and 10% of bacteria have been properly described.

Correct microorganism identification is essential for appro-

priate classification, and the criteria for the identification of

diverse microorganism species are still equivocal. Some

strains can be misidentified as closely related species. This

is the case, for example, for Cronobacterspp.130 that can easily

be misidentified as apathogenic Enterobacter turices, E.

helveticusand E. pulveris. Cronobacter spp. are Gram-negative

opportunistic food-borne pathogens and are known as rare but

important causes of neonatal infections. To overcome this

problem, MALDI-MS was successfully used for rapid genus and

species-specific identification. Moreover, multi-isotope ima-

ging mass spectrometry is contributing to understanding thebacterial diversity of bacterioplankton and helping to link

microbial diversity to the biogeochemistry of the pelagic zone

of the aquatic system.131

The marine environment has also been proven to be a

source of diverse arrays of bioactive metabolites with great

potential for pharmaceutical and other applications. In fact,

MALDI-MS was applied to classify environmental

Sphingomonadaceae using ribosomal subunit proteins coded

in the S10-spc-alpha operon as biomarkers.132 In particular,

sponges have a largely unexplored biosynthetic potential. Sets

of bacteria were cultured from marine sponges (Isops phlegraei,

Haliclonasp., Phakellia ventilabrum and Plakortis sp. growing

on the Norwegian coast). Intact cell MALDI-MS was used forthe rapid screening and proteometric clustering of a subset of

the strain collection comprising 456 isolates. The 11 resolved

groups were also verified by 16S rDNA analyses. The results

indicated that MALDI-MS is effective for the rapid identifica-

tion of isolates, for the selection of strains representing rare

species and for their dereplication, i.e., rapid grouping of

bacterial isolates for subsequent characterization.133

Screening for microbial population complexity and diversity

in the sediment of contaminated environments has also

received increased interest, particularly due to the significance

of these microbes for environmental protection. The taxono-

mical identification of microbial isolates obtained from

sediment samples contaminated with polychlorinated biphe-

nyls was successfully performed with MALDI-MS with minimal

time demand and reduced costs.134

The contribution of MS to worldwide biodefence has also

been substantial.135,136 In fact, some potential agents for

biological attacks are microorganisms and biotoxins. MS was

demonstrated to be a valid tool for the rapid identification of

potential bioagents. Confident identification of an organismcan be achieved by top-down proteomics following identifica-

tion of individual protein biomarkers from their tandem mass

spectra. In bottom-up proteomics, the rapid digestion of intact

protein biomarkers is again followed by MS to provide

unambiguous bioagent identification and characteriza-

tion.133,134

Accurate discrimination between species of filamentous

fungi is also essential because some species have specific

antifungal susceptibility patterns, and misidentification may

result in inappropriate therapy. Direct surface analysis of

fungal cultures90,137,138 and yeasts139 by MALDI-MS has been

evaluated for species identification. The protein profiles of

intact fungal spores83,140 such as Aspergillus, Fusarium andMucoralesdemonstrated that MALDI-MS is appropriate for the

routine identification of filamentous fungi in medical micro-

biology laboratories.73,138

Culture collection strains representing 55 species of

Aspergillus, Fusarium and Mucorales were used to establish

one reference database for MALDI-MS-based species identifi-

cation with the MALDI BioTyper 2.0 software. To evaluate the

database, 103 blind-coded fungal isolates collected in a

routine clinical microbiology laboratory were tested, and

96.8% of the isolates were correctly identified to the species

level in agreement with reference methods. Eight technical

replicates of 15 strains were also obtained to study the

variation of mass spectra. Little variation was observed for

each spectrum, whereas enough MS variation could be

observed to separate each strain (Fig. 5).90

Yeast infections cause significant mortality in critically ill

and immunocompromised patients. In particular, Candida

spp. are the 4th most common cause of nosocomial blood-

stream infections in the United States, and Cryptococcus

neoformans is the most common cause of fungal meningitis

worldwide.141,142 Candida species were reliably identified by

MALDI-MS, which had superior performance over conven-

tional methods, and it was possible to discriminate between

different molecular types of Cryptococcus neoformans and

Cryptococcus gattiiwith the same technique.143,144

Non-fermenting bacteria

Non-fermenting bacteria are a taxonomically heterogeneous

group of bacteria of the Proteobacteria division, which cannot

catabolize glucose. The genera Pseudomonas, Burkholderia,

Stenotrophomonasand others belong to this large group. They

are ubiquitous environmental opportunists, and some species

can cause severe infections,145 particularly in immunocom-

promised or cystic fibrosis (CF) patients.146 It has been

demonstrated that classical phenotypic methods can fre-

quently misidentify non-fermenters.147,148

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For this class of bacteria, molecular tools such as 16S rDNA

gene sequencing provide reliable results, but less accurate

results have been obtained at the species level. Therefore, a

reference database for MALDI-MS based on the identification

of non-fermenters was established by Mellmann et al.149,150

and 16S rRNA gene sequencing was used for comparison.

Different cultivation conditions and mass spectrometer

instruments were used, and the methodology was evaluated

with 80 blind-coded clinical non-fermenter strains. The study

demonstrated that the MALDI-MS method provides fast and

thorough identification of non-fermenting bacteria, even more

accurately than partial 16S rRNA gene sequencing for species

identification of members of theBurkholderia cepaciacomplex.

A large international multicenter study has also demonstrated

the high reproducibility of the identification of non-ferment-

ing bacteria by MALDI-MS, with 98.75% correct species

identification. This study demonstrated the suitability of the

technique as an alternative to partial 16S rRNA platforms. Avery recent study also evaluated the ability of the technique to

identify non-fermenting bacteria from among a total of 182

isolates from 70 CF patients because non-fermenters are the

main cause of mortality in this type of patient.151 MALDI- MS

was found to improve routine identification, particularly by

enlarging the Biotyper 2.0 database to include the rare and

infrequent microorganisms recovered from CF patients.152

Non-culture-based identification of bacteria from blood and

milk

In addition to being increasingly used for the rapid identifica-

tion of bacteria and fungi, MALDI-MS also holds promise for

bacterial identification from blood culture (BC) broths inhospital laboratories153155 and bacterial identification directly

from milk samples.156

A MALDI-MS-based approach has been shown to perform

rapid (,20 min) bacterial identification directly from positive

BCs with high accuracy. Positive predictive values for the direct

identification of both Gram-positive and Gram-negative

bacteria from monomicrobial blood culture broths were

100% to the genus level. A diagnostic algorithm for positive

blood culture broths that incorporates Gram staining and

MALDI-MS should be able to identify the majority of

pathogens, particularly at the genus level.153,154

The routine identification of microorganisms that contam-

inate milk is mostly based on phenotypic characteristics such

as colony morphology, hemolytic potential and several

biochemical reactions, which are time-consuming and

costly.157

Additionally, these tests may fail to correctly identifyall agents, and even though methods based on PCR are

developing rapidly, there may be no agreement among the

techniques that phenotypically and genotypically differentiate

bacterial species, resulting in the false identification of agents.

Non-culture MALDI-MS identification based on protein finger-

printing from bacteria (E. coli, S. aureus and E. faecalis)

inoculated and recovered directly from milk samples has been

successful (Fig. 6). Although relatively high bacterial loads (106

to 107 bacteria mL21 of milk) must be present, the simple

incubation of an initial load of 104 bacteria mL21 of milk can

be used to facilitate bacterial replication and successful

Fig. 5 Three-dimensional principal component analysis (PCA) plot of the technical replicates of selected reference strains of ( a)Aspergillus(seven strains), (b)Fusarium

(four strains) and (c) Mucorales (four strains). Reproduced from ref. 90 with permission from John Wiley and Sons.

Fig. 6 MALDI-MS ribosomal protein fingerprints for the identification of

bacteria in whole milk. Data were collected in them/z400022 000 range after

processing 900 mL of whole milk that had been experimentally contaminated

withE. coliat (a) 103, (b) 104, (c) 105, (d) 106, (e) 107, (f) 108, or (g) 109 cfu ml21.

Reproduced from Proteomics ref. 156 with permission from John Wiley and

Sons.


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identification. Fast, reliable and sensitive protocols for the

analysis of relatively low concentrations of bacteria present in

milk could be of great value for the dairy industry.

Testing for antibiotic resistance with MS and polymicrobial

culture analysis

Antibiotic resistance is the ability of a microorganism to

withstand the effects of an antibiotic drug, and this ability

represents a huge health concern in the medical andveterinary fields. Recent studies have shown that individuals

are at risk of carrying antibiotic-resistant bacteria after a series

of antibiotic treatments due to the resulting selection for

antibiotic-resistant microorganisms. The most common

mechanisms of antibiotic resistance can be divided in three

classes: alteration of the antibiotic target, restriction of

antibiotic access to the target and inactivation of the

antibiotic.158160

Recently, the ability of MALDI MS to effectively discriminate

bacteria strains which have acquired resistance to a variety of

antibiotics has been demonstrated, indicating that this

technique has the potential to differentiate bacterial strains

with varying degrees of antibiotic resistance.161

The rapid detection of resistance type is necessary to select

the best antibiotic therapy. b-Lactam antibiotics represent a

broad class of antibiotics that contain a b-lactam nucleus in

their molecular structure. These antibiotics include penicillin

derivatives (penams), cephalosporins (cephems), monobac-

tams, and carbapenems.b-Lactam antibiotics act by inhibiting

the synthesis of the peptidoglycan layer of bacterial cell walls,

which is important for cell wall structural integrity.b-Lactam

antibiotics mainly affect Gram-positive organisms because

peptidoglycan is the outermost and primary component of

their cell wall. This antibiotic class binds in the active site of

penicillin-binding proteins (PBPs), preventing the final cross-

linking (transpeptidation) of the nascent peptidoglycan layer

and disrupting cell wall synthesis.162 Since these antibiotics

are widely administered to treat infections in human and

domestic animals, many Gram-positive bacteria have alreadydeveloped resistance mechanisms.

Antimicrobial susceptibility has classically been determined

using a variety ofin vitromethods such as disk diffusion and

broth microdilution as well as automated instrument-based

methods. These methods may require from a few hours, such

as for the antimicrobial susceptibility test (AST), to 2496 h for

a pure culture of the suspected pathogen to be obtained and

subjected to disk diffusion assays.3,163 A novel MALDI-MS

method for the detection of b-lactamase resistance has

recently been reported. Resistance to b-lactam antibiotics

can be easily monitored by MS because hydrolysis of the

central b-lactam ring by b-lactamases results in the disap-

pearance of the original ion, which is shifted 18 m/z unitshigher in the spectrum of the antibiotic. In many cases,

hydrolysis is directly followed by a decarboxylation of the

hydrolyzed product, resulting in a further shift of 244 m/z

units due to detection of the hydrolyzed form. Because MS

easily monitors such m/zshifts, a MALDI-MS assay was set up

to analyze the hydrolysis reactions of different b-lactam

antibiotics (Fig. 7).164

MALDI-MS can also be applied to study bacterial resistance

to antibiotics or antimicrobial compounds secreted by other

bacterial species.68,165,166 Reportedly, antibiotic-resistant and

non-resistant strains of an important human pathogen, S.

aureus, can be differentiated by MALDI-MS by rapid and

accurate discrimination between methicillin-sensitive andmethicillin-resistant strains of this organism, which can could

lead to major improvements in the treatment strategies for

infected patients.167 The same microorganism has been

identifiedviabiomarker analysis as one of the main pathogens

responsible for prosthetic joint infections, indicating reliable

differentiation between S. aureus and coagulase-negative

staphylococci.168

The resistance mechanism of colistin-resistant variants of

Acinetobacter baumannii was elucidated by MALDI-MS169 by

determining the phosphoethanolamine modification of lipid

A. For Bacteroides fragilis, it was recently shown that the

differentiation of cfiA gene-encoded class B metallo-b-lacta-

mase was possible by direct MALDI-MS.170,171

The carbapenemresistance of B. fragilis is due to a species-specific metallo-

b-lactamase, which is encoded by the cfiA (ccrA) gene of the

organism. Almost 100% of the carbapenem-resistant bacteroid

strains were cfiA-positive B. fragilis isolates. However, such

clonal differentiation for resistant and susceptible clones

cannot be expected for the majority of bacteria. Antibiotic

resistance in Gram-negative rods, particularly

Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp.,

has been an increasing problem worldwide. Infections by

multidrug-resistant Gram-negative bacteria are usually treated

with carbapenems. This resistance is caused by an alteration

Fig. 7 (A and B) MALDI-MS of ampicillin after incubation with a b-lactamase-

producing strain (B). (C) Inhibition of hydrolysis by a b-lactamase-producing

strain was performed in the presence of clavulanic acid. Peaks corresponding to

the non-hydrolyzed form of ampicillin are highlighted in gray. Peaks

corresponding to the hydrolyzed form of ampicillin are indicated with an arrow.

Reproduced from ref. 164 with permission from the American Society for

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in the outer membrane of the cell wall and by the production

of carbapenemases.172 Carbapenamase activity was detected in

124 strains following comparison between well-typed bacterial

carbapenem non-susceptible isolates and clinical isolates

susceptible to carbapenems. Antibiotic resistance was demon-

strated by detecting the disrupted amide bond and its

cationized forms.173

Although the microbiological methods of microorganism

culture and isolation are successful, mixed cultures orpolymicrobial cultures may occur. For example, one bacterial

isolate from subclinical bovine mastitis was identified as

Staphylococcus aureusby an enzymatic assay, and yet the same

sample, when analyzed by Biotyper software after MALDI-MS,

displayed a signature of Enterococcus faecalis mixed with S.

aureus, which was confirmed by 16S RNA sequencing.174

When bacterial identification of human clinical isolates was

performed by MALDI-MS directly on 500 blood broth cultures,

there were 27 polymicrobial cultures, and 25 (92.6%) had at

least one species correctly identified by the instrument

database. Using the same instrument and software platform,

similar results have been observed by Moussaoui et al.175 and

Christner et al.,176 who reported that 80.9% and 81.2%,respectively, of at least one of the species present were

identified in polymicrobial cultures obtained directly from

blood culture vials.175,176

Conclusions and real-world perspectives

Diverse ionization methods and MS techniques can be

successfully used for microorganism identification and

research. MALDI-MS is the leading MS technique for clinical

and commercial microbiological use, and there are consistent

reports demonstrating the confidence of this approach incomparison to other non-MS based gold-standard approaches

to identify a large range of microorganisms. The broad use of

this technique now appears to only be dependent on

regulatory issues in various countries, including the United

States.

Limitations of MS-based microorganism identification are

dependent on the initial cost of the technology, which is

related to the instrument configurations. Also, most labora-

tories will need an internal validation period in which the

transition between the traditional methods to MS-based

microorganism identification and staff training is performed.

Since one instrument is sufficient for a high number of

samples/day, technical problems can interrupt the routine and

impact clinical decisions, especially in septicemic conditions.

These issues should be managed with appropriate planning

such as the long-term benefits related to the cost/sample. The

identification of bacteria isolated from 928 human clinical

samples in a routine microbiology setting has been recently

compared using the BD Phoenix, API panels and other

recommended procedures and MALDI-MS using a TOF

analyzer and the Biotyper software. Besides the velocity of

the diagnosis, MALDI-MS showed substantial savings of

around U$ 23 per isolate, depending on the cost of the

instrument.

Therefore, microbiology is ready to enter into a new era in

which molecular rather than morphological or biochemical

characteristics of microorganisms will be rapidly assessed

(directly from biological fluids) by MS for identification. The

use of this technique is not only limited to clinical diagnosis,

but it has been shown to have successful application for

detecting antibiotic resistance, characterizing microorganismsthat are difficult to isolate and culture and exploring the

biodiversity of microorganisms present in the environment

and in the digestive tracts of animals and humans. Mass

spectrometry-based proteomics approaches have also been

applied to gain a greater understanding of the pathophysiol-

ogy and virulence of microorganisms. This approach is

providing key insights to better understand the molecular

processes involved in protein secretion, modification, synth-

esis and degradation.177179

A new era has indeed emerged in which bacterial

identification seems fully prepared to make the transition

from the agar plate and visual inspection to the massspectrometer for characterization at the more accurate

molecular level.

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