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Cite this: DOI: 10.1039/c6an00131a

Received 20th January 2016,Accepted 6th April 2016

DOI: 10.1039/c6an00131a

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Applications of MALDI-TOF MS in environmentalmicrobiology

Inês C. Santos,a Zacariah L. Hildenbrandb,c and Kevin A. Schug*a,c

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is an

emerging technique for microbial identification, characterization, and typing. The single colony method

can be used for obtaining a protein fingerprint or profile unique to each microorganism. This technique

has been mainly used in the clinical field, but it also has significant potential in the environmental field.

The applications of MALDI-TOF MS in environmental microbiology are discussed in this review. An over-

view on the use of MALDI-TOF MS for environmental proteomics and metabolomics is given as well as its

use for bacterial strain typing and bioremediation research. A more detailed review on the use of this

technique for the identification, differentiation, and categorization of environmental microorganisms is

given. Some of the parameters that can influence the results and reproducibility of MALDI-TOF MS are

also discussed.

Introduction

The identification of microorganisms is very important indifferent fields. Usually this identification is made by morpho-logical (e.g. cell shape), phenotypic (e.g. Gram staining), andgenetic tests (e.g. polymerase chain reaction (PCR)). The mor-phological and phenotypic tests are time consuming, while thegenetic tests require a high level of expertise and can be quiteexpensive. Therefore, these techniques are not ideally suitablefor routine identification and alternatives would be welcomefor the rapid and low cost identification of microorganisms.1

Matrix-assisted laser desorption ionization time-of-flight(MALDI-TOF) mass spectrometry (MS) is becoming a reliabletool for microorganism identification.2 Despite high initialacquisition costs, this technique can identify bacteria in a fewminutes and provide a low cost per sample analysis when com-pared to conventional methods.

To perform MALDI-TOF identification, as summarized inFig. 1, microorganisms are placed on a target plate where theyare overlaid with a matrix solution, which co-crystalizes withthe sample and lyses the cells. The plate is placed in theinstrument where a laser converts the bacteria constituents,mainly ribosomal protein molecules, into gas-phase ions thatare separated and identified according to their mass/charge

ratio. The mass spectrometer gives a spectral fingerprint thatis unique to the microorganism being analyzed. The organismis then identified by comparing its spectral profile with a refer-ence database (fingerprint-based approach). Correlations ofpeak positions and intensities between experimental and data-base spectra are used to generate a match score. This matchscore is a level of confidence that the unknown isolate is arepresentative of the candidate microorganism(s) matchedfrom the database. Protein mass pattern spectra can be usedto identify bacteria on the genus, species, and even on the sub-species level.1,3–5

Currently, two main identification databases are available:Bruker BioTyper from Bruker Daltonics, Inc. and SARAMISfrom bioMérieux. The databases use the Bruker MainSpectrum analysis (MSP) and the bioMérieux SuperSpectrumapproaches, respectively.1,2,5 These approaches differ in thealgorithms used to identify the microorganisms. The MSPtechnique consists of a collection of reference spectra obtainedfrom single reference strains, while the SuperSpectrumtechnique consists of spectra obtained from various clinicaland reference strains grown under different conditions.2

Matrix solutions used in MALDI-TOF MS analysis can affectthe quality and reproducibility of the protein fingerprints.Sinapinic acid and α-cyano-4-hydroxycinnamic acid (CHCA) arethe most commonly reported matrix compounds used toobtain good-quality spectra in microbial identification.6 Fur-thermore, formic acid (0.1%) can be added to the matrix solu-tion to suppress salt-containing adduct ions, originated fromculture media, and to improve the resolution of spectra.6

Different sample preparation methods are available formicroorganism identification.7 The simpler “whole cell”

aDepartment of Chemistry and Biochemistry, The University of Texas at Arlington,

Arlington, TX, USA. E-mail: kschug@uta.edu; Fax: +1 817 272 3808;

Tel: +1 817 272 3541bInform Environmental, LLC, Dallas, TX, USAcAffiliate of the Collaborative Laboratories for Environmental Analysis and

Remediation, The University of Texas at Arlington, Arlington, TX, USA

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method consists of depositing a single colony on the MALDIplate which is consequently overlaid with matrix. The othermethods require a chemical protein extraction step before ana-lysis using an organic acid such as trifluoroacetic acid (TFA) orformic acid. With protein extraction methods, a more detailedprotein fingerprint can be obtained; however, these are moretime consuming when compared to the whole cell method,which does not require any chemical treatment. Nevertheless,the addition of ethanol or methanol has been shown toenhance the signals of higher mass proteins.3 The identifi-cation of microorganisms by MALDI-TOF MS requires a cellculturing step to obtain pure cultures. Enough bacterial cells(105–107 bacterial cells) must be grown for extraction and ana-lysis, and this may be considered a drawback. However, theneed for culturing is also a common step for every other con-ventional identification method.

MALDI-TOF MS has been extensively applied and is stillmainly used in the clinical field as shown by the number ofreviews published.8–12 In the last years, hundreds of systemshave been installed worldwide in clinical microbiology labora-tories. The importance of rapidly identifying microorganismsinvolved in human infections and consequently, applying theright therapeutic, is unquestionable. However, MALDI-TOF MScan also provide a significant contribution in environmentalmicrobiology.

In this review, the applications of MALDI-TOF MS in thefield of environmental microbiology are explored. An overviewof the works that describe the use of this technique for proteo-mics (fingerprinting and profiling) in microbial identification,differentiation, and categorization is given. The potential forMALDI-TOF MS in metabolomics profiling is also discussed.

Additionally, the use of MALDI-TOF MS for bioremediationresearch, mainly in identifying site-specific bacteria andassociated enzymes, is also discussed. In the end, the para-meters that may influence the reproducibility of the massspectra are viewed as well as some approaches to improve thequality of the MS results in the field of environmentalmicrobiology.

MALDI-TOF MS environmentalmicrobiology applications

Environmental microbiology is an area of research where theuse of MALDI-TOF MS remains to be comprehensivelyexplored. This technique is a popular tool for proteomics,including microbial typing, but it may also be used in otherstudies such as metabolomics. These two areas provide infor-mation on the expression of proteins and on the diversity ofproduced metabolites, respectively, in an organism under aspecific set of conditions.2,13 Overviews of these differentapplications are given below.

Proteomics

Protein fingerprinting. The identification of microorganismsfrom environmental sources is necessary to understand themicrobial community, for environmental monitoring, and toidentify possible pathogenic microorganisms. MALDI-TOF MShas been an important advance in the field of environmentalproteomics as the protein fingerprint of each microorganismcan be used for identification. The identification can be per-formed by comparing the unknown protein profile to a data-

Fig. 1 MALDI-TOF MS for bacterial identification. The sample colony is placed on a metal plate and overlaid with matrix. The molecules are ionizedby the laser and accelerated by an electric field. The ions are separated according to their m/z (flight time), while subjected to vacuum, until theyreach the detector, where the abundance of each signal is registered.

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base of reference profiles or by co-analyzing the unknownprofile with profiles of known bacteria. Table 1 presents someexamples of microorganisms identified from environmentaland clinical sources using MALDI-TOF MS. It is noteworthythat the same microorganisms can be found in both clinicaland environmental samples. In fact, some human pathogensderive from environmental reservoirs and are thus denomi-nated as environmental pathogens. Nevertheless, environ-mental microorganisms are more diverse and so theiridentification and characterization pose a significant challengeas the field grows.

Previous findings have described the application of MALDI-TOF MS in the identification of microorganisms in environ-mental samples such as sewage sludge, marine sponges,water, soil, roots, and the rhizosphere.14–21 For example, in thework by Emami et al.,15 MALDI-TOF MS was used to character-ize bacteria in ballast water. Thirty-six isolates were identified,at the genus-level, and the results were similar to thoseobtained by 16S rRNA gene sequencing. For higher qualityspectra, the authors used cell lysates from actively growingcolonies instead of crude cells and α-cyano-4-hydroxycinnamicacid (HCCA) as matrix, which they found to be an importantfactor when trying to differentiate between closely related iso-lates. Additionally, Ferreira et al.18 and Štursa et al.19 studiedthe use of MALDI-TOF MS for the identification and character-ization of microorganisms in the rhizosphere of plants. In theformer work, the authors built a database containing proteinprofiles of 56 species of fast growing rhizobia and were able toidentify large populations of isolates from nodules with a

100% effectiveness. Furthermore, they concluded that MALDI-TOF MS is a very useful tool for diversity and ecologicalstudies.

Overall, the works mentioned above describe MALDI-TOFMS as a powerful technique in the field of environmentalmicrobiology for the rapid screening and identification of bac-teria and for ecological studies. However, this technique stillpresents some drawbacks.14,16,17 Kopcakova et al.16 studied theability of MALDI-TOF MS to identify cultivable microflora fromtwo waste disposal sites from the non-ferrous metal industry.High quality mass spectra were obtained but most of thebacteria isolates could be not identified. The overallidentification rate was lower than 20%. The authors empha-sized the need to expand and refine the reference databasespectra to improve the ability of MALDI-TOF MS to identifyenvironmental bacteria, especially those acquired fromextreme environments. In the works by Lovecka et al. andKoubek et al.,14,17 the ability of MALDI-TOF MS to identifybacterial isolates obtained from contaminated soil was investi-gated. They were unable to identify all the isolates and werealso unable to identify them down to the species level. Theseworks indicate the need of a universal sample pre-treatmentprotocol and a more complete and environmentally-focuseddatabase to improve the ability of MALDI-TOF MS to success-fully identify environmental bacteria.

To overcome these limitations, researchers have attemptedto identify microorganisms by detecting and identifyingspecific protein markers (biomarkers) in the protein finger-print.24 A biomarker is a particular molecule that is specific toa microorganism and confirms its detection and/or identifi-cation.1 Peptides and proteins are the molecules most used asbiomarkers in MS-based microorganism identification, due totheir high abundance compared to other classes of moleculesand their relatively high ionization efficiencies. Furthermore,proteins represent the genetic status of a bacteria and are,therefore, more informative and characteristic than lipids andmetabolites. These protein biomarkers can be, for example,virulence factors, toxins, and other strain-specific proteins thatare uniquely indicative of a certain microorganism and can beused to facilitate the identification. For instance, by knowingthe molecular weight of a toxin, targeted searches for signalsat the corresponding m/z ratio can be made to aid the identifi-cation of the microorganism.12 In fact, when studying MALDI-TOF MS reproducibility, Wang et al.25 found a number ofsignals that were conserved under different experimental con-ditions and that have the highest potential for use as bio-markers for bacterial identification. Therefore, these and otherauthors proposed the use of specific conserved biomarker pro-teins for bacterial identification as they are not altered by exter-nal conditions. For example, in the work by Ruelle et al.,20

bacterial identification was based on the observation of a setof biomarkers as shown in Fig. 2. Furthermore, the authorsstudied the conditions that led to good-quality and reproduci-ble spectra for the rapid identification of environmental bac-teria. A protocol using a matrix solution composed of α-cyano-4-hydroxycinnamic acid and an ethanol treatment for the

Table 1 Examples of bacteria identified by MALDI-TOF MS in environ-mental and clinical fields

Environmental14–22 Clinical5,22,23

Achromobacter sp. Acinetobacter baumanniiAcinetobacter sp. Campylobacter spp.Aeromonas sp. Enterohemorrhagic

Escherichia coliArthrobacter sp. Enterococcus faecium,

Enterococcus faecalisBacillus mycoide Haemophilus influenzaBurkholderia xenovorans Klebsiella pneumonia,

Klebsiella oxytocaEscherichia coli Listeria spp.Enterococcus faecium, Enterococcusfaecalis

Neisseria spp.

Microbacterium sp. Nocardia spp.Pseudoalteromonas sp. Proteus mirabilisPseudomonas aeruginosa,Pseudomonas stutzeri, Pseudomonasputida, Pseudomonas gessardii

Pseudomonas aeruginosa

Rhizobium Salmonella thyphimurium,Salmonella enterica

Rhodococcus sp. Shigella spp.Salmonella spp. Staphylococcus aureus,

Staphylococcus epidermidisSerratia fonticola Streptococcus pneumoniaeStenotrophomonas sp. Vibrio parahemolyticusVibrio spp. Yersinia enterocolitica

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extraction of more and higher mass cell compounds wasadopted. With this standardized protocol, environmental bac-terial strains of Escherichia coli, Salmonella, and Acinetobacterwere identified from sewage sludge using MALDI-TOF MS. Asanother example, Dieckmann et al.,21 performed the strain-level identification of diverse strains isolated from marinesponges by implementing weighted pattern matching, wherethe peaks with higher importance for discriminating strainswere given more weight in the analysis.

When compared to conventional identification methods,MALDI-TOF MS provides higher throughput and a lower costof analysis. However, only cultivable bacteria are identifiedby this technique. As it is known, less than 1% of microbialspecies in the environment are able to grow in rich growthmedia under laboratory conditions.26 This may be seen as a

major drawback of this technique when applied in the studyof environmental communities. Nevertheless, recent newapproaches to cultivate previously uncultivated bacteria havebeen studied by using dilute nutrient media or simulatednatural environments,27 which may help overcome limit-ations for identifying the limited number of cultivablebacteria.

Protein profiling. Nowadays, two approaches for bacterialidentification through protein identification and discovery ofnew biomarkers are used: top-down28 and bottom-up29 proteo-mics. In the former approach, intact proteins are identified byanalyzing their primary structure, while in the latter approach,the proteins are enzymatically digested and their peptidesused for identification. Traditionally, these approaches are per-formed by gel electrophoresis and immunoassays such aswestern blot, ELISA, immunohistochemistry, and proteinmicroarrays.30 In immunoassays, protein identification is per-formed by using specific antibodies that are labeled with anenzyme or tagged to a fluorophore for detection. Nevertheless,these techniques present some drawbacks such as being laborintensive and they can sometimes provide poor reproducibilityand resolution. Mass spectrometry is now considered to be themost powerful tool in proteomics, as it provides higherthroughput and it is very sensitive and reproducible. In thetop-down approach, intact proteins are separated and intro-duced in the mass spectrometer where they are fragmented(MS/MS). The proteins can be identified by comparing MS/MSspectra with fragmentations predicted from protein sequencesin existing proteome databases. In the bottom-up approach,the proteins are first separated by chromatography or gel elec-trophoresis, then digested into constituent peptides by specificenzymes. The peptide mixtures are then introduced in themass spectrometer for further interrogation.31,32 Peptideidentification is performed by comparing the masses of pep-tides and MS/MS fragment ions with theoretical sequencesderived from genome sequence data. When using MS, top-down proteomics presents some limitations compared tobottom-up proteomics as peptides are more easily fractionated,ionized, and fragmented compared to intact proteins.33 Bio-informatics approaches for automated protein identificationusing mass spectral data are important tools to support top-down and bottom-up proteomics. The informatics approachesfor protein identification have already been extensivelyreviewed by Johnson et al. and Zhang et al.32,34 and so will notbe further discussed here.

Both MALDI and electrospray ionization (ESI) have beenextensively used for proteomic analyses. However, MALDI pro-vides some advantages, such as simple sample preparationand formation of singly charged ions. Therefore, MALDI-TOFMS can be an important tool in the identification of proteinsfor microorganism identification.35,36 Demirev et al.,35 demon-strated the ability of MALDI-TOF/TOF MS to rapidly identifyintact Bacillus spore species using a top-down approach.Protein spore biomarkers were fragmented and identified bycomparing their spectra with a proteome database. Fragmention spectra of whole protein biomarkers were obtained

Fig. 2 MALDI spectra of (A) Salmonella 2B5, (B) Acinetobacter 14B5 and(C) Escherichia coli 1B1 showing the potential genus- (.) and strain- (*)specific biomarkers. (Reprinted from Ruelle et al.20 with permission fromWiley. Copyright 2004.)

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without the need for digestion, separation, and cleanup. Theidentification of protein spore biomarkers allowed the identifi-cation of Bacillus spore species such as Bacillus globigii andBacillus cereus. Bacillus spores were also identified in a bottom-up approach using MALDI-TOF MS in work by Warscheid andFenselau.36 Spore proteins were enzymatically digested andanalyzed by MALDI-TOF MS using a curved-field reflectroninstrument. Proteins were identified by partial sequencing ofdistinctive tryptic peptides from Bacillus spores via post-sourcedecay analysis combined with genome-based databasesearches by Mascot Sequence Query. Various spore proteinswere identified which allowed the characterization of Bacillusspecies. By performing the protein identification, microorgan-isms were also identified. As shown, when using MALDI-TOFMS, top-down and bottom-up approaches provide a muchhigher level of identification specificity for individual micro-organisms when compared to the identification of micro-organisms using their protein fingerprint.

Top-down and bottom-up proteomics are used not only forbacterial identification, but also in microbial ecology fordifferent applications,37 where the bottom-up approach, inspite of being more laborious, is more commonly used thanthe top-down approach. MALDI-TOF MS can be used to tracknew functional genes and metabolic pathways. For example,Kurian et al.38 used two-dimensional electrophoresis (2-DE)and MALDI-TOF MS to provide insight into the heterotrophicmetabolism of Synechocystis sp. PCC 6803. They were able tocharacterize the cellular metabolic shift due to a trophicchange in Synechocystis by identifying the proteins producedin autotrophic and heterotrophic conditions.

MALDI-TOF MS can also be used to map the proteins of anecosystem in a time-resolved fashion. Kan et al.39 used proteo-mic approaches to study time-dependent protein expressionprofiles of Chesapeake Bay microbial communities. Theauthors reported that the MALDI-TOF MS analysis of highlyexpressed proteins produced no significant matches to knownproteins. They conjectured that it is unlikely that many pro-teins in environmental samples will share a high level of iden-tity with proteins in sequence databases derived from culturedorganisms. De Vriendt et al.40 used 2-DE and MALDI-TOF/TOFMS to describe the changes in protein expression of Shewanellaoneidensis MR-1 when growing as a biofilm.

Additionally, MALDI-TOF MS can be used in the identifi-cation of proteins associated with specific stresses. Whensubjected to changes in environmental parameters, micro-organisms change their protein expression profiles, as aresponse to overcome these changes. These differences in theprotein expression profiles can be used as an indicator ofenvironmental pollution. In the work by Heim et al.,41 MALDI-TOF MS was used to identify proteins produced by Pseudo-monas putida due to iron limitation stress. The authors were ableto identify 25 proteins that were up- and downregulated due toiron deprivation. Lacerda et al.42 used 2-DE, MALDI-TOF MS/MS, and de novo sequencing to identify proteins differentiallyexpressed over time following exposure of a bacterial commu-nity to an inhibitory level of cadmium.

Clearly, MALDI-TOF MS is an important tool in microbialecology, allowing the identification of stress-related proteinsand an insight into the microbial community proteome. Never-theless, an improvement in proteome databases is needed todrive this application forward.

Microbial typing. Another interesting application of MALDI-TOF MS is for the typing of bacteria, to identify microorgan-isms at the strain level.43 The methods most commonly usedfor microbial typing are 16S rRNA sequencing, pulsed field gelelectrophoresis (PFGE), multilocus sequence typing (MLST),and repetitive extragenic palindromic-polymerase chain reac-tion (rep-PCR).43–45 Brief descriptions and the main advan-tages and disadvantages of the methods often used foridentification and typing are presented in Table 2. In spite oftheir recognized resolution, many of these approaches areexpensive, laborious, and time consuming. These are certainlyundesirable attributes, specifically in the identification of con-tamination sources as is, for example, described in the worksby Giebel et al. and Siegrist et al.46,47 When dealing with patho-gens, a rapid identification of the contamination source isnecessary to rapidly resolve the problem. Due to these reasons,MALDI-TOF MS is gaining more attention in this field as itrequires less sample preparation and provides higher analysisthroughput.

When using MALDI-TOF MS, microbial typing can be per-formed either for taxonomy, bacterial differentiation, or categ-orization.43 In taxonomy, the microorganisms are classifiedinto genera, species, or strains, based on their protein finger-print or profile similarities, as explained previously. For bac-terial differentiation and also identification, MALDI-TOF MScan be used to cluster, in the form of a dendrogram, theprotein profiles according to their similarities. By comparingthe percentage of similarity of an unknown profile withknown bacteria profiles, the taxonomy of the unknown bac-teria can be deduced. Furthermore, strain-specific differencescan be determined and used to study taxonomic and inter-and intra-species diversity. Several works have been describedthat use MALDI-TOF MS for bacterial differentiation andidentification and some are discussed in this manuscript. Inthe work by Dieckmann et al.,21 Pseudoalteromonas sp. iso-lated from marine sponges and differing in only 1 bp out of400 bp or by 3–4 bp out of 1500 bp of their 16S rRNA genesequences, were readily discriminated by their MALDI-TOFMS spectra. In fact, the authors described considerable intra-and inter-species classification problems, which 16S rRNAsequencing failed to resolve. MALDI-TOF MS, by introducingadditional phenotypic markers, was able to discriminate veryclosely related species with high reliability. In anotherstudy,48 MALDI-TOF MS was used to characterize seven pet-roleum microorganisms. The authors showed the discrimi-nation power of this technique as these seven petroleumstrains were clustered into three groups, consistent with themolecular identification using the gyrB gene sequence as thephylogenetic marker. MALDI-TOF MS was able to discriminatestrains that, due to their similarities, were not discriminatedby 16S rRNA.

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Additionally, Munoz et al.49 verified that MALDI-TOF MSanalysis of whole cells is a powerful tool for studying the culti-vable fraction of hypersaline environments. The authors usedMALDI-TOF MS to classify bacterial isolates into 25 phenotypicclusters at 52% similarity, which was validated by 16S rRNAsequencing to indicate that each phenotypic cluster consistedof a homogeneous set of strains. In the work by Donohueet al.,50 MALDI-TOF MS was used for the speciation ofunknown environmental water isolates of Aeromonas using them/z signature of known strains of that microorganism. Due toits analysis speed and its capability for handling a largenumber of samples, the authors proffered that this techniquecould be useful for environmental monitoring.

Eddabra et al.51 described the use of MALDI-TOF MS to dis-criminate 30 Vibrio strains isolated from two wastewater treat-ment plants. The dendrogram obtained by MALDI-TOF MSwas different from the one obtained by PFGE which could beexplained by the different targets analyzed by the twomethods. Nevertheless, the results showed the ability ofMALDI-TOF MS to differentiate closely related Vibrio spp.,which presented a high congruence of strain grouping. Inanother work, Stets et al.52 were able to identify and group bac-terial isolates from wheat roots. By comparing the dendro-grams obtained with whole-cell MALDI-TOF MS analysis and16S rRNA gene sequence phylogeny, they observed that theformer had a higher resolution within the genus level than thelatter, as it was able to separate the isolates sharing high 16SrRNA sequence identity in different clusters. Therefore, theauthors concluded that whole-cell MALDI-TOF MS analysis canbe used as a rapid and efficient screening method for groupingbacterial isolates from environmental samples, independently

of databases. Furthermore, they determined that this tech-nique may have the potential to differentiate bacterial isolatesat the strain level.

All the above works described the successful application ofMALDI-TOF MS in grouping bacterial isolates. Nevertheless,the ability of this technique to differentiate at the strain leveldoes not always overcome the ability of conventional methodsas shown by Ghyselinck et al.53 The authors examined the taxo-nomic resolution of MALDI-TOF MS profiling for bacteria iso-lated from the rhizosphere of potato plants. In fact, thistechnique was able to differentiate bacterial strains but theconventional technique, rep-PCR, facilitated strain differen-tiation more readily than MALDI-TOF MS when members ofthe genera Rhizobium, Streptomyces, Paenibacillus, Arthrobacter,and Pseudomonas were considered.

Bacterial strain typing by MALDI-TOF MS can be used todetermine the origin of a specific strain by grouping the iso-late’s protein profile by source. This information can be usedto determine the source of, for example, a microbial contami-nant. This is an approach termed bacterial source-tracking(BST). The works by Giebel et al. and Siegrist et al.46,47 describethe potential use of MALDI-TOF MS to identify the sourcesof pathogenic bacteria (fecal contamination) found in re-creational and surface water, respectively. Siegrist et al.47 usedMALDI-TOF MS-based fingerprinting and the Dice similaritycoefficient to cluster several E. coli isolates from canine,bovine, and avian sources. Using six avian, three bovine, twocanine, and four human E. coli isolates, the authors observedthat only the human isolates did not group completely bysource. In the work by Giebel et al.,46 the Pearson product-moment correlation coefficient was used. As with the Dice

Table 2 Review of the methods often used for bacterial identification and typing43–45

Method Description Pros Cons

Pulsed field gelelectrophoresis (PFGE)

Gel electrophoresis technique where thepolarity of the current changes forseparation of very large DNA fragments

High discriminatoryability and reproducibility

Relatively costly and timeconsuming

Amplified fragment lengthpolymorphism (AFLP)

PCR amplification of a subset of DNAfragments generated by restrictionenzyme digestion

Reproducible Labor-intensive and costly

Random amplification ofpolymorphic DNA (RAPD)

PCR amplification of random DNA. Theamplification is followed by electrophoresis

Cheap, rapid, and easy toperform

Lack of reproducibility

Variable-number tandemrepeat (VNTR)

PCR amplification of polymorphic regions ofDNA containing the VNTRs

High discriminatoryability

Lack of reproducibility

Repetitive extragenicpalindromic PCR (rep-PCR)

PCR amplification of repetitive DNAelements

Easy to perform Discriminatory power andreproducibility is lower comparedto PFGE and MLST

16S rRNA sequencing PCR amplification and sequencing of 16SrRNA gene sequences

Useful for thediscrimination until theGenus level

Relatively costly and limiteddiscriminatory power

Multilocus sequence analysisand multilocus sequencetyping (MLST)

PCR amplification and sequencing ofmultiple housekeeping genes

Good discriminatoryability

Time consuming and costly

MALDI-TOF MS Molecular weights of proteins are used toidentify microorganisms

High throughput, easy toperform, low cost peranalysis

High initial instrumentation cost,limited resolution, and databasediscordances

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similarity coefficient, they found intrareplicate repeatability tobe lower than that of the former software. Fig. 3 shows thecluster analysis of 21 Enterococcus isolates from seven sources(human, bovine, canine, chicken, duck, goose, and gull). All21 isolates were not grouped by source, but the gull andchicken isolates composed their own discrete cluster, and twoout of the three human (human77 and human80) isolatesgrouped together. Overall, both works recognize the ability ofMALDI-TOF MS to group isolates by source and the potentialof this technique to fingerprint environmental isolates andtherefore be applied as a rapid and accurate BST tool.

In spite of its recognized benefits in bacterial strain typing,MALDI-TOF MS still needs to be further explored in the fieldof environmental chemistry. The identification at the strainlevel requires higher resolution which may be more challen-ging as strains of the same species are quite similar.43 Also,some factors may influence the reproducibility of the tech-nique, as explained below, which is very critical when theidentification of a bacterium’s strain is intended.

Applications in bioremediation. Environmental pollution isof great concern and environmentally friendly alternatives forremediation are necessary. The use of microorganisms toreduce or eliminate hazardous compounds from the environ-ment, so called bioremediation, is a promising approach.54

MALDI-TOF MS can be used for the rapid screening and

identification of site-specific microorganisms present incontaminated environments using their global proteinexpression.24 This allows researchers then to focus on specificmicroorganism species to evaluate their potential for degra-dation of chemical hazards. After evaluating their capability,the isolated microorganisms can be used for bioremediationof contaminated and polluted sites. As an example, in theworks by Lovecka et al. and Uhlik et al.,14,55 the identificationof bacteria isolated from contaminated soil for bioremediationpurposes was performed using MALDI-TOF MS. In both works,the bacteria were isolated from contaminated soil by usingtheir ability to grow on solid mineral medium with the chemi-cal hazards, pesticides or biphenyl, as a sole carbon source.Afterwards, the isolates were identified, some to the strain-level, using MALDI-TOF MS. The results obtained were inagreement with the results of 16S rRNA sequencing. However,in both works there were additional microorganisms that werenot successfully identified by MALDI-TOF MS, which theauthors believe may be due to their absence in the database.Nevertheless, the authors believe that MALDI-TOF MS is animportant tool for bioremediation research as it allows therapid and accurate identification of site-specific microorgan-isms. As the isolated microorganisms have the ability to growusing the hazardous exogenous compounds as sole carbonsource, they can act as potential degraders and therefore be

Fig. 3 Cluster analysis of mass spectra from Enterococcus isolates from seven sources. Similarity coefficients were calculated using the method ofPearson, and the dendrogram was constructed using the UPGMA approach. (Reprinted from Giebel et al.46 with permission from Elsevier. Copyright2008.)

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used for on-site bioremediation. However, only the work byLovecka et al.14 specifically studied the capability for theidentified bacteria to degrade the contaminants by measuringthe degradation of pesticides and the formation of degradationproducts.

Top-down and bottom-up proteomics can also be used inbioremediation studies to identify the proteins and enzymesthat catalyze the mineralization of chemical hazards. Theseenzymes can be used as catalysts for cell-free remediation.24

Tomás-Gallardo et al.,56 used MALDI-TOF MS(/MS) for theidentification of proteins specifically involved in phthalate andprotocatechuic acid (PCA) degradation by Rhodococcus sp.through de novo peptide sequencing. In the work by Kimet al.,57 eighty unique proteins were identified by 2-DE/MALDI-TOF MS from Pseudomonas putida KT 2440 cultured in thepresence of six different organic compounds. The authorsrefer that the proteomics analysis was laborious and accuratequantification was not easy to perform. To overcome this draw-back, cleavable isotope-coded affinity tag (ICAT) analysis wasperformed. The information gained from this experiment com-plemented the results, confirming some proteins ID andrevealing additional ones. In another work,58 the Acinetobacterradioresistens S13 membrane proteome was profiled during

aromatic exposure using 2-DE/MALDI-TOF MS. The developedmethod allowed the identification of proteins that were onlyexpressed in the presence of aromatic substrates.

Furthermore, bottom-up proteomics has also been used tomonitor bioremediation. In work by Wilmes et al.,59 2-DE andMALDI-TOF MS were used to identify highly expressed proteinsduring microbial transformations from a mixed culture acti-vated sludge system important for phosphorus removal. Theauthors were able to identify proteins related to the chemicaltransformations.

MALDI-TOF MS, as a high-throughput technique, is aninteresting tool for bioremediation studies. This techniqueallows the rapid identification of site-specific microorganismspresent in contaminated environments. Additionally, theidentification of enzymes for the mineralization of chemicalhazards is also possible without the need of cultivation.MALDI-TOF MS can also be used to monitor the bioremedia-tion strategy in situ. Undoubtedly, this is a technique for whichmuch more can be explored.

Metabolomics

Metabolomics involves the investigation of all metabolites pro-duced and liberated by an organism under certain conditions.

Fig. 4 Metabolite analysis E. coli extract by MALDI-TOF-MS. Top: Mass spectrum of methanol lysed E. coli strain DH5-R. Bottom: Magnificationfrom 700 to 800 m/z. Lower trace is blank. Upper trace is E. coli sample, offset 10% for clarity. * indicates peaks in matrix or blank. (Reprinted fromEdwards and Kennedy,62 with permission from the American Chemical Society. Copyright 2005.)

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MALDI-TOF MS has been extensively used for proteomic fin-gerprinting but it is also a sensitive tool for untargeted meta-bolomics, more specifically in metabolic profiling andfingerprinting.60 Metabolic profiling consists of studying aspecific group of metabolites related to a specific metabolicpathway. This approach can be an important tool for bioreme-diation studies. In bioremediation, it is important to deter-mine if the isolated bacteria from contaminated sites are ableto degrade chemical hazards into less hazardous, and ulti-mately, completely innocuous byproducts. Mass spectrometrycan be used to follow the biodegradation pathways either inthe environment or in laboratory assays by identifying the cata-bolic products, therefore determining the bacteria’s capabilityfor degrading toxic compounds.24 In laboratory assays, themicroorganism is incubated in mineral media with the pollu-tant, and MALDI-TOF MS can be used to identify the expectedmetabolites that are formed as a result of the biodegradation.

Due to its high sensitivity, MALDI-TOF MS can be used as atool to predict environmental contaminations as the metab-olite fingerprint may change when the microorganism isexposed to such types of stress.61 This approach is usually per-formed by direct injection mass spectrometry (DIMS) but theuse of MALDI-TOF MS could be explored for metabolic finger-printing as it allows minimal sample preparation and high-throughput analysis. In fact, in the work by Edwards andKennedy,62 MALDI-TOF MS was used for metabolomics whereover 100 metabolites from E. coli were detected as shown inFig. 4. The same authors acknowledged that MALDI-TOF MShas been less commonly used for the characterization of smallmolecules. In fact, when analyzed by this technique, smallmolecules suffer from matrix ion interferences and detectorsaturation in the low mass range. However, Edwards andKennedy62 demonstrated that negative ionization modeMALDI-TOF MS with 9-aminoacridine (9-AA) as the matrixappears to be a promising tool for metabolic profiling. Forfuture work, the authors propose the use of separations toimprove the number of compounds detected and detectionlimits by minimizing competitive ionization.

Furthermore, MALDI-TOF MS can be used for metabolicprofiling by detecting specific metabolic biomarkers that arean indicator of the presence of a particular microorganism.4

For example, Persson et al.63 were able to detect bacteriochloro-phyll a and homologs of bacteriochlorophyll c that arecharacteristic of the bacteria Chlorobium tepidum. The authorssuggested that MALDI-TOF MS can provide taxonomic infor-mation by studying the pigment composition of photo-synthetic bacteria.

Improving MALDI-TOF MSreproducibility

Applications of MALDI-TOF MS in environmental micro-biology, either for identification or typing, still present somelimitations as different factors can influence the reproducibil-ity of the method or lead to the misidentification of bacteria.25

These limitations may be due to different reasons such as thecomposition of the bacteria’s cell wall or the choice of theappropriate sample pre-treatment method. According to Wanget al. and Valentine et al.,25,64 the same species can producedifferent mass spectra if different chemical extraction methodsor growth conditions are used. Furthermore, Toh-Boyo et al.,65

demonstrated that matrix surface morphology heterogeneity isan important factor contributing to mass spectra profile repro-ducibility in bacteria MALDI-TOF MS analysis. The authorspoint out the importance of the sample preparation strategy toreduce or eliminate the MALDI matrix morphology heterogen-eity, thereby reducing the variability of the bacteria mass spec-tral profiles. Also, the growth age can have an influence onMALDI-TOF MS results. At early stages, bacteria produceincreased amounts of proteins as these are needed for growth.Therefore, the quantity of proteins varies with age. Due tothese reasons, it is very important to identify bacteria grownunder similar conditions and, when performing comparativestudies, it is important to use bacteria that have entered thesame growth phase.7

As previously discussed and according to Havlicek et al.,2

there are different databases available in the market for micro-organism identification using MALDI-TOF MS. The identifi-cation of bacteria is performed with the aid of a proteindatabase, where the unknown protein spectrum is comparedto reference spectra. Therefore, this database must be, asmuch as possible, complete. However, these platforms do notcurrently include the broad range of microorganisms found inthe environment.14 The protein databases are biased towardsclinical organisms. In fact, some of the previously describedworks that use MALDI-TOF MS for microorganism identifi-cation in environmental samples argue that a more environ-mentally-oriented database should be a priority.14,52 Moreover,if a microorganism is exposed to a stressful environment, itsprotein profile can change due to the production of stress-related proteins which may lead to its misidentification. Thus,the way in which the databases are populated with spectra formatching purposes may require special considerations relativeto clinical databases, since the range of environmental stressescan be quite a bit more variable.

Conclusions

MALDI-TOF MS can be a powerful tool in the field of environ-mental microbiology and bioremediation research. Thistechnique has already been proven to be a workhorse inproteomics. A significant number of papers describe the use ofthis technique for the identification and differentiation ofenvironmental microorganisms through their protein profile.In fact, MALDI-TOF MS can be used, not only for microorgan-ism identification and differentiation but also for detection ofprotein or metabolite biomarkers that can be used as an indi-cator of the presence of a specific bacterium. This identifi-cation is important to detect possible microbial pathogens, tostudy the bacterial community within the environment, or to

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identify contaminant-degrading bacteria. The application ofMALDI-TOF MS for metabolomics still has room for improve-ment specifically when applied to bioremediation studies toconfirm the bacteria’s capacity to degrade toxic pollutants.Furthermore, this technique can be used as an important toolfor bacterial source-tracking as identification to the strain levelis possible.

In spite of its recognized advantages, MALDI-TOF MS repro-ducibility is influenced by culture conditions and instrumentalparameters. Therefore, it is very important to use the samesample preparation protocol and to ensure that the analyzedbacteria are all at the same growth stage. Additionally,improvements in the protein database should be made toinclude more environmental microorganisms and to take intoaccount different protein profiles that can be obtained due toenvironmental stress.

We believe that the potential of MALDI-TOF MS in the fieldof environmental microbiology has yet much to be explored.Currently, MALDI-TOF MS methods for bacterial identificationstill require validation from molecular methods due to thelimitations described. Additionally, challenges are encoun-tered due to the complexity of environmental samples and dueto the diversity of environmental microorganisms. However,future advancements such as the improvement of the proteindatabases and sample preparation will allow this technique tobe implemented as a routine method for environmental micro-biology, eventually replacing conventional methods. In fact,MALDI-TOF MS is simple to perform allowing a low cost andfast analysis. This technique can be further explored in bio-remediation research as an important tool for the rapid identi-fication of site-specific bacteria present in a contaminatedenvironment or the identification of enzymes responsible forthe production or removal of chemical hazards.

Acknowledgements

Support for this work is acknowledged from the CollaborativeLaboratories for Environmental Analysis and Remediation atThe University of Texas at Arlington. This consortium is largelysupported by philanthropic contributions by landowners andcitizens concerned about the potential environmental impactof industrial processes.

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