Trends in Analytical Chemistry 52 (2013) 16–22

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Trends in Analytical Chemistry

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Review

Foodomics in microbial safety

Jasminka Giacometti a, Djuro Josic a,b,⇑a Department of Biotechnology, University of Rijeka, Radmile Matejcic 2, HR-51000 Rijeka, Croatiab Warren Alpert Medical Scholl, Brown University, Providence, RI, USA

a r t i c l e i n f o

Keywords:Biomarker

Cellular markerFood analysisFoodomicsFood pathogenFood safetyMicrobial contaminationMicrobial proteomicsMicrobial safetyToxins

0165-9936/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.trac.2013.09.003

⇑ Corresponding author at: Department of BiotechRadmile Matejcic 2, HR-51000 Rijeka, Croatia. Tel.: +3

E-mail address: djuro_josic@brown.edu (D. Josic).

a b s t r a c t

With traditional methods of food analysis, foodomics offers considerable opportunities to assess produc-tion, delivery and monitoring of the quality and the safety of food. Omics analyses of food pathogens, andof food that is contaminated with microbial agents and toxins provide reliable information about micro-bial activities during the infection, the outbreak of disease and the recovery period. Omics methods areeffective tools for identifying cellular markers for the adaptive behavior of pathogenic microorganismsunder different stress conditions and markers for microbial contamination of food.

� 2013 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162. Use of omics methods to identify microbial contaminants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183. Use of omics to follow microbial adaptation to stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3.1. Temperature shock and microbial adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183.2. Osmotic stress adaptation and the influence of high hydrostatic pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193.3. Influence of other stress factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

4. Formation of biofilms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205. Microbial toxins in food. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

1. Introduction

Microorganisms are essential for the function of the digestivetract of humans and animals, and, for millennia, they have beenused by human kind as necessary tools for fermentation processesin food technology and biotechnology [1]. There are also more than250 microbial pathogens known to cause food-borne illnesses.New risks are being encountered because of changing characteris-tics of the relevant microorganisms and food-production method-ologies, and changes in the environment and ecology, and theincrease in the global trade of foodstuffs. In addition, demandson food safety increase steadily. Due to the nature of both foodpathogens and our food chain, measures to ensure food safety have

ll rights reserved.

nology, University of Rijeka,85 51 58 45 60.

to be implemented on a global scale, necessitating also a generalapproach to this important topic [2]. The number of outbreaks ofillness caused by food pathogens was recently reported as risingin some industrialized countries. There is also a shift from thetraditional problems with food from mostly animal origin (e.g.,meat, eggs, and milk products) to food of plant origin, shellfish,and traditional fermented food products. In recent years, numerousincidents have occurred in contamination of fresh and processedfood with foodborne pathogens [3].

Proteomics, peptidomics and metabolomics techniques offerconsiderable opportunities to assess animal and plant health andproduction, and to monitor the quality and the safety of food ofanimal and plant origin. Some of these methods and the strategiesfor their use are listed in Table 1.

Genome, proteome, lipidome and metabolome analyses ofpathogens and their metabolites in contaminated food providereliable information about their activities during infection, out-

J. Giacometti, D. Josic / Trends in Analytical Chemistry 52 (2013) 16–22 17

breaks of disease and healing periods. Importantly, functionallyrelevant proteins and products of metabolism are identified inorder to trace microbial infection, so in-vivo proteome and metab-olome data can help to guide further functional analysis efficiently[4–6].

Technical shortcomings in the past limited quantification, iden-tification of post-translational modifications (PTMs), coverage ofsecreted and low-abundance proteins, and identification of lowconcentrations of microbial metabolites in foodstuffs. New omicsmethods introduced into food analysis, so called foodomics thatincludes proteomics, peptidomics, glycomics and metabolomics,

Table 1Some omics data on-line repositories. {Modified according to [53], with permission} *

Data types Online resource

Genomics Genomes OnLine Database (GOLD) Repository of completed andMicrobial Genome Database(MBGD)

MBGD is a database for commicrobial genomes (orthologanalysis and gene order com

National Microbial Pathogen DataResource (NMPDR)

The NMPDR provided curatecomparative analysis of genoemphasis on the food-borne

Transcriptomics Gene Expression Omnibus (GEO) Microarray and SAGE-based

Stanford Microarray Database(SMD)

Microarray-based genome-w

ArrayExpress -functional genomicsdata

Functional genomics experimmicroarray and high through

ExPASy – Bioinformatics ResourcePortal

Links to transcriptomics

Proteomics World-2DPAGE Links to 2D-PAGE data

ExPASy – Bioinformatics ResourcePortal

Link to protein sequences an

ExPASy – Bioinformatics ResourcePortal

Links to mass spectrometry

BIOBASE BKL PROTEOME™ is a databamanuallycurated details fromand easily searchable format

PRIDE – Proteomics IdentificationsDatabase

The PRIDE PRoteomics IDEntcompliant, public data reposandpeptide identifications, psupporting spectral evidence

Metabolomics MetaCyc MetaCyc is a database of nonmetabolic pathways

The Molecular Ancestry Network(MANET)

MANET database traces evolbiomolecular networks

Kyoto Encyclopedia of Genes andGenomes (KEGG)

KEGG is a database resourceutilities of the biological sysecosystem, from molecular-lmolecular datasets generatedhighthroughput experimenta

Metabolite and Tandem MSdatabase (METLIN)

The METLIN Metabolite Dataas well as tandem mass spec

Lipidomics Lipid Metabolites and PathwaysStrategy (LIPID MAPS)

Genome-scale lipids databas

The official database of JapaneseConference on the Biochemistry ofLipids (JCBL)

Lipid class database

Avanti,Polar Lipids,Inc. Services in research and pharphospholipids, sphingolipids

The AOCS Lipid Library The AOCS Lipid Library

Cyberlipid Lipid databases and encyclopLipidHome ‘‘LipidHome’ theoretically ge

* This table presents some of the databases that store and distribute genome-scale omicshave associated data-dissemination resources such as glycomics and fluxomics, and are thomics data resources, but, rather, provides a reasonably broad sample of the data that a

together with already established genomic methods, are emergingto overcome most of these limitations and challenges. This will fur-ther improve in-vivo investigations and enhance the value of thesetechniques as an important resource to investigate and to combatinfectious diseases [4–6]. This perspective brings the opportunityto develop new targets in order to ensure food safety that is impor-tant for human health and for agriculture, food processing and foodstorage. Ensuring food safety in the future will require new meth-ods for identification, monitoring and assessing food-borne haz-ards during production, storage, delivery and consumption.

Description URL

ongoing genome projects http://www.genomesonline.orgparative analysis of completely sequenced

identification, paralog clustering, motifparison)

http://mbgd.genome.ad.jp/

d annotations in an environment formes and biological subsystems, with anpathogens

http://www.nmpdr.org/FIG/wiki/view.cgi

genome-wide expression profiles http://www.ncbi.nlm.nih.gov/geo

ide expression data http://smd.princeton.edu/

ents include gene expression data fromput sequencing studies

http://www.ebi.ac.uk/arrayexpress/http://www.expasy.org/transcriptomics

http://us.expasy.org/ch2d/2d-index.html

d identification http://www.expasy.org/proteomics/protein_sequences_and_identification

and 2-DE data http://www.expasy.org/proteomics/mass_spectrometry_and _2-DE_data

se and data analysis platform containingthe PubMed literature in a highly structured

http://www.proteinscience.com/databases.htm

ifications database is a centralized, standardsitory for proteomics data, including proteinost-translational modifications and

http://www.ebi.ac.uk/pride/

redundant, experimentally elucidated http://metacyc.org/

ution of protein architecture onto http://www.manet.uiuc.edu/

for understanding high-level functions andtem, such as the cell, the organism and theevel information, especially largescale

by genome sequencing and otherl technologie

http://www.genome.jp/kegg/pathway.html

base is a repository of metabolite informationtrometry data

http://metlin.scripps.edu/index.php

e http://www.lipidmaps.org

http://www.lipidbank.jp/

maceutical scientists with the highest qualityand sterols

http://avantilipids.com

http://lipidlibrary.aocs.org/index.html

edia http://www.cyberlipid.orgnerate lipidc molecules and useful metadata http://www.ebi.ac.uk/apweiler-

srv/lipidhome

data sets through publicly accessible Web sites. Some omics technologies do not yeterefore not included in this table. This table does not represent all publicly availablere readily accessible to researchers today.

18 J. Giacometti, D. Josic / Trends in Analytical Chemistry 52 (2013) 16–22

In addition, there has been considerable public interest in thesafety of the investigation of fungal toxins, toxic chemicals in food,and possible changes during food processing [7]. Microbialinfection may cause contamination with mycotoxins, bacterialand other toxins in the food, soil, water and air (such as algae).Poisonings caused by toxic components are sometimes difficultto link to a particular food, as the onset of the effects may begradual and undetected until chronic or permanent damage occurs[3,8].

This article focuses on the role of foodomics in assuring foodsafety. Pathogenic bacteria (i.e. Escherichia coli strains, Salmonella,some Bacillus strains, and Campylobacter) are of the greatestcurrent concern in fresh products, while Listeria monocytogenescontamination is of primary concern in processed products [9].The pathogenic fungi, Aspergillus, Fusarium, Penicillium, Alternariaand Claviceps, and some algae are the eukaryotic food pathogenswith the highest toxigenic potential [10,11]. Viruses and prionswere also recently discussed as further causes for concern forsafety, especially in food of animal origin [5], but this topic is outof the scope of this review.

2. Use of omics methods to identify microbial contaminants

There are well-established, sensitive methods available that aremostly based on immunochemical techniques for detection of bac-teria and their toxins. Omics technologies, mostly proteomics,genomics, glycomics, lipidomics and metabolomics, are further,more sensitive and specific ways for identifying microbial foodcontaminants and their toxins, and for monitoring cleaning andsanitation [4,5]. Among others, DNA microarray technology,GC-MS based metabolomics, LC-MS/MS-based proteomics and lip-idomics methods were applied [6,12–14]. In order to followchanges during food processing, omics investigations of modelmicroorganisms under stress conditions, such as cold and heat,osmotic pressure, high pressure, availability of nutrients and otherenvironmental factors, are essential in order to follow theiradaptation and reaction to extreme conditions [11].

There are investigations of several model microorganisms bothbacteria and fungi. Their adaptive stress response is a crucial modeof cellular protection towards environmental and food-relevantstress. Cellular and metabolic biomarkers quantitatively correlateto the adaptive stress and can also predict the impact of theenvironmental change on ability of microbes to resist and to sur-vive. Minimally processed, so-called ‘‘ready to eat’’ food that hasbecome very popular in recent years, and antimicrobial washingby using different disinfectants and natural antimicrobial agentsare other important factors that dramatically change populationsof microorganisms in fresh food.

Such food can retain some potential pathogenic bacteria, suchas Aeromonas spp. and Yersinia spp., and even some unexpectedcontaminants [15]. It is also documented that the recent seriousoutbreak of food poisoning in Germany, caused by a novel strainof E. coli appeared after consuming such minimally-processed food[German Federal Institute of Risk Assessment, 30 June 2011].

3. Use of omics to follow microbial adaptation to stress

The adaptive stress response of a microbial population,especially bacteria, is a crucial mode of cellular protection towardsenvironmental and food-related stresses. Some cellularcomponents, mostly specific proteins and metabolites, quantita-tively correlate to adaptive stress and can also predict the impactof changing environments on microbial ability to resist and to sur-vive [11,15].

The ability of microorganisms to develop resistance uponactivation of adaptive stress responses is also beneficial for variousindustrial applications [16]. Den Besten et al. [17] identified severalmolecular biomarkers for stress-adaptive behavior at transcrip-tome, proteome and metabolome levels. In Bacillus cereus, as amodel microorganism, potential candidate biomarkers to stressresponse were identified, such as transcriptional regulator rB,catalases involved in H2O2-scavenging, chaperones and ATP-dependent Clp proteases involved in protein repair andmaintenance.

In an overview, Abee et al. [18] integrated three different omicsstrategies, namely use of information from transcriptomics andproteomics data, and determination of the activity of marker en-zymes. This approach led to the identification of biomarkersimportant for B. cereus cells in prediction of the robustness levelof adaption of this microorganism towards the lethal stresses.

Investigations using a wide variety of environmental changeswere further performed with B. cereus, and were extended tothe other bacterial species [10–14,17,18] and different fungi[19,20]. Such quantitative approaches using several omics meth-ods led to prediction of microbial performance using molecularbiomarkers for the early detection of food pathogens and to con-trol of their adaptive behavior that results in enhanced resistance[11–14,17–20].

3.1. Temperature shock and microbial adaptation

Temperature stress impairs DNA replication, transcription, andtranslation processes [21]. After cold shock, microbial cells haveto cope with several recognized problems, such as low membranefluidity, high superhelical density of the DNA for opening thedouble helix, decrease in enzyme activities and adjusted proteinlevels, adaptation of ribosomes function at low temperatures andinitiation of translation of secondary structures in RNA. Upon theseconditions, appropriate signal-transduction processes are activatedand subsequently lead to the mobilization of necessary stress-pro-tection measures through modifications in gene-expression andprotein-function activities [22]. Insights into gene-expressionchanges mobilized during stress-adaptation responses were re-cently gained through transcriptome and proteome stress analysisin bacteria suggesting that these bacteria have developed differentstress regulatory networks [23]. In B. cereus, heat shock also had astrong rB-activating effect, but other stresses (such as ethanolshock, osmotic shock, and acid stress) were also found to lead tothe activation of rB [24]. A mechanistic understanding of therB-activation processes and assessment of its regulons couldprovide tools for pathogen control and inactivation in the foodindustry and clinical settings.

Some investigations studied the cold-shock response and cold-shock proteins (Csps) using E. coli and B. subtilis as model systems.Using a combined transcriptomics and proteomics approach, Bud-de et al. [25] identified the members of the chill-stress stimulon ofB. subtilis on a genome-wide scale. A total of 580 genes, represent-ing approximately 14% of the protein-coding capacity of B. subtilis,displayed several temperature-dependent alterations at genome,proteome and metabolome levels.

These authors suggest that the heat-shock proteins have a ma-jor function in cell protection during stress situations. Betterunderstanding of the cold-shock response of food-borne pathogensis necessary to prevent food spoilage and poisoning. Using compar-ative proteomics analysis of hypo-thermally adapted E. coli O157wild-type and rpoS mutant strains, Vidovic et al. [26] identifiedproteins that were differentially expressed upon cold temperature,e.g., RpoS, a 37.8 kDa protein that regulates the expression of pro-teins involved in homeoviscous adaptation during cold shock, and

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various proteins involved in central metabolic pathways of thisfood-borne pathogen.

Bacterial thermosensing functions probably stem from changesin the plasma-membrane fluidity and localization of some key pro-teins in this organelle. They are compatible with the proposedmembrane location of peptidyl glutamyl peptide hydrolase (PgpH).Liu et al. [27] suggest that the PgpH multi-drug-resistance proteinis a potential mediator of cold signaling in L. monocytogenes.Adaptation processes related to proteins implicated in metabolicpathways for energy production were present to a higher level inthe cells grown at 4�C. Increased Csp synthesis and activity atlow temperatures provides DNA and RNA chaperone functions,which are needed in cold-exposed L. monocytogenes cells to helpresolve structural hurdles of nucleic acids [21].

In order to survive stress caused by processing, food-bornepathogens have developed distinct regulatory mechanisms asadaptation to new environmental conditions (e.g., a diverse rangeof PTMs) and change in fatty-acid composition and membrane flu-idity after cold and heat stress factors can further regulate theactivity and the abundance of specific proteins in bacterial, yeastand fungal cells, thereby controlling key processes that contributeto the virulence of these organisms [11,14].

3.2. Osmotic stress adaptation and the influence of high hydrostaticpressure

Sodium chloride is one of the most commonly employed agentsfor food preservation. Osmotic stress of pathogens is linked tosurvival and growth at high salt levels, mostly NaCl, and low wateractivity. Determination of changes following cellular stress adapta-tion can contribute to a better understanding of strategies and pos-sible cross-protection mechanisms on the basis of transcriptomicsand phenotype characteristics [17,18]. The transcriptome profilesfor two salt conditions were compared in order to investigate theoverlap and to identify specific responses of mildly and severelysalt-stressed cells. The whole-genome expression analyses ofmildly and severely salt-stressed B. cereus cells revealed an overlapin the transcriptome response.

The physiological response of the model bacterium B. subtilis tochanging osmolality has been analyzed in substantial detail at thelevel of single genes or proteins [28]. It is known that many mem-brane proteins, especially transporters of compatible solutes andions, play a crucial role in adaptation of the microbial cells to saltstress. Transcriptomics and proteomics approaches can provide aview of the dynamic changes occurring in salt-stressed B. subtiliscultures because these studies provide an unbiased view of cellscopying with high salinity. Furthermore, salt stress induced thetranscription of osmoprotectant transporters and can also provideprotection against other stresses, such as change of temperature[29,30].

Hahne et al. [31] applied whole genome-microarray technologyand metabolic labeling for quantitative proteomics analysis. Theirresults indicated a well-coordinated induction of gene expressionsubsequent to an osmotic upshift, which involved large parts ofthe genes that are regulated by the same regulatory protein. About500 osmotic up-regulation genes of B. subtilis genes were detectedand they are rich basis for further in-depth investigation of thephysiological and genetic responses of microbial cells to hyperos-motic stress.

L. monocytogenes, e.g., can survive a variety of environmentalstresses, such as high salt levels, and a range of temperatures,and it can also tolerate acidic conditions. Consequently, thehigh degree of bacterial resistance is a reason for difficulties incontrolling this pathogen in the food. Another problem is the L.monocytogenes resistance related to treatment during food pro-cessing and preservation [32]. Extended studies of this bacterium

also revealed a complex process in understanding the mechanismsof action of both salt shock proteins (Ssps) and stress acclimationproteins (Saps) [33].

High-pressure hydrostatic processing (HHP) is a non-thermalprocess used in inactivation and elimination of pathogenic andfood-spoilage microorganisms. The use of HHP technology is oneof the recently developed methods for food preservation [34].

Pressure treatment induced various changes in microbial cells,including alterations in the cell membrane, cell morphology, effectson proteins, including enzymes and effects on genetic mechanismsof microorganisms. However, the mechanisms of microbial inacti-vation by HHP are still not fully understood. Microorganisms varyin their response to pressure [35,36]. Generally, Gram-positive bac-teria are more resistant to pressure than Gram-negatives and cocci.Certain bacteria and strains, such as E. coli O157:H7 and threestrains of L. monocytogenes can be exceptionally pressure resistant.The HHP treatment ranges is 200–800 MPa for a period of5–30 min, depending on the food matrices and microorganisms.In food matrices, such as mussel, HHP treatment causes disruptionof lysosomes, and both changes of sarcoplasmatic proteins andactivities of some enzymes degraditive enzymes. In B. cereus, flagel-lin, a protein that forms the filament in bacterial flagellum, was theprotein that quantitatively changed. Interestingly, a significant de-crease of flagellin concentration was also observed after stresscaused by bactericide agents [6,35–37]. Little is known about thepressure sensitivity of toxic mold growth (e.g., the inactivation ofAspergillus niger was often combined with shock coming from oth-ers processes, such as post-heat shock) [38].

Vanlint et al. [39] examined and compared the HHP resistanceamong strains of E. coli, Shigella flexneri, Salmonella typhimurium,Salmonella enteritidis, Yersinia enterocolitica, Aeromonas hydrophila,Pseudomonas aeruginosa and Listeria innocua. It was found thatHHP resistance could only be detected in some of the E. coli strains,indicating that a specific genetic predisposition might be requiredfor resistance development. HHP resistance proved to be a verystable trait that was maintained for >80 generations in the absenceof HHP exposure. These authors suggested that, at the mechanisticlevel, HHP resistance is not necessarily linked to derepression ofthe heat-shock genes, and is not related to the phenomenon ofpersistence.

C. jejuni had a specific response to HHP treatment, which couldnot be anticipated from the responses of other bacterial species tothis stress. Examining the response of C. jejuni to HHP shock, onlyproteins specific to oxidative stress were induced. This could ex-plain why C. jejuni is rather more sensitive to this treatment thanother Gram-negative bacteria [40]. In most cases, when HHP wasused as a tool to study protein function, other changes, such asmicrobial production of conjugated fatty acids after this treatment,were also observed. These changes have a significant effect on plas-ma-membrane fluidity, and so on the viability and the virulence offood pathogens [41].

3.3. Influence of other stress factors

Antimicrobial chemicals, such as potent oxidants, are widelyapplied to clean and to disinfect food-contact surfaces. However,the cellular response of bacteria to various disinfectants is unclear.Using a genome-wide comparative transcriptional approach,Ceragioli et al. [42] analyzed the general and specific transcriptomeresponses of the model organism B. cereus upon exposure to vari-ous disinfectant treatments. The exposure of these bacteria to dif-ferent antimicrobial agents resulted in up-regulation of genesinvolved in fatty-acid metabolism, metabolism of the quaternaryammonium compounds (QACs), and resistance membrane–bondedproteins, which are able to enhance bacterial tolerance towardother antibacterial compounds. Furthermore, in increased levels

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of genes that activated DNA, a damage-repair system was ob-served, including the SOS response. These results may affect therecontamination capacity of B. cereus and, in this way, food qualityand safety.

Catalase production and activation is the important line of de-fense of bacteria against H2O2, the agent that is frequently usedfor chemical decontamination. Most bacteria produce one or morecatalases, which are a by-product of their aerobic growth [43].

Abee et al. [18] clearly displayed a pattern of organization of thesamples using a tree of the transcriptome samples and clusteredtranscriptome data, including stressors, such as detergents (ben-zalkonium chloride, hydrogen peroxide, peracetic acid, sodiumhypochlorite), ethanol, heat, various acids (HCl, lactic acid, aceticacid) and salt stress at different time intervals and concentrations.

Acquisition of resistance in E. coli has been related mainly tochanges in the composition of lipopolysaccharide (LPS) and fattyacids in the membrane [44]. In contrast to antibiotics, which havevery specific targets within the bacterial cell, the biocides affectmultiple cellular components [45].

Prolonged exposure to a high concentration of propionate (PA)during the food-processing systems and within the gut of infectedhosts have a stress effect on the proteome of S. enteritidis. Calhounet al. [46] noted a significant difference in the proteomes of PAadapted and unadapted S. enteritidis and affirmed the contributionof several proteins as biomarker candidates for PA-induced acidresistance.

In conclusion, new strategies and technologies have beendeveloped in order to reduce to a minimum number of cases offood contamination with pathogen agents. However, there are alsonew risks, caused by the adaptation of food-borne pathogens anddevelopment of new surviving strategies. And, finally, the changesduring stress adaptation in microbial transcriptome, proteome andmetabolome can be a further guide for detection of food-bornepathogens [6,18].

Fig. 1. Use of foodomics in the development pathway for food production, and as

4. Formation of biofilms

Formation of a microbial biofilm is the process by which amicrobial cell attaches to abiotic surfaces, the surfaces of otherunicellular organisms, the epithelia of multicellular organisms,and interfaces, such as that between air and water. The type of sur-face adhesion enables microbial cells, such as bacteria, to adaptthemselves favorably to their environment and to survive. Sur-face-attached bacteria, which may form a monolayer, and multi-layer biofilms use distinct structures for adhesion in biofilms, anddevelop distinct transcriptional profiles within these two structures[47,48]. Extracellular polymeric substances (EPSs) comprise biolog-ical polymers, such as exopolysaccharide, protein and DNA [49].These polymers mediate bacterial aggregation and surface attach-ment. They may also serve as a reservoir for extracellular, degrada-tive enzymes and the nutrients released by their function.

EPSs (or matrix) are important in both the formation and thestructure of the biofilm. They also protect the cells by preventingthe crossing of the antimicrobial and xenobiotics to the cells inthe biofilm and confer protection against environmental stresses,such as UV radiation, pH change, osmotic shock and desiccation(1,47–49).

The phenomenon of resistance within biofilms can be explainedby several mechanisms, including delayed penetration of the anti-microbial agents into the extracellular matrix, slowing of growthrate of organisms or other changes that include interaction of theorganisms with a surface. Better knowledge of biofilm formationand the mechanism of its degradation will provide important infor-mation for successful development of sanitation plans. The ulti-mate composite is a biofilm that is resistant to cleaners andsanitizers and is extremely difficult to remove [1,11,49].

Filamentous fungi and some yeasts also form biofilms. Villenaet al. [50] investigated an initial profiling of the intracellular prote-ome of A. niger ATCC 10864 biofilm cultures developed on polyes-

sessing food safety, origin and quality. {Adapted from [5], with permission}.

J. Giacometti, D. Josic / Trends in Analytical Chemistry 52 (2013) 16–22 21

ter cloth. Proteomics maps showed different expression patterns inboth culture systems with differentially expressed proteins in eachcase, where in biofilm cultures some proteins were overexpressedand differentially expressed. These results show significant differ-ences between the proteomes of A. niger and Candida albicans bio-film and free-living microbes, and indicate that cell adhesion is themost important stimulus responsible for biofilm development[1,11,50,51].

Omics methods have been very helpful to gain some insight intoincreased resistance of microorganisms in biofilms. Despite theremarkable improvements in these technologies in the past fewyears, the number of these studies is still insufficient.

5. Microbial toxins in food

Food toxins are cellular components or metabolic products offood pathogens. These substances are a severe threat to humanhealth. Bacterial and fungal toxins (mycotoxins) may be acutelyharmful, but the mycotoxins especially may also cause chronicdamage, such as teratogenic, immunotoxic, nephrotoxic and estro-genic effects [52]. Most intracellular and secreted bacterial toxinsthat cause food poisoning are intensively studied together withbacterial food poisoning.

The investigations of fungal toxins, toxic chemicals in food andpossible changes during the processing of foodstuffs are importantpublic health affairs [7]. Poisonings caused by toxic components,such as mycotoxins that come in the soil, water and air, are some-times difficult to link to a particular food. The effects may be subtleand may not be detected until chronic and/or permanent damageoccurs. Contamination with mycotoxins is a universal problem. Itfrequently occurs in developing countries, where suitable cultiva-tion, processing and storage technologies are implemented withdifficulty [8]. Identification of biomarkers of the most-relevant tox-ins that have been detected in this type of environment and theirmonitoring will yield a more accurate risk assessment.

Around 400 mycotoxins have been registered so far [8]. Theycan be found in a variety of foods, ranging from cereals, peanuts,spices, fruits and vegetables. In most cases, the mycotoxins foundin products of animal origin, mostly in meat, milk and eggs,originated from animal feed. The classes of mycotoxins areaflatoxins, ochratoxins, trichtothecenes, zearalenone, fumonisinstremorgenic toxins and ergot alkaloids. After suitable samplepreparation, even low concentrations of mycotoxins can bedetected using metabolomics techniques [10].

New safety risks have to be taken into consideration because ofthe continuous adaptation of the relevant foodborne pathogensand other potentially pathogen microorganisms to changes inproduction methodologies, changes in the environment and theincrease in the global trade of foodstuffs. Increased demand fortraditional food in industrial countries and improvements in livingstandards in some developing countries have to be taken intoconsideration [1,52].

6. Conclusions

Assuring food safety, especially microbial food safety, is still avery complex task. As shown in Fig. 1, the safety hazards can bepresent at any stage of the food chain, starting with productionof raw materials, and ending with food delivery and consumption.The effectiveness of food control should focus on identifying theissues that pose the greatest hazards, as well as the controls withinthe food-production systems and delivery in order to minimize therisk of microbial contamination and contamination with microbialand other toxins.

Foodomics will bring innovations into this field, and the use ofthese new techniques will play an important role in understandingthe approaches to solve the problems and to deal with newfood-safety challenges. These include the use of high-throughputfoodomics technologies for rapid identification of pathogens andpathogen metabolites, characterization of the interaction betweenthe pathogen and the food matrix, and determination of virulenceand microbial resistance. Usually, the use of only one of the abovelisted methods is insufficient for conclusive identification of one,especially new, food pathogen, and a thorough validation strategyusing complementary methods is necessary. For this reason, moreaggressive, innovative, high-throughput, global-level omics studiescombined with bioinformatics are required. These studies shouldbe performed with not only model microbial systems, but alsoin-vivo clinical sources and food safety. It will result in betteracceptance of omics methods and their more frequent use in rou-tine food analysis.

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