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Page 1: Biofilm-associated persistence of food-borne pathogens

Accepted Manuscript

Biofilm-associated persistence of food-borne pathogens

A. Bridier, P. Sanchez-Vizuete, M. Guilbaud, J.-C. Piard, M. Naïtali, R. Briandet

PII: S0740-0020(14)00090-2

DOI: 10.1016/j.fm.2014.04.015

Reference: YFMIC 2156

To appear in: Food Microbiology

Received Date: 9 February 2014

Revised Date: 15 April 2014

Accepted Date: 27 April 2014

Please cite this article as: Bridier, A., Sanchez-Vizuete, P., Guilbaud, M., Piard, J.-C., Naïtali, M.,Briandet, R., Biofilm-associated persistence of food-borne pathogens, Food Microbiology (2014), doi:10.1016/j.fm.2014.04.015.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.

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Biofilm-associated persistence of food-borne pathogens

A. Bridier1, P. Sanchez-Vizuete2,3, M. Guilbaud2,3, J.-C. Piard2,3, M. Naïtali2,3 & R.

Briandet2,3*

1. Irstea, UR HBAN, Antony, France; 2. Inra, UMR 1319 Micalis, Jouy-en-Josas, France; 3.

AgroParisTech, UMR Micalis, Massy, France.

Abstract

Microbial life abounds on surfaces in both natural and industrial environments, one of which

is the food industry. A solid substrate, water and some nutrients are sufficient to allow the

construction of a microbial fortress, a so-called biofilm. Survival strategies developed by

these surface-associated ecosystems are beginning to be deciphered in the context of

rudimentary laboratory biofilms. Gelatinous organic matrices consisting of complex mixtures

of self-produced biopolymers ensure the cohesion of these biological structures and contribute

to their resistance and persistence. Moreover, far from being just simple three-dimensional

assemblies of identical cells, biofilms are composed of heterogeneous sub-populations with

distinctive behaviours that contribute to their global ecological success. In the clinical field,

biofilm-associated infections (BAI) are known to trigger chronic infections that require

dedicated therapies. A similar belief emerging in the food industry, where biofilm tolerance to

environmental stresses, including cleaning and disinfection/sanitation, can result in the

persistence of bacterial pathogens and the recurrent cross-contamination of food products.

The present review focuses on the principal mechanisms involved in the formation of biofilms

of food-borne pathogens, where biofilm behaviour is driven by its three-dimensional

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ACCEPTED MANUSCRIPTheterogeneity and by species interactions within these biostructures, and we look at some

emergent control strategies.

Keywords: biofilm, food-borne pathogens, interspecies interactions, biocide tolerance, in-situ

microscopy, spatial modelling.

*Corresponding author:

Romain Briandet, UMR1319 Micalis, 25 avenue de la république, 91300 Massy France.

Telephone: 33 1 69 53 64 77 E-mail: [email protected]

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ACCEPTED MANUSCRIPT1. Industrial biofilms and food safety

Throughout the food chain, wet industrial surfaces can provide a solid substrate for the

development and persistence of spatially-organized vibrant ecosystems called biofilms, which

may contain pathogenic micro-organisms (Figure 1). Bacillus cereus, Escherichia coli,

Shigella spp. and Staphylococcus aureus have been detected in biofilms developing in the

dairy and egg processing industries (Jan et al., 2011; Sharma and Anand, 2002; Shi and Zhu,

2009), and Listeria spp., Staphylococcus spp., and Vibrio spp. have been isolated from

industrial equipment surfaces in seafood processing plants (Bagge-Ravn et al., 2003;

Gutierrez et al., 2012; Shi and Zhu, 2009). The bacterial contamination of food-contact

surfaces is a major factor in pathogen persistence in food processing environments, and is

believed to have a significant public health impact. Indeed, in industrialized countries, the

percentage of people suffering from food-borne diseases each year has been reported to be up

to 30% (WHO, 2002). Every year in France, more than 250,000 people acquire an infection

related to food contaminated by pathogenic micro-organisms. About 400 of these infections,

most of which are due to bacteria, have a fatal outcome. A French survey indicated that ca.

60% of food-borne infections occurred as a result of microbial transfer from equipment

surfaces to processed foods (Anonymous, 2011).

The settlement and persistence of food-borne pathogens in food processing environment is

intimately related to their response to both biotic and abiotic factors (Davey and O'Toole,

2000; Donlan, 2002; Dunne, 2002; Mariani et al., 2007; Stoodley et al., 2002; Winkelstroter et

al., 2013). Most of the pathogens involved in food-borne diseases are able to adhere to and

form biofilms on most materials and under almost all the environmental conditions

encountered in food production plants (Figure 2). Listeria monocytogenes 10403S was shown

to adhere to 17 different, food-use approved materials including metals, rubbers and polymers

(Beresford et al., 2001). The initial attachment of Salmonella enterica ser. Typhimurium to

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ACCEPTED MANUSCRIPTAISI 316 stainless steel surfaces was four times lower on those that had been polished using

bright-alum or electropolishing, when compared to untreated surfaces (Schlisselberg and

Yaron, 2013). The attachment of E. coli O157:H7 to beef-contact surfaces (i.e. stainless steel

and high-density polypropylene) was influenced by both the type of soiling substrate and

temperature; attachment occurred not only at 15°C (a temperature representative of beef

processing areas during non-production hours) but also during cold storage (Dourou et al.,

2011).

When a planktonically grown pathogen lands on a surface, it will most likely encounter the

interface of a resident biofilm rather than a sterile material, and this is similar to the case in

some food-related environments. Habimana et al.(2009) demonstrated that L. monocytogenes

recruitment in Lactococcus lactis biofilms was governed by biofilm rugosity and the nature of

the matrix. Once integrated into the surface community, bacteria may encounter a broad

diversity of local environments that generate heterogeneous cell populations with different

patterns of stress resistance (Stewart and Franklin, 2008). Specific local micro-environments

may be favourable to the fitness of pathogens with demanding growth conditions. This was

recently shown in the case of microaerophilic Campylobacter jejuni bacteria. When cultured

alone under atmospheric pressure, surface-associated contamination was essentially composed

of non-culturable bacteria, while C. jejuni in a mixed culture together with a Pseudomonas

strain remained in a culturable physiological state (Ica et al., 2012). Given the spatial

proximity between their inhabitants, biofilms in the food industry have recently been

suggested to be hot-spots for plasmid transfers, including antibiotic multi-resistant plasmids

(Madsen et al., 2012; Van Meervenne et al., 2014). Today, complementary microscopic,

analytical and spatial modelling technologies offer an opportunity to dissect at different scales

the influence and interplay of spatial organization and bacterial interactions on pathogen

behaviour (Wessel et al., 2013; Xavier et al., 2004).

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ACCEPTED MANUSCRIPTTo deal with this colonization of surfaces by unwanted organisms, industrial operators

implement a series of hygiene procedures which include cleaning and disinfection. Although

the efficiency of the latter has been demonstrated with respect towith planktonic bacteria, this

is far from being the case with biofilms that may be much more tolerant to these treatments

(Bridier et al., 2011a). It is therefore now recognized that biofilm resistance to sanitizing

procedures is multifactorial, resulting from an accumulation of different mechanisms related

to biofilm architecture. The use of chemical biocides, as well as polluting the environment,

has been also shown to result in only a transient perturbation of surface-associated ecosystems

(Leriche et al., 2003; Mettler and Carpentier, 1998). Food manufacturers therefore need to

address conflicting concerns such as consumer safety and environmental protection while

suffering from a lack of objective information to guide their technological choices.

2. How pathogens conquer industrial surfaces

The microbial colonization of surfaces is a process that involves both physico-chemical and

biological phenomena and is possibly implemented through a developmental model (Monds

and O’Toole, 2009). It starts with cell-surface contact that triggers a reversible adhesion of the

two corpuses through van der Waals, electrostatic and Lewis acid-base interactions (Bellon-

Fontaine et al., 1990). In the case of L. monocytogenes, the electrostatic charge of bacterial

cell walls (conferred by peptidoglycan anionic teichoic acids) and cell surface hydrophobicity

(enhanced by the presence of lactic acid) have been shown to govern its attachment to

stainless steel (Briandet et al., 1999a and 1999b). The presence of proteins anchored to the

cell wall may drastically affect microbial behaviour at the interfaces. In S. enterica, the BapA

surface protein is directly involved in biofilm formation at the liquid-air interface (Latasa et

al., 2005). As for L. monocytogenes, the inactivation of the SecA2 protein export pathway has

been shown to impact biofilm formation and architecture (Renier et al., 2013). Interestingly,

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ACCEPTED MANUSCRIPTan ubiquitous protein cell wall anchoring system mediated by transpeptidases (termed

sortases) has been characterized in Gram-positive bacteria (Mazmanian et al., 1999). This

allows the covalent linkage of cell surface proteins harbouring an LPXTG amino-acid

signature to the cell wall, some of them being involved in both adhesion and biofilm

development (Guiton et al., 2009).

A broad diversity of extracellular appendages such as flagella, pili or curli may be involved in

the initial stages of biofilm formation (Van Houdt and Michiels, 2010). Flagella are not only

the principal components for bacterial motility, but also play an important role in cell-surface

and cell-cell interactions (Figure 3). Flagella have been shown to be critical to the initial

contact and structuring of E. coli and Yersinia enterocolitica biofilms (Kim et al., 2008; Pratt

and Kolter, 1998; Van Houdt and Michiels, 2005). This was also reported in the case of a

flagella-deficient mutant of L. monocytogenes which displayed reduced initial adhesion but

hyper-biofilm development under flow conditions (Todhanakasem and Young, 2008). In the

case of B. cereus, flagella were shown to be required for static biofilm initiation at the liquid-

air interface, but not at the surface-liquid interface (Houry et al., 2010). Fimbriae (Latin for

‘thread’ or ‘fibre’) or pili (Latin for ‘hair’) are filamentous surface appendages which are

thinner and shorter than flagella and involved in cell-cell and cell-surface contacts. Pili have

long been characterized in Gram-negative bacteria but only recently in a number of Gram-

positive bacteria (Linke et al., 2008). In Gram-negative bacteria, pili have been implicated in

diverse functions e.g. adhesion, phage binding, DNA transfer, biofilm formation, cell

aggregation, host cell invasion and twitching motility (Proft and Baker, 2009). Type 1 pili are

the most common adhesion factors found in E. coli and their presence is critical to achieve a

stable initial attachment (Cookson et al., 2002). They also play an important role in Klebsiella

pneumoniae biofilm formation on polystyrene (Schembri et al., 2005) and in S. enterica ser.

Enteritidis adherence to polymers and stainless steel (Austin et al., 1998). Sexual pili,

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ACCEPTED MANUSCRIPTresponsible for gene transfer by conjugation (Ghigo, 2001; Reisner et al., 2003), are also

involved in adhesion, biofilm formation and virulence since they act as adhesion factors

allowing cell-surface interaction. Curli fibres are another type of pili present in Gram-negative

bacteria. They are amyloid-like proteins involved in attachment to surfaces and biofilm

formation. First described in E. coli (Chapman et al., 2002), curli have a variable effect on

bacterial attachment and biofilm formation, which is usually strain dependent (Boyer et al.,

2007; Cookson et al., 2002; Ryu et al., 2004). These fibres, which are also produced by S.

enterica biofilm cells, were shown to enhance sanitizer tolerance in mono and multi-species

conditions (Ryu and Beuchat, 2005; Wang et al., 2012; Wang et al., 2013). In Gram-positive

bacteria also, pili have been definitely assigned as important players in the initial adhesion of

bacteria to surfaces, in bacterial aggregation as well as in biofilm formation (Mandlik et al.,

2008). This applies to Gram-positive food-borne pathogens, as shown by recent studies

in Enterococcus spp. (Nallapareddy et al., 2006; Sillanpaa et al., 2010; Garsin and Willems,

2010).

The complex matrix of mature biofilms acts as a “shield” against environmental stresses and

antimicrobial agents. The principal components of this gelatinous material are water and

biopolymers (Flemming and Wingender, 2010; Flemming, 2011). Cellulose has been

identified as the main matrix component in S. enterica, E. coli and Cronobacter sakazakii

(Grimm et al., 2008; Healy et al., 2010). St. aureus poly-N-acetylglucosamine (PNAG), which

is biochemically close to the polyglucosamine (PGA) produced by E. coli, is important to

maintaining the architectural integrity of biofilms and their associated protection against the

action of biocides (Izano et al., 2008; Wang et al., 2004). Extracellular DNA has been more

recently identified as a contributor to the matrix and has been detected in large quantities in

both Gram-negative and Gram-positive biofilms (Izano et al., 2008; Rice et al., 2007). Recent

studies have shown that food-borne pathogens such as L. monocytogenes and B. cereus

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ACCEPTED MANUSCRIPTrelease DNA as an adhesion mediator or a major component in their biofilm matrix (Harmsen

et al., 2010; Vilain et al., 2009). Recent reports have described proteinaceous amyloid fibres

as a common structural component in biofilm matrices (Dueholm et al., 2010; Romero et al.,

2010). In St. aureus, small peptides called phenol soluble modulins (PMS) can self-assemble

into amyloid-like fibres that participate in biofilm assembly and integrity (Schwartz et al.,

2012).

The different stages of surface contamination are driven by quorum-sensing (QS) and other

genetic regulators. For S. enterica and many other pathogens, cellular transition between

motility and sessility involves the second messenger cyclic-di-GMP (Cotter and Stibitz, 2007;

Simm et al., 2007; Ahmad et al., 2013). In St. aureus, the accessory gene regulator (agr)

system plays a central role in QS signalling which is essential to complex 3D architectures

and mature biofilm phenotypes (Arvidson and Tegmark, 2001; Kong et al., 2006). However,

its complex role in biofilm formation is probably strain- and growth condition-dependent

(Yarwood et al., 2004). In L. monocytogenes, a similar system is involved in bacterial

adhesion and virulence (Autret et al., 2003; Rieu et al., 2007). As for B. cereus, multicellular

behaviour is controlled by the CodY regulator which induces biofilm formation under nutrient

starvation conditions but enhances the expression of motility and virulence factors when

nutrients are abundant (Lindback et al., 2012).

The end of this cycle is cell dispersion: bacteria are able to actively detach from the

community and start another colonization cycle in a new habitat. QS signals are often

involved in this active process. The aforementioned agr system activates dispersion in St.

aureus biofilms via protease production (Boles and Horswill, 2008). Cyclic-di-GMP

indirectly represses PGA synthesis by controlling CsrA repressor activity in E. coli (Wang et

al., 2005; Jonas et al., 2008). The norspermidine and norspermine polyamides produced by B.

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ACCEPTED MANUSCRIPTsubtilis biofilm cells are able to disrupt St. aureus and E. coli biofilms (Kolodkin-Gal et al.,

2012).

During recent decades, the development of mathematical modelling tools such as individual-

based model (IBM) has constituted a promising approach to better understanding and

predicting the processes that lead to the development of bacterial populations, including

pathogens (Wang and Zhang, 2010). Indeed, using IBM approaches, a bacterial community is

modelled by describing the actions and properties of its individual parts (i.e. bacterial cells),

so that the dynamics and variability of the response of each cell in the structure can be

considered (Kreft et al., 2001). IBM was combined to food micro-environment descriptions in

order to predict growth of the pathogen L. monocytogenes on cheese (Ferrier et al., 2013). The

results obtained showed that the model generated consistent bacterial counts when compared

to those observed experimentally, and suggested the reliability of this approach in predicting

pathogen development. IBM was also used successfully to predict biofilm structure and

identify the processes governing biofilm development (Xavier et al., 2004). For instance, it

was shown that the structure of a nitrifying biofilm was dramatically influenced by the

production of extracellular polymeric substances and capsule formation (Kreft and

Wimpenny, 2001). More recently, a multi-scale model incorporating both IBM and constraint-

based metabolic modelling was developed to study the formation of Pseudomonas aeruginosa

biofilms (Biggs and Papin, 2013). Interestingly, the authors demonstrated that the resulting

model framework correctly recapitulated known biofilm characteristics and yielded useful

predictions.

In general, the development of improved computational approaches (Lardon et al., 2011) in

the coming years is expected to facilitate the generation of data and hence provide a clearer

understanding of the development and three-dimensional organization of biofilms.

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ACCEPTED MANUSCRIPT3. 3D-driven heterogeneities

In a bacterial population, even in a homogeneous environment in terms of the

physicochemical conditions prevailing, it has been shown that isogenic cells can exhibit

stochastic fluctuations in their gene expression levels (Elowitz et al., 2002; Thattai and van

Oudenaarden, 2004; Chalancon et al., 2012). This “noise” in gene expression is important

because it generates phenotypic variations and cellular differentiation within the population,

regardless of environmental conditions (Stewart and Franklin, 2008). The resulting

heterogeneity may lead to an increase in the fitness of the community because of its greater

adaptability to adverse conditions such as antimicrobial stress, due to the presence of different

phenotypes.

In addition, by living in biofilms, bacteria will specifically experience multiple micro-

environments depending on their location in the spatial structure (Stewart and Franklin,

2008). Such local heterogeneity directly impacts global biofilm traits such as mechanical

cohesiveness or resistance to biocide treatment for example and therefore plays a key role in

the ability of pathogens to persist in the food chain (Verraes et al., 2013). The origins of this

heterogeneity are now starting to be better understood and they appear to be intimately related

to the three-dimensional architecture of biofilms.

The presence of an oxygen gradient in biofilms, including totally oxygen-depleted areas in the

internal layers of these structures, was revealed at an early stage using microelectrodes (Xu et

al., 1998). Working on P. aeruginosa biofilms with a thickness of approximately 150 µm, the

authors demonstrated that oxygen concentrations fell dramatically in the upper 30 µm of the

structure. This phenomenon appeared be associated with the consumption of oxygen by

respiring cells in these upper layers, rather than a limitation on penetration related to physical

exclusion (Stewart and Franklin, 2008). Such observations could also be extended to other

nutrients (glucose, nitrate, etc.) and inverted patterns for metabolites such as organic acids, the

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ACCEPTED MANUSCRIPTlatter accumulating in internal layers (Stewart, 2003). The direct consequence of this chemical

heterogeneity is the adaptation of bacteria to their direct local micro-environment and the

emergence of a physiological heterogeneity within the biofilm (Bordi and Bentzmann, 2011).

Indeed, the stratified growth or metabolic activity of P. aeruginosa in a biofilm has been

demonstrated by different studies (Werner et al., 2004; Xu et al., 1998). Using fluorescent

gene fusion reporters and “anatomical” hallmarks, Serra et al. (2013) visualized the

physiological heterogeneity in macrocolony biofilms of E. coli K-12 as a function of known

global regulatory networks in response to biofilm-intrinsic nutrient gradients. This

heterogeneous pattern of physiological activity may play a key role in persistence of the

biofilm throughout the expression of specific protective factors, especially in internal biofilm

layers that are depleted in terms of nutrients and oxygen (Davies, 2003; Stewart and Franklin,

2008). Numerous research studies have revealed the specific gene expression profiles of

bacteria in biofilms compared to planktonic suspensions, in different pathogenic species that

include E. coli, P. aeruginosa, L. monocytogenes, St. aureus or S. enterica (Beloin et al., 2008;

He and Ahn, 2011; Ren et al., 2004; Sauer et al., 2002; Tremoulet et al., 2002a and 2002b).

These studies reported for example that genes involved in oxidative or acidic stress responses

were induced in biofilms and could participate in the increased resistance observed to some

oxidizing agents.

As well as this physiological heterogeneity, the way of life of biofilms may also promote the

emergence of genetic diversity within the community (Figure 4). The harsh conditions that

prevail in a biofilm, and particularly local oxidative stress, may enhance genetic mutations

and thus favour the emergence of variants (Ciofu et al., 2005; Mai-Prochnow et al., 2008;

Boles and Singh, 2008; Conibear et al., 2009). When focusing on Pseudomonas fluorescens, a

bacterium involved in food spoilage, Rajmohan et al. (2002) and Workentine et al. (2013)

demonstrated that the emergence of variants is specific to surface-associated growth and not

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ACCEPTED MANUSCRIPTobserved in planktonic cultures. Moreover, using strains expressing different fluorescent

proteins, these authors analysed the spatial distribution of colony morphological variants in

mixed-culture biofilms with the wild-type phenotype, and revealed that two variants displayed

a significant growth advantage when compared to the wild-type in a biofilm. Indeed, biofilm-

emerging variants may exhibit specific characteristics such as a modified ability to form a

biofilm, or resistance to antimicrobials, which finally impact the stress tolerance of the whole

community, as has been shown for pathogens like P. aeruginosa or St. aureus (Boles et al.,

2004; Savage et al., 2013) (Figure 5).

The close relationship between the 3D-driven heterogeneities and functional properties of

biofilms imply the need to develop and apply suitable methods and techniques so that these

heterogeneities can be studied. In the area of gene expression, Lenz et al. (2008) developed a

technique based on a combination of laser capture micro-dissection (LCM) microscopy and

multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR), which was used to

isolate and quantify RNA transcripts from small localized areas in a P. aeruginosa biofilm.

They were thus able to see that mRNA levels of individual genes were not uniformly

distributed throughout the three-dimensional structure but could vary by several orders of

magnitude over small distances. LCM was subsequently combined with Affymetrix

microarrays to analyse the transcriptome of targeted local sub-populations in a P. aeruginosa

biofilm (Williamson et al., 2012). Such methodological developments, together with

development of a global computational model of spatio-temporal gene and protein expression

in biofilms (Zhang et al., 2013) have provided access to a map of the heterogeneous gene

expression prevailing in these structures and thus greatly improved our representation of these

complex biological edifices.

With respect to the greater resistance to biocides of bacteria in biofilms compared to

planktonic suspensions (Mah and O’Toole, 2001; Bridier et al., 2011a; Vazquez-Sanchez et

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ACCEPTED MANUSCRIPTal., 2014), it is also crucial to consider the heterogeneity of bacterial susceptibility in the

structure if we are to understand the mechanisms governing this resistance. Time-lapse

methods combining confocal laser scanning microscopy and specific fluorescent markers of

cell viability were recently developed and applied, enabling non-invasive monitoring of the

activity of antimicrobials within 3D biofilm structures (Takenaka et al., 2008; Davison et al.,

2010; Bridier et al., 2011b; Bridier et al., 2012). The data thus obtained on the spatio-temporal

patterns of biocide action, and their potential utilization in mathematical models (Zhang,

2012) may be crucial to identifying the phenomena which lead to a limitation of biocidal

efficacy in biofilms. Such knowledge constitutes a prerequisite to developing efficient

disinfection treatments and controlling the microbiological quality of surfaces in food

processing environments.

4. Species interactions and the persistence of microbial contaminants

A wide variety of bacterial species are present in food processing environments and known to

form biofilms on surfaces. Because of this broad diversity, surface-associated communities

are usually complex associations of different species, which interact in different ways to

constitute a complex and dynamic network (Yang et al., 2011) (Figure 6). Such interactions

play a key role in shaping biofilm architecture and are responsible for specific functions

(Burmolle et al., 2014). In particular, an increasing number of studies have reported that

multispecies biofilms appear to be more resistant to antimicrobial activity than their mono-

species counterparts (Burmolle et al., 2006; Luppens et al., 2008; Simoes et al., 2009; VanVan

der Veen and Abee, 2011; Schwering et al., 2013; Giaouris et al., 2013). For instance, it was

shown that under most conditions, the pathogen L. monocytogenes in a mixed biofilm with

Lactobacillus plantarum exhibited higher resistance to benzalkonium chloride and peracetic

acid than single species biofilms (van der Veen and Abee, 2011). Similarly, Lee et al. (2013)

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Pseudomonas protegens were more resistant to the antimicrobials sodium dodecyl sulphate

and tobramycin than single-species biofilms. This enhanced resistance was linked to the

protection offered by the resistant species to the whole community, rather than selection for

the resistant species. Other studies have highlighted the fact that pathogens may be protected

when living in association with other strains in a mixed biofilm. For example, it has been

suggested that Staphylococcus sciuri is protected against chlorine because of its aggregation

within microcolonies that have formed with a more resistant Kocuria sp. strain (Leriche et al.,

2003). It has also been proposed that this spatial protection could play a role in the persistence

of St. aureus in a mixed biofilm with a Bacillus subtilis strain, with a hyper-biofilm phenotype

being isolated from a medical environment (Bridier et al., 2012). Indeed, St. aureus displayed

a higher degree of resistance to peracetic acid treatment in a mixed biofilm, and 3D confocal

images revealed that cells of the pathogen were entrapped in protruding structures formed by

the B. subtilis strain and could thus benefit from its abundant protective matrix. The sharing of

extracellular polymeric substances, and its role in resistance to sanitization, was also recently

observed in a mixed biofilm formed by Shiga toxin-producing E. coli and S. Typhimurium

(Wang et al., 2013). Such observations indicate that the spatial organization of species, and

the sharing of matrix components, may therefore determine the degree of persistence of

pathogens in mixed communities as well as their resistance to cleaning and disinfection

treatments (i.e. poorer accessibility of cleaning/disinfection agents to cells located in the

internal part of a biofilm).

In addition to such unspecific processes, some findings have suggested that microbial species

may actively cooperate. Using an individual-based modelling approach, Xavier and Foster

(2007) showed that the presence of multiple strains in a biofilm could promote the production

of extra-polymeric substances, which are key components in the persistence of biofilms on a

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ACCEPTED MANUSCRIPTsurface, and their resistance. Several experimental studies reported that an association of

strains could lead to an increase in biomass production and pathogen persistence (Burmolle et

al., 2006; Rieu et al., 2008). It was reported that the pathogen E. coli O157:H7 produced 400

times more biomass when it was co-existing in a biofilm with Acinetobacter calcoaceticus

when compared to a monoculture under the same conditions (Habimana et al., 2010).

Moreover, it was proposed that the physicochemical interactions of the polymeric substances

secreted by different species might cause a modification to matrix properties and contribute to

the resistance of the whole mixed community against disinfection (Allison and Matthews,

1992; Skillman et al., 1999). A recent article also showed that E. coli and S. Typhimurium

were able to share functional amyloids, which are subunits secreted into the extracellular

environment prior to assembly into curli, the latter being important components in their

extracellular matrix (Zhou et al., 2012). The authors reported that this interspecies interaction

promoted bacterial surface attachment and the development of mixed biofilms.

The recent finding that even evolutionarily distant neighbouring bacteria such as B. subtilis

and E. coli are capable of directly communicating and exchanging cytoplasmic materials

(such as DNA, proteins, etc.) via intercellular connecting appendices called nanotubes,

highlighted the fact that a biofilm does indeed constitute an environment that facilitates

sharing and hence the acquisition of new phenotypic traits (Dubey and Ben-Yehuda, 2011).

Throughout this communication and cooperation between different cells and species, a

biofilm thus offers an environment that can promote the emergence and dissemination of

specific functional properties that will safeguard the community in the face of adverse

conditions (Sachs and Hollowell, 2012). Based on these observations, it would seem that

targeting the mechanisms by which species interact in mixed communities might be a better

solution to control biofilms that include pathogens (Boyle et al., 2013), but achieving this

requires a clearer understanding of the nature of these interactions.

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ACCEPTED MANUSCRIPTIn recent years, modelling approaches focused on species interactions within biofilms have

emerged, and should contribute to developing a comprehensive view of these complex

microbial ecosystems (Xavier et al., 2005; Alpkvist and Klapper, 2007). Using a computer

simulation that could capture key features of the biology and the physical environment of

structured bacterial sub-populations, Mitri et al. (2011) tried to describe the impact of the

presence of multiple species in the evolution of cooperative secretions. Their model revealed

the important influence of nutrient competition on the nature of the interactions between

secreting and non-secreting strains, and the resulting spatial organization of species as a

function of nutrient conditions. When focusing on development of the pathogen L.

monocytogenes in a mixed biofilm with Lc. lactis, Habimana et al. (2011) developed a

simplified individual-based model that could simulate bacterial growth in a three-dimensional

space. They demonstrated that the slower growth rate of the pathogen compared to Lc. lactis

during the initial stages of dual-species biofilm formation was probably the source of the

inhibition of pathogen development, a finding that agreed with microscopic observations.

The development of modelling tools to identify parameters of importance to biofilm

architecture and functions, and particularly to better understand the interactions between

bacterial species interactions in mixed communities, therefore offers a promising approach to

deciphering the microbial communities present in food processingfood environments.

5. Emerging strategies to control biofilms in the food industry

The regular application of cleaning and disinfection procedures is a common strategy

employed to control pathogen implantation on either industrial equipment or the products

themselves (Jahid and Ha, 2012). However, such procedures are not fully effective on biofilm

structures and can induce the selection of resistant phenotypes (Simoes et al., 2010). For

more than twenty years, therefore, the international scientific community has been focusing

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context, the increasing account being taken of environmental impacts has led to the

exploration of biocides generated on-site, such as ozone or acidic electrolyzed water. These

are generally considered as green (or eco-friendly) biocides and they are chemical residue-

free (Ashraf et al., 2014). Natural compounds extracted from bacterial cultures or aromatic

plants, and “generally recognized as safe”, are now being evaluated for their potential to

eradicate biofilms. They may exert a high level of lethal activity against pathogens, be

efficient in penetrating the structure of a biofilm, and be easily degraded in the environment.

For example, oregano oil, thymol and carvacrol displayed clear efficacy on Staphylococcus

biofilms, achieving cell reduction levels that were quite competitive with those found with

most chemical biocides (Nostro et al., 2007). Essential oils, including thymol, carvacrol, and

eugenol and their combinations, were found to be as active against industrial paper mill

biofilms as against planktonic cells (Neyret et al., 2014).

Bacteriophages have also been identified as potential candidates to attack pathogens in

bacterial biofilms. They can diffuse through the biofilm matrix while retaining their

antibacterial efficacy (Briandet et al., 2008; Donlan, 2009). Natural and engineered phages

were found to be effective against food-borne pathogen biofilms (Simoes et al., 2010). For

example, an engineered phage expressing a biofilm-degrading enzyme enabled a 99.997%

reduction in the cell count of an E. coli biofilm (Lu and Collins, 2007). Phages were also

found to be effective in controlling L. monocytogenes on stainless steel which was either clean

or had been soiled with fish proteins. However, their efficacy was greater on cells dislodged

from the surface than on those organised within ain biofilm (Lu and Collins, 2007).

LISTEX™ P100, a natural phage product active against Listeria, has been recognized by the

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(Goodridge and Bisha, 2011; Pereira Da Silva and Pereira De Martinis, 2013).

The use of enzyme-based detergents may offer additional tools to improve disinfection

processes. Enzymes can target and loosen the matrix as well being able to trigger cell release

from biofilms. This allows conventional disinfection agents to reach their bacterial targets

embedded in a biofilm or dispersed as planktonic cells. Depending on the composition of the

biofilm matrix, different enzymes may be preferred, including proteases, cellulases,

polysaccharide depolymerases, alginate lyases, dispersin B or DNAses (Xavier et al., 2005;

Orgaz et al., 2007; Jabbouri and Sadovskaya, 2010; Lequette et al., 2010). In industrial or

medical environments, numerous microbial species coexist within the same biofilm, thus

increasing the biochemical heterogeneity of the matrix. Efficient formulations may therefore

be composed of mixtures of enzymes with different substrate spectra. Under these strategies,

enzyme interactions with food components need to be taken into account. For instance, the

action of proteolytic enzymes is reduced in the presence of milk (Augustin et al., 2004).

Another innovative approach to sensitizing industrial biofilms to biocides is to use hyper-

swimming tunnelling bacteria. A recent study demonstrated that planktonic bacilli propelled

by flagella were able to tunnel deep into a biofilm structure (Figure 7) (Houry et al., 2012).

The transient pores created in the matrix by these bacterial stealth swimmers increased

macromolecular transfer within the biofilm and thus enhanced the killing of biofilm cells by

facilitating the penetration and action of disinfectants from the environment (Houry et al.,

2012).

In addition to biological and/or chemical alternatives, new physical processes (e.g. pulsed-

light or laser decontamination devices) are promising option for specific industrial

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2007; Kamgang-Youbi et al., 2009; Joaquin et al., 2009) and their application in food

preservation is now being considered (Hati, 2012; Wiktor et al., 2013). Preliminary studies

have been performed in the treatment of food surfaces associated microbial contaminations

(Froehling et al., 2012; Baier et al., 2013). Although there is still insufficient information on

concomitant physical and chemical processes, as well as on the changes induced in food by

these plasma technologies (Schluter et al., 2013), it has been demonstrated that they can

generate long-term chemical by-products (Naitali et al., 2010). This phenomenon, associated

with the high energy demands of these technologies, have until now limited their widespread

utilization. Faced with the limitations of curative biofilm treatments, other approaches have

been developed in order to prevent the establishment of structured biofilms. Modifications to

inert surfaces so as to reduce their interactions with microbial cells and subsequent adhesion

have been proposed (Simoes et al., 2010). In this regard, modified highly hydrophobic

polystyrene demonstrated their antibacterial efficacy against different micro-organisms,

including L. monocytogenes (Poncin-Epaillard et al., 2013). Functionalized carbohydrates

acting as lectin inhibitors have been used to coat materials and prevent the production of

microbial adhesins or their interactions with the surface (Korea et al., 2011). Biosurfactants

have also been reported as promising anti-adhesive and anti-microbial coating molecules

(Meylheuc et al., 2006; Rodrigues, 2011). The use of chemical or natural antimicrobials as

alternative coating molecules has also been found to be effective in preventing both adhesion

and bacterial development on inert and food surfaces (Zhang et al., 2006; Duan et al., 2007).

Surface modifications using nano-materials such as silver, cobalt and iron mixed oxides are

interesting candidates for the prevention of biofouling, notably in the context of treatment

technologies for drinking water (Ashraf et al., 2014).

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interfering with bacterial communication, these antagonists should prevent biofilm

implementation or maturation. Brominated furanones were shown to have successfully

reduced biofilm formation (Ni et al., 2009; Sintim et al., 2010). Similarly, the cyclic-di-GMP

pathway that has been shown to regulate diverse cellular processes involved in biofilm

formation (notably matrix synthesis) may be a promising antimicrobial target (Romling and

Amikam, 2006; Sintim et al., 2010; Landini et al., 2010).

According to the concept of guided microbial ecology, protective biofilms have been

proposed as barrier microflora against the potential establishment of food-borne pathogens;

for example in poultry farms to control Salmonella. Some work in this area has focused on the

ecology of wooden shelves used for cheese ripening (Mariani et al., 2007). This showed that

the resident microflora on these shelves enabled inhibition of the pathogen L. monocytogenes

by up to 2 log10 (CFU/cm²) during 12 days of ripening, while inactivation of the

autochthonous microflora enabled an increase in the L. monocytogenes population by 4 log10

(CFU/cm²) during the same period (Mariani et al., 2011). Three types of activity, i.e.

competition, exclusion and displacement, may be mediated by protective biofilms acting

against the formation of food-borne pathogen biofilms (Woo and Ahn, 2013). Competition

studies between food grade Lc. lactis bacteria and L. monocytogenes in dual-species biofilms

revealed that the shorter generation time of Lc. lactis than that of L. monocytogenes was the

key factor involved in inhibition of the pathogen (Habimana et al., 2011). As for competitive

exclusion, the genetic background of the protective biofilm bacterium Lc. lactis, which

impacts both its physiology and its physicochemical surface properties, was shown to be

critical to its ability to prevent the fixation of L. monocytogenes (Habimana et al., 2009).

Other factors as bacteriocins which are produced by such protective biofilms also display

valuable bacterial traits that may potentialize competitive exclusion or the displacement of

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6. Future needs

Because of the high tolerance/resistance of pathogen-associated biofilms, it is necessary to

obtain knowledge on the state of microbial contaminants in terms of planktonic or biofilm-

associated cells, in order to optimize cleaning and disinfection procedures in the food

industry. Hall-Stoodley et al. (2012) proposed diagnostic guidelines for clinical biofilms

implicated in BAI. Similar criteria could be proposed regarding the detection of food-borne

biofilm-associated contamination. Some diagnostic tools are available, but others still need to

be developed (Table 1).

Because some bacteria in biofilms may be non-culturable, immunological or molecular tools

are necessary to detect all the pathogens present during a given process and enable the online

monitoring of industrial biofilms (Tan et al., 2014). It is therefore necessary to develop

models that can predict the potential settlement of pathogens and their development as a

biofilm in the specific environment of food processing. Biomarkers for biofilm bacteria and

universal matrix probes will also be valuable tools to determine the status of biofilms.

The ex-situ analysis of coupons that have been placed for a given period within a processing

line can enable the observation and characterization of industrial biofilms. FISH (fluorescence

in situ hybridisation) can then be used to directly detect a given pathogen within the biofilm.

In situ and non-invasive probes are also needed to directly evaluate the implantation of

pathogens in the form of a biofilm in processing lines.

Endemic flora in a given food environment probably persist in the form of a biofilm.

Carpentier and Cerf (2011) attempted to define L. monocytogenes persistent strains as

“isolated on at least three sampling dates in a one-year period”. The molecular typing of

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amplified polymorphism DNA) or PFGE (pulsed field gel electrophoresis) enables

determination of the origin of contamination and a distinction between persistent (endemic)

and non-persistent (sporadic) strains. The systematic whole genome sequencing (WGS) of

microbial contaminants could enablecould higher resolution in that context.

Finally, it is necessary to standardise the methods used to evaluate biocide efficacy on

surface-associated cells so that new and efficient anti-biofilm strategies can be developed.

Indeed, in most cases, the efficiency of a disinfection procedure is assessed after cells have

been recovered from the surface using ultrasonic and beads or swabs. However, it is well

known that such sampling procedures only enable the partial recovery of adhering cells, and

that the cells sampled may not be representative of the global physiological state of the whole

community. For example, sampling efficiencies as poor as 10% have sometimes been found

on porous substrates. In this context, the effectiveness of surface disinfection is possibly

overestimated (Grand et al., 2011).

Acknowledgements

A. Bridier is a recipient of Agence Nationale de la Recherche (ANR) funding in the context of

the Investments for the Future (Programme Investissements d’Avenir) programme ANR-10-

BTBR-02. P. Sanchez-Vizuete is a recipient of Ile-de-France Regional Council «DIM Astrea»

PhD funding. Financial support was also provided by the French National Research Agency

ANR-12-ALID-0006 programme and the European FP7-SUSCLEAN programme. Our thanks

to R. Losick for the gift of strain TMN547. Julien Deschamps, Thierry Meylheuc, Margareth

Renault, Elsa Trotier (from the MICALIS UMR 1319Micalis UMR1319 INRA-

AgroParisTech) and the associated MIMA2 imaging platform are warmly thanked for their

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English revision.

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ACCEPTED MANUSCRIPTTable 1 Suggested criteria and methods for the detection of food-borne biofilm-associated contamination (adapted from the criteria proposed for Biofilm Associated Infection (Hall-Stoodley and Stoodley, 2009; Hall-Stoodley et al., 2012) Criteria Current diagnostic tools Needs

Culture or non-culture methods evidence the presence of food-borne pathogens known to form biofilms in specific environments of the food process

Plates, molecular identification tools (q-PCR)

Models to predict pathogen settlement according to cell properties and environmental conditions (surface properties, fluid velocity, etc.), predictive microbiology to evaluate potential growth or persistence as a biofilm

Microscopic examination of equipment (when possible), of disposable coupons, of processed foods or of fluids demonstrates clustered cells embedded in a matrix

Photonic microscopy (with fluorescence for opaque material), atomic force microscopy, scanning electron microscopy, specific matrix probe (labelled lectins or antibodies for EPS, Syto 9 or SyberGreen for DNA)

In-situ and non invasive observation of biofilms, food grade microscopic cell probes, universal microscopic matrix probes

Pathogens are associated with a surface

Sampling (with swabs, sponges, contact plates, disposable coupons), ex-situ microscopy or numbering methods

In-situ and non invasive observation of biofilms, food grade microscopic probes, specific genetic or biochemical biomarkers of biofilm bacteria

Contamination is localized to particular sites throughout the process (however there may be dispersion of cells within processing lines)

Mapping of surface contamination using disposable coupons, biofilm detection kits

In-situ and non invasive observation of biofilms, food grade microscopic probes, universal microscopic matrix probes

Recurrence of food contamination at multiple time points (particularly with the same micro-organisms)

Genetic fingerprint (PFGE, ribotyping, FISH microscopy

Systematic sequencing of microbial contaminants

Recalcitrance to disinfection treatment in spite of the susceptibility of planktonic cells

Disinfection standards for sample cells, minimum biofilm eradication concentration (MBEC) assay, fluorescent probes of viability (Chemchrome V8, Live-dead)

Normalisation of the evaluation of biocide efficacy for surface-associated cells, quantification of biocide penetration and reactivity in biofilm matrix

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Figure 1: Diversity of the spatial architecture of E. coli biofilms. Biofilms formed on polystyrene by four isolates of E. coli were observed by confocal microscopy (green= live cells; red= dead cell). Images size is 180x180 µm. Figure 2: Spatial assemblage of an LL. monocytogenes LO28 biofilm as visualized by scanning electron microscopy. The white dot bar scale indicates 6 µm. Figure 3: Heterogeneity of gene expression in a BB. subtilis isogenic population. The fate of biofilm cells is controlled by stochastic regulation through which cells can switch between two alternative states, i.e.individual swimming or chained sessile cells. Here, a green sub-population expressing flagella coexists with a red sub-population expressing matrix genes (strain TMN547). Image size is 240x240 µm. Figure 4: Genetic diversification in a macrocolony biofilm of Salmonella sp. grown on congo red and comassie blue agar. Image size 1.7x1.7 cm. Figure 5: Biofilms generate variants (bottom) with different properties when compared with the parental strain (upper panel). Example of an increase in A) the biofilm matrix (a red, dry and rough morphotype characterized by the production of cellulose; width 3.3 cm), B) virulence (weaker phagocytosis after 14 days by the amoeba Dictyostelium discoideum may be linked to greater virulence) and C) biofilm formation (white bar is 30 µm) in SS. enterica strains. Figure 6: Diversity of the spatial patterns of interaction between E. coli expressing the green fluorescent protein and three red-labelled Gram-negative strains isolated from food processing surfaces. Images size is 140x140 µm. Figure 7: Green bacilli swimmers create transitory pores in the biofilm matrix of St. aureus (here in red). Left image size is 140x140 µm (right image is a zoom).

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Highlights

- Ability to form/join microbial biofilms impacts foodborne pathogen tolerance

- Biofilm tolerance to biocides requires the development of new hygienic

strategies

- Interspecific interactions influence pathogen fitness/survival in biofilms

- Spatial modeling as a tool to decipher biofilm structure/function relationships