**3. Genes involved in the biofilm formation**

Over time, beside the conditions that favor the biofilm formation in food processing plants**,** the genetic background of biofilm forming microorganisms was also intensively studied. At each step of biofilm development and dispersal, there is a specific genetic signal control.

The *L. monocytogenes* pattern of the microarray gene expression was analyzed at different time intervals (4, 12, and 24 h) in order to depict genes' expression at different stages of biofilm formation. The results showed that more than 150 genes were upregulated after 4 h of biofilm formation and a total of 836 genes highlighted a slow increase in expression with time [23]. Although for many bacterial species the genome sequencing allowed the identification of genes that were involved in biofilm synthesis, for *L. monocytogenes*, these genes could not be identified using just the bioinformatics analysis.

In the biofilm formation, the attachment step is a prerequisite in which flagella and type I pili-mediated motilities are critical for the initial interaction between the cells and surface.

In order to find out the roles of the genes and regulatory pathway controlling the biofilm formation, researchers applied one or two genome-wide approaches, like transposon insertion mutagenesis or/and transcriptome analyses. With a transposon mutagenesis library, it was possible to identify 70 *L. monocytogenes* mutants, with Himar1 mariner transposon insertion, which produced less biofilms [24]. From a total of 38 genetic loci identified, 4 of them (**Table 1**) were found to be involved in bacterial motility (*fli*D, *fli*Q, *fla*A, and *mot*A), a required property for initial surface


**217**

**Gene/KEGG/protein encoded**

**Microcolonies development**

*dlt*A/LMOf2365\_099/D-alanine-D-alanyl carrier protein ligase

Catalyzes the first step in the D-alanylation of lipoteichoic acid (LTA) Carrier protein involved in the D-alanylation of LTA

Involved in the transport of activated D-alanine through the membrane

Cell wall biogenesis

*L. monocytogenes S. aureus*

[24]

*dlt*C/LMOf2365\_099/D-alanyl carrier protein

*dlt*B/lmo0973/DltB

*sdrC/*NWMN\_0523/Serine-aspartate repeat-

containing protein C

 *sdrH*/SAUSA300\_1985 Serine-aspartate repeat family protein

*bhs*A/STY1254/Multiple stress resistance protein

*bsm*A/*yjf*O/Lipoprotein

*csg*D/b1040/CsgBAC operon transcriptional

regulatory protein

*mlr*A/b2127/HTH-type transcriptional regulator

*sin*R/BSU24610/HTH-type transcriptional

regulator

*eps*G (*yve*Q *eps*H (*yve*R)/BSU34300/Putative

glycosyl-transferase

*ymd*B/BSU16970/2′,3′-cyclic-nucleotide

Regulatory role. Induces genes involved in

Directing the early stages of colony

development

Exopolysaccharide synthesis

[167]

biofilm formation

Catalyzes the interconversion between glucose-6-

phosphate and alpha-glucose-1-phosphate

2′-phospho-diesterase

*pgc*A/Phosphoglucomutase

)/BSU34310/Transmembrane protein

Production of exopolysaccharide

DNA-binding transcription factor activity

Negatively regulates transcription of the *eps*

operon

Stress response, response to copper ion

Regulation of biofilm formation. May repress cell–cell interaction and cell surface interaction

*E. coli*

[159]

Single-species biofilm formation; enhanced

*E. coli, S. enterica*

[160]

[161]

[162]

flagellar motility

The master regulator for adhesive curli

fimbriae expression

Activates transcription of *csg*D

DNA-binding protein master regulator of

*B. subtilis*, *B. cereus*

[163,

164]

[165]

[166]

biofilm formation

Biofilm maintenance

Stress response to hydrogen peroxide and to DNA

DNA-binding transcription activator activity

damage

Cell adhesion

Mediates interactions with components of the extracellular matrix to promote bacterial adhesion

**Gene function**

**Role**

**Bacterium**

**Ref.**

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

*S. aureus, B. subtilis*

*S. aureus*

[158]

### *Bacterial Biofilms*


### *Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

*Bacterial Biofilms*

**216**

**Gene/KEGG/protein encoded**

*Initial attachment*

*fli*

*fla*A/lmo0690/Flagellin

*fli*D/Flagellar hook-associated protein 2

*mot*A/BN418\_0793/Flagellar motor protein

*prf*A/IJ09\_09365/Listeriolysin positive regulatory

*fimA/*JW4277/Type-1 fimbrial protein, A chain

*fhiA/*ECUMN\_0250*/*Flagellar biosynthesis protein

*yadL/*ECs0141/*yadM*/*yad*K/*yad*C/Fimbrial protein

*tab*A/yjgK/b4252/toxin-antitoxin biofilm protein

*icaA/*Poly-beta-1,6-N-acetyl-D-glucosamine

synthase from *ica*ADBCR operon

*tpi*A/SAR0830/Triosephosphate isomerase

*sra*P**/**SAOUHSC\_02990/ Serine-rich adhesin for

*Spo*0A/BSU24220/Stage 0 sporulation protein A

*deg*S/BSU35500/Signal transduction histidine-

protein kinase/phosphatase

*fli*L/STM1975/Flagellar protein

*ycf*R/Outer membrane protein

Regulatory role in sporulation

Transition to growth phase; flagellum formation

Controls the rotational direction of flagella

Promotes the attachment to the surface

Motility, cell adhesion

*S. enterica*

[156]

[157]

Biofilm formation

platelets

Involved in gluconeogenesis pathway

Mediates binding to human platelets

Fimbrial bio-synthesis

Represses fimbria genes

Acetylglucosaminyl transferase activity, cell

adhesion

Single-species biofilm

Involved in the polymerization of a biofilm

*S. aureus*

[149]

[150]

[151]

adhesin polysaccharide

Role in adherence

Plays a positive role in biofilm formation

Single-species surface biofilm formation

*B. cereus, B. subtilis*

[152]

[153]

[154, 155]

Enable bacteria to colonize the host epithelium

Motility bacterial-type flagellum assembly

Cell adhesion

factor A

Q*/*LMON\_0682*/*Flagellar biosynthesis protein

Motility

Flagella bio-synthesis

Enable the polymerization of the flagellin

monomers; flagellar capping protein

Flagellar motor rotation

DNA-binding transcription factor activity

formation

Positive regulation of single species biofilm

*L. monocytogenes*

*E. coli*

[30,

31]

**Gene function**

**Role** Cell adhesion and bacterial attachment

*L. monocytogenes*

[23–25]

[148]

**Bacterium**

**Ref.**


**219**

**Gene/KEGG/protein encoded**

*clfA/* / Clumping factor A; *clfB/* NWMN\_2529/ Clumping factor B

*ica*C/SAOUHSC\_03005/poly-beta-1,6-N-acetyl-D-

glucosamine export protein (PNAG)

*pflA/*SAOUHSC\_00188/Pyruvate formate lyase-

activating enzyme

 *pfl*B/SACOL020/Formate acetyltransferase

*sar*A/Transcriptional regulator

*agr*D/LMM7\_0043/Putative autoinducing peptide

lmo0048/Putative AgrB-like protein

*agr*C/Accessory gene regulator

*agr*A/CQ02\_00305/BN389\_00610/

Accessory gene regulator

*agr*B/MF\_00784/Accessory gene regulator

luxS/lmo1288/S-ribosyl-homo-cysteine lyase

*lux*Q/Autoinducer 2 sensor kinase/phosphatase

**Table 1.**

*List of genes with significant role in biofilm formation within pathogenic microorganisms (UniprotKB database).*

Phospho-relay sensor kinase activity

Proteolytic processing of *Agr*D

Catalysis of precursor molecules of AI-2

Histidine kinase activity

A response regulator

Involved in proteolytic processing

Involved in proteolytic processing

Global regulator of a few genes with important roles in biofilm development

Enzymes that catalyze the first step in the acetogenesis from pyruvate

Organic free radical synthesis

Biofilm formation process in a cell density-

dependent manner

Quorum Sensing

*S. aureus*

[179]

Export of PNAG across the cell membrane

**Gene function** Cell surface-associated protein implicated in bacterial attachment

**Role** Aggregation of unicellular organisms; cell adhesion

**Bacterium**

*S. aureus* *E. coli, S. aureus*

[149]

**Ref.** [178]

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

[180]

*L. monocytogenes*

*L. monocytogenes*

*B. cereus* *S. aureus*

[181]

[182]

[183]

[184]

*S. aureus* *L. monocytogenes* 

[48]

[49]

*E. coli, B. cereus, S.* 

*enterica*

*E. coli, B. cereus, S.* 

*enterica*

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

[29]

*Bacterial Biofilms*


### *Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

*Bacterial Biofilms*

**218**

**Gene/KEGG/protein encoded**

*gcp*A/SL1344\_191/Biofilm formation in nutrient-

deficient medium

*Biofilm maturation*

*tas*A/ BSU24620/major biofilm matrix component

*tap*A/ BSU24640/TasA anchoring/assembly

protein

*sip*W/BSU24630/Signal peptidase IW

*bsl*A (*yua*B)/BSU31080/Biofilm-surface layer

*wcaF/*b2054*/*Putative colanic acid biosynthesis

acetyl-transferase

*wca*L/STM2100/Putative colanic acid biosynthesis

glycosyl-transferase

*bss*R (*yli*H)/JW0820/Biofilm regulator

*mqs*R/b3022/mRNA interferase toxin

*tqs*A/b1601/AI-2 transport protein

*bdc*A/b4249/Cyclic-di-GMP-binding biofilm

dispersal mediator protein

*ihf*AB/Integration host factor

*bap*A/biofilm-associated protein

Regulation of biofilm formation

Motility-quorum sensing cell proliferation

Efflux transmembrane transporter activity

Controls cell motility, size, aggregation,

and production of extracellular DNA and

extracellular polysaccharides

Specific DNA-binding protein

Large surface proteins family

Matrix density

*S. enterica, S. aureus*

[175–

177]

Cellulose production

Bacterial adhesion

Biofilm maturation

In the glucose presences, cells showed

*E. coli*

[33]

[172]

[31]

increased biofilm formation

Biofilm architecture

Represses biofilm formation and motility

Biofilm dispersal

*E. coli, S. enterica*

[174]

protein A

TapA

Identical protein binding

Important for proper anchoring and

polymerization of TasA fibers at the cell surface

Cleavage of the signal sequence of TasA and

Confers a specific microstructure to the biofilm

Confers hydrophobicity to the biofilm

Involved in the pathway slime

polysaccharide biosynthesis

*B. subtilis,* No paralog

[170–

171]

[172]

in *B. cereus* genome

*E. coli* *S. enterica*

[173]

surface

Synthesis of colanic acid

Major component of the biofilm

*B. cereus*

[168]

extracellular matrix

Essential for biofilm formation

*B. subtilis* No paralog in

[169]

*B. cereus* genome

*B. cereus*

**Gene function** Biofilm production under low-nutrient concentrations

**Role**

**Bacterium**

*S. enterica*

**Ref.**

[156]

**Table 1.**

*List of genes with significant role in biofilm formation within pathogenic microorganisms (UniprotKB database).*

### *Bacterial Biofilms*

attachment. Another gene with increased expression at 4 h and decreased expression after 12 h from biofilm initiation was *prf*A, the listeriolysin positive regulatory factor A. It seems that this regulatory factor is necessary just in the initial stages of biofilm formation and aggregation but not in the colonization stage [23, 25, 26].

Extracellular and surface proteins such as internalin A and BapL, respectively, have been found to be involved in the initial bacterial adhesion in *L. monocytogenes* EGD-e [27]. Moreover, its mobility is ensured by flagella and is temperaturedependent affecting the biofilm formation. As such, above 30°C, the transcription of *flaA* is stopped.

*S. aureus* genes responsible for cell adhesion to the surface are included in the icaADBC operon with functions in biosynthesis of the glucosamine polymer and polysaccharide intercellular adhesins [28]. Therefore, other genes encoding a number of transporter proteins (*pro*P, *opu*D, *aap*A, and *dlt*A) were upregulated after 8 hours from the biofilm initiation [29]. For *E. coli*, the genes involved in the cell adhesion, like *fim*A, *yad*K, *yad*N, *yad*M, and *yad*C-encoding fimbriae-like proteinsare coexpressed with the integral cell membrane genes, with outer membrane proteins (*htr*E), with transcriptional regulators (*mng*R and *nha*R), or other genes, but this network appears to be strain specific [30, 31].

In the case of *S. enterica*, differential expression analysis revealed that *ycf*R is highly conserved as in many Gram-negative bacteria, being upregulated under chlorine stress and responsible for the virulence and attachment of bacterium to the glass or polystyrene [32, 33].

Moreover, *Salmonella* spp.-related biofilms are driven by a transcriptional regulatory CsgD protein that activates the expression of curli and cellulose. The transcription of *csg*BAC operon, which encodes the structural subunits for curli, indirectly activates the transcription of the second mechanism, *adr*A, associated with cellulose production [10]. Important factors in the activation of *Salmonella* spp. biofilms are the c-di-GMP that is behaving like a secondary messenger molecule when the CsgD content is elevated [34].

Microcolonies are formed by cell proliferation, and many genes involved in cell division, cell wall biogenesis, virulence and motility, stress response, and transcriptional regulation factors are expressed.

**Table 1** shows a selection of the genes that are expressed in all the steps of biofilm formation or are upregulated under influence of different biotic or abiotic factors. It was reported that the ∆*dlt*ABC *L. monocytogenes* strains are defective in biofilm formation, validating by transposon mutagenesis, the critical role of d-alanylation of teichoic acids, for biofilm synthesis [24]. So, the mutants without d-alanine on the surface of teichoic acids have a higher negative charge and develop a biofilm-negative phenotype.

The mature biofilm evolves from microcolonies and this development is associated with EPS production. The biofilm matrix of *B. cereus* is similar to other *Bacillus* sp., but the *eps* genes, responsible for the EPS synthesis, are not mandatory for *B. cereus* compared to *B. subtilis* [35]. Little is known about the regulatory networks in *B. cereus*, but studies have shown that CodY and SpoOA may as well play a crucial role in biofilm formation [36].

Furthermore, the structural proteins encoded by *tap*A and *bsl*A from *B. subtilis* genome are absent in the matrix of *B. cereus* because these genes have no paralog in *B. cereus* genome. Instead the *tas*A gene is essential for *B. cereus* biofilm development, being responsible for the matrix fiber synthesis [37].

An important polysaccharide identified in the matrix biofilm of many pathogenic bacteria is the colanic acid, which plays an important physiological role for bacteria living in biofilm. This EPS is synthesized by specific enzymes encoded by *wca*L gene (*S. enterica*) or *wca*F (*E. coli*). It has been also shown that *rpo*S gene,

**221**

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

the main regulator of the general stress response, may be seen as a key factor in the

Consequently, the transition from the planktonic state to the biofilm state is critical and it is subjected to a strict gene regulation, essential for matrix synthesis,

Nevertheless, bacteria of multiple genetic backgrounds communicate by regulating their relationship of cooperativeness through a mechanism called quorum sensing (QS) in which the bacterial cells are having social interactions with each other through small diffusible signal molecules called autoinducers, thus contribut-

Quorum sensing process described in the 1970s is involved in the control of various gene expressions through chemical signaling molecules that are synthesized in response to cell population density [39]. When bacteria start to sense their critical biomass, they answer by activating or repressing genes from 10% of bacteria genome [40]. The system has been described for both Gram-negative and Gram-

Among QS, other two important regulators are known to control biofilm shape and structure: cyclic diguanosine-5′-monophosphate (c-di-GMP) and small RNAs. For example, *S. aureus* biofilm development is regulated by many environmental conditions and genetic signals. A significant constituent in biofilm formation is mediated by the polysaccharide intercellular adhesin composed mainly of polymeric N-acetyl-glucosamine (PNAG) and eDNA, encoded by the ica operon [41]. In certain cases, such as *S. aureus*, biofilm-associated protein (Bap) is involved in biofilm maturation rather than polysaccharide intercellular adhesion (polysaccha-

The c-di-GMP involvement in *S. aureus* is an important biofilm regulator that allosterically switches on enzymes of exopolysaccharide biosynthesis [43], while the function of small RNA genes involved is still not yet studied in detail [44]. Although it has been noticed to show an increased susceptibility to disinfectants in planktonic state, however, in biofilm state, it may be among the most resistant ones equally

Gram-positive bacteria such as *S. aureus*, *B. subtilis*, and *L. monocytogenes* are communicating through inducers encoded by accessory gene regulator (*Agr*) system (**Table 1**). It seems like the *Agr* complex regulates more than 100 genes in the *S. aureus* genome [45], and its deletion from *L. monocytogenes* genome affects

The accessory gene regulator of *S. aureus* modulates the expression of virulence factors and toxins in response to autoinducing peptides (AIPs) while luxS synthesizes AI-2, which inhibits exopolysaccharide synthesis through an unknown QS

For *S. enterica* and *E. coli*, the QS system is mediated by two genes, *lux*S and

Since biofilms act as a barrier that protects the embedded cells against cleaning and disinfecting agents [51], the control of biofilm is an issue that is currently

The *L. monocytogenes* QS signaling triggers the transcriptional activation of one of the virulence PrfA-regulated genes a*ctA*, resulting in the bacterial aggregation and biofilm formation [10]. Another gene involved in the cell-to-cell interactions is *secA2* gene. Its deletion may inactivate the SecA2 pathway with an increased cell

*lux*R, homolog to *Sdi*A in order to reach intercellular signaling [48, 49].

**4. Fighting against biofilms with nonconventional methods**

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

cell aggregation, and cell signaling.

ing to the biofilm development [10].

ride intercellular adhesins) expression [42].

more than 600 genes [46].

aggregation and sedimentation [50].

cascade [47].

important for food as well as for the medical sectors.

positive bacteria.

development of mature biofilms in *E. coli* [38].

### *Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

*Bacterial Biofilms*

of *flaA* is stopped.

glass or polystyrene [32, 33].

attachment. Another gene with increased expression at 4 h and decreased expression after 12 h from biofilm initiation was *prf*A, the listeriolysin positive regulatory factor A. It seems that this regulatory factor is necessary just in the initial stages of biofilm

Extracellular and surface proteins such as internalin A and BapL, respectively, have been found to be involved in the initial bacterial adhesion in *L. monocytogenes* EGD-e [27]. Moreover, its mobility is ensured by flagella and is temperaturedependent affecting the biofilm formation. As such, above 30°C, the transcription

*S. aureus* genes responsible for cell adhesion to the surface are included in the icaADBC operon with functions in biosynthesis of the glucosamine polymer and polysaccharide intercellular adhesins [28]. Therefore, other genes encoding a number of transporter proteins (*pro*P, *opu*D, *aap*A, and *dlt*A) were upregulated after 8 hours from the biofilm initiation [29]. For *E. coli*, the genes involved in the cell adhesion, like *fim*A, *yad*K, *yad*N, *yad*M, and *yad*C-encoding fimbriae-like proteinsare coexpressed with the integral cell membrane genes, with outer membrane proteins (*htr*E), with transcriptional regulators (*mng*R and *nha*R), or other genes,

In the case of *S. enterica*, differential expression analysis revealed that *ycf*R is highly conserved as in many Gram-negative bacteria, being upregulated under chlorine stress and responsible for the virulence and attachment of bacterium to the

Moreover, *Salmonella* spp.-related biofilms are driven by a transcriptional regulatory CsgD protein that activates the expression of curli and cellulose. The transcription of *csg*BAC operon, which encodes the structural subunits for curli, indirectly activates the transcription of the second mechanism, *adr*A, associated with cellulose production [10]. Important factors in the activation of *Salmonella* spp. biofilms are the c-di-GMP that is behaving like a secondary messenger mol-

Microcolonies are formed by cell proliferation, and many genes involved in cell division, cell wall biogenesis, virulence and motility, stress response, and transcrip-

The mature biofilm evolves from microcolonies and this development is associated with EPS production. The biofilm matrix of *B. cereus* is similar to other *Bacillus* sp., but the *eps* genes, responsible for the EPS synthesis, are not mandatory for *B. cereus* compared to *B. subtilis* [35]. Little is known about the regulatory networks in *B. cereus*, but studies have shown that CodY and SpoOA may as well play a crucial

Furthermore, the structural proteins encoded by *tap*A and *bsl*A from *B. subtilis* genome are absent in the matrix of *B. cereus* because these genes have no paralog in *B. cereus* genome. Instead the *tas*A gene is essential for *B. cereus* biofilm develop-

An important polysaccharide identified in the matrix biofilm of many pathogenic bacteria is the colanic acid, which plays an important physiological role for bacteria living in biofilm. This EPS is synthesized by specific enzymes encoded by *wca*L gene (*S. enterica*) or *wca*F (*E. coli*). It has been also shown that *rpo*S gene,

ment, being responsible for the matrix fiber synthesis [37].

**Table 1** shows a selection of the genes that are expressed in all the steps of biofilm formation or are upregulated under influence of different biotic or abiotic factors. It was reported that the ∆*dlt*ABC *L. monocytogenes* strains are defective in biofilm formation, validating by transposon mutagenesis, the critical role of d-alanylation of teichoic acids, for biofilm synthesis [24]. So, the mutants without d-alanine on the surface of teichoic acids have a higher negative charge and develop

formation and aggregation but not in the colonization stage [23, 25, 26].

but this network appears to be strain specific [30, 31].

ecule when the CsgD content is elevated [34].

tional regulation factors are expressed.

a biofilm-negative phenotype.

role in biofilm formation [36].

**220**

the main regulator of the general stress response, may be seen as a key factor in the development of mature biofilms in *E. coli* [38].

Consequently, the transition from the planktonic state to the biofilm state is critical and it is subjected to a strict gene regulation, essential for matrix synthesis, cell aggregation, and cell signaling.

Nevertheless, bacteria of multiple genetic backgrounds communicate by regulating their relationship of cooperativeness through a mechanism called quorum sensing (QS) in which the bacterial cells are having social interactions with each other through small diffusible signal molecules called autoinducers, thus contributing to the biofilm development [10].

Quorum sensing process described in the 1970s is involved in the control of various gene expressions through chemical signaling molecules that are synthesized in response to cell population density [39]. When bacteria start to sense their critical biomass, they answer by activating or repressing genes from 10% of bacteria genome [40]. The system has been described for both Gram-negative and Grampositive bacteria.

Among QS, other two important regulators are known to control biofilm shape and structure: cyclic diguanosine-5′-monophosphate (c-di-GMP) and small RNAs. For example, *S. aureus* biofilm development is regulated by many environmental conditions and genetic signals. A significant constituent in biofilm formation is mediated by the polysaccharide intercellular adhesin composed mainly of polymeric N-acetyl-glucosamine (PNAG) and eDNA, encoded by the ica operon [41]. In certain cases, such as *S. aureus*, biofilm-associated protein (Bap) is involved in biofilm maturation rather than polysaccharide intercellular adhesion (polysaccharide intercellular adhesins) expression [42].

The c-di-GMP involvement in *S. aureus* is an important biofilm regulator that allosterically switches on enzymes of exopolysaccharide biosynthesis [43], while the function of small RNA genes involved is still not yet studied in detail [44]. Although it has been noticed to show an increased susceptibility to disinfectants in planktonic state, however, in biofilm state, it may be among the most resistant ones equally important for food as well as for the medical sectors.

Gram-positive bacteria such as *S. aureus*, *B. subtilis*, and *L. monocytogenes* are communicating through inducers encoded by accessory gene regulator (*Agr*) system (**Table 1**). It seems like the *Agr* complex regulates more than 100 genes in the *S. aureus* genome [45], and its deletion from *L. monocytogenes* genome affects more than 600 genes [46].

The accessory gene regulator of *S. aureus* modulates the expression of virulence factors and toxins in response to autoinducing peptides (AIPs) while luxS synthesizes AI-2, which inhibits exopolysaccharide synthesis through an unknown QS cascade [47].

For *S. enterica* and *E. coli*, the QS system is mediated by two genes, *lux*S and *lux*R, homolog to *Sdi*A in order to reach intercellular signaling [48, 49].

The *L. monocytogenes* QS signaling triggers the transcriptional activation of one of the virulence PrfA-regulated genes a*ctA*, resulting in the bacterial aggregation and biofilm formation [10]. Another gene involved in the cell-to-cell interactions is *secA2* gene. Its deletion may inactivate the SecA2 pathway with an increased cell aggregation and sedimentation [50].
